ORCID Profile
0000-0003-0150-123X
Current Organisation
University of Melbourne
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Biodiscovery | Natural Products Chemistry | Microbiology | Biologically Active Molecules | Genetics | Data mining and knowledge discovery | Microbiology | Infectious Agents | Enzymes | Microbial Genetics | Microbial Ecology | Genomics | Environmental Biotechnology | Bioinformatic methods development | Bacteriology | Cell Development, Proliferation and Death | Bacteriology
Expanding Knowledge in the Biological Sciences | Ecosystem Adaptation to Climate Change | Infectious Diseases | Respiratory System and Diseases (incl. Asthma) | Environmentally Sustainable Manufacturing not elsewhere classified | Biofuel (Biomass) Energy | Expanding Knowledge in the Chemical Sciences | Health not elsewhere classified |
Publisher: Springer Science and Business Media LLC
Date: 19-06-2012
Abstract: Mycobacterium ulcerans is an unusual bacterial pathogen with elusive origins. While closely related to the aquatic dwelling M. marinum , M. ulcerans has evolved the ability to produce the immunosuppressive polyketide toxin mycolactone and cause the neglected tropical disease Buruli ulcer. Other mycolactone-producing mycobacteria (MPM) have been identified in fish and frogs and given distinct species designations ( M. pseudoshottsii, M. shinshuense , M. liflandii and M. marinum ), however the evolution of M. ulcerans and its relationship to other MPM has not been defined. Here we report the comparative analysis of whole genome sequences from 30 MPM and five M. marinum . A high-resolution phylogeny based on genome-wide single nucleotide polymorphisms (SNPs) showed that M. ulcerans and all other MPM represent a single clonal group that evolved from a common M. marinum progenitor. The emergence of the MPM was driven by the acquisition of the pMUM plasmid encoding genes for the biosynthesis of mycolactones. This change was accompanied by the loss of at least 185 genes, with a significant overrepresentation of genes associated with cell wall functions. Cell wall associated genes also showed evidence of substantial adaptive selection, suggesting cell wall remodeling has been critical for the survival of MPM. Fine-grain analysis of the MPM complex revealed at least three distinct lineages, one of which comprised a highly clonal group, responsible for Buruli ulcer in Africa and Australia. This indicates relatively recent transfer of M. ulcerans between these continents, which represent the vast majority of the global Buruli ulcer burden. Our data provide SNPs and gene sequences that can differentiate M. ulcerans lineages, suitable for use in the diagnosis and surveillance of Buruli ulcer. M. ulcerans and all mycolactone-producing mycobacteria are specialized variants of a common Mycobacterium marinum progenitor that have adapted to live in restricted environments. Examination of genes lost or retained and now under selective pressure suggests these environments might be aerobic, and extracellular, where slow growth, production of an immune suppressor, cell wall remodeling, loss or modification of cell wall antigens, and biofilm-forming ability provide a survival advantage. These insights will guide our efforts to find the elusive reservoir(s) of M. ulcerans and to understand transmission of Buruli ulcer.
Publisher: Microbiology Society
Date: 06-11-2015
Publisher: MDPI AG
Date: 11-06-2018
DOI: 10.3390/V10060319
Publisher: eLife Sciences Publications, Ltd
Date: 14-06-2022
DOI: 10.7554/ELIFE.77195
Abstract: During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues and is exposed to new selective pressures, thus, potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation however, a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2590 S . aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr -mediated adaptation, we identified non-canonical, genome-wide significant loci including sucA-sucB and stp1 . The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.
Publisher: Cold Spring Harbor Laboratory
Date: 22-03-2023
DOI: 10.1101/2023.03.19.532693
Abstract: Fundamental to effective Legionnaires’ disease outbreak control is the ability to rapidly identify the environmental source(s) of the causative agent, Legionella pneumophila . Genomics has revolutionised pathogen surveillance but L. pneumophila has a complex ecology and population structure that can limit source inference based on standard core genome phylogenetics. Here we present a powerful machine learning approach that assigns the geographical source of Legionnaires’ disease outbreaks more accurately than current core genome comparisons. Models were developed upon 534 L. pneumophila genome sequences, including 149 genomes linked to 20 previously reported Legionnaires’ disease outbreaks through detailed case investigations. Our classification models were developed in a cross-validation framework using only environmental L. pneumophila genomes. Assignments of clinical isolate geographic origins demonstrated high predictive sensitivity and specificity of the models, with no false positives or false negatives for 13 out of 20 outbreak groups, despite the presence of within-outbreak polyclonal population structure. Analysis of the same 534-genome panel with a conventional phylogenomic tree and a core genome multi-locus sequence type allelic distance-based classification approach revealed that our machine learning method had the highest overall classification performance – agreement with epidemiological information. Our multivariate statistical learning approach maximises use of genomic variation data and is thus well-suited for supporting Legionnaires’ disease outbreak investigations.
Publisher: Wiley
Date: 13-11-2007
Publisher: Public Library of Science (PLoS)
Date: 26-04-2005
Publisher: American Society for Microbiology
Date: 16-10-2023
Publisher: American Society for Microbiology
Date: 24-12-2019
Abstract: Staphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus . Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.
Publisher: Elsevier BV
Date: 10-2021
Publisher: eLife Sciences Publications, Ltd
Date: 17-05-2022
Publisher: Cold Spring Harbor Laboratory
Date: 08-08-2023
DOI: 10.1101/2023.08.07.552371
Abstract: Ross River Virus and Barmah Forest Virus infections (alphaviruses) have short incubation periods and are transmitted to humans by mosquitoes. Mycobacterium ulcerans infection (Buruli ulcer) has a much longer incubation period and its mode of transmission is contested. We studied the relationship between month of notification of alphavirus infections and Buruli ulcer in the temperate Australian state of Victoria over the six-year period, 2017-2022. Using cross-correlation, a signal processing technique, we found that a five-month temporal shift in month of Buruli ulcer notification provided optimal alignment with month of alphavirus notification over the six-year period. This closely matches the previously determined 5-month Buruli ulcer incubation period. Inferred transmission of both conditions showed coordinated maxima in summer and autumn and coordinated minima in winter and spring. The close alignment in season of transmission of alphavirus infection and Buruli ulcer in Victoria supports mosquitoes as the primary local vector of M. ulcerans.
Publisher: Public Library of Science (PLoS)
Date: 05-03-2015
Publisher: Springer Science and Business Media LLC
Date: 03-05-2021
DOI: 10.1038/S41467-021-22760-6
Abstract: Mycobacterium kansasii can cause serious pulmonary disease. It belongs to a group of closely-related species of non-tuberculous mycobacteria known as the M. kansasii complex (MKC). Here, we report a population genomics analysis of 358 MKC isolates from worldwide water and clinical sources. We find that recombination, likely mediated by distributive conjugative transfer, has contributed to speciation and on-going ersification of the MKC. Our analyses support municipal water as a main source of MKC infections. Furthermore, nearly 80% of the MKC infections are due to closely-related M. kansasii strains, forming a main cluster that apparently originated in the 1900s and subsequently expanded globally. Bioinformatic analyses indicate that several genes involved in metabolism (e.g., maintenance of the methylcitrate cycle), ESX-I secretion, metal ion homeostasis and cell surface remodelling may have contributed to M. kansasii ’s success and its ongoing adaptation to the human host.
Publisher: Cold Spring Harbor Laboratory
Date: 16-05-2020
DOI: 10.1101/2020.05.12.20099929
Abstract: BACKGROUND: Whole-genome sequencing of pathogens can improve resolution of outbreak clusters and define possible transmission networks. We applied high-throughput genome sequencing of SARS-CoV-2 to 75% of cases in the State of Victoria (population 6.24 million) in Australia. METHODS: Cases of SARS-CoV-2 infection were detected through active case finding and contact tracing. A dedicated SARS-CoV-2 multidisciplinary genomic response team was formed to enable rapid integration of epidemiological and genomic data. Phylodynamic analysis was performed to assess the putative impact of social restrictions. RESULTS: Between 25 January and 14 April 2020, 1,333 COVID-19 cases were reported in Victoria, with a peak in late March. After applying internal quality control parameters, 903 s les were included in genomic analyses. Sequenced s les from Australia were representative of the global ersity of SARS-CoV-2, consistent with epidemiological findings of multiple importations and limited onward transmission. In total, 76 distinct genomic clusters were identified these included large clusters associated with social venues, healthcare facilities and cruise ships. Sequencing of sequential s les from 98 patients revealed minimal intra-patient SARS-CoV-2 genomic ersity. Phylodynamic modelling indicated a significant reduction in the effective viral reproductive number (Re) from 1.63 to 0.48 after the implementation of travel restrictions and population-level physical distancing. CONCLUSIONS: Our data provide a comprehensive framework for the use of SARS-CoV-2 genomics in public health responses. The application of genomics to rapidly identify SARS-CoV-2 transmission chains will become critically important as social restrictions ease globally. Public health responses to emergent cases must be swift, highly focused and effective.
Publisher: PeerJ
Date: 04-05-2018
DOI: 10.7717/PEERJ.4784
Abstract: Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites ( attP and attB , respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages ΦC31 and ΦBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the ΦBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on ΦBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a ΦBT1 integrase-based vector can insert at multiple pseudo- attB sites within a single strain and we determine the sequence of a pseudo- attB motif. These data suggest that ΦBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.
Publisher: American Society for Microbiology
Date: 11-2018
DOI: 10.1128/AAC.00898-18
Abstract: Coagulase-negative staphylococci (CoNS), such as Staphylococcus capitis , are major causes of bloodstream infections in neonatal intensive care units (NICUs). Recently, a distinct clone of S. capitis (designated S. capitis NRCS-A) has emerged as an important pathogen in NICUs internationally.
Publisher: Public Library of Science (PLoS)
Date: 26-07-2021
DOI: 10.1371/JOURNAL.PNTD.0009636
Abstract: Nontuberculosis mycobacterial (NTM) infections are increasing in prevalence across the world. In many cases, treatment options for these infections are limited. However, there has been progress in recent years in the development of new antimycobacterial drugs. Here, we investigate the in vitro activity of SPR719, a novel aminobenzimidazole antibiotic and the active form of the clinical-stage compound, SPR720, against several isolates of Mycobacterium ulcerans , Mycobacterium marinum and Mycobacterium chimaera . We show that SPR719 is active against these NTM species with a MIC range of 0.125–4 μg/ml and that this compares favorably with the commonly utilized antimycobacterial antibiotics, rif icin and clarithromycin. Our findings suggest that SPR720 should be further evaluated for the treatment of NTM infections.
Publisher: American Society for Microbiology
Date: 22-12-2021
DOI: 10.1128/SPECTRUM.01273-21
Abstract: Recent years have seen a surge in interest in the roles of BMVs in host-microbe interactions and interbacterial communication. As a result of such rapid growth in the field, there is a lack of uniformity in BMV enumeration.
Publisher: Massachusetts Medical Society
Date: 09-02-2017
DOI: 10.1056/NEJMC1612023
Publisher: American Society for Microbiology
Date: 02-1997
DOI: 10.1128/AEM.63.2.815-815D.1997
Abstract: Volume 62, no. 9, p. 3386, column 1, last line: "(specific gravity, 2.10) and centrifuging the mixture at 2,000 x g for 10 min" should read "(specific gravity, 1.10) and centrifuging the mixture at 1,050 x g for 10 min." Page 3387, column 1, line 16: "25 cycles at 90(deg)C for 5 min" should read "25 cycles at 90(deg)C for 5 s." [This corrects the article on p. 3385 in vol. 62.].
Publisher: Microbiology Society
Date: 07-2019
Abstract: Vancomycin-resistant Enterococcus faecium (VREfm) is a globally significant public health threat and was listed on the World Health Organization’s 2017 list of high-priority pathogens for which new treatments are urgently needed. Treatment options for invasive VREfm infections are very limited, and outcomes are often poor. Whole-genome sequencing is providing important new insights into VREfm evolution, drug resistance and hospital adaptation, and is increasingly being used to track VREfm transmission within hospitals to detect outbreaks and inform infection control practices. This mini-review provides an overview of recent data on the use of genomics to understand and respond to the global problem of VREfm.
Publisher: Cold Spring Harbor Laboratory
Date: 22-12-2020
DOI: 10.1101/2020.12.22.423893
Abstract: SARS-CoV-2 uses subgenomic (sg)RNA to produce viral proteins for replication and immune evasion. We applied long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA was upregulated earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of ORF1ab containing nsp1 joined to ORF10 and 3’UTR was upregulated at 48 hours post infection in human cell lines. We identified double-junction sgRNA containing both TRS-dependent and independent junctions. We found multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA, and that sgRNA modifications are stable across transcript clusters, host cells and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle. Our results are available via an interactive web-app at coinlab.mdhs.unimelb.edu.au/ .
Publisher: Springer Science and Business Media LLC
Date: 16-07-2010
Abstract: Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I AES-I) has been found to be widespread in CF patients in eastern Australia. Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum s les. We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA ® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum.
Publisher: Springer Science and Business Media LLC
Date: 11-07-2019
DOI: 10.1038/S41467-019-10932-4
Abstract: WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus . WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PAS CYT ) domain of the histidine kinase WalK. Introducing the WalK H271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PAS CYT domain reveal a metal-binding site, in which a zinc ion (Zn 2+ ) is tetrahedrally-coordinated by four amino acids including H271. The WalK H271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn 2+ -binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn 2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.
Publisher: PeerJ
Date: 24-01-2017
DOI: 10.7717/PEERJ.2916
Abstract: From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn 1549 and Tn 916 , transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.
Publisher: American Society for Microbiology
Date: 27-02-2019
Abstract: The study results reported here perfectly demonstrate the power and promise of clinical metagenomics to recover genome sequences of important drug-resistant bacteria and to rapidly provide rich data that inform outbreak investigations and treatment decisions, independently of the need to culture the organisms.
Publisher: Springer Science and Business Media LLC
Date: 22-03-2018
DOI: 10.1186/S13756-018-0335-Z
Abstract: Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of hospital-acquired infections. New, presumably better-adapted strains of VRE appear unpredictably it is uncertain how they spread despite improved infection control. We aimed to investigate the relatedness of a novel sequence type (ST) of vanB E. faecium - ST796 - very near its time of origin from hospitals in three Australian states and New Zealand. Following near-simultaneous outbreaks of ST796 in multiple institutions, we gathered then tested colonization and bloodstream infection isolates’ antimicrobial resistance (AMR) phenotypes, and phylogenomic relationships using whole genome sequencing (WGS). Patient meta-data was explored to trace the spread of ST796. A novel clone of vanB E. faecium (ST796) was first detected at one Australian hospital in late 2011, then in two New Zealand hospitals linked by inter-hospital transfers from separate Melbourne hospitals. ST796 also appeared in hospitals in South Australia and New South Wales and was responsible for at least one major colonization outbreak in a Neonatal Intensive Care Unit without identifiable links between centers. No exceptional AMR was detected in the isolates. While WGS analysis showed very limited ersity at the core genome, consistent with recent emergence of the clone, clustering by institution was observed. Evolution of new E. faecium clones, followed by recognized or unrecognized movement of colonized in iduals then rapid intra-institutional cross-transmission best explain the multi-center, multistate and international outbreak we observed.
Publisher: Cold Spring Harbor Laboratory
Date: 16-07-2021
DOI: 10.1101/2021.07.16.452633
Abstract: Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily toxin driven, leading to the suggestion that hyper-virulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the toxic activity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced toxicity. Interestingly, the majority of isolates that were attenuated in toxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.
Publisher: Wiley
Date: 06-04-2005
Publisher: American Association for the Advancement of Science (AAAS)
Date: 03-01-2023
DOI: 10.1126/SCISIGNAL.ABJ8194
Abstract: Staphylococcus aureus can cause infections that are often chronic and difficult to treat, even when the bacteria are not antibiotic resistant because most antibiotics act only on metabolically active cells. Subpopulations of persister cells are metabolically quiescent, a state associated with delayed growth, reduced protein synthesis, and increased tolerance to antibiotics. Serine-threonine kinases and phosphatases similar to those found in eukaryotes can fine-tune essential bacterial cellular processes, such as metabolism and stress signaling. We found that acid stress–mimicking conditions that S. aureus experiences in host tissues delayed growth, globally altered the serine and threonine phosphoproteome, and increased threonine phosphorylation of the activation loop of the serine-threonine protein kinase B (PknB). The deletion of stp , which encodes the only annotated functional serine-threonine phosphatase in S. aureus , increased the growth delay and phenotypic heterogeneity under different stress challenges, including growth in acidic conditions, the intracellular milieu of human cells, and abscesses in mice. This growth delay was associated with reduced protein translation and intracellular ATP concentrations and increased antibiotic tolerance. Using phosphopeptide enrichment and mass spectrometry–based proteomics, we identified targets of serine-threonine phosphorylation that may regulate bacterial growth and metabolism. Together, our findings highlight the importance of phosphoregulation in mediating bacterial quiescence and antibiotic tolerance and suggest that targeting PknB or Stp might offer a future therapeutic strategy to prevent persister formation during S. aureus infections.
Publisher: Cold Spring Harbor Laboratory
Date: 10-10-2019
DOI: 10.1101/800250
Abstract: The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans . There is no effective BU vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl-reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described TLR-2 agonist-based lipopeptide adjuvant, R 4 Pam2Cys. Mice were vaccinated and then challenged via tail inoculation with 14-20 colony forming units (CFU) of an engineered bioluminescent strain of M. ulcerans . Mice receiving either the experimental ER vaccine or Mycobacterium bovis Bacille Calmette-Guérin (BCG) were equally well protected, with both groups faring significantly better than non-vaccinated animals ( p .05). A suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were then used to interrogate the immune response data to develop disease-prognostic models. High levels of IL-2 and low IFN -γ produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression then revealed vaccine-specific profiles of protection. High titres of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the experimental ER vaccine. In contrast, high titres of IL-6, TNF -α , IFN -γ and IL-10 in the DLN and low IFN γ titres in the spleen were associated with protection following BCG vaccination. This study suggests an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.
Publisher: Springer Science and Business Media LLC
Date: 22-06-2022
DOI: 10.1038/S41467-022-31177-8
Abstract: Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA–RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA–RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA–mRNA interactions are recovered and we find that an mRNA encoding a long 3′ untranslated region (UTR) (termed vigR 3′UTR) functions as a regulatory ‘hub’ within the RNA–RNA interaction network. We demonstrate that the vigR 3′UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA–mRNA base-pairing. Deletion of the vigR 3′UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3′UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.
Publisher: Cold Spring Harbor Laboratory
Date: 18-11-2015
DOI: 10.1101/032151
Abstract: The lipopolysaccharide (O) and flagellar (H) surface antigens of Escherichia coli are targets for serotyping that have traditionally been used to identify pathogenic lineages of E. coli. As serotyping has several limitations, public health reference laboratories are increasingly moving towards whole genome sequencing (WGS) for the rapid characterisation of bacterial isolates. Here we present a method to rapidly and accurately serotype E. coli isolates from raw, short read sequence data, leveraging the known genetic basis for the biosynthesis of O- and H-antigens. Our approach bypasses the need for de novo genome assembly by directly screening WGS reads against a curated database of alleles linked to known E. coli O-groups and H-types (the EcOH database) using the software package SRST2. We validated our approach by comparing in silico results with those obtained via serological phenotyping of 197 enteropathogenic (EPEC) isolates. We also demonstrated the utility of our method to characterise enterotoxigenic E. coli (ETEC) and the uropathogenic E. coli (UPEC) epidemic clone ST131, and for in silico serotyping of foodborne outbreak-related isolates in the public GenomeTrakr database.
Publisher: Wiley
Date: 11-10-2010
DOI: 10.1111/J.1365-2958.2010.07401.X
Abstract: Mycobacterium ulcerans is the causative agent of the debilitating skin disease Buruli ulcer, which is most prevalent in Western and Central Africa. M. ulcerans shares >98% DNA sequence identity with Mycobacterium marinum, however, M. marinum produces granulomatous, but not ulcerative, lesions in humans and animals. Here we report the differential expression of a small heat shock protein (Hsp18) between strains of M. ulcerans (Hsp18(+) ) and M. marinum (Hsp18(-) ) and describe the molecular basis for this difference. We show by gene deletion and GFP reporter assays in M. marinum that a ergently transcribed gene called hspR_2, immediately upstream of hsp18, encodes a MerR-like regulatory protein that represses hsp18 transcription while promoting its own expression. Naturally occurring mutations within a 70 bp segment of the 144 bp hspR_2-hsp18 intergenic region among M. ulcerans strains inhibit hspR_2 transcription and explain the Hsp18(+) phenotype. We also propose a biological role for Hsp18, as we show that this protein significantly enhances bacterial attachment or aggregation during biofilm formation. This study has uncovered a new member of the MerR family of transcriptional regulators and suggests that upregulation of hsp18 expression was an important pathoadaptive response in the evolution of M. ulcerans from a M. marinum-like ancestor.
Publisher: Public Library of Science (PLoS)
Date: 10-06-2010
Publisher: eLife Sciences Publications, Ltd
Date: 14-04-2023
DOI: 10.7554/ELIFE.84983
Abstract: Buruli ulcer (BU) is a neglected tropical disease caused by infection of subcutaneous tissue with Mycobacterium ulcerans . BU is commonly reported across rural regions of Central and West Africa but has been increasing dramatically in temperate southeast Australia around the major metropolitan city of Melbourne, with most disease transmission occurring in the summer months. Previous research has shown that Australian native possums are reservoirs of M. ulcerans and that they shed the bacteria in their fecal material (excreta). Field surveys show that locales where possums harbor M. ulcerans overlap with human cases of BU, raising the possibility of using possum excreta surveys to predict the risk of disease occurrence in humans. We thus established a highly structured 12 month possum excreta surveillance program across an area of 350 km 2 in the Mornington Peninsula area 70 km south of Melbourne, Australia. The primary objective of our study was to assess using statistical modeling if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations. Over two s ling c aigns in summer and winter, we collected 2,282 possum excreta specimens of which 11% were PCR positive for M. ulcerans -specific DNA. Using the spatial scanning statistical tool SaTScan , we observed non-random, co-correlated clustering of both M. ulcerans positive possum excreta and human BU cases. We next trained a statistical model with the Mornington Peninsula excreta survey data to predict the future likelihood of human BU cases occurring in the region. By observing where human BU cases subsequently occurred, we show that the excreta model performance was superior to a null model trained using the previous year’s human BU case incidence data (AUC 0.66 vs 0.55). We then used data unseen by the excreta-informed model from a new survey of 661 possum excreta specimens in Geelong, a geographically separate BU endemic area to the southwest of Melbourne, to prospectively predict the location of human BU cases in that region. As for the Mornington Peninsula, the excreta-based BU prediction model outperformed the null model (AUC 0.75 vs 0.50) and pinpointed specific locations in Geelong where interventions could be deployed to interrupt disease spread. This study highlights the One Health nature of BU by confirming a quantitative relationship between possum excreta shedding of M. ulcerans and humans developing BU. The excreta survey-informed modeling we have described will be a powerful tool for the efficient targeting of public health responses to stop BU. This research was supported by the National Health and Medical Research Council of Australia and the Victorian Government Department of Health (GNT1152807 and GNT1196396).
Publisher: PeerJ
Date: 03-01-2028
DOI: 10.7717/PEERJ.4210
Abstract: Until recently, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks. All KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids. Initial investigations indicated that the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates ( K. pneumoniae 92%) identified from 35 different locations across metropolitan and rural Victoria between 2012 and 2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host ersity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation. A real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase-producing Enterobacteriaceae surveillance and management.
Publisher: Wiley
Date: 13-09-2023
Abstract: Rhizonin A and B are hepatotoxic cyclopeptides produced by bacterial endosymbionts (Mycetohabitans endofungorum) of the fungus Rhizopus microsporus. Their toxicity critically depends on the presence of 3‐furylalanine (Fua) residues, which also occur in pharmaceutically relevant cyclopeptides of the endolide and bingchamide families. The biosynthesis and incorporation of Fua by non‐ribosomal peptide synthetases (NRPS), however, has remained elusive. By genome sequencing and gene inactivation we elucidated the gene cluster responsible for rhizonin biosynthesis. A suite of isotope labeling experiments identified tyrosine and l‐DOPA as Fua precursors and provided the first mechanistic insights. Bioinformatics, mutational analysis and heterologous reconstitution identified dioxygenase RhzB as necessary and sufficient for Fua formation. RhzB is a novel type of heme‐dependent aromatic oxygenases (HDAO) that enabled the discovery of the bingchamide biosynthesis gene cluster through genome mining.
Publisher: Oxford University Press (OUP)
Date: 06-03-2018
DOI: 10.1093/JAC/DKY064
Abstract: Enterococcus faecium is an important nosocomial pathogen. It has a high propensity for horizontal gene transfer, which has resulted in the emergence of MDR strains that are difficult to treat. The most notorious of these, vancomycin-resistant E. faecium, are usually treated with linezolid or daptomycin. Resistance has, however, been reported, meaning that new therapeutics are urgently needed. The 1,2,4-oxadiazoles are a recently discovered family of antimicrobials that are active against Gram-positive pathogens and therefore have therapeutic potential for treating E. faecium. However, only limited data are available on the activity of these antimicrobials against E. faecium. To determine whether the 1,2,4-oxadiazole antimicrobials are active against MDR and daptomycin-non-susceptible E. faecium. The activity of the 1,2,4-oxadiazole antimicrobials against vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium was determined using susceptibility testing, time-kill assays and synergy assays. Toxicity was also evaluated against human cells by XTT and haemolysis assays. The 1,2,4-oxadiazoles are active against a range of MDR E. faecium, including isolates that display non-susceptibility to vancomycin and daptomycin. This class of antimicrobial displays rapid bactericidal activity and demonstrates superior killing of E. faecium compared with daptomycin. Finally, the 1,2,4-oxadiazoles act synergistically with daptomycin against E. faecium, with subinhibitory concentrations reducing the MIC of daptomycin for non-susceptible isolates to a level below the clinical breakpoint. The 1,2,4-oxadiazoles are active against MDR and daptomycin-non-susceptible E. faecium and hold great promise as future therapeutics for treating infections caused by these difficult-to-treat isolates.
Publisher: AMPCo
Date: 2007
DOI: 10.5694/J.1326-5377.2007.TB00801.X
Abstract: Mycobacterium ulcerans is a slow-growing environmental bacterium that causes Buruli ulcer (also known as Bairnsdale ulcer in Victoria and Daintree ulcer in northern Queensland). We describe two patients with laboratory-confirmed Buruli ulcer who were infected either in New South Wales or overseas. A molecular epidemiological investigation demonstrated that, while one case was probably acquired in Papua New Guinea, the other was most likely to have been acquired in southern NSW. To our knowledge, this is the first case of M. ulcerans infection acquired in NSW.
Publisher: Elsevier BV
Date: 02-2023
Publisher: Wiley
Date: 11-02-2019
Abstract: The nargenicin family of antibiotics are macrolides containing a rare ether-bridged cis-decalin motif. Several of these compounds are highly active against multi-drug resistant organisms. Despite the identification of the first members of this family almost 40 years ago, the genetic basis for the production of these molecules and the enzyme responsible for formation of the oxa bridge, remain unknown. Here, the 85 kb nargenicin biosynthetic gene cluster was identified from a human pathogenic Nocardia arthritidis isolate and this locus is solely responsible for nargenicin production. Further investigation of this locus revealed a putative iron-α-ketoglutarate-dependent dioxygenase, which was found to be responsible for the formation of the ether bridge from the newly identified deoxygenated precursor, 8,13-deoxynargenicin. Uncovering the nargenicin biosynthetic locus provides a molecular basis for the rational bioengineering of these interesting antibiotic macrolides.
Publisher: Proceedings of the National Academy of Sciences
Date: 09-09-2021
Abstract: Soil is a complex and competitive environment, forcing its inhabitants to develop strategies against competitors, predators, and pathogens. Identifying and understanding the molecular mechanisms has translational value for medicine, ecology, and agriculture. In this study, we show that a member of important soil-dwelling fungi ( Mortierella ) forms a tight alliance with toxin-producing bacteria ( Mycoavidus ) that live within the fungal hyphae and protect their host from nematode attack. This discovery is relevant since Mortierella species correlate with healthy soils and are used as plant growth–promoting fungi in agriculture. Unraveling an ecological role for fungal endosymbionts in Mortierella , our results contribute to the understanding of a mainspring in fungal–endobacterial symbioses and open the possibility for the development of new biocontrol agents.
Publisher: Cold Spring Harbor Laboratory
Date: 22-05-2019
DOI: 10.1101/644856
Abstract: Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been h ered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here we establish the complete genomes and methylomes for seven clinically significant, genetically erse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus . Our analyses revealed marked differences in the gene arrangement, chromosomal location and movement of type I RM systems between the two species. Unlike S. aureus , S. epidermidis type I RM systems demonstrate extensive ersity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis . Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidis hsdMS , we readily overcame restriction barriers in S. epidermidis , and achieved transformation efficiencies equivalent to those of modification deficient mutants. With these functional experiments we demonstrate how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be transformation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis from erse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction defective, clinically significant, multidrug-resistant ST2 isolate as an ideal candidate for molecular studies. Staphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections has been hindered by an inability to genetically manipulate “hospital-adapted” strains that cause clinical disease. Here we provide the first comprehensive analyses of the mechanisms whereby S. epidermidis resists the uptake of foreign DNA and demonstrate that these are distinct from those described for S. aureus. Until now it had been assumed that these are the same. Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinically relevant isolates for the first time.
Publisher: Public Library of Science (PLoS)
Date: 10-11-2011
Publisher: Cold Spring Harbor Laboratory
Date: 30-04-2020
DOI: 10.1101/2020.04.28.067363
Abstract: 2. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. To establish and validate a reverse transcription loop-mediated isothermal lification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. We used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 μL of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65°C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical s les of 14 minutes (SD ±7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID 50 mL −1 ), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. With a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19. 3. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.
Publisher: American Society for Microbiology
Date: 11-02-2020
DOI: 10.1128/MSYSTEMS.00665-19
Abstract: Bacterial small RNAs (sRNAs) are RNA molecules that can have important regulatory roles across gene expression networks. There is a growing understanding of the scope and potential breadth of impact of sRNAs on global gene expression patterns in Staphylococcus aureus , a major human pathogen. Here, transcriptome comparisons were used to examine the roles of sRNA genes with a potential role in the response of S. aureus to antibiotic exposure. Although no measurable impact on key bacterial phenotypes was observed after deleting each of 18 sRNAs identified by these comparisons, this research is significant because it underscores the subtle modes of action of these sometimes abundant molecules within the bacterium.
Publisher: Oxford University Press (OUP)
Date: 20-12-2017
DOI: 10.1093/JAC/DKW473
Publisher: American Society for Microbiology
Date: 27-02-2019
Abstract: Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans . The disease is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Currently, the major hurdles facing disease control are incomplete understandings of both the mode of transmission and environmental reservoirs of M. ulcerans . As decades of spasmodic environmental s ling surveys have not brought us much closer to overcoming these hurdles, the Buruli ulcer research community has recently switched to using comparative genomics. The significance of our research is in how we used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our approach shows that it might be possible to use bacterial population genomics to assess the impact of health interventions, providing valuable feedback for managers of disease control programs in areas where health surveillance infrastructure is poor.
Publisher: American Society for Microbiology
Date: 09-1996
DOI: 10.1128/AEM.62.9.3385-3390.1996
Abstract: Current methods for detection of Cryptosporidium parvum oocysts in water are time-consuming and difficult. We have developed a reverse transcription (RT)-PCR which can detect the presence of a single viable oocyst spiked into concentrated environmental water s les. The test is based on the detection of mRNA from a C. parvum heat shock protein (hsp). The synthesis of hsp was induced by a short 45 degrees C incubation followed by oocyst lysis by a freeze-thaw process. Hsp70 mRNA, produced only from viable oocysts, was then isolated by hybridization to oligo(dT)25-coated magnetic beads. Detection was achieved by RT-PCR lification of a 590-bp region of hsp70 mRNA specific for C. parvum. To test the method, s les of reticulated, reservoir, bore, and river water were concentrated by chemical flocculation and Percoll-sucrose gradient centrifugation and then spiked with dilutions of oocysts. In all four of the water types examined, the detection of single oocysts was possible by RT-PCR combined with Southern hybridization. RT-PCR products were not obtained from formalin-inactivated oocysts. An RNA internal positive control fragment was synthesized that was included with each reaction to guard against RT-PCR false-negative results that may be caused by the presence of inhibitory substances. However, when the magnetic beads were used to extract and concentrate mRNA, no inhibition was observed. The technique is versatile, straightforward, and rapid (1 day) and provides a sensitive and economic means of screening concentrated water s les for the presence of C. parvum.
Publisher: Elsevier BV
Date: 04-2012
DOI: 10.1016/J.MEEGID.2012.01.018
Abstract: Mycobacterium ulcerans (M. ulcerans) causes a devastating infection of the skin and underlying tissue commonly known as Buruli ulcer (BU). Genetic analyses indicate that M. ulcerans has a common ancestor with Mycobacterium marinum (M. marinum) and has erged from this fish and human pathogen perhaps around a million years ago. M. ulcerans is characterized by minimal genetic ersity and since it has a highly clonal population structure, genetic differences between in idual isolates reflect changes that have occurred sequentially from their respective progenitors. This feature, which is shared by other bacterial pathogens with low sequence ersity, such as Yersinia pestis and Bordetella pertussis renders M. ulcerans a promising model to reveal evolutionary mechanisms. Until today transmission pathways and environmental reservoirs of M. ulcerans are not entirely explored. However, comparative genome analysis of closely related M. ulcerans isolates is anticipated to give deeper insights into the population structure of this enigmatic mycobacterium.
Publisher: Wiley
Date: 03-08-2014
DOI: 10.1111/MMI.12720
Abstract: Recent advances in genomics and molecular biology are providing an excellent opportunity to get a glimpse into the past, to examine the present, and to predict the future evolution of pathogenic mycobacteria, and in particular that of Mycobacterium tuberculosis, the agent of human tuberculosis. The recent availability of genome sequences of several Mycobacterium canettii strains, representing evolutionary early-branching tubercle bacilli, has allowed the genomic and molecular features of the putative ancestor of the M. tuberculosis complex (MTBC) to be reconstituted. Analyses have identified extensive lateral gene transfer and recombination events in M. canettii and/or the MTBC, leading to suggestions of a past environmental reservoir where the ancestor(s) of the tubercle bacilli might have adapted to an intracellular lifestyle. The daily increases in M. tuberculosis genome data and the remaining urgent Public Health problem of tuberculosis make it more important than ever to try and understand the origins and the future evolution of the MTBC. Here we critically discuss a series of questions on gene-loss, acquisition, recombination, mutation and conservation that have recently arisen and which are key to better understand the outstanding evolutionary success of one of the most widespread and most deadly bacterial pathogens in the history of humankind.
Publisher: Elsevier BV
Date: 09-2016
Publisher: Cold Spring Harbor Laboratory
Date: 21-07-2023
DOI: 10.1101/2023.07.21.548488
Abstract: Hepatitis A virus (HAV) infections are an increasing public health concern in low-endemicity regions due to outbreaks from foodborne infections and sustained transmission among vulnerable groups, including persons experiencing homelessness, those who inject drugs, and men who have sex with men (MSM), which is further compounded by aging, unvaccinated populations. DNA sequence characterisation of HAV for source tracking is performed by comparing small subgenomic regions of the virus. While this approach has been successful when robust epidemiological data are available, poor genetic resolution can lead to conflation of outbreaks with sporadic cases. HAV outbreak investigations would greatly benefit from the additional phylogenetic resolution obtained by whole virus genome sequence comparisons. However, HAV genomic approaches can be difficult because of challenges in isolating the virus, low sensitivity of direct metagenomic sequencing in complex s le matrices like various foods such as fruits, vegetables and molluscs, and difficulty designing highly multiplexed PCR primers across erse HAV genotypes. Here, we introduce a proof-of-concept pan- HAV oligonucleotide hybrid capture enrichment assay from serum and frozen berry specimens that yields complete and near-complete HAV genomes from as few as four input HAV genome copies. We used this method to recover HAV genomes from human serum specimens with high Cτ values (34·7—42·7), with high assay performance for all six human HAV sub-genotypes, both contemporary and historical. Our approach provides a highly sensitive and streamlined workflow for HAV WGS from erse s le types, that can be the basis for harmonised and high-resolution molecular epidemiology during HAV outbreak surveillance. This proof-of-concept study introduces a hybrid capture oligo panel for whole genome sequencing (WGS) of all six human pathogenic hepatitis A virus (HAV) sub-genotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging s le matrices can be accommodated, making it suitable for broad implementation in clinical and public health laboratories. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations, as well as our understanding of the global ersity and transmission dynamics of HAV.
Publisher: American Chemical Society (ACS)
Date: 04-12-2019
DOI: 10.1021/ACSCHEMBIO.9B00805
Abstract: Genome mining identified the fungal-bacterial endosymbiosis
Publisher: American Society for Microbiology
Date: 07-2015
Abstract: Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates of S. aureus . Here, we describe the construction and characterization of the IMXXB series of Escherichia coli strains that mimic the type I adenine methylation profiles of S. aureus clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages. IMPORTANCE The genetic manipulation of clinical S. aureus isolates has been h ered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus . The IMXXB series of E. coli strains should expedite the process of mutant construction in erse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus .
Publisher: Cold Spring Harbor Laboratory
Date: 28-02-2018
DOI: 10.1101/273904
Abstract: Large-scale genomic studies of within-host evolution during Staphylococcus aureus bacteraemia (SAB) are needed to understanding bacterial adaptation underlying persistence and thus refining the role of genomics in management of SAB. However, available comparative genomic studies of sequential SAB isolates have tended to focus on selected cases of unusually prolonged bacteraemia, where secondary antimicrobial resistance has developed. To understand the bacterial genomic evolution during SAB more broadly, we applied whole genome sequencing to a large collection of sequential isolates obtained from patients with persistent or relapsing bacteraemia. We show that, while adapation pathways are heterogenous and episode-specific, isolates from persistent bacteraemia have a distinctive molecular signature, characterised by a low mutation frequency and high proportion of non-silent mutations. By performing an extensive analysis of structural genomic variants in addition to point mutations, we found that these often overlooked genetic events are commonly acquired during SAB. We discovered that IS 256 insertion may represent the most effective driver of within-host microevolution in selected lineages, with up to three new insertion events per isolate even in the absence of other mutations. Genetic mechanisms resulting in significant phenotypic changes, such as increases in vancomycin resistance, development of small colony phenotypes, and decreases in cytotoxicity, included mutations in key genes ( rpoB, stp, agrA ) and an IS256 insertion upstream of the walKR operon. This study provides for the first time a large-scale analysis of within-host evolution during invasive S. aureus infection and describes specific patterns of adaptation that will be informative for both understanding S. aureus pathoadaptation and utilising genomics for management of complicated S. aureus infections.
Publisher: Microbiology Society
Date: 03-08-2015
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.MEEGID.2013.03.047
Abstract: Resistance to new antimicrobials is generally recognized in Staphylococcus aureus soon after they are released for clinical use. In the case of vancomycin, which was first released in the 1950s, resistance was not reported until the mid 1990s, with the description of vancomycin-intermediate S. aureus (VISA), and heterogenous-VISA (hVISA). Unraveling the complex genetic and cell wall structural changes conferring low-level vancomycin resistance in S. aureus has proved challenging. However the recent advances in high throughput whole-genome sequencing has played a key role in determining the breadth of bacterial chromosomal changes linked with resistance. Diverse mutations in a small number of staphylococcal regulatory genes, in particular walKR, graRS, vraSR and rpoB, have been associated with hVISA and VISA. Only a small number of these mutations have been experimentally proven to confer the resistance phenotype and some of these only partially contribute to resistance. It also appears that the evolution of VISA from VSSA is a step-wise process. Transcriptomics studies, and analysis of host pathogen interactions, indicate that the evolution of vancomycin-susceptible S. aureus to VISA is associated not only with antibiotic resistance, but with other changes likely to promote persistent infection. These include predicted alterations in central metabolism, altered expression of virulence associated factors, attenuated virulence in vivo, and alterations in susceptibility to host innate immune responses, together with reduced susceptibility to other antibiotics. In fact, current data suggests that hVISA and VISA represent a bacterial evolutionary state favoring persistence in the face of not only antibiotics, but also the host environment. The additional knowledge of staphylococcal biology that has been uncovered during the study of hVISA and VISA is significant. The present review will detail the current understanding of the evolutionary process in the generation of hVISA and VISA, and explore the erse additional changes that occur in these strains.
Publisher: Wiley
Date: 17-09-2020
Publisher: Cold Spring Harbor Laboratory
Date: 16-05-2016
DOI: 10.1101/053728
Abstract: Alcohol-based hand rubs are international pillars of hospital infection control, restricting transmission of pathogens such as Staphylococcus aureus . Despite this success, health care infections caused by Enterococcus faecium (Efm) - another multidrug resistant pathogen - are increasing. We tested alcohol tolerance of 139 hospital Efm isolates, obtained between 1997 and 2015 and found Efm post-2010 were 10-fold more tolerant to alcohol killing than older isolates. Using a mouse infection control model, we then showed that alcohol tolerant Efm resisted standard 70% isopropanol surface disinfection and led to gastrointestinal colonization significantly more often than alcohol sensitive Efm. We next looked for bacterial genomic signatures of adaptation. Tolerant Efm have independently accumulated mutations modifying genes involved in carbohydrate uptake and metabolism. Mutagenesis confirmed their roles in isopropanol tolerance. These findings suggest bacterial adaptation and complicate infection control recommendations. Additional policies and procedures to prevent Efm spread are required.
Publisher: Springer Science and Business Media LLC
Date: 09-2020
DOI: 10.1038/S41467-020-18314-X
Abstract: Genomic sequencing has significant potential to inform public health management for SARS-CoV-2. Here we report high-throughput genomics for SARS-CoV-2, sequencing 80% of cases in Victoria, Australia (population 6.24 million) between 6 January and 14 April 2020 (total 1,333 COVID-19 cases). We integrate epidemiological, genomic and phylodynamic data to identify clusters and impact of interventions. The global ersity of SARS-CoV-2 is represented, consistent with multiple importations. Seventy-six distinct genomic clusters were identified, including large clusters associated with social venues, healthcare and cruise ships. Sequencing sequential s les from 98 patients reveals minimal intra-patient SARS-CoV-2 genomic ersity. Phylodynamic modelling indicates a significant reduction in the effective viral reproductive number ( R e ) from 1.63 to 0.48 after implementing travel restrictions and physical distancing. Our data provide a concrete framework for the use of SARS-CoV-2 genomics in public health responses, including its use to rapidly identify SARS-CoV-2 transmission chains, increasingly important as social restrictions ease globally.
Publisher: Wiley
Date: 20-03-2009
Abstract: In contrast to the great majority of mycobacterial species that are harmless saprophytes, Mycobacterium tuberculosis and other closely related tubercle bacilli have evolved to be among the most important human and animal pathogens. The need to develop new strategies in the fight against tuberculosis (TB) and related diseases has fuelled research into the evolutionary success of the M. tuberculosis complex members. Amongst the various disciplines, genomics and functional genomics have been instrumental in improving our understanding of these organisms. In this review we will present some of the recent key findings on molecular determinants of mycobacterial pathogenicity and attenuation, the evolution of M. tuberculosis, genome dynamics, antigen mining for improved diagnostic and subunit antigens, and finally the identification of novel drug targets. The genomics revolution has changed the landscape of TB research, and now underpins our renewed efforts to defeat this deadly pathogen.
Publisher: Public Library of Science (PLoS)
Date: 13-05-2015
Publisher: Cold Spring Harbor Laboratory
Date: 17-02-2020
DOI: 10.1101/2020.02.16.951533
Abstract: Mycobacterium ulcerans is the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenic M. ulcerans proteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU. Recombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged with M. ulcerans via subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice. The MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG 1 . However, all mice developed ulcers by day-40 post- M. ulcerans challenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals. These data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU.
Publisher: Wiley
Date: 08-2008
Abstract: Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel-based and gel-free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein-coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild-type strain and an isogenic mycolactone-deficient mutant, by 2-DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.
Publisher: Cold Spring Harbor Laboratory
Date: 14-08-2017
DOI: 10.1101/175950
Abstract: Until recently, KPC-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks. All KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing (WGS) was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids. Initial investigations indicated the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates ( K. pneumoniae 92%) identified from 35 different locations across metropolitan and rural Victoria between 2012-2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host ersity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation. A real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase producing Enterobacteriaceae management.
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 07-2007
Publisher: American Society for Microbiology
Date: 05-2012
DOI: 10.1128/JCM.06460-11
Abstract: This work reports the design and evaluation of a rapid loop-mediated isothermal lification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS 2404 . The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions.
Publisher: Wiley
Date: 05-08-2019
Abstract: Siderophores are key players in bacteria–host interactions, with the main function to provide soluble iron for their producers. Gramibactin from rhizosphere bacteria expands siderophore function and ersity as it delivers iron to the host plant and features an unusual diazeniumdiolate moiety for iron chelation. By mutational analysis of the grb gene cluster, we identified genes (grbD and grbE) necessary for diazeniumdiolate formation. Genome mining using a GrbD‐based network revealed a broad range of orthologous gene clusters in mainly plant‐associated Burkholderia/Paraburkholderia species. Two new types of diazeniumdiolate siderophores, megapolibactins and plantaribactin were fully characterized. In vitro assays and in vivo monitoring experiments revealed that the iron chelators also liberate nitric oxide (NO) in plant roots. This finding is important since NO donors are considered as biofertilizers that maintain iron homeostasis and increase overall plant fitness.
Publisher: Elsevier BV
Date: 2021
DOI: 10.2139/SSRN.3750715
Publisher: Microbiology Society
Date: 03-2005
Abstract: The presence of a 174 kb plasmid called pMUM001 in Mycobacterium ulcerans , the first ex le of a mycobacterial plasmid encoding a virulence determinant, was recently reported. Over half of pMUM001 is devoted to six genes, three of which encode giant polyketide synthases (PKS) that produce mycolactone, an unusual cytotoxic lipid produced by M. ulcerans . In this present study the remaining 75 non-PKS-associated protein-coding sequences (CDS) are analysed and it is shown that pMUM001 is a low-copy-number element with a functional ori that supports replication in Mycobacterium marinum but not in the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum . Sequence analyses revealed a highly mosaic plasmid gene structure that is reminiscent of other large plasmids. Insertion sequences (IS) and fragments of IS, some previously unreported, are interspersed among functional gene clusters, such as those genes involved in plasmid replication, the synthesis of mycolactone, and a potential phosphorelay signal transduction system. Among the IS present on pMUM001 were multiple copies of the high-copy-number M. ulcerans elements IS 2404 and IS 2606 . No plasmid transfer systems were identified, suggesting that trans -acting factors are required for mobilization. The results presented here provide important insights into this unusual virulence plasmid from an emerging but neglected human pathogen.
Publisher: Springer Science and Business Media LLC
Date: 27-09-2022
Publisher: Cold Spring Harbor Laboratory
Date: 08-03-2023
DOI: 10.1101/2023.03.06.531458
Abstract: Daptomycin is a last-resort antibiotic used for treatment of infections caused by Gram-positive antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations, however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and β-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that the improved survival of the clpP mutant strain during daptomycin treatment was associated with reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10 × MIC daptomycin resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP , favor antibiotic resistance over virulence gene expression. The bacterium Staphylococcus aureus is a leading cause of life-threatening infections and treatment is challenged by the worldwide dissemination of methicillin-resistant Staphylococcus aureus (MRSA) that are multi-drug resistant. Daptomycin, a cell membrane-targeting cationic lipopeptide, is one of the few antibiotics with activity against MRSA, however, the killing mechanism of daptomycin and the mechanisms leading to resistance are not fully understood. Here we show than an MRSA strain, isolated from the blood of a patient treated with daptomycin, has acquired a mutation that inactivates the ClpXP protease resulting in increased resistance to several antibiotics and diminished expression of virulence genes. Super resolution microscopy showed that the mutant avoids daptomycin-elicited killing by preventing the binding of the antibiotic to the septal site and by growing into a rod-shaped morphology. In summary, this study discloses new perspectives on the mechanism of killing and the mechanism of resistance to an antibiotic of last resort.
Publisher: Research Square Platform LLC
Date: 10-2020
DOI: 10.21203/RS.3.RS-78954/V1
Abstract: Mycobacterium kansasii is a nontuberculous mycobacterium that can cause serious pulmonary disease. Genotyping suggested that the species is composed of at least six subtypes that vary in clinical significance, with subtype I being clinically dominant but less commonly isolated from environmental sources. Here we report a population genomics study of 358 M. kansasii isolates obtained from global water and clinical sources. Phylogenomic analyses revealed that the six subtypes are more accurately designated as closely related subspecies. These subspecies show le evidence of recombination mediated by distributive conjugative transfer that has contributed to subspeciation and on-going ersification. Water was confirmed as a source of clinical infections by showing that genomes of clinical strains from an Australian outbreak were almost indistinguishable from strains contaminating the drinking water supply. Most clinical infections (nearly 80%) were due to a recently emerged group of strains designated the M. kansasii main complex (MKMC), which appears to have originated in Europe in 1900s and expanded globally over the past century. Comparative genomic analyses revealed that the MKMC has maintained the methylcitrate cycle and expanded ESX-I secretion-associated proteins, perhaps facilitating metabolic adaptation and pathogenicity for human hosts. Evidence of on-going positive selection in isolates of the MKMC was found in genes involved in carbon and secondary metabolism, metal ion homeostasis and cell surface remodeling that could represent adaptation to human hosts. These results further our understanding of the epidemiology and pathogenicity of M. kansasii and emphasize the importance of monitoring its potential transition to a more human-adapted pathogen.
Publisher: Microbiology Society
Date: 20-09-2016
Publisher: Public Library of Science (PLoS)
Date: 29-07-2021
DOI: 10.1371/JOURNAL.PNTD.0009601
Abstract: Mosquito control interventions are widely used to reduce mosquito-borne diseases. It is unclear what combination of interventions are most effective in reducing human disease. A novel intervention study for Buruli ulcer targeting mosquito vectors was proposed for a Buruli ulcer-endemic area of Victoria, Australia. The local community expressed a preference for avoiding widespread residual spraying of pyrethroids. To inform the design of a future cluster randomised control study (cRCT) for Buruli ulcer prevention in Victoria, we conducted a systematic literature review. The aim was to describe cRCT designs which investigated interventions other than non-targeted insecticide for reducing mosquito-borne disease transmission, and comment on the strengths and weaknesses of these study designs. Five medical research databases were searched for eligible literature from the earliest available sources up to 5 July 2019 (Medline, Embase, Web of Science, EBM Reviews, CAB Direct). Reference lists of identified studies were hand searched. Eligible studies were cRCTs using targeted chemical or biological mosquito control interventions, or mosquito breeding source reduction, with the occurrence of mosquito-borne disease as an outcome. Eight eligible cRCTs, conducted between 1994–2013 were identified in a variety of settings in the Americas and Asia. Interventions to reduce dengue transmission were mass adult trapping and source reduction. Interventions to reduce malaria transmission were largescale larvicide administration and (topical and spatial) repellent use. Three studies showed the intervention was associated with statistically significant reductions in the disease of interest and entomological indicators. High community engagement with the intervention were common to all three. In two studies, large buffer zones reduced contamination between study arms. Heterogeneity was reduced through increasing study cluster numbers, cluster matching and randomisation. High community engagement is vital for a cRCT reducing mosquito-borne disease with a mosquito control intervention. These findings support a mosquito breeding source reduction intervention for Aedes control in a future study of Buruli ulcer prevention if local communities are supportive and very engaged. Regular administration of larvicide to sites unsuited to source reduction may supplement the intervention.
Publisher: Wiley
Date: 30-03-2017
Abstract: The natural products isatropolone A-C (1-3) were reisolated from Streptomyces Gö66, with 1 and 3 showing potent activity against Leishmania donovani. They contain a rare tropolone ring derived from a type II polyketide biosynthesis pathway. Their biosynthesis was elucidated by labeling experiments, analysis of the biosynthesis gene cluster, its partial heterologous expression, and structural characterization of various intermediates. Owing to their 1,5-diketone moiety, they can react with ammonia, amines, lysine, and lysine-containing peptides and proteins, which results in the formation of a covalent bond and subsequent pyridine ring formation. Their fluorescence properties change upon amine binding, enabling the simple visualization of reacted amines including proteins.
Publisher: American Chemical Society (ACS)
Date: 27-07-2023
Publisher: Springer Science and Business Media LLC
Date: 2009
DOI: 10.1038/NRMICRO2077
Abstract: Buruli ulcer is an emerging human disease caused by infection with a slow-growing pathogen, Mycobacterium ulcerans, that produces mycolactone, a cytotoxin with immunomodulatory properties. The disease is associated with wetlands in certain tropical countries, and evidence for a role of insects in transmission of this pathogen is growing. Comparative genomic analysis has revealed that M. ulcerans arose from Mycobacterium marinum, a ubiquitous fast-growing aquatic species, by horizontal transfer of a virulence plasmid that carries a cluster of genes for mycolactone production, followed by reductive evolution. Here, the ecology, microbiology, evolutionary genomics and immunopathology of Buruli ulcer are reviewed.
Publisher: Cold Spring Harbor Laboratory
Date: 14-06-2016
DOI: 10.1101/058842
Publisher: American Society for Microbiology
Date: 12-10-2023
DOI: 10.1128/AAC.00785-23
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 05-2017
Publisher: Springer Science and Business Media LLC
Date: 25-04-2022
DOI: 10.1038/S41557-022-00923-2
Abstract: Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus ( XP ) living in mutualistic symbiosis with entomopathogenic nematodes generate natural products to mediate bacteria–nematode–insect interactions. However, a lack of systematic analysis of the XP biosynthetic gene clusters (BGCs) has limited the understanding of how natural products affect interactions between the organisms. Here we combine pangenome and sequence similarity networks to analyse BGCs from 45 XP strains that cover all sequenced strains in our collection and represent almost all XP taxonomy. The identified 1,000 BGCs belong to 176 families. The most conserved families are denoted by 11 BGC classes. We homologously (over)express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, virulence factors against insects, metallophores and insect immunosuppressants. These findings explain the functional basis of bacterial natural products in this tripartite relationship.
Publisher: American Society for Microbiology
Date: 05-2019
DOI: 10.1128/AAC.02356-18
Abstract: Staphylococcus aureus is a significant human pathogen whose evolution and adaptation have been shaped in part by mobile genetic elements (MGEs), facilitating the global spread of extensive antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding MGEs, in particular, how changes in the structure of multidrug resistance (MDR) plasmids may influence important staphylococcal phenotypes, is incomplete.
Publisher: Public Library of Science (PLoS)
Date: 30-11-2015
Publisher: Microbiology Society
Date: 25-08-2016
Publisher: American Society for Microbiology
Date: 09-2019
DOI: 10.1128/JCM.00573-19
Abstract: Carbapenemase-producing Enterobacterales (CPE) are being increasingly reported in Australia, and integrated clinical and genomic surveillance is critical to effectively manage this threat. We sought to systematically characterize CPE in Victoria, Australia, from 2012 to 2016.
Publisher: Cold Spring Harbor Laboratory
Date: 17-11-2022
DOI: 10.1101/2022.11.16.516821
Abstract: Buruli ulcer (BU) is a neglected tropical disease caused by infection of subcutaneous tissue with Mycobacterium ulcerans . BU is commonly reported across rural regions of Central and West Africa but has been increasing dramatically in temperate southeast Australia around the major metropolitan city of Melbourne. Previous research has shown that Australian native possums are reservoirs of M. ulcerans and that they shed the bacteria in their fecal material (excreta). Field surveys show that locales where possums harbor M. ulcerans overlap with human cases of BU, raising the possibility of using possum excreta surveys to predict the risk of disease occurrence in humans. We thus established a highly structured 12-month possum excreta surveillance program across an area of 350 km 2 in the Mornington Peninsula area 70 km south of Melbourne, Australia. The primary objective of our study was to assess if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations. Over two s ling c aigns in summer and winter, we collected 2282 possum excreta specimens of which 11% were PCR positive for M. ulcerans -specific DNA. Using the spatial scanning statistical tool SatScan , we observed non-random, co-correlated clustering of both M. ulcerans positive possum excreta and human BU cases. We next trained a statistical model with the Mornington Peninsula excreta survey data to predict the future likelihood of human BU cases occurring in the region. By observing where human BU cases subsequently occurred, we show that the excreta model performance was superior to a null model trained using the previous year’s human BU case incidence data (AUC 0.66 vs 0.55). We then used data unseen by the excreta-informed model from a new survey of 661 possum excreta specimens in Geelong, a geographically separate BU endemic area to the southwest of Melbourne, to prospectively predict the location of human BU cases in that region. As for the Mornington Peninsula, the excreta-based BU prediction model outperformed the null model (AUC 0.75 vs 0.50) and pinpointed specific locations in Geelong where interventions could be deployed to interrupt disease spread. This study highlights the One Health nature of BU by confirming a quantitative relationship between possum excreta shedding of M. ulcerans and humans developing BU. The excreta survey-informed modeling we have described will be a powerful tool for efficient targeting of public health responses to stop BU.
Publisher: Springer Science and Business Media LLC
Date: 07-10-2008
Abstract: Mycolactones are immunosuppressive and cytotoxic polyketides, comprising five naturally occurring structural variants (named A/B, C, D, E and F), produced by different species of very closely related mycobacteria including the human pathogen, Mycobacterium ulcerans . In M. ulcerans strain Agy99, mycolactone A/B is produced by three highly homologous type I polyketide megasynthases (PKS), whose genes ( mlsA1 : 51 kb, mlsA2 : 7.2 kb and mlsB : 42 kb) are found on a 174 kb plasmid, known as pMUM001. We report here comparative genomic analysis of pMUM001, the complete DNA sequence of a 190 kb megaplasmid (pMUM002) from Mycobacterium liflandii 128FXT and partial sequence of two additional pMUM replicons, combined with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These data reveal how PKS module and domain differences affecting MlsB correlate with the production of mycolactones E and F. For mycolactone E these differences from MlsB in M. ulcerans Agy99 include replacement of the AT domain of the loading module (acetate to propionate) and the absence of an entire extension module. For mycolactone F there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone F The mycolactone PKS locus on pMUM002 revealed the same large, three-gene structure and extraordinary pattern of near-identical PKS domain sequence repetition as observed in pMUM001 with greater than 98.5% nucleotide identity among domains of the same function. Intra- and inter-strain comparisons suggest that the extreme sequence homogeneity seen among the mls PKS genes is caused by frequent recombination-mediated domain replacement. This work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial PKS biochemistry.
Publisher: Springer Science and Business Media LLC
Date: 02-11-2020
Publisher: Public Library of Science (PLoS)
Date: 10-08-2012
Publisher: Oxford University Press (OUP)
Date: 18-12-2013
Publisher: Royal Society of Chemistry (RSC)
Date: 2003
DOI: 10.1039/B308163J
Abstract: LC-MSn analysis of mycolactone toxin from extracts of Mycobacterium ulcerans has shown that minor co-metabolites, including two previously unreported, differ structurally from mycolactone only in a small portion of the polyketide side-chain.
Publisher: Cold Spring Harbor Laboratory
Date: 02-02-2023
DOI: 10.1101/2023.02.01.526694
Abstract: The capacity to undertake whole genome sequencing (WGS) in public health laboratories (PHLs) has grown rapidly in response to COVID-19, and SARS-CoV-2 genomic data has been invaluable for managing the pandemic. The public health response has been further supported by the rapid upgrade and implementation of laboratory and bioinformatic resources. However, there remains a high degree of variability in methods and capabilities between laboratories. In addition to evolving methodology and improved understanding of SARS-CoV-2, public health laboratories have become strained during surges in case numbers, adding to the difficulty of ensuring the highest data accuracy. Here, we formed a national working group comprised of laboratory scientists and bioinformaticians from Australia and New Zealand to improve data concordance across PHLs. Through investigating discordant sequence data from Australia’s first external SARS-CoV-2 WGS proficiency testing program (PTP), we show that most discrepancies in genome assessment arose from intrahost variation. While others could be remedied using reasonable, parsimonious bioinformatic quality control. Furthermore, we demonstrate how multidisciplinary national working groups can inform guidelines in real time for bioinformatic quality acceptance criteria. Provision of technical feedback allows laboratory improvement during a pandemic in real time, enhancing public health responses. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. The COVID-19 pandemic has brought the utility of genomics to the forefront of microbial surveillance in public health. Our findings provide recommendations for monitoring bioinformatic quality controls in a pandemic context and how multidisciplinary national working groups can provide technical feedback for actionable improvement towards sequence data concordance.
Publisher: Cold Spring Harbor Laboratory
Date: 08-06-2016
DOI: 10.1101/057760
Abstract: Whole-genome sequencing (WGS) provides the highest resolution analysis for comparison of bacterial isolates in public health microbiology. However, although increasingly being used routinely for some pathogens such as Listeria monocytogenes and Salmonella enterica , the use of WGS is still limited for other organisms, such as Neisseria gonorrhoeae . Multi-antigen sequence typing (NG-MAST) is the most widely performed typing method for epidemiologic surveillance of gonorrhoea. Here, we present NGMASTER – a command-line software tool for performing in silico NG-MAST on assembled genome data. NGMASTER rapidly and accurately determined the NG-MAST of 630 assembled genomes, facilitating comparisons between WGS and previously published gonorrhoea epidemiological studies. The source code and user documentation are available at github.com/MDU-PHL/ngmaster .
Publisher: American Society for Microbiology
Date: 03-2005
DOI: 10.1128/JB.187.5.1668-1676.2005
Abstract: The 174-kb virulence plasmid pMUM001 in Mycobacterium ulcerans epidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation of M. ulcerans Agy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine erse M. ulcerans strains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in all M. ulcerans strains. Physical mapping of these plasmids revealed that like M. ulcerans Agy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis that M. ulcerans erged from the closely related organism Mycobacterium marinum by acquiring pMUM. Our results suggest that pMUM is a defining characteristic of M. ulcerans but that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.
Publisher: PeerJ
Date: 07-08-2020
DOI: 10.7717/PEERJ.9659
Abstract: Mycobacterium ulcerans is the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenic M. ulcerans proteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU. Recombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged with M. ulcerans via subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice. The MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG 1 . However, all mice developed ulcers by day-40 post- M. ulcerans challenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals. These data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU .
Publisher: Cold Spring Harbor Laboratory
Date: 21-11-2019
DOI: 10.1101/849539
Abstract: Vancomycin resistant Enterococcus faecium (VREfm) is an emerging antibiotic resistant pathogen. Strain-level investigations are beginning to reveal the molecular mechanisms used by VREfm to colonize regions of the human bowel. However, the role of commensal bacteria during VREfm colonization, in particular following antibiotic treatment, remains largely unknown. We employed licon 16S rRNA gene sequencing and metabolomics in a murine model system to try and investigate functional roles of the gut microbiome during VREfm colonization. First-order taxonomic shifts between Bacteroidetes and Tenerricutes within the gut microbial community composition were detected both in response to pretreatment using ceftriaxone, and to subsequent VREfm challenge. Using neural networking approaches to find co-occurrence profiles of bacteria and metabolites, we detected key metabolome features associated with butyric acid during and after VREfm colonization. These metabolite features were associated with Bacteroides , indicative of a transition towards a pre-antibiotic naïve microbiome. This study shows the impacts of antibiotics on the gut ecosystem, and the progression of the microbiome in response to colonisation with VREfm. Our results offer insights towards identifying potential non-antibiotic alternatives to eliminate VREfm through metabolic re-engineering to preferentially select for Bacteroides . This study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions between commensal gut bacteria and a specific pathogen. Knowledge from this research will inform gut microbiome engineering strategies, with the aim of translating observations from animal models to human-relevant therapeutic applications.
Publisher: American Chemical Society (ACS)
Date: 26-08-2021
Publisher: Oxford University Press (OUP)
Date: 04-09-2018
DOI: 10.1093/JAC/DKY331
Abstract: Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, there has been a recent concerning increase in bacteraemia associated with the vanA genotype, prompting investigation into the genomic epidemiology of VREfm. A population-level study of VREfm (10 November-9 December 2015) was conducted. A total of 321 VREfm isolates (from 286 patients) across Victoria State were collected and sequenced with Illumina NextSeq. SNPs were used to assess relatedness. STs and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data. Illumina reads from remaining isolates were then mapped to these assemblies to identify their probable vanA-harbouring plasmid. vanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2%, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm were more closely related within hospitals versus between them [core SNPs 10 (IQR 1-357) versus 356 (179-416), respectively], suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within versus between hospitals, due to widespread dissemination of ST796. Different vanA-harbouring plasmids were found across STs. With the exception of ST78 and ST796, Tn1546 transposons also varied. Phylogenetic analysis revealed Australian strains were often interspersed with those from other countries, suggesting ongoing cross-continental transmission. Emerging vanA-VREfm in Australia is polyclonal, indicating repeat introductions of vanA-VREfm into hospitals and subsequent dissemination. The close relationship to global strains reinforces the need for ongoing screening and control of VREfm in Australia and abroad.
Publisher: Public Library of Science (PLoS)
Date: 23-07-2013
Publisher: Elsevier BV
Date: 10-2021
Publisher: Public Library of Science (PLoS)
Date: 04-09-2020
Publisher: American Society for Microbiology
Date: 03-2007
DOI: 10.1128/JB.01442-06
Abstract: It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans , the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum . Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more erse assemblage of M. marinum strains. Like M. ulcerans , the fish and frog MPM show multiple copies of the insertion sequence IS 2404 . Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly ided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.
Publisher: Wiley
Date: 03-1996
DOI: 10.1111/J.1365-2672.1996.TB03227.X
Abstract: The isolation of pathogenic strains of Yersinia enterocolitica from food and water s les by culture is time-consuming and unreliable. A two-step PCR procedure has been developed which, after a period of bacterial enrichment, can detect and confirm the presence of pathogenic Y. enterocolitica within a single day. This PCR method works effectively for a range of environmental water types, including reticulated waters, reservoirs and creeks. A survey of environmental waters in Victoria, Australia, showed that the PCR method detected pathogenic Y. enterocolitica in water s led from four separate sites (two creeks and two reservoirs). Repeat s lings of the two reservoirs yielded PCR-positive results on all but one occasion. Culture analysis of the same s les detected pathogenic Y. enterocolitica in only one s le, indicating that the PCR can detect pathogenic Y. enterocolitica which are undetectable by culture. Results from this study confirm that potentially pathogenic strains of Y. enterocolitica can exist in environmental waters.
Publisher: Elsevier BV
Date: 08-2021
Publisher: Royal Society of Chemistry (RSC)
Date: 2008
DOI: 10.1039/B803101K
Publisher: American Society for Microbiology
Date: 22-12-2020
Abstract: The majority of Staphylococcus aureus strains causing human disease are methicillin-susceptible (MSSA) and can be treated with antistaphylococcal penicillins (such as oxacillin). While acquisition of the mec gene represents the main resistance mechanism to oxacillin, S. aureus can acquire low-level resistance through adaptive mutations in other genes. In this study, we used genomic approaches to understand the basis of S. aureus adaption to oxacillin and its dynamic at the population level. By combining a genome analysis of clinical isolates from persistent MSSA infections, in vitro selection of oxacillin resistance, and genome-wide association analysis on a large collection of isolates, we identified 21 genes linked to secondary oxacillin resistance. Adaptive mutations in these genes were easy to select when S. aureus was exposed to oxacillin, but they also came at a substantial cost in terms of bacterial fitness, suggesting that this phenotype emerges preferentially in the setting of sustained antibiotic exposure.
Publisher: American Society for Microbiology
Date: 03-2014
DOI: 10.1128/JCM.03286-13
Abstract: Enterococci are a major cause of health care-associated infections and account for approximately 10% of all bacteremias globally. The aim of this study was to determine the proportion of enterococcal bacteremia isolates in Australia that are antimicrobial resistant, with particular emphasis on susceptibility to icillin and the glycopeptides, and to characterize the molecular epidemiology of the Enterococcus faecalis and Enterococcus faecium isolates. From 1 January to 31 December 2011, 1,079 unique episodes of bacteremia were investigated, of which 95.8% were caused by either E. faecalis (61.0%) or E. faecium (34.8%). The majority of bacteremias were health care associated, and approximately one-third were polymicrobial. Ampicillin resistance was detected in 90.4% of E. faecium isolates but was not detected in E. faecalis isolates. Vancomycin nonsusceptibility was reported in 0.6% and 36.5% of E. faecalis and E. faecium isolates, respectively. Unlike Europe and the United States, where vancomycin resistance in E. faecium is predominately due to the acquisition of the vanA operon, 98.4% of E. faecium isolates harboring van genes carried the vanB operon, and 16.1% of the vanB E. faecium isolates had vancomycin MICs at or below the susceptible breakpoint of the CLSI. Although molecular typing identified 126 E. faecalis pulsed-field gel electrophoresis pulsotypes, % belonged to two pulsotypes that were isolated across Australia. E. faecium consisted of 73 pulsotypes from which 43 multilocus sequence types were identified. Almost 90% of the E. faecium isolates were identified as CC17 clones, of which approximately half were characterized as ST203, which was isolated Australia-wide. In conclusion, the Australian Enterococcal Sepsis Outcome Programme (AESOP) study has shown that although they are polyclonal, enterococcal bacteremias in Australia are frequently caused by icillin-resistant vanB E. faecium .
Publisher: Microbiology Society
Date: 02-2021
Abstract: In the last 10 years, the barriers preventing the uptake of foreign DNA by clinical Staphylococcus aureus isolates have been identified and powerful mutagenesis techniques such as allelic exchange are now possible in most genotypes. However, these targeted approaches can still be cumbersome, and the construction of unmarked deletions oint mutations may take many weeks or months. Here, we introduce a streamlined allelic exchange protocol using IMxxB Escherichia coli and the plasmid pIMAY-Z. With this optimized approach, a site-specific mutation can be introduced into S. aureus in 5 days, from the start of cloning to isolation of genomic DNA for confirmatory whole-genome sequencing. This streamlined protocol considerably reduces the time required to introduce a specific, unmarked mutation in S. aureus and should dramatically improve the scalability of gene-function studies.
Publisher: American Society for Microbiology
Date: 15-04-2018
DOI: 10.1128/AEM.02612-17
Abstract: Since 2000, cases of the neglected tropical disease Buruli ulcer, caused by infection with Mycobacterium ulcerans , have increased 100-fold around Melbourne (population 4.4 million), the capital of Victoria, in temperate southeastern Australia. The reasons for this increase are unclear. Here, we used whole-genome sequence comparisons of 178 M. ulcerans isolates obtained primarily from human clinical specimens, spanning 70 years, to model the population dynamics of this pathogen from this region. Using phylogeographic and advanced Bayesian phylogenetic approaches, we found that there has been a migration of the pathogen from the east end of the state, beginning in the 1980s, 300 km west to the major human population center around Melbourne. This move was then followed by a significant increase in M. ulcerans population size. These analyses inform our thinking around Buruli ulcer transmission and control, indicating that M. ulcerans is introduced to a new environment and then expands, rather than it being from the awakening of a quiescent pathogen reservoir. IMPORTANCE Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans and is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Despite the majority of disease burden occurring in regions of West and central Africa, Buruli ulcer is also becoming increasingly common in southeastern Australia. Major impediments to controlling disease spread are incomplete understandings of the environmental reservoirs and modes of transmission of M. ulcerans . The significance of our research is that we used genomics to assess the population structure of this pathogen at the Australian continental scale. We have then reconstructed a historical bacterial spread and modeled demographic dynamics to reveal bacterial population expansion across southeastern Australia. These findings provide explanations for the observed epidemiological trends with Buruli ulcer and suggest possible management to control disease spread.
Publisher: Public Library of Science (PLoS)
Date: 13-09-2022
DOI: 10.1371/JOURNAL.PONE.0274627
Abstract: In recent years reported cases of Buruli ulcer, caused by Mycobacterium ulcerans , have increased substantially in Victoria, Australia, with the epidemic also expanding geographically. To develop an understanding of how M . ulcerans circulates in the environment and transmits to humans we analyzed environmental s les collected from 115 properties of recent Buruli ulcer cases and from 115 postcode-matched control properties, for the presence of M . ulcerans . Environmental factors associated with increased odds of M . ulcerans presence at a property included certain native plant species and native vegetation in general, more alkaline soil, lower altitude, the presence of common ringtail possums ( Pseudocheirus peregrinus ) and overhead powerlines. However, only overhead powerlines and the absence of the native plant Melaleuca lanceolata were associated with Buruli ulcer case properties. S les positive for M . ulcerans were more likely to be found at case properties and were associated with detections of M . ulcerans in ringtail possum feces, supporting the hypothesis that M . ulcerans is zoonotic, with ringtail possums the strongest reservoir host candidate. However, the disparity in environmental risk factors associated with M . ulcerans positive properties versus case properties indicates the involvement of human behavior or the influence of other environmental factors in disease acquisition that requires further study.
Publisher: Proceedings of the National Academy of Sciences
Date: 21-01-2004
Abstract: Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized ex le of plasmid-mediated virulence in a Mycobacterium , and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster ( % amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.
Publisher: American Society for Microbiology
Date: 16-02-2023
DOI: 10.1128/MRA.01129-22
Abstract: Staphylococcus aureus strain JKD6159 represents a prominent community-acquired methicillin-resistant S. aureus (MRSA) clone in Australia. Here, we report an improved assembly of the original S. aureus JKD6159 genome sequence. By using deep sequencing with multiple technologies combined with carefully curated assembly and polishing, we believe the assembly to contain zero errors.
Publisher: Oxford University Press (OUP)
Date: 30-01-2017
DOI: 10.1093/GBE/EVX003
Publisher: American Society for Microbiology
Date: 09-2006
DOI: 10.1128/AAC.00422-06
Abstract: Low-level vancomycin-resistant Staphylococcus aureus (vancomycin-intermediate S. aureus [VISA] and heterogenous VISA [hVISA]) is increasingly reported and leads to glycopeptide treatment failure. Various phenotypic features have been reported for these isolates, but the genetic changes leading to hVISA and VISA have yet to be clearly determined. We assessed phenotypic, antibiotic resistance, and genomic changes by using genomic DNA microarray comparison and sequencing of selected loci in five pairs of clinical hVISA/VISA strains and the initial methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained prior to vancomycin therapy. The isolates were from adult patients in Australia and New Zealand who had persistent MRSA bacteremia ( days) while receiving vancomycin therapy. In all cases, the initial isolates were found to be fully vancomycin-susceptible Staphylococcus aureus (VSSA). The hVISA/VISA phenotype was associated with increased cell wall thickness, reduced autolytic activity in four of five hVISA/VISA strains, and a striking reduction in biofilm formation compared to the parent strains in all pairs. All five pairs appeared to be isogenic, and genomic DNA microarray comparison suggested that major genetic changes are not required for the development of the resistant phenotype in these strains. No sequence differences were found in the agr locus or the tcaRA genes for any pair, but a marked reduction in RNAIII expression was found in four pairs. In summary, hVISA/VISA arises from fully VSSA during persistent infection that fails to respond to glycopeptide therapy and is associated with significant phenotypic changes, including a marked reduction in biofilm-forming ability. These clinically derived pairs of isolates will be a useful resource to elucidate the genetic mechanism of resistance in hVISA/VISA strains.
Publisher: Microbiology Society
Date: 24-03-2023
Abstract: In early 2020, the Medical Biology Laboratory of the Pasteur Institute of Cambodia isolated an unusually high number of fluoroquinolone-resistant Salmonella enterica subspecies enterica serovar Paratyphi A strains during its routine bacteriological surveillance activities in Phnom Penh, Cambodia. A public-health investigation was supported by genome sequencing of these Paratyphi A strains to gain insights into the genetic ersity and population structure of a potential outbreak of fluoroquinolone-resistant paratyphoid fever. Comparative genomic and phylodynamic analyses revealed the 2020 strains were descended from a previously described 2013–2015 outbreak of Paratyphi A infections. Our analysis showed sub-lineage 2.3.1 had remained largely susceptible to fluoroquinolone drugs until 2015, but acquired chromosomal resistance to these drugs during six separate events between late 2012 and 2015. The emergence of fluoroquinolone resistance was rapidly followed by the replacement of the original susceptible Paratyphi A population, which led to a dramatic increase of fluoroquinolone-resistant blood-culture-confirmed cases in subsequent years (2016–2020). The rapid acquisition of resistance-conferring mutations in the Paratyphi A population over a 3 year period is suggestive of a strong selective pressure on that population, likely linked with fluoroquinolone use. In turn, emergence of fluoroquinolone resistance has led to increased use of extended-spectrum cephalosporins like ceftriaxone that are becoming the drug of choice for empirical treatment of paratyphoid fever in Cambodia.
Publisher: Elsevier BV
Date: 04-2021
Publisher: Microbiology Society
Date: 04-10-2023
Publisher: Elsevier BV
Date: 04-2022
Publisher: American Society for Microbiology
Date: 15-06-2023
DOI: 10.1128/AAC.00328-23
Abstract: Daptomycin is a last-resort antibiotic used for the treatment of infections caused by Gram-positive antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here, we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and β-lactam antibiotics as well as decreased expression of virulence factors.
Publisher: Springer Science and Business Media LLC
Date: 06-02-2017
DOI: 10.1038/NCOMMS14403
Publisher: Springer Science and Business Media LLC
Date: 26-01-2022
DOI: 10.1038/S41467-022-28156-4
Abstract: Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen. Identifying VREfm transmission dynamics permits targeted interventions, and while genomics is increasingly being utilised, methods are not yet standardised or optimised for accuracy. We aimed to develop a standardized genomic method for identifying putative VREfm transmission links. Using comprehensive genomic and epidemiological data from a cohort of 308 VREfm infection or colonization cases, we compared multiple approaches for quantifying genetic relatedness. We showed that clustering by core genome multilocus sequence type (cgMLST) was more informative of population structure than traditional MLST. Pairwise genome comparisons using split k-mer analysis (SKA) provided the high-level resolution needed to infer patient-to-patient transmission. The more common mapping to a reference genome was not sufficiently discriminatory, defining more than three times more genomic transmission events than SKA (3729 compared to 1079 events). Here, we show a standardized genomic framework for inferring VREfm transmission that can be the basis for global deployment of VREfm genomics into routine outbreak detection and investigation.
Publisher: Oxford University Press (OUP)
Date: 04-04-2014
DOI: 10.1093/CID/CIU203
Abstract: We identified 12 patients with Clostridium difficile infection between July 2011 and March 2012 from whom an unusual C. difficile strain was isolated. This strain had a single-nucleotide deletion of the tcdC gene at position 117 and binary toxin genes, which are characteristic of the hypervirulent ribotype (RT) 027 strain. A retrospective cohort study of 12 patients infected with C. difficile RT244 and 24 patients infected with non-RT244/non-RT027 strains matched for place of diagnosis and time of collection of specimen was performed. We performed whole-genome sequencing to understand the relationship of the RT244 strain to other C. difficile strains and further understand its virulence potential. Clostridium difficile RT244 was associated with more severe disease and a higher mortality rate. Phylogenomic analysis using core genome single-nucleotide polymorphisms showed that RT244 is in the same genetic clade (clade 2) as RT027 but is distinct from all RT027 strains. The pathogenicity locus of the RT244 strain encodes a variant toxin B, and this was confirmed by demonstration of Clostridium sordellii-like cytopathic effect on Vero cells. Toxin B production in culture supernatants was lower than that seen with a RT027 strain. Our findings demonstrate the pathogenic potential of this RT244 C. difficile strain and emphasize the importance of ongoing surveillance for emergent strains.
Publisher: American Society for Microbiology
Date: 08-2007
DOI: 10.1128/AEM.02971-06
Abstract: Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS 2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS 2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS 2404 PCR for the analysis of a variety of environmental s les. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.
Publisher: American Society for Microbiology
Date: 08-2013
DOI: 10.1128/AAC.00263-13
Abstract: The critical role of noncoding small RNAs (sRNAs) in the bacterial response to changing conditions is increasingly recognized. However, a specific role for sRNAs during antibiotic exposure has not been investigated in Staphylococcus aureus . Here, we used Illumina RNA-Seq to examine the sRNA response of multiresistant sequence type 239 (ST239) S. aureus after exposure to four antibiotics (vancomycin, linezolid, ceftobiprole, and tigecycline) representing the major classes of antimicrobials used to treat methicillin-resistant S. aureus (MRSA) infections. We identified 409 potential sRNAs and then compared global sRNA and mRNA expression profiles at 2 and 6 h, without antibiotic exposure and after exposure to each antibiotic, for a vancomycin-susceptible strain (JKD6009) and a vancomycin-intermediate strain (JKD6008). Exploration of this data set by multivariate analysis using a novel implementation of nonnegative matrix factorization (NMF) revealed very different responses for mRNA and sRNA. Where mRNA responses clustered with strain or growth phase conditions, the sRNA responses were predominantly linked to antibiotic exposure, including sRNA responses that were specific for particular antibiotics. A remarkable feature of the antimicrobial response was the prominence of antisense sRNAs to genes encoding proteins involved in protein synthesis and ribosomal function. This study has defined a large sRNA repertoire in epidemic ST239 MRSA and shown for the first time that a subset of sRNAs are part of a coordinated transcriptional response to specific antimicrobial exposures in S. aureus . These data provide a framework for interrogating the role of staphylococcal sRNAs in antimicrobial resistance and exploring new avenues for sRNA-based antimicrobial therapies.
Publisher: Elsevier BV
Date: 03-2001
Publisher: Springer Science and Business Media LLC
Date: 27-01-2023
Publisher: Cold Spring Harbor Laboratory
Date: 28-03-2018
DOI: 10.1101/289975
Abstract: Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, the vanB genotype is dominant however there has been a recent increase in the predominantly plasmid-encoded vanA genotype, prompting investigation into the genomic epidemiology of VREfm in this context. A cross-sectional study of VREfm in Victoria, Australia (Nov.10 th - Dec.9 th , 2015). A total of 321 VREfm isolates (from 286 patients) were collected and whole-genome sequenced with Illumina NextSeq. Single nucleotide polymorphisms (SNPs) were used to assess relatedness. Multi-locus sequence types (STs), and genes associated with resistance and virulence were identified. The vanA -harbouring plasmid from an isolate from each ST was assembled using long-read data. vanA -VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS (-) clade, ST1421, predominated (30.5%, 30.5% and 37.2% of vanA -VREfm, respectively). Most vanB- VREfm were ST796 (77.7%). vanA -VREfm isolates were closely-related within hospitals vs. between them (core SNPs 10 [interquartile range 1-357] vs. 356 [179-416] respectively), suggesting discrete introductions of vanA -VREfm, with subsequent intra-hospital transmission. In contrast, vanB -VREfm had similar core SNP distributions within vs. between hospitals, due to widespread dissemination of ST796. Overall, vanA -harbouring plasmids differed across STs, and with exception of ST78 and ST796, Tn 1546 transposons also varied. vanA -VREfm in Victoria is associated with multiple STs, and is not solely mediated by a single shared plasmid/Tn 1546 transposon clonal transmission appears to play an important role, predominantly within, rather than between, hospitals. In contrast, vanB- VREfm appears to be well-established and widespread across Victorian healthcare institutions.
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.MIB.2017.11.030
Abstract: Enterococci are long-standing members of the human microbiome and they are also widely distributed in nature. However, with the surge of antibiotic-resistance in recent decades, two enterococcal species (Enterococcus faecalis and Enterococcus faecium) have emerged to become significant nosocomial pathogens, acquiring extensive antibiotic resistance. In this review, we summarize what is known about the evolution of virulence in E. faecium, highlighting a specific clone of E. faecium called ST796 that has emerged recently and spread globally.
Publisher: Hindawi Limited
Date: 24-02-2021
DOI: 10.1111/CMI.13317
Abstract: Staphylococcus aureus is a major opportunistic human pathogen that is globally prevalent. Although S. aureus and humans may have co-evolved to the point of commensalism, the bacterium is equipped with virulence factors causing devastating infections. The adoption of an intracellular lifestyle by S. aureus is an important facet of its pathogenesis. Occupying a privileged intracellular compartment permits evasion from the bactericidal actions of host immunity and antibiotics. However, this localization exposes S. aureus to cell-intrinsic processes comprising autophagy, metabolic challenges and clearance mechanisms orchestrated by host programmed cell death pathways (PCDs), including apoptosis, pyroptosis and necroptosis. Mounting evidence suggests that S. aureus deploys pathoadaptive mechanisms that modulate the expression of its virulence factors to prevent elimination through PCD pathways. In this review, we critically analyse the current literature on the interplay between S. aureus virulence factors with the key, intertwined nodes of PCD. We discuss how S. aureus adaptation to the human host plays an essential role in the evasion of PCD, and we consider future directions to study S. aureus-PCD interactions.
Publisher: Springer Science and Business Media LLC
Date: 08-2017
DOI: 10.1038/S41598-017-07288-4
Abstract: Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines.
Publisher: Frontiers Media SA
Date: 14-10-2015
Publisher: PeerJ
Date: 31-07-2018
DOI: 10.7717/PEERJ.5294
Abstract: Buruli ulcer is a disease of the skin and soft tissues caused by infection with a slow growing pathogen, Mycobacterium ulcerans . A vaccine for this disease is not available but M. ulcerans possesses a giant plasmid pMUM001 that harbours the polyketide synthase (PKS) genes encoding a multi-enzyme complex needed for the production of its unique lipid toxin called mycolactone, which is central to the pathogenesis of Buruli ulcer. We have studied the immunogenicity of enzymatic domains in humans with M. ulcerans disease, their contacts, as well as non-endemic areas controls. Between March 2013 and August 2015, heparinized whole blood was obtained from patients confirmed with Buruli ulcer. The blood s les were diluted 1 in 10 in Roswell Park Memorial Institute (RPMI) medium and incubated for 5 days with recombinant mycolactone PKS domains and mycolyltransferase antigen 85A (Ag85A). Blood s les were obtained before and at completion of antibiotic treatment for 8 weeks and again 8 weeks after completion of treatment. Supernatants were assayed for interferon-γ (IFN-γ) and interleukin-5 (IL-5) by enzyme-linked immunosorbent assay. Responses were compared with those of contacts and non-endemic controls. More than 80% of patients and contacts from endemic areas produced IFN-γ in response to all the antigens except acyl carrier protein type 3 (ACP3) to which only 47% of active Buruli ulcer cases and 71% of contacts responded. The highest proportion of responders in cases and contacts was to load module ketosynthase domain (Ksalt) (100%) and enoylreductase (100%). Lower IL-5 responses were induced in a smaller proportion of patients ranging from 54% after ketoreductase type B stimulation to only 21% with ketosynthase type C (KS C). Among endemic area contacts, the, highest proportion was 73% responding to KS C and the lowest was 40% responding to acyltransferase with acetate specificity type 2. Contacts of Buruli ulcer patients produced significantly higher IFN-γ and IL-5 responses compared with those of patients to PKS domain antigens and to mycolyltransferase Ag85A of M. ulcerans . There was low or no response to all the antigens in non-endemic areas controls. IFN-γ and IL-5 responses of patients improved after treatment when compared to baseline results. The major response to PKS antigen stimulation was IFN-γ and the strongest responses were observed in healthy contacts of patients living in areas endemic for Buruli ulcer. Patients elicited lower responses than healthy contacts, possibly due to the immunosuppressive effect of mycolactone, but the responses were enhanced after antibiotic treatment. A vaccine made up of the most immunogenic PKS domains combined with the mycolyltransferase Ag85A warrants further investigation.
Publisher: Cold Spring Harbor Laboratory
Date: 31-01-2018
DOI: 10.1101/257915
Abstract: Acquired mutations are a major mechanism of bacterial antibiotic resistance generation and dissemination, and can arise during treatment of infections. Early detection of sub-populations of resistant bacteria harbouring defined resistance mutations could prevent inappropriate antibiotic prescription. Here we present RM-seq, a new licon-based DNA sequencing workflow based on single molecule barcoding coupled with deep-sequencing that enables the high-throughput characterisation and sensitive detection of resistance mutations from complex mixed populations of bacteria. We show that RM-seq reduces both background sequencing noise and PCR lification bias and allows highly sensitive identification and accurate quantification of antibiotic resistant sub-populations, with relative allele frequencies as low as 10 -4 . We applied RM-seq to identify and quantify rif icin resistance mutations in Staphylococcus aureus using pools of 10,000 in vitro selected clones and identified a large number of previously unknown resistance-associated mutations. Targeted mutagenesis and phenotypic resistance testing was used to validate the technique and demonstrate that RM-seq can be used to link subsets of mutations with clinical resistance breakpoints at high-throughput using large pools of in vitro selected resistant clones. Differential analysis of the abundance of resistance mutations after a selection bottleneck detected antimicrobial cross-resistance and collateral sensitivity-conferring mutations. Using a mouse infection model and human clinical s les, we also demonstrate that RM-seq can be effectively applied in vivo to track complex mixed populations of S. aureus and another major human pathogen, Mycobacterium tuberculosis during infections. RM-seq is a powerful new tool to both detect and functionally characterise mutational antibiotic resistance.
Publisher: Cold Spring Harbor Laboratory
Date: 10-04-2008
Abstract: Mycobacterium marinum , a ubiquitous pathogen of fish and hibia, is a near relative of Mycobacterium tuberculosis , the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum . M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis . The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.
Publisher: American Society for Microbiology
Date: 07-2013
DOI: 10.1128/AAC.00279-13
Abstract: Vancomycin-intermediate Staphylococcus aureus (VISA) strains often arise by mutations in the essential two-component regulator walKR however their impact on walKR function has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptible S. aureus (VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS 256 insertions in the walKR 5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction in walKR gene expression in the IS 256 mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS 256 insertions had replaced two SigA-like walKR promoters with weaker, hybrid promoters. Removal of IS 256 reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation of ssaA but no change in expression of sak and sceD in both IS 256 mutants. Whole-genome sequencing of the two mutants revealed an additional IS 256 insertion within agrC for one mutant, and we confirmed that this mutation abolished agr function. These data provide the first substantial analysis of walKR promoter function and show that prolonged vancomycin exposure can result in VISA through an IS 256 -mediated reduction in walKR expression however, the mechanisms by which this occurs remain to be determined.
Publisher: Frontiers Media SA
Date: 06-04-2022
DOI: 10.3389/FIMMU.2022.832223
Abstract: Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2 , which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.
Publisher: Cold Spring Harbor Laboratory
Date: 12-02-2022
DOI: 10.1101/2022.02.11.480068
Abstract: During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues, and is exposed to new selective pressures, thus potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation, however a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2,590 S. aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single-nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr -mediated adaptation we identified non-canonical, genome-wide significant loci including sucA - sucB and stp1 . The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.
Publisher: Microbiology Society
Date: 11-07-2016
Publisher: American Society for Microbiology
Date: 04-2000
DOI: 10.1128/JCM.38.4.1482-1487.2000
Abstract: Two high-copy-number insertion sequences, IS 2404 and IS 2606 , were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR lification of the region between adjacent copies of IS 2404 and IS 2606 . We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans , and it has confirmed previous studies suggesting a clonal population structure of M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulcerans isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans -infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype.
Publisher: Oxford University Press (OUP)
Date: 10-2008
DOI: 10.1111/J.1574-6968.2008.01328.X
Abstract: Reliable molecular detection of Mycobacterium ulcerans in environmental s les is essential to study the ecology and transmission of this important human pathogen. Variable number tandem repeat (VNTR) typing is a valuable method for distinguishing M. ulcerans isolates from different geographic regions and for distinguishing M. ulcerans from other members of the Mycobacterium marinum/M. ulcerans complex, but its application to environmental s les has not yet been evaluated systematically. This study compares the sensitivity and specificity of PCR detection of 13 VNTR loci to determine the best loci for the analysis of environmental s les. This study demonstrates that VNTR typing using selected loci can be a useful addition to established molecular methods for detecting M. ulcerans in the environment and highlights some of the issues encountered when using molecular methods to detect microorganisms in environmental s les. When applied to environmental s les collected from an endemic region in Victoria, Australia, VNTR typing confirmed that the strain of M. ulcerans being detected was indistinguishable from the strain causing disease in humans in that region.
Publisher: Cold Spring Harbor Laboratory
Date: 29-11-2019
DOI: 10.1101/856955
Abstract: Enzymes that cleave ATP to activate carboxylic acids play essential roles in primary and secondary metabolism in all domains of life. Class I adenylate-forming enzymes share a conserved structural fold but act on a wide range of substrates to catalyze reactions involved in bioluminescence, nonribosomal peptide biosynthesis, fatty acid activation, and β-lactone formation. Despite their metabolic importance, the substrates and catalytic functions of the vast majority of adenylate-forming enzymes are unknown without tools available to accurately predict them. Given the crucial roles of adenylate-forming enzymes in biosynthesis, this also severely limits our ability to predict natural product structures from biosynthetic gene clusters. Here we used machine learning to predict adenylate-forming enzyme function and substrate specificity from protein sequence. We built a web-based predictive tool and used it to comprehensively map the biochemical ersity of adenylate-forming enzymes across ,000 candidate biosynthetic gene clusters in bacterial, fungal, and plant genomes. Ancestral enzyme reconstruction and sequence similarity networking revealed a ‘hub’ topology suggesting radial ergence of the adenylate-forming superfamily from a core enzyme scaffold most related to contemporary aryl-CoA ligases. Our classifier also predicted β-lactone synthetases in novel biosynthetic gene clusters conserved across different strains of Nocardia . To test our computational predictions, we purified a candidate β-lactone synthetase from Nocardia brasiliensis and reconstituted the biosynthetic pathway in vitro to link the gene cluster to the β-lactone natural product, nocardiolactone. We anticipate our machine learning approach will aid in functional classification of enzymes and advance natural product discovery.
Publisher: Wiley
Date: 20-04-2022
DOI: 10.5694/MJA2.51505
Publisher: Public Library of Science (PLoS)
Date: 04-05-2011
Publisher: American Society for Microbiology
Date: 05-2015
Abstract: Infections caused by highly successful clones of hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) are a major public health burden. The globally dominant sequence type 239 (ST239) HA-MRSA clone has persisted in the health care setting for decades, but the basis of its success has not been identified. Taking a collection of 123 ST239 isolates spanning 32 years, we have used population-based functional genomics to investigate the evolution of this highly persistent and successful clone. Phylogenetic reconstruction and population modeling uncovered a previously unrecognized distinct clade of ST239 that was introduced into Australia from Asia and has perpetuated the epidemic in this region. Functional analysis demonstrated attenuated virulence and enhanced resistance to last-line antimicrobials, the result of two different phenomena, adaptive evolution within the original Australian ST239 clade and the introduction of a new clade displaying shifts in both phenotypes. The genetic ersity between the clades allowed us to employ genome-wide association testing and identify mutations in other essential regulatory systems, including walKR , that significantly associate with and may explain these key phenotypes. The phenotypic convergence of two independently evolving ST239 clades highlights the very strong selective pressures acting on HA-MRSA, showing that hospital environments have favored the accumulation of mutations in essential MRSA genes that increase resistance to antimicrobials, attenuate virulence, and promote persistence in the health care environment. Combinations of comparative genomics and careful phenotypic measurements of longitudinal collections of clinical isolates are giving us the knowledge to intelligently address the impact of current and future antibiotic usage policies and practices on hospital pathogens globally. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for innumerable drug-resistant health care-associated infections globally. This study, the first to investigate the evolutionary response of hospital-associated MRSA (HA-MRSA) over many decades, demonstrates how MRSA can persist in a region through the reintroduction of a previously unrecognized distinct clade. This study also demonstrates the crucial adaptive responses of HA-MRSA to the highly selective environment of the health care system, the evolution of MRSA isolates to even higher levels of antibiotic resistance at the cost of attenuated virulence. However, in vivo persistence is maintained, resulting in a clone of HA-MRSA able to resist almost all antimicrobial agents and still cause invasive disease in the heavily compromised hosts found in modern health care settings.
Publisher: Cold Spring Harbor Laboratory
Date: 29-03-2020
DOI: 10.1101/2020.03.28.013177
Abstract: The association of the agriculturally significant phytopathogenic fungus Rhizopus microsporus with the bacterial endosymbiont Burkholderia rhizoxinica is a remarkable ex le of bacteria controlling host physiology and reproduction. Here, we show that a group of transcription activator-like effectors (TALEs) called Burkholderia TALE-like proteins (BATs) from B. rhizoxinica are essential for the establishment of the symbiosis. Mutants lacking BAT proteins are unable to induce host sporulation. Utilising novel microfluidic devices in combination with fluorescence microscopy we observed the accumulation of BAT-deficient mutants in specific fungal side-hyphae with accompanying increased fungal re-infection. High-resolution live imaging revealed septa biogenesis at the base of infected hyphae leading to compartmental trapping of BATdeficient endobacteria. Trapped endosymbionts showed reduced intracellular survival, suggesting a protective response from the fungal host against bacteria lacking specific effectors. These findings underscore the involvement of BAT proteins in maintaining a balance between mutualism and antagonism in bacterial-fungal interactions and provide deeper insights into the dynamic interactions between bacteria and eukaryotes.
Publisher: American Society for Microbiology
Date: 13-04-2023
DOI: 10.1128/SPECTRUM.00447-23
Abstract: Staphylococcus aureus is a human pathogen that upon invasion of a host encounters stresses, such as nutritional restriction. The bacteria respond by switching on a signaling cascade controlled by the nucleotides (p)ppGpp.
Publisher: Wiley
Date: 08-06-2020
DOI: 10.1111/IMCB.12343
Publisher: Cold Spring Harbor Laboratory
Date: 21-01-2022
DOI: 10.1101/2022.01.21.477171
Abstract: Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus ( XP ) living in mutualistic symbiosis with entomopathogenic nematodes produce a myriad of natural products to mediate bacteria–nematode–insect interactions. However, a lack of systematic analysis of the biosynthetic gene clusters (BGCs) has limited the understanding of how natural products justify the bacterial niche specificity. Here we combine pangenome and sequence similarity networks to analyze BGCs from 45 XP species. The identified 1,000 BGCs belong to 176 families, over half of which are unknown. Eleven BGCs represent the most conserved families. We then homologously express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, insect virulence factors, or insect immune suppressors. These findings account for the functional basis of bacterial natural products in this tripartite relationship.
Publisher: Microbiology Society
Date: 06-2009
Abstract: The human pathogen Mycobacterium ulcerans produces a polyketide metabolite called mycolactone with potent immunomodulatory activity. M. ulcerans strain Agy99 has a 174 kb plasmid called pMUM001 with three large genes ( mlsA1 , 51 kb mlsA2 , 7.2 kb mlsB , 43 kb) that encode type I polyketide synthases (PKS) required for the biosynthesis of mycolactone, as demonstrated by transposon mutagenesis. However, there have been no reports of transfer of the mls locus to another mycobacterium to demonstrate that these genes are sufficient for mycolactone production because in addition to their large size, the mls genes contain a high level of internal sequence repetition, such that the entire 102 kb locus is composed of only 9.5 kb of unique DNA. The combination of their large size and lack of stability during laboratory passage makes them a challenging prospect for transfer to a more rapidly growing and genetically tractable host. Here we describe the construction of two bacterial artificial chromosome Escherichia coli / Mycobacterium shuttle vectors, one based on the pMUM001 origin of replication bearing mlsB , and the other based on the mycobacteriophage L5 integrase, bearing mlsA1 and mlsA2 . The combination of these two constructs permitted the two-step transfer of the entire 174 kb pMUM001 plasmid to Mycobacterium marinum , a rapidly growing non-mycolactone-producing mycobacterium that is a close genetic relative of M. ulcerans . To improve the stability of the mls locus in M. marinum , recA was inactivated by insertion of a hygromycin-resistance gene using double-crossover allelic exchange. As expected, the Δ recA mutant displayed increased susceptibility to UV killing and a decreased frequency of homologous recombination. Southern hybridization and RT-PCR confirmed the stable transfer and expression of the mls genes in both wild-type M. marinum and the recA mutant. However, neither mycolactone nor its predicted precursor metabolites were detected in either strain. These experiments show that it is possible to successfully manipulate and stably transfer the large mls genes, but that other bacterial host factors appear to be required to facilitate mycolactone production.
Publisher: Cold Spring Harbor Laboratory
Date: 08-05-2019
DOI: 10.1101/631127
Abstract: Our inability to predict whether certain mutations will confer antibiotic resistance has made it difficult to rapidly detect the emergence of resistance, identify pre-existing resistant populations and manage our use of antibiotics to effective treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis , the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F 420 H 2 -dependent reaction. We show that the native menaquinone-reductase activity of Ddn is important in aerobic respiration and essential for emergence from dormancy, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, even though it was never exposed to pretomanid. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.
Publisher: Elsevier
Date: 2022
DOI: 10.1016/BS.AMPBS.2022.06.004
Abstract: Bacteria have developed resistance against every antimicrobial in clinical use at an alarming rate. There is a critical need for more effective use of antimicrobials to both extend their shelf life and prevent resistance from arising. Significantly, antimicrobial tolerance, i.e., the ability to survive but not proliferate during antimicrobial exposure, has been shown to precede the development of bona fide antimicrobial resistance (AMR), sparking a renewed and rapidly increasing interest in this field. As a consequence, problematic infections for the first time are now being investigated for antimicrobial tolerance, with increasing reports demonstrating in-host evolution of antimicrobial tolerance. Tolerance has been identified in a wide array of bacterial species to all bactericidal antimicrobials. Of particular interest are enterococci, which contain the opportunistic bacterial pathogens Enterococcus faecalis and Enterococcus faecium. Enterococci are one of the leading causes of hospital-acquired infection and possess intrinsic tolerance to a number of antimicrobial classes. Persistence of these infections in the clinic is of growing concern, particularly for the immunocompromised. Here, we review current known mechanisms of antimicrobial tolerance, and include an in-depth analysis of those identified in enterococci with implications for both the development and prevention of AMR.
Publisher: Public Library of Science (PLoS)
Date: 03-10-2011
Publisher: American Society for Microbiology
Date: 11-1998
Publisher: Elsevier BV
Date: 07-2023
Publisher: Public Library of Science (PLoS)
Date: 04-09-2014
Publisher: Wiley
Date: 04-2021
DOI: 10.1002/JEV2.12080
Abstract: Gram‐positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory‐adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe‐associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV‐derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll‐like receptor (TLR) 2, 7, 8, 9 and nucleotide‐binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV‐associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram‐positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host.
Publisher: Public Library of Science (PLoS)
Date: 24-11-2009
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 11-2018
Publisher: Elsevier BV
Date: 12-2019
DOI: 10.1016/J.CELREP.2019.11.103
Abstract: Tissue-resident memory T (Trm) cells are described as having a "sensing and alarming" function, meaning they can rapidly release cytokines in response to local cognate antigen recognition, which in turn, draws circulating immune cells into the tissue. Here, we show noncognate, bystander activation can also trigger the sensing and alarming function of pulmonary CD8
Publisher: American Chemical Society (ACS)
Date: 30-10-2015
DOI: 10.1021/PR5010086
Abstract: Interactions between a host and a bacterial pathogen are mediated by cross-talk between molecules present on, or secreted by, pathogens and host binding-molecules. Identifying proteins involved at this interface would provide substantial insights into this interaction. Although numerous studies have examined in vitro models of infection at the level of transcriptional change and proteomic profiling, there is virtually no information available on naturally occurring host-pathogen interactions in vivo. We employed membrane shaving to identify peptide fragments cleaved from surface-expressed bacterial proteins and also detected proteins originating from the infected host. We optimized this technique for media-cultured Corynebacterium pseudotuberculosis, a sheep pathogen, revealing a set of 247 surface proteins. We then studied a natural host-pathogen interaction by performing membrane shaving on C. pseudotuberculosis harvested directly from naturally infected sheep lymph nodes. Thirty-one bacterial surface proteins were identified, including 13 not identified in culture media, suggesting that a different surface protein repertoire is expressed in this hostile environment. Forty-nine host proteins were identified, including immune mediators and antimicrobial peptides such as cathelicidin. This novel application of proteolytic shaving has documented sets of host and pathogen proteins present at the bacterial surface in an infection of the native host.
Publisher: American Society for Microbiology
Date: 26-06-2019
Abstract: Teixobactin is a new antimicrobial with no known mechanisms of resistance. Understanding how resistance could develop will be crucial to the success and longevity of teixobactin as a new potent antimicrobial. Antimicrobial tolerance has been shown to facilitate the development of resistance, and we show that E. faecalis is intrinsically tolerant to teixobactin at high concentrations. We subsequently chose E. faecalis as a model to elucidate the molecular mechanism underpinning teixobactin tolerance and how this may contribute to the development of teixobactin resistance.
Publisher: Wiley
Date: 05-08-2019
Abstract: Siderophores are key players in bacteria–host interactions, with the main function to provide soluble iron for their producers. Gramibactin from rhizosphere bacteria expands siderophore function and ersity as it delivers iron to the host plant and features an unusual diazeniumdiolate moiety for iron chelation. By mutational analysis of the grb gene cluster, we identified genes (grbD and grbE) necessary for diazeniumdiolate formation. Genome mining using a GrbD‐based network revealed a broad range of orthologous gene clusters in mainly plant‐associated Burkholderia/Paraburkholderia species. Two new types of diazeniumdiolate siderophores, megapolibactins and plantaribactin were fully characterized. In vitro assays and in vivo monitoring experiments revealed that the iron chelators also liberate nitric oxide (NO) in plant roots. This finding is important since NO donors are considered as biofertilizers that maintain iron homeostasis and increase overall plant fitness.
Publisher: American Society for Microbiology
Date: 10-1997
DOI: 10.1128/AEM.63.10.4135-4138.1997
Abstract: Mycobacterium ulcerans is an environmental bacterium which causes chronic skin ulcers. Despite significant epidemiological evidence to suggest that water is the source of infection, the organism has never been identified in the environment. Environmental water s les were collected from a small town in which an outbreak of 29 cases had occurred in a 3-year period. These were examined by mycobacterial culture and PCR lification. Similar to previous studies, M. ulcerans was not cultured from the water s les. However, five s les were positive for M. ulcerans by PCR. These s les were collected from a sw and a golf course irrigation system within the outbreak area. This is the first time that M. ulcerans has been demonstrated to be present in the environment and supports the postulated epidemiology of disease due to this organism.
Publisher: Cold Spring Harbor Laboratory
Date: 05-03-2023
DOI: 10.1101/2023.03.01.23286614
Abstract: Bacterial pathogens such as vancomycin-resistant Enterococcus faecium (VREfm) that are resistant to almost all antibiotics are among the top global threats to human health. Daptomycin is a new last-resort antibiotic for VREfm infections with a novel mode-of-action, but for which resistance has surprisingly and alarmingly been widely reported. The causes of such a rapid emergence of resistance to this novel antibiotic have been unclear. Here we show that the use of rifaximin, an unrelated antibiotic used prophylactically to prevent hepatic encephalopathy in liver disease patients, is causing resistance to this last-resort antibiotic in VREfm. We show that mutations within the bacterial RNA polymerase complex confer cross- resistance to both rifaximin and daptomycin. Furthermore, VREfm with these mutations are spread globally across at least 5 continents and 20 countries, making this a major yet previously unrecognised mechanism of resistance. Until now, rifaximin has been considered ‘low-risk’ for development of antibiotic resistance. Our study shows this is not the case and that widespread rifaximin use may be compromising the clinical efficacy of daptomycin, one of the major last-resort interventions for multidrug resistant pathogens. These findings demonstrate that unanticipated antibiotic cross-resistance may potentially undermine global strategies designed to preserve the clinical use of last-resort antibiotics.
Publisher: Springer Science and Business Media LLC
Date: 03-08-2016
Publisher: Cold Spring Harbor Laboratory
Date: 08-01-2007
DOI: 10.1101/GR.5942807
Abstract: Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is r ant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed % nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS 2404 , 91 copies of IS 2606 , 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans -specific by comparative genomics were verified as such by PCR screening a erse collection of 33 strains of M. ulcerans and M. marinum . This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.MEEGID.2013.04.026
Abstract: Staphylococcus aureus is one of the most important human pathogens, causing life-threatening infection in the community and hospital setting. The population genetics of S. aureus and the evolution of virulence is the focus of this review. We describe the various techniques in determining S. aureus population structure and discuss the insights gained from whole genome sequencing of various S. aureus strains. The emergence of community-acquired, methicillin-resistant S. aureus provides a framework for the discussion on evolution of virulence, and the role of horizontal gene transfer in the development of virulence and antibiotic resistance is explored. The knowledge generated from population genetics has the potential to inform strategies to assist in the prevention or treatment of this highly successful human pathogen.
Publisher: Wiley
Date: 13-09-2023
Abstract: Rhizonin A and B are hepatotoxic cyclopeptides produced by bacterial endosymbionts (Mycetohabitans endofungorum) of the fungus Rhizopus microsporus. Their toxicity critically depends on the presence of 3‐furylalanine (Fua) residues, which also occur in pharmaceutically relevant cyclopeptides of the endolide and bingchamide families. The biosynthesis and incorporation of Fua by non‐ribosomal peptide synthetases (NRPS), however, has remained elusive. By genome sequencing and gene inactivation we elucidated the gene cluster responsible for rhizonin biosynthesis. A suite of isotope labeling experiments identified tyrosine and l‐DOPA as Fua precursors and provided the first mechanistic insights. Bioinformatics, mutational analysis and heterologous reconstitution identified dioxygenase RhzB as necessary and sufficient for Fua formation. RhzB is a novel type of heme‐dependent aromatic oxygenases (HDAO) that enabled the discovery of the bingchamide biosynthesis gene cluster through genome mining.
Publisher: Cold Spring Harbor Laboratory
Date: 22-06-2017
DOI: 10.1101/153874
Abstract: Whole-genome sequencing of microbial pathogens is revolutionising modern approaches to outbreaks of infectious diseases and is reliant upon organism culture. Culture-independent methods have shown promise in identifying pathogens, but high level reconstruction of microbial genomes from microbiologically complex s les for more in-depth analyses remains a challenge. Here, using metagenomic sequencing of a human faecal s le and analysis by tetranucleotide frequency profiling projected onto emergent self-organising maps, we were able to reconstruct the underlying populations of two extensively-drug resistant pathogens, Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae and vancomycin-resistant Enterococcus faecium. From these genomes, we were able to ascertain molecular typing results, such as MLST, and identify highly discriminatory mutations in the metagenome to distinguish closely related strains. These proof-of-principle results demonstrate the utility of clinical s le metagenomics to recover sequences of important drug-resistant bacteria and application of the approach in outbreak investigations, independent of the need to culture the organisms.
Publisher: Oxford University Press (OUP)
Date: 29-01-2014
DOI: 10.1093/GBE/EVU022
Publisher: Wiley
Date: 18-03-2020
Publisher: Cold Spring Harbor Laboratory
Date: 15-12-2021
DOI: 10.1101/2021.12.14.472725
Abstract: Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analysed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~136 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2 , which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.
Publisher: Royal Society of Chemistry (RSC)
Date: 2005
DOI: 10.1039/B506835E
Abstract: Structures are proposed, based on LC-ion trap MSn analysis and high-resolution FTICR MS/MS analysis, for a novel family of mycolactone toxins isolated from the frog pathogen MU128FXT and differing from those produced by the human pathogen M. ulcerans MUAgy99 in having an altered polyketide side chain.
Publisher: Oxford University Press (OUP)
Date: 19-08-2019
DOI: 10.1093/JAC/DKZ341
Abstract: Oral azithromycin given during labour reduces carriage of bacteria responsible for neonatal sepsis, including Staphylococcus aureus. However, there is concern that this may promote drug resistance. Here, we combine genomic and epidemiological data on S. aureus isolated from mothers and babies in a randomized intra-partum azithromycin trial (PregnAnZI) to describe bacterial population dynamics and resistance mechanisms. Participants from both arms of the trial, who carried S. aureus in day 3 and day 28 s les post-intervention, were included. Sixty-six S. aureus isolates (from 7 mothers and 10 babies) underwent comparative genome analyses and the data were then combined with epidemiological data. Trial registration (main trial): ClinicalTrials.gov Identifier NCT01800942. Seven S. aureus STs were identified, with ST5 dominant (n = 40, 61.0%), followed by ST15 (n = 11, 17.0%). ST5 predominated in the placebo arm (73.0% versus 49.0%, P = 0.039) and ST15 in the azithromycin arm (27.0% versus 6.0%, P = 0.022). In azithromycin-resistant isolates, msr(A) was the main macrolide resistance gene (n = 36, 80%). Ten study participants, from both trial arms, acquired azithromycin-resistant S. aureus after initially harbouring a susceptible isolate. In nine (90%) of these cases, the acquired clone was an msr(A)-containing ST5 S. aureus. Long-read sequencing demonstrated that in ST5, msr(A) was found on an MDR plasmid. Our data reveal in this Gambian population the presence of a dominant clone of S. aureus harbouring plasmid-encoded azithromycin resistance, which was acquired by participants in both arms of the study. Understanding these resistance dynamics is crucial to defining the public health drug resistance impacts of azithromycin prophylaxis given during labour in Africa.
Publisher: Informa UK Limited
Date: 21-09-2016
DOI: 10.1080/14787210.2016.1233815
Abstract: Management of invasive Staphylococcus aureus infections is complex. Dramatic improvements in bacterial whole genome sequencing (WGS) offer new opportunities for personalising the treatment of S. aureus infections. Areas covered: We address recent achievements in S. aureus genomics, describe genetic determinants of antibiotic resistance and summarise studies that have defined molecular characteristics associated with risk and outcome of S. aureus invasive infections. Potential clinical use of WGS for resistance prediction, infection outcome stratification and management of persistent /relapsing infections is critically discussed. Expert commentary: WGS is not only providing invaluable information to track the emergence and spread of important S. aureus clones, but also allows rapid determination of resistance genotypes in the clinical environment. An evolving opportunity is to infer clinically important outcomes and optimal therapeutic approaches from widely available S. aureus genome data, with the goal of in idualizing management of invasive S. aureus infections.
Publisher: Cold Spring Harbor Laboratory
Date: 19-05-2023
DOI: 10.1101/2023.05.16.541063
Abstract: Staphylococcus aureus is an adaptable human pathogen causing life-threatening endocarditis and bacteraemia. Methicillin-resistant S. aureus (MRSA) is alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteraemia and vancomycin treatment failure is often associated with vancomycin-intermediate S. aureus strains termed VISA. The regulatory 3’ UTR of vigR mRNA contributes to vancomycin tolerance in the clinical VISA isolate JKD6008 and upregulates the lytic transglycosylase IsaA. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we find that the vigR 3’ UTR also interacts with mRNAs involved in carbon metabolism, amino acid biogenesis, cell wall biogenesis, and virulence. The vigR 3’ UTR was found to repress dapE , a succinyl-diaminopimelate desuccinylase required for lysine and cell wall peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the vigR 3’ UTR increased VISA virulence in a wax moth larvae model, and we find that an isaA mutant is completely attenuated in the larvae model. Sequence and structural analysis of the vigR 3’ UTR indicates that the UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions (STAR repeats) that partly contribute sequence for the isaA interaction seed and may functionalise the 3’ UTR. Our findings reveal an extended regulatory network for vigR , uncovering a novel mechanism of regulation of cell wall metabolism and virulence in a clinical S. aureus isolate.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 11-11-2016
Abstract: Red squirrels are an unexpected reservoir of otherwise human-restricted leprosy bacilli
Publisher: Springer Science and Business Media LLC
Date: 18-03-2023
DOI: 10.1038/S41467-023-37200-W
Abstract: Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20–40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli , Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes . Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.
Publisher: American Society for Microbiology
Date: 10-2008
DOI: 10.1128/AAC.01613-07
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA), once restricted to hospitals, is spreading rapidly through the wider community. Resistance to vancomycin, the principal drug used to treat MRSA infections, has only recently emerged, is mainly low level, and characteristically appears during vancomycin therapy (vancomycin-intermediate S. aureus [VISA] and hetero-resistant VISA). This phenomenon suggests the adaptation of MRSA through mutation, although defining the mutations leading to resistance in clinical isolates has been difficult. We studied a vancomycin-susceptible clinical MRSA isolate (MIC of 1 μg/ml) and compared it with an isogenic blood culture isolate from the same patient, despite 42 days of vancomycin treatment (MIC of 4 μg/ml). A whole-genome sequencing approach allowed the nearly complete assembly of the genome sequences of the two isolates and revealed only six nucleotide substitutions in the VISA strain compared with the parent strain. One mutation occurred in graS , encoding a putative two-component regulatory sensor, leading to a change from a polar to a nonpolar amino acid (T136I) in the conserved histidine region of the predicted protein. Replacing the graS allele of the vancomycin-susceptible parent strain with the graS allele from the VISA derivative resulted in increased vancomycin resistance at a level between those of the vancomycin-susceptible S. aureus and VISA clinical isolates, confirming a role for graRS in VISA. Our study suggests that MRSA is able to develop clinically significant vancomycin resistance via a single point mutation, and the two-component regulatory system graRS is a key mediator of this resistance. However, additional mutations are likely required to express the full VISA phenotype.
Publisher: Public Library of Science (PLoS)
Date: 27-07-2010
Publisher: American Society for Microbiology
Date: 17-01-2014
DOI: 10.1128/MICROBIOLSPEC.MGM2-0025-2013
Abstract: Most mycobacterial species are harmless saprophytes, often found in aquatic environments. A few species seem to have evolved from this pool of environmental mycobacteria into major human pathogens, such as Mycobacterium tuberculosis , the agent of tuberculosis, Mycobacterium leprae , the leprosy bacillus, and Mycobacterium ulcerans , the agent of Buruli ulcer. While the pathogenicity of M. ulcerans relates to the acquisition of a large plasmid encoding a polyketide-derived toxin, the molecular mechanisms by which M. leprae or M. tuberculosis have evolved to cause disease are complex and involve the interaction between the pathogen and the host. Here we focus on M. tuberculosis and closely related mycobacteria and discuss insights gained from recent genomic and functional studies. Comparison of M. tuberculosis genome data with sequences from nontuberculous mycobacteria, such as Mycobacterium marinum or Mycobacterium kansasii , provides a perception of the more distant evolution of M. tuberculosis , while the recently accomplished genome sequences of multiple tubercle bacilli with smooth colony morphology, named Mycobacterium canettii , have allowed the ancestral gene pool of tubercle bacilli to be estimated. The resulting findings are instrumental for our understanding of the pathogenomic evolution of tuberculosis-causing mycobacteria. Comparison of virulent and attenuated members of the M. tuberculosis complex has further contributed to identification of a specific secretion pathway, named ESX or Type VII secretion. The molecular machines involved are key elements for mycobacterial pathogenicity, strongly influencing the ability of M. tuberculosis to cope with the immune defense mounted by the host.
Publisher: Cold Spring Harbor Laboratory
Date: 09-02-2017
DOI: 10.1101/105213
Abstract: Mycobacterium chimaera is an opportunistic environmental mycobacterium, belonging to the Mycobacterium intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest world-wide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays, specific for M. chimaera . Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera . We targeted one of these regions and developed a TaqMan qPCR assay for M. chimaera with a detection limit of 10 CFU in whole blood. In vitro screening against DNA extracted from 40 other mycobacteria and 22 bacterial species from 21 erse genera confirmed in silico predicted specificity for M. chimaera . Screening 33 water s les from heater cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture negative s les positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera .
Publisher: Frontiers Media SA
Date: 20-10-2021
DOI: 10.3389/FMICB.2021.749935
Abstract: Healthcare associated infections caused by vancomycin-resistant Enterococcus faecium (VREfm) have a major impact on health outcomes. VREfm is difficult to treat because of intrinsic and acquired resistance to many clinically used antimicrobials, with daptomycin being one of the few last line therapeutic options for treating multidrug-resistant VREfm. The emergence of daptomycin-resistant VREfm is therefore of serious clinical concern. Despite this, the impact that daptomycin-resistant VREfm have on patient health outcomes is not clearly defined and knowledge on the mechanisms and genetic signatures linked with daptomycin resistance in VREfm remains incomplete. To address these knowledge gaps, phenotypic daptomycin susceptibility testing was undertaken on 324 E. faecium isolates from Australia and New Zealand. Approximately 15% of study isolates were phenotypically resistant to daptomycin. Whole genome sequencing revealed a strong association between vanA -VREfm and daptomycin resistance, with 95% of daptomycin-resistant study isolates harbouring vanA . Genomic analyses showed that daptomycin-resistant VREfm isolates were polyclonal and carried several previously characterised mutations in the liaR and liaS genes as well as several novel mutations within the rpoB, rpoC , and dltC genes. Overall, 70% of daptomycin-resistant study isolates were found to carry mutations within the liaR, rpoB, rpoC , or dltC genes. Finally, in a mouse model of VREfm bacteraemia, infection with the locally dominant daptomycin-resistant clone led to reduced daptomycin treatment efficacy in comparison to daptomycin-susceptible E. faecium . These findings have important implications for ongoing VREfm surveillance activities and the treatment of VREfm infections.
Publisher: Cold Spring Harbor Laboratory
Date: 06-03-2023
DOI: 10.1101/2023.03.04.531075
Abstract: Assessing the impact of SARS-CoV-2 variants on the host is crucial with continuous emergence of new variants. We employed single-cell sequencing to investigate host transcriptomic response to ancestral and Alpha-strain SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from adults and adolescents. Strong innate immune responses were observed across lowly-infected and bystander cell-types, and heightened in Alpha-infection. Contrastingly, the innate immune response of highly-infected cells was like mock-control cells. Alpha highly-infected cells showed increased expression of protein refolding genes compared with ancestral-strain-infected adolescent cells. Oxidative phosphorylation- and translation-related genes were down-regulated in bystander cells versus infected and mock-control cells, suggesting that the down-regulation is protective and up-regulation supports viral activity. Infected adult cells revealed up-regulation of these pathways compared with infected adolescents, implying enhanced pro-viral states in infected adults. Overall, this highlights the complexity of cell-type-, age- and viral-strain-dependent host epithelial responses to SARS-CoV-2 and the value of air-liquid-interface cultures.
Publisher: Public Library of Science (PLoS)
Date: 14-04-2017
Publisher: American Association for the Advancement of Science (AAAS)
Date: 08-2018
DOI: 10.1126/SCITRANSLMED.AAR6115
Abstract: Alcohol-based disinfectants and particularly hand rubs are a key way to control hospital infections worldwide. Such disinfectants restrict transmission of pathogens, such as multidrug-resistant
Publisher: American Thoracic Society
Date: 15-08-2018
Publisher: Cold Spring Harbor Laboratory
Date: 22-04-2022
DOI: 10.1101/2022.04.21.22273941
Abstract: Outcomes for patients with severe bacterial infections are determined by the interplay between host, pathogen, and treatments. Most notably, patient age and antibiotic resistance contributes significantly to poor outcomes. While human genomics studies have provided insights into the host genetic factors impacting outcomes of Staphylococcus aureus infections, comparatively little is known about S. aureus genotypes and disease severity. Building on the idea that bacterial pathoadaptation is a key driver of clinical outcomes, we develop a new genome-wide association study (GWAS) framework to identify adaptive bacterial mutations associated with clinical treatment failure and mortality in three large and independent S. aureus bacteraemia cohorts, comprising 1358 episodes. We discovered S. aureus loci with previously undescribed convergent mutations linked to both poorer infection outcomes and reduced susceptibility to vancomycin. Our research highlights the potential of vancomycin-selected mutations and vancomycin MIC as key explanatory variables to predict SAB severity. The contribution of bacterial variation was much lower for clinical outcomes (heritability 5%), however, GWAS allowed us to identify additional, MIC-independent candidate pathogenesis loci. Using supervised machine-learning, we were able to quantify the predictive potential of these adaptive S. aureus signatures, along with host determinants of bacteraemia outcomes. The statistical genomics framework we have developed is a powerful means to capture adaptive mutations and find bacterial factors that influence and predict severe infections. Our findings underscore the importance of systematically collected, rich clinical and microbiological data to understand bacterial mechanisms promoting treatment failure.
Publisher: Elsevier BV
Date: 10-2020
Publisher: Cold Spring Harbor Laboratory
Date: 13-12-2022
DOI: 10.1101/2022.12.11.519971
Abstract: Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic ersity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa ( In tracellular Tox icity of S. a ureus ) to quantify intracellula cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.
Publisher: American Society for Microbiology
Date: 09-2013
DOI: 10.1128/JCM.01393-13
Publisher: Springer Science and Business Media LLC
Date: 17-10-2016
DOI: 10.1007/S00249-016-1178-2
Abstract: Atomic force microscopy measurements of capsule thickness revealed that that the wild-type Klebsiella pneumoniae AJ218 capsular polysaccharides were rearranged by exposure to colistin. The increase in capsule thickness measured near minimum inhibitory/bactericidal concentration (MIC/MBC) is consistent with the idea that colistin displaces the alent cations that cross-bridge adjacent lipopolysaccharide (LPS) molecules through the capsule network. Cryo-electron microscopy demonstrated that the measured capsule thickness at near MIC/MBC of 1.2 μM was inflated by the disrupted outer membrane, through which the capsule is excreted and LPS is bound. Since wild-type and capsule-deficient strains of K. pneumoniae AJ218 have equivalent MICs and MBCs, the presence of the capsule appeared to confer no protection against colistin in AJ218. A spontaneously arising colistin mutant showed a tenfold increase in resistance to colistin genetic analysis identified a single amino acid substitution (Q95P) in the PmrB sensor kinase in this colistin-resistant K. pneumoniae AJ218. Modification of the lipid A component of the LPS could result in a reduction of the net-negative charge of the outer membrane, which could hinder binding of colistin to the outer membrane and displacement of the alent cations that bridge adjacent LPS molecules throughout the capsular polysaccharide network. Retention of the cross-linking alent cations may explain why measurements of capsule thickness did not change significantly in the colistin-resistant strain after colistin exposure. These results contrast with those for other K. pneumoniae strains that suggest that the capsule confers colistin resistance.
Publisher: Elsevier BV
Date: 08-2020
Publisher: Elsevier BV
Date: 05-2022
Publisher: PeerJ
Date: 28-02-2017
DOI: 10.7717/PEERJ.3047
Abstract: The emergence and evolution of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains in Africa is poorly understood. However, one particular MRSA lineage called ST88, appears to be rapidly establishing itself as an “African” CA-MRSA clone. In this study, we employed whole genome sequencing to provide more information on the genetic background of ST88 CA-MRSA isolates from Ghana and to describe in detail ST88 CA-MRSA isolates in comparison with other MRSA lineages worldwide. We first established a complete ST88 reference genome (AUS0325) using PacBio SMRT sequencing. We then used comparative genomics to assess relatedness among 17 ST88 CA-MRSA isolates recovered from patients attending Buruli ulcer treatment centres in Ghana, three non-African ST88s and 15 other MRSA lineages. We show that Ghanaian ST88 forms a discrete MRSA lineage (harbouring SCC mec- IV [2B]). Gene content analysis identified five distinct genomic regions enriched among ST88 isolates compared with the other S. aureus lineages. The Ghanaian ST88 isolates had only 658 core genome SNPs and there was no correlation between phylogeny and geography, suggesting the recent spread of this clone. The lineage was also resistant to multiple classes of antibiotics including β -lactams, tetracycline and chlor henicol. This study reveals that S. aureus ST88-IV is a recently emerging and rapidly spreading CA-MRSA clone in Ghana. The study highlights the capacity of small snapshot genomic studies to provide actionable public health information in resource limited settings. To our knowledge this is the first genomic assessment of the ST88 CA-MRSA clone.
Publisher: Proceedings of the National Academy of Sciences
Date: 15-08-2016
Abstract: Whereas most of the more than 130 described mycobacterial species are harmless saprophytes, Mycobacterium tuberculosis , the human tuberculosis-causing agent, represents one of the deadliest bacterial pathogens in the history of humankind. To explore the mechanisms behind this spectacular evolutionary trajectory toward pathogenicity, we have experimentally investigated the faculty of different tuberculosis-causing mycobacteria in conducting horizontal gene transfer (HGT). Our studies identified unique chromosomal DNA transfer between strains of the Mycobacterium canettii clade, which resemble most closely the putative common ancestor of the M. tuberculosis complex. This outstanding feature suggests that during the evolution of M. tuberculosis , HGT might have represented the major mechanism for acquisition of genes that helped these mycobacteria to increasingly resist host defenses and become major pathogens.
Publisher: Cold Spring Harbor Laboratory
Date: 03-10-2018
DOI: 10.1101/434506
Abstract: Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans . In this study we used a previously reported strain of M. ulcerans , genetically engineered to constitutively produce bioluminescence, to follow the progression of Buruli ulcer in mice using an in-vivo imaging (IVIS ® ) system. We aimed to characterize a mouse tail infection model for pathogenesis, as well as for pre-clinical vaccine and drug development research for Buruli ulcer. Immune parameters, such as antibody titers and cytokine levels, were determined throughout the course of the infection and histology specimens were examined for comparison with human pathology. Nine out of ten (90%) BALB/c mice infected subcutaneously with 10 5 M. ulcerans JKD8049 (containing pMV306 hsp16+luxG13) exhibited light emission from the site of infection over the course of the experiment indicating M. ulcerans growth in-vivo . Five out of ten (50%) animals developed clinical signs of disease. Antibody titers were overall low and their onset was late, as measured by responses to both heterogenous (bacterial whole cell lysate) and single antigen (Hsp18) targets. IFN-γ, and IL-10 are reported to play a vital role in host control of Buruli ulcer and these cytokines were elevated in animals with pathology. For mice with advanced pathology, histology revealed clusters of acid-fast bacilli within subcutaneous tissue 300-400 μm beneath the epidermis of the tail, with macrophage infiltration and granuloma-formation resembling human Buruli ulcer. This study has shown the utility of using bioluminescent M. ulcerans and IVIS ® in a mouse tail infection model to study Buruli ulcer infection. Buruli ulcer is one of the so called neglected tropical diseases. It is an infectious disease, mainly occurring in West Africa but also in Australia. It manifests as skin lesion and ulcer. Up to date, the way of transmission is inadequately understood. Also, there is no vaccine to protect against the disease. Buruli ulcer is treatable with a course of antibiotics that need to be given for the duration of two months. More laboratory research is needed to elucidate the mechanism of transmission, develop a vaccine and improve and shorten antibiotic therapy. For this, animal (mouse) models of disease are used. The aim of this study was to refine and improve the mouse tail infection model of Buruli ulcer. For this, we used a genetically modified Mycobacterium ulcerans strain that emits light. After infection of animals, light emitted from the bacteria was read out with an in-vivo imaging (IVIS) camera. This allowed us to monitor the location of bacteria in the living animal over time without the need to kill the animal. We also measured parameters of the immune system such as antibodies and cytokines as a baseline for future studies into immunology, vaccine development and pathology of Buruli ulcer. We successfully improved and characterized the mouse tail infection model in Buruli ulcer with the use of modern technology using light emitting bacteria and the IVIS camera.
Publisher: Springer Science and Business Media LLC
Date: 04-01-2023
DOI: 10.1038/S41467-022-35713-4
Abstract: Realising the promise of genomics to revolutionise identification and surveillance of antimicrobial resistance (AMR) has been a long-standing challenge in clinical and public health microbiology. Here, we report the creation and validation of abritAMR , an ISO-certified bioinformatics platform for genomics-based bacterial AMR gene detection. The abritAMR platform utilises NCBI’s AMRFinderPlus , as well as additional features that classify AMR determinants into antibiotic classes and provide customised reports. We validate abritAMR by comparing with PCR or reference genomes, representing 1500 different bacteria and 415 resistance alleles. In these analyses, abritAMR displays 99.9% accuracy, 97.9% sensitivity and 100% specificity. We also compared genomic predictions of phenotype for 864 Salmonella spp. against agar dilution results, showing 98.9% accuracy. The implementation of abritAMR in our institution has resulted in streamlined bioinformatics and reporting pathways, and has been readily updated and re-verified. The abritAMR tool and validation datasets are publicly available to assist laboratories everywhere harness the power of AMR genomics in professional practice.
Publisher: Cold Spring Harbor Laboratory
Date: 24-08-2020
DOI: 10.1101/2020.08.24.265355
Abstract: Rapid modulation of gene expression is a key feature for the success of bacteria, particularly for those that rapidly have to adapt to different niches. The lifecycles of Photorhabdus and Xenorhabdus involve a mutualistic association with nematodes as well as an entomopathogenic phase 1,2 , both of which rely on the production of numerous specialized metabolites (SMs) 3,4 . Several regulators have been previously implicated in the regulation of SM production in these genera 3,4 . However, the molecular underpinnings regulating SM production and the role of small regulatory RNAs (sRNAs) in this process are unknown. Here we describe the mechanism underlying RNA-mediated control of SM synthesis. We show that the Hfq-dependent sRNA, ArcZ, is an essential requirement for SM production. We discovered that ArcZ directly base-pairs with the mRNA encoding HexA, a key repressor of SM genes. We further demonstrate that the ArcZ regulon is not restricted to SM production, but rather modulates up to ~15% of the transcriptional output in both Photorhabdus and Xenorhabdus . Together, our study shows that sRNAs are crucial for SM production in these species, reveals previously unknown targets for biosynthetic pathway manipulations, and offers a new tool for the (over)production, isolation and identification of unknown natural products.
Publisher: Microbiology Society
Date: 09-2020
DOI: 10.1099/JMM.0.001238
Abstract: Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. Aim. To establish and validate a reverse transcription loop-mediated isothermal lification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals. Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical s les of 14 min ( sd ±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID 50 ml −1 ), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.
Publisher: American Society for Microbiology
Date: 02-2017
DOI: 10.1128/AAC.02000-17
Abstract: Topical antibiotics, such as mupirocin and fusidic acid, are commonly used in the prevention and treatment of skin infections, particularly those caused by staphylococci. However, the widespread use of these agents is associated with increased resistance to these agents, potentially limiting their efficacy. Of particular concern is the observation that resistance to topical antibiotics is often associated with multidrug resistance, suggesting that topical antibiotics may play a role in the emergence of multidrug-resistant (MDR) strains. New Zealand (NZ) has some of the highest globally recorded rates of topical antibiotic usage and resistance. Using a combination of Pacific Biosciences single-molecule real-time (SMRT) whole-genome sequencing, Illumina short-read sequencing, and Bayesian phylogenomic modeling on 118 new multilocus sequence type 1 (ST1) community Staphylococcus aureus isolates from New Zealand and 61 publically available international ST1 genome sequences, we demonstrate a strong correlation between the clinical introduction of topical antibiotics and the emergence of MDR ST1 S. aureus . We also provide in vitro experimental evidence showing that exposure to topical antibiotics can lead to the rapid selection of MDR S. aureus isolates carrying plasmids that confer resistance to multiple unrelated antibiotics, from within a mixed population of competitor strains. These findings have important implications regarding the impact of the indiscriminate use of topical antibiotics.
Publisher: Springer Science and Business Media LLC
Date: 27-01-2016
DOI: 10.1038/NMICROBIOL.2015.19
Abstract: Mycobacterium tuberculosis is a major, globally spread, aerosol-transmitted human pathogen, thought to have evolved by clonal expansion from a Mycobacterium canettii-like progenitor. In contrast, extant M. canettii strains are rare, genetically erse, and geographically restricted mycobacteria of only marginal epidemiological importance. Here, we show that the contrasting evolutionary success of these two groups is linked to loss of lipooligosaccharide biosynthesis and subsequent morphotype changes. Spontaneous smooth-to-rough M. canettii variants were found to be mutated in the polyketide-synthase-encoding pks5 locus and deficient in lipooligosaccharide synthesis, a phenotype restored by complementation. Importantly, these rough variants showed an altered host-pathogen interaction and increased virulence in cellular- and animal-infection models. In one variant, lipooligosaccharide deficiency occurred via homologous recombination between two pks5 genes and removal of the intervening acyltransferase-encoding gene. The resulting single pks5 configuration is similar to that fixed in M. tuberculosis, which is known to lack lipooligosaccharides. Our results suggest that pks5-recombination-mediated bacterial surface remodelling increased virulence, driving evolution from putative generalist mycobacteria towards professional pathogens of mammalian hosts.
Publisher: MDPI AG
Date: 09-06-2020
DOI: 10.3390/MICROORGANISMS8060870
Abstract: Staphylococcus aureus is a facultative pathogen that can encode numerous antibiotic resistance and immune evasion genes and can cause severe infections. Reduced susceptibility to last resort antibiotics such as vancomycin and daptomycin is often associated with mutations in walRK, an essential two-component regulatory system (TCS). This study focuses on the WalK accessory membrane proteins YycH and YycI and their influence on WalRK phosphorylation. Depletion of YycH and YycI by antisense RNA caused an impaired autolysis, indicating a positive regulatory function on WalK as has been previously described. Phosphorylation assays with full-length recombinant proteins in phospholipid liposomes showed that YycH and YycI stimulate WalK activity and that both regulatory proteins are needed for full activation of the WalK kinase. This was validated in vivo through examining the phosphorylation status of WalR using Phos-tag SDS-PAGE with a yycHI deletion mutant exhibiting reduced levels of phosphorylated WalR. In the yycHI knockdown strain, muropeptide composition of the cell wall was not affected, however, the wall teichoic acid content was increased. In conclusion, a direct modulation of WalRK phosphorylation activity by the accessory proteins YycH and YycI is reported both in vitro and in vivo. Taken together, our results show that YycH and YycI are important in the direct regulation of WalRK-dependent cell wall metabolism.
Publisher: Public Library of Science (PLoS)
Date: 26-03-2008
Publisher: American Society for Microbiology
Date: 05-1998
DOI: 10.1128/AEM.64.5.1743-1749.1998
Abstract: We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable Cryptosporidium parvum oocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary s le concentrates with simultaneous detection of viable C. parvum oocysts, Giardia cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-μl packed pellet volumes of concentrates from creek and river water s les. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water s les. S le volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable Giardia cysts increased from 24% by IF microscopy to 69% by RT-PCR. Viable C. parvum oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable Cryptosporidium spp. in four s les by IF microscopy (14%), suggesting that Cryptosporidium species other than C. parvum were present in the water. This combination of the large-volume s ling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology.
Publisher: American Society for Microbiology
Date: 11-2017
DOI: 10.1128/AEM.01482-17
Abstract: Public health agencies are increasingly relying on genomics during Legionnaires' disease investigations. However, the causative bacterium ( Legionella pneumophila ) has an unusual population structure, with extreme temporal and spatial genome sequence conservation. Furthermore, Legionnaires' disease outbreaks can be caused by multiple L. pneumophila genotypes in a single source. These factors can confound cluster identification using standard phylogenomic methods. Here, we show that a statistical learning approach based on L. pneumophila core genome single nucleotide polymorphism (SNP) comparisons eliminates ambiguity for defining outbreak clusters and accurately predicts exposure sources for clinical cases. We illustrate the performance of our method by genome comparisons of 234 L. pneumophila isolates obtained from patients and cooling towers in Melbourne, Australia, between 1994 and 2014. This collection included one of the largest reported Legionnaires' disease outbreaks, which involved 125 cases at an aquarium. Using only sequence data from L. pneumophila cooling tower isolates and including all core genome variation, we built a multivariate model using discriminant analysis of principal components (DAPC) to find cooling tower-specific genomic signatures and then used it to predict the origin of clinical isolates. Model assignments were 93% congruent with epidemiological data, including the aquarium Legionnaires' disease outbreak and three other unrelated outbreak investigations. We applied the same approach to a recently described investigation of Legionnaires' disease within a UK hospital and observed a model predictive ability of 86%. We have developed a promising means to breach L. pneumophila genetic ersity extremes and provide objective source attribution data for outbreak investigations. IMPORTANCE Microbial outbreak investigations are moving to a paradigm where whole-genome sequencing and phylogenetic trees are used to support epidemiological investigations. It is critical that outbreak source predictions are accurate, particularly for pathogens, like Legionella pneumophila , which can spread widely and rapidly via cooling system aerosols, causing Legionnaires' disease. Here, by studying hundreds of Legionella pneumophila genomes collected over 21 years around a major Australian city, we uncovered limitations with the phylogenetic approach that could lead to a misidentification of outbreak sources. We implement instead a statistical learning technique that eliminates the ambiguity of inferring disease transmission from phylogenies. Our approach takes geolocation information and core genome variation from environmental L. pneumophila isolates to build statistical models that predict with high confidence the environmental source of clinical L. pneumophila during disease outbreaks. We show the versatility of the technique by applying it to unrelated Legionnaires' disease outbreaks in Australia and the UK.
Publisher: Cold Spring Harbor Laboratory
Date: 25-09-2020
DOI: 10.1101/2020.09.24.310821
Abstract: Pairwise single nucleotide polymorphisms (SNPs) are a cornerstone for genomic approaches to multidrug-resistant organisms (MDROs) transmission inference in hospitals. However, the impact of key analysis parameters on these inferences has not been systematically analysed. We conducted a multi-hospital 15-month prospective study, sequencing 1537 MDRO genomes for comparison methicillin-resistant Staphylococcus aureus , vancomycin-resistant Enterococcus faecium , and extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae . We systematically assessed the impact of s le and reference genome ersity, masking of prophage and regions of recombination, cumulative genome analysis compared to a three-month sliding-window, and the comparative effects each of these had when applying a SNP threshold for inferring likely transmission (≤15 SNPs for S. aureus , ≤25 for other species). Across the species, using a reference genome of the same sequence type provided a greater degree of pairwise SNP resolution, compared to species and outgroup-reference alignments that typically resulted in inflated SNP distances and the possibility of missed transmission events. Omitting prophage regions had minimal impacts, however, omitting recombination regions a highly variable effect, often inflating the number of closely related pairs. Estimating pairwise SNP distances was more consistent using a sliding-window than a cumulative approach. The use of a closely-related reference genome, without masking of prophage or recombination regions, and a sliding-window for isolate inclusion is best for accurate and consistent MDRO transmission inference. The increased stability and resolution provided by these approaches means SNP thresholds for putative transmission inference can be more reliably applied among erse MDROs. This work was supported by the Melbourne Genomics Health Alliance (funded by the State Government of Victoria, Department of Health and Human Services, and the ten member organizations) an National Health and Medical Research Council (Australia) Partnership grant (GNT1149991) and in idual grants from National Health and Medical Research Council (Australia) to NLS (GNT1093468), JCK (GNT1008549) and BPH (GNT1105905).
Publisher: eLife Sciences Publications, Ltd
Date: 20-03-2023
Publisher: Oxford University Press (OUP)
Date: 26-07-2022
Abstract: Enterococcus faecalis and Enterococcus faecium are Gram-positive commensal gut bacteria that can also cause fatal infections. To study clinically relevant multi-drug resistant E. faecalis and E. faecium strains, methods are needed to overcome physical (thick cell wall) and enzymatic barriers that limit the transfer of foreign DNA and thus prevent facile genetic manipulation. Enzymatic barriers to DNA uptake identified in E. faecalis and E. faecium include type I, II and IV restriction modification systems and CRISPR-Cas. This review examines E. faecalis and E. faecium DNA defence systems and the methods with potential to overcome these barriers. DNA defence system bypass will allow the application of innovative genetic techniques to expedite molecular-level understanding of these important, but somewhat neglected, pathogens.
Publisher: Public Library of Science (PLoS)
Date: 07-02-2020
Publisher: Springer Science and Business Media LLC
Date: 03-09-2018
Publisher: Oxford University Press (OUP)
Date: 21-02-2013
DOI: 10.1093/BFGP/ELT006
Abstract: Staphylococcus aureus remains a major opportunistic human pathogen, and while in many in iduals it is associated with asymptomatic colonization, it is also capable of causing a range of clinical syndromes from minor skin infections to life-threatening septicemia. Staphylococcus aureus has also demonstrated a remarkable capacity to acquire antimicrobial resistance. Recent technological advances in genomics have led to an avalanche of studies providing deep insights into how S. aureus is evolving globally and within the human host. However, there are still significant experimental barriers in using these insights to try and better understand the biology of S. aureus. Here, we summarize recent advances in the understanding of S. aureus through the use of genomic approaches, and contemplate what the near future holds for truly functional genomics that will allow us to better understand the biology of this pathogen.
Publisher: Frontiers Media SA
Date: 12-08-2021
DOI: 10.3389/FMICB.2021.703711
Abstract: Mosquito breeding sites are complex aquatic environments with wide microbial ersity and physicochemical parameters that can change over time during the development of immature insect stages. Changes in biotic and abiotic conditions in water can alter life-history traits of adult mosquitos but this area remains understudied. Here, using microbial genomic and metabolomics analyses, we explored the metabolites associated with Aedes aegypti breeding sites as well as the potential contribution of Klebsiella sp., symbiotic bacteria highly associated with mosquitoes. We sought to address whether breeding sites have a signature metabolic profile and understand the metabolite contribution of the bacteria in the aquatic niches where Ae. aegypti larvae develop. An analysis of 32 mosquito-associated bacterial genomes, including Klebsiella , allowed us to identify gene clusters involved in primary metabolic pathways. From them, we inferred metabolites that could impact larval development (e.g., spermidine), as well as influence the quality assessment of a breeding site by a gravid female (e.g., putrescine), if produced by bacteria in the water. We also detected significant variance in metabolite presence profiles between water s les representing a decoupled oviposition event (oviposition by single females and manually deposited eggs) versus a control where no mosquito interactions occurred (PERMANOVA: p & 0.05 R 2 = 24.64% and R 2 = 30.07%). Five Klebsiella metabolites were exclusively linked to water s les where oviposition and development occurred. These data suggest metabolomics can be applied to identify compounds potentially used by female Ae. aegypti to evaluate the quality of a breeding site. Elucidating the physiological mechanisms by which the females could integrate these sensory cues while ovipositing constitutes a growing field of interest, which could benefit from a more depurated list of candidate molecules.
Publisher: American Society for Microbiology
Date: 08-2000
DOI: 10.1128/AEM.66.8.3206-3213.2000
Abstract: We recently described the use of PCR to identify the environmental source of Mycobacterium ulcerans during an outbreak of ulcerative disease that occurred in a localized region of southeast Australia. The PCR used was based on lification of the M. ulcerans -specific insertion sequence, IS 2404 . In this study we developed a new test that is a substantial improvement over the original PCR method in terms of sensitivity, reliability, and ease of use. In the new method magnetic bead sequence capture-PCR is used to detect two M. ulcerans sequences (IS 2404 and IS 2606 ) and total mycobacterial 16S ribosomal DNA. We used sequence capture-PCR to test water and plant material collected over a 12-month period during 1998 and 1999 from sites near the centers of two distinct foci of M. ulcerans infections. A golf course irrigation system in one area and a small shallow lake in another area repeatedly were PCR positive for M. ulcerans . Nearby sites and sites unrelated to the endemic areas were negative. Based on the PCR data, a most-probable-number method was used to estimate the concentration of M. ulcerans cells in positive s les from both regions. This procedure resulted in average concentrations of 0.5 cell per 100 ml of water and 40 cells per 100 g of detritus. Loss of the PCR signal coincided with a decrease in ulcerative disease in each area. These results provide further evidence that M. ulcerans may be transmitted from a point environmental source and demonstrate the utility of magnetic bead sequence capture-PCR for identification of nonculturable microbial pathogens in the environment.
Publisher: Public Library of Science (PLoS)
Date: 14-11-2013
Publisher: Springer Science and Business Media LLC
Date: 23-08-2018
Publisher: Elsevier BV
Date: 10-2016
DOI: 10.1016/J.CMI.2017.03.027
Abstract: Vancomycin-intermediate Staphylococcus aureus (VISA) is associated with genetic changes that may also impact upon pathogenicity. In the current study, we compared the virulence of clinical VISA strains with their isogenic vancomycin-susceptible progenitors (VSSA). Production of the critical virulence protein, α toxin, was assessed using Western blot analysis and was correlated to agr activity using a bioluminescent agr-reporter. Cytotoxicity and intracellular persistence were compared ex vivo for VSSA and VISA within non-professional phagocytes (NPP). Virulence and host immune responses were further explored in vivo using a murine model of bacteraemia. VISA isolates produced up to 20-fold less α toxin compared with VSSA, and this was corroborated by either loss of agr activity due to agr mutation, or altered agr activity in the absence of mutation. VISA were less cytotoxic towards NPP and were associated with enhanced intracellular persistence, suggesting that NPP may act as a reservoir for VISA. Infection with VSSA strains produced higher mortality in a murine bacteraemia model (≥90% 7-day mortality) compared with infection with VISA isolates (20% to 50%, p <0.001). Mice infected with VISA produced a d ened immune response (4.6-fold reduction in interleukin-6, p <0.001) and persistent organ bacterial growth was observed for VISA strains out to 7 days. These findings highlight the remarkable adaptability of S. aureus, whereby, in addition to having reduced antibiotic susceptibility, VISA alter the expression of pathogenic factors to circumvent the host immune response to favour persistent infection over acute virulence.
Publisher: American Chemical Society (ACS)
Date: 07-11-2019
DOI: 10.1021/ACSCHEMBIO.9B00763
Abstract: The genus
Publisher: American Chemical Society (ACS)
Date: 08-07-2019
DOI: 10.1021/ACSCHEMBIO.9B00406
Abstract: The bacterial endosymbiont (
Publisher: American Society for Microbiology
Date: 11-2009
DOI: 10.1128/JCM.00760-09
Abstract: Mycobacterium ulcerans causes the devastating infectious skin disease Buruli ulcer and has a monomorphic population structure. The resolution of conventional genetic fingerprinting methods is therefore not sufficient for microepidemiological studies aiming to characterize transmission pathways. In a previous comparative genomic hybridization analysis with a microarray covering part of the M. ulcerans genome, we have found extensive insertional-deletional sequence polymorphisms among M. ulcerans isolates of erse geographic origins that allowed us to distinguish between strains coming from different continents. Since large numbers of insertion sequences are spread over the genome of African M. ulcerans strains, we reasoned that these may drive large sequence polymorphisms in otherwise clonal local mycobacterial populations. In this study, we used a printed DNA microarray covering the whole genome of the Ghanaian M. ulcerans reference strain Agy99 for comparative genomic hybridization. The assay identified multiple regions of difference when DNA of a Japanese M. ulcerans strain was analyzed. In contrast, not a single insertional-deletional genomic variation was found within a panel of disease isolates coming from an area of Ghana where Buruli ulcer is endemic. These results indicate that, despite the expectations deduced from other mycobacterial pathogens, only analyses of single nucleotide polymorphisms will have the potential to differentiate local populations of M. ulcerans .
Publisher: American Society for Microbiology
Date: 25-08-2020
DOI: 10.1128/MSYSTEMS.00452-20
Abstract: This study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions between commensal gut bacteria and a specific pathogen. Knowledge from this research will inform gut microbiome engineering strategies, with the aim of translating observations from animal models to human-relevant therapeutic applications.
Publisher: Cold Spring Harbor Laboratory
Date: 02-05-2017
DOI: 10.1101/133033
Abstract: Public health agencies are increasingly relying on genomics during Legionnaires’ disease investigations. However, the causative bacterium ( Legionella pneumophila ) has an unusual population structure with extreme temporal and spatial genome sequence conservation. Furthermore, Legionnaires’ disease outbreaks can be caused by multiple L. pneumophila genotypes in a single source. These factors can confound cluster identification using standard phylogenomic methods. Here, we show that a statistical learning approach based on L. pneumophila core genome single nucleotide polymorphism (SNP) comparisons eliminates ambiguity for defining outbreak clusters and accurately predicts exposure sources for clinical cases. We illustrate the performance of our method by genome comparisons of 234 L. pneumophila isolates obtained from patients and cooling towers in Melbourne, Australia between 1994 and 2014. This collection included one of the largest reported Legionnaires’ disease outbreaks, involving 125 cases at an aquarium. Using only sequence data from L. pneumophila cooling tower isolates and including all core genome variation, we built a multivariate model using discriminant analysis of principal components (DAPC) to find cooling tower-specific genomic signatures, and then used it to predict the origin of clinical isolates. Model assignments were 93% congruent with epidemiological data, including the aquarium Legionnaires’ outbreak and three other unrelated outbreak investigations. We applied the same approach to a recently described investigation of Legionnaires’ disease within a UK hospital and observed model predictive ability of 86%. We have developed a promising means to breach L. pneumophila genetic ersity extremes and provide objective source attribution data for outbreak investigations.
Publisher: Springer Science and Business Media LLC
Date: 10-02-2014
Publisher: Microbiology Society
Date: 19-04-2022
Abstract: Background. Australia’s response to the coronavirus disease 2019 (COVID-19) pandemic relies on widespread availability of rapid, accurate testing and reporting of results to facilitate contact tracing. The extensive geographical area of Australia presents a logistical challenge, with many of the population located distant from a laboratory capable of robust severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. A strategy to address this is the deployment of a mobile facility utilizing novel diagnostic platforms. This study aimed to evaluate the feasibility of a fully contained transportable SARS-CoV-2 testing laboratory using a range of rapid point-of-care tests. Method. A 20 ft (6.1 m) shipping container was refurbished (GeneWorks, Adelaide, South Australia) with climate controls, laboratory benches, hand-wash station and a class II biosafety cabinet. Portable marquees situated adjacent to the container served as stations for registration, s le acquisition and personal protective equipment for staff. Specimens were collected and tested on-site utilizing either the Abbott ID NOW or Abbott Panbio rapid tests. SARS-CoV-2 positive results from the rapid platforms or any participants reporting symptoms consistent with COVID-19 were tested on-site by GeneXpert Xpress RT-PCR. All s les were tested in parallel with a standard-of-care RT-PCR test (Panther Fusion SARS-CoV-2 assay) performed at the public health reference laboratory. In-laboratory environmental conditions and data management-related factors were also recorded. Results. Over a 3 week period, 415 participants were recruited for point-of-care SARS-CoV-2 testing. From time of enrolment, the median result turnaround time was 26 min for the Abbott ID NOW, 32 min for the Abbott Panbio and 75 min for the Xpert Xpress. The environmental conditions of the refurbished shipping container were found to be suitable for all platforms tested, although humidity may have produced condensation within the container. Available software enabled turnaround times to be recorded, although technical malfunction resulted in incomplete data capture. Conclusion. Transportable container laboratories can enable rapid COVID-19 results at the point of care and may be useful during outbreak settings, particularly in environments that are physically distant from centralized laboratories. They may also be appropriate in resource-limited settings. The results of this pilot study confirm feasibility, although larger trials to validate in idual rapid point-of-care testing platforms in this environment are required.
Publisher: Wiley
Date: 18-03-2020
Publisher: Oxford University Press (OUP)
Date: 08-08-2022
DOI: 10.1093/PNASNEXUS/PGAC152
Abstract: The tight association of the pathogenic fungus Rhizopus microsporus and its toxin-producing, bacterial endosymbionts (Mycetohabitans spp.) is distributed worldwide and has significance for agriculture, food production, and human health. Intriguingly, the endofungal bacteria are essential for the propagation of the fungal host. Yet, little is known about chemical mediators fostering the symbiosis, and universal metabolites that support the mutualistic relationship have remained elusive. Here, we describe the discovery of a complex of specialized metabolites produced by endofungal bacteria under symbiotic conditions. Through full genome sequencing and comparative genomics of eight endofungal symbiont strains from geographically distant regions, we discovered a conserved gene locus (hab) for a nonribosomal peptide synthetase as a unifying trait. Bioinformatics analyses, targeted gene deletions, and chemical profiling uncovered unprecedented depsipeptides (habitasporins) whose structures were fully elucidated. Computational network analysis and labeling experiments granted insight into the biosynthesis of their nonproteinogenic building blocks (pipecolic acid and β-phenylalanine). Deletion of the hab gene locus was shown to impair the ability of the bacteria to enter their fungal host. Our study unveils a common principle of the endosymbiotic lifestyle of Mycetohabitans species and expands the repertoire of characterized chemical mediators of a globally occurring mutualistic association.
Publisher: eLife Sciences Publications, Ltd
Date: 13-04-2023
Publisher: Public Library of Science (PLoS)
Date: 02-11-2010
Publisher: Public Library of Science (PLoS)
Date: 20-07-2010
Publisher: Frontiers Media SA
Date: 06-10-2017
Publisher: Microbiology Society
Date: 12-2020
Abstract: Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health laboratory activities. Here, we announce a BioProject (PRJNA556438) dedicated to sharing complete genomes chosen to represent a range of pathogenic bacteria with regional importance to Australia and the Southwest Pacific enriching the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health microbiology laboratories. In this first step, we present 26 complete high-quality bacterial genomes. Additionally, we describe here a framework for reconstructing complete microbial genomes and highlight some of the challenges and considerations for accurate and reproducible genome reconstruction.
Publisher: American Society for Microbiology
Date: 04-2016
DOI: 10.1128/AAC.03020-15
Abstract: The prevalence of fusidic acid (FA) resistance among Staphylococcus aureus strains in New Zealand (NZ) is among the highest reported globally, with a recent study describing a resistance rate of approximately 28%. Three FA-resistant S. aureus clones (ST5 MRSA, ST1 MSSA, and ST1 MRSA) have emerged over the past decade and now predominate in NZ, and in all three clones FA resistance is mediated by the fusC gene. In particular, ST5 MRSA has rapidly become the dominant MRSA clone in NZ, although the origin of FA-resistant ST5 MRSA has not been explored, and the genetic context of fusC in FA-resistant NZ isolates is unknown. To better understand the rapid emergence of FA-resistant S. aureus , we used population-based comparative genomics to characterize a collection of FA-resistant and FA-susceptible isolates from NZ. FA-resistant NZ ST5 MRSA displayed minimal genetic ersity and represented a phylogenetically distinct clade within a global population model of clonal complex 5 (CC5) S. aureus . In all lineages, fusC was invariably located within staphylococcal cassette chromosome (SCC) elements, suggesting that SCC-mediated horizontal transfer is the primary mechanism of fusC dissemination. The genotypic association of fusC with mecA has important implications for the emergence of MRSA clones in populations with high usage of fusidic acid. In addition, we found that fusC was colocated with a recently described virulence factor ( tirS ) in dominant NZ S. aureus clones, suggesting a fitness advantage. This study points to the likely molecular mechanisms responsible for the successful emergence and spread of FA-resistant S. aureus .
Publisher: Cold Spring Harbor Laboratory
Date: 05-11-2019
DOI: 10.1101/829663
Abstract: Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health lab activities. Here, we present complete genomes of pathogen strains of regional importance to the Southwest Pacific and Australia. These enrich the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health labs. Whole-genome sequence (WGS) data is increasingly important in public health microbiology (1–4). The data can be used to replicate many of the basic bacterial sub-typing approaches, as well as support epidemiological investigations, such as surveillance and outbreak investigation (5–7). The appeal of WGS data comes from the promise of a single workflow to process all microbial pathogens that can provide easily portable data that promotes deeper integration of surveillance and investigation efforts across jurisdictions. This promise is leading to a concerted effort to move microbial public health to a primarily genome-based workflow at numerous jurisdictions (8–10), including Australia (11).
Publisher: Elsevier BV
Date: 12-2021
DOI: 10.1016/J.VACCINE.2021.05.092
Abstract: Buruli ulcer, caused by Mycobacterium ulcerans, is a neglected tropical disease endemic to over 30 countries, with increasing incidence in temperate, coastal Victoria, Australia. Strategies to control transmission are urgently required. This study systematically reviews the literature to identify and describe candidate prophylactic Buruli ulcer vaccines. This review highlights that Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine studied in randomised controlled trials and confirms its importance as a benchmark for comparison against putative vaccines in pre-clinical studies. Nevertheless, BCG alone is unable to offer long-term protection in humans. A number of experimental vaccines that exceed the protection provided by BCG in mice have emerged, particularly those utilising recombinant BCG expressing immunogenic M. ulcerans proteins. Although progress is promising, there remain key questions about the optimal approach to characterising the immunological correlates of protection in humans and strategies to investigate the safety and efficacy of such vaccines in humans.
Publisher: Cold Spring Harbor Laboratory
Date: 11-07-2023
DOI: 10.1101/2023.07.11.548529
Abstract: Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, ‘unproductive’ inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extra- and intracellular burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intracellular bacterial populations, and appears to involve interruption of NFkB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been u. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection resulting in release of pro-inflammatory cytokines (IL8, TNFα). Our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation.
Publisher: Elsevier BV
Date: 12-2012
Publisher: BMJ
Date: 15-12-2018
DOI: 10.1136/SEXTRANS-2017-053287
Abstract: Drug-resistant Neisseria gonorrhoeae are now a global public health threat. Direct transmission of antibiotic-resistant gonococci between in iduals has been proposed as a driver for the increased transmission of resistance, but direct evidence of such transmission is limited. Whole-genome sequencing (WGS) has superior resolution to investigate outbreaks and disease transmission compared with traditional molecular typing methods such as multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence (NG-MAST). We therefore aimed to systematically investigate the transmission of N. gonorrhoeae between men in sexual partnerships using WGS to compare isolates and their resistance to antibiotics at a genome level. 458 couples from a large prospective cohort of men who have sex with men (MSM) tested for gonorrhoea together between 2005 and 2014 were included, and WGS was conducted on all isolates from couples where both men were culture-positive for N. gonorrhoeae . Resistance-determining sequences were identified from genome assemblies, and comparison of isolates between and within in iduals was performed by pairwise single nucleotide polymorphism and pangenome comparisons, and in silico predictions of NG-MAST and MLST. For 33 of 34 (97% 95% CI 85% to 100%) couples where both partners were positive for gonorrhoea, the resistance-determining genes and mutations were identical in isolates from each partner (94 isolates in total). Resistance determinants in isolates from 23 of 23 (100% 95% CI 86% to 100%) men with multisite infections were also identical within an in idual. These partner and within-host isolates were indistinguishable by NG-MAST, MLST and whole genomic comparisons. These data support the transmission of antibiotic-resistant strains between sexual partners as a key driver of resistance rates in gonorrhoea among MSM. This improved understanding of the transmission dynamics of N. gonorrhoeae between sexual partners will inform treatment and prevention guidelines.
Publisher: American Society for Microbiology
Date: 04-2007
Publisher: Springer Science and Business Media LLC
Date: 09-10-2017
DOI: 10.1038/S41564-017-0039-9
Abstract: Xenorhabdus and Photorhabdus species dedicate a large amount of resources to the production of specialized metabolites derived from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS). Both bacteria undergo symbiosis with nematodes, which is followed by an insect pathogenic phase. So far, the molecular basis of this tripartite relationship and the exact roles that in idual metabolites and metabolic pathways play have not been well understood. To close this gap, we have significantly expanded the database for comparative genomics studies in these bacteria. Clustering the genes encoded in the in idual genomes into hierarchical orthologous groups reveals a high-resolution picture of functional evolution in this clade. It identifies groups of genes-many of which are involved in secondary metabolite production-that may account for the niche specificity of these bacteria. Photorhabdus and Xenorhabdus appear very similar at the DNA sequence level, which indicates their close evolutionary relationship. Yet, high-resolution mass spectrometry analyses reveal a huge chemical ersity in the two taxa. Molecular network reconstruction identified a large number of previously unidentified metabolite classes, including the xefo eptides and tilivalline. Here, we apply genomic and metabolomic methods in a complementary manner to identify and elucidate additional classes of natural products. We also highlight the ability to rapidly and simultaneously identify potentially interesting bioactive products from NRPSs and PKSs, thereby augmenting the contribution of molecular biology techniques to the acceleration of natural product discovery.
Publisher: Elsevier BV
Date: 2019
DOI: 10.1016/J.IJID.2018.10.012
Abstract: Buruli ulcer (BU) is a chronic and destructive infection of the skin and soft tissues caused by Mycobacterium ulcerans. Recently, population flows have triggered the appearance of several sporadic cases of BU in non-endemic countries. This represents a significant diagnostic challenge for clinicians and microbiologists. We describe the first case of BU imported to Spain. The patient was a Spanish woman who had stayed 5 months in the jungle of Peru.
Publisher: Microbiology Society
Date: 20-12-2021
DOI: 10.1099/MIC.0.001119
Abstract: Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.
Publisher: Public Library of Science (PLoS)
Date: 26-08-2021
DOI: 10.1371/JOURNAL.PNTD.0009678
Abstract: Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rare but chronic debilitating skin and soft tissue disease found predominantly in West Africa and Southeast Australia. While a moderate body of research has examined the distribution of M . ulcerans , the specific route(s) of transmission of this bacterium remain unknown, hindering control efforts. M . ulcerans is considered an environmental pathogen given it is associated with lentic ecosystems and human-to-human spread is negligible. However, the pathogen is also carried by various mammals and invertebrates, which may serve as key reservoirs and mechanical vectors, respectively. Here, we examine and review recent evidence from these endemic regions on potential transmission pathways, noting differences in findings between Africa and Australia, and summarising the risk and protective factors associated with Buruli ulcer transmission. We also discuss evidence suggesting that environmental disturbance and human population changes precede outbreaks. We note five key research priorities, including adoption of One Health frameworks, to resolve transmission pathways and inform control strategies to reduce the spread of Buruli ulcer.
Publisher: American Society for Microbiology
Date: 04-05-2016
Publisher: American Chemical Society (ACS)
Date: 15-09-2021
DOI: 10.1021/ACSBIOMATERIALS.1C01105
Abstract: The ability to detect SARS-CoV-2 is critical to implementing evidence-based strategies to address the COVID-19 global pandemic. Expanding SARS-CoV-2 diagnostic ability beyond well-equipped laboratories widens the opportunity for surveillance and control efforts. However, such advances are predicated on the availability of rapid, scalable, accessible, yet high-performance diagnostic platforms. Methods to detect viral RNA using reverse transcription loop-mediated isothermal lification (RT-LAMP) show promise as rapid and field-deployable tests however, the per-unit costs of the required diagnostic hardware can be a barrier for scaled deployment. Here, we describe a diagnostic hardware configuration for LAMP technology, named the
Publisher: Cold Spring Harbor Laboratory
Date: 07-05-2023
DOI: 10.1101/2023.05.07.539718
Abstract: In temperate southeastern Australia over the past two decades there has been a marked progressive increase in human cases of Buruli ulcer, an infection of subcutaneous tissue caused by Mycobacterium ulcerans . Native possums are the major local environmental reservoir of M. uclerans as they not only develop Buruli lesions but they also shed M. ulcerans in their excreta. However the way humans acquire M. ulcerans from possums has not been determined. Previous case-control studies, insect field surveys and vector competence studies have suggested a role for mosquitoes in M. ulcerans transmission between possums and humans. To explore these links we conducted an extensive, 4-month structured mosquito field survey and four ad hoc field surveys across an area of 350km 2 on the Mornington Peninsula, an area endemic for Buruli ulcer. We then compared spatial and temporal patterns of M. ulcerans -positive mosquito occurrence with M. ulcerans -positive possums (established by previous possum excreta surveys) and human Buruli ulcer cases across the region. We used metabarcoding to assess mosquito blood-feeding host preference and to reconstruct M. ulcerans genomes from positive mosquitoes to test epidemiological inferences. We collected 66,325 mosquitoes spanning 26 different species from 180 repeatedly s led traps over a 4-month peri o C d ul . ex molestus and Aedes notoscriptus were the dominant species (42% and 35% of trapped mosquitoes, respectively). PCR screening 25% of trapped mosquitoes revealed a significant association between M. ulcerans and Ae. notoscriptus (p .0001) with a maximum likelihood estimate (MLE) of 5.88 M. ulcerans positive mosquitoes per 1,000 tested. Using spatial scanning statistics, we also observed significant overlap between clusters of M. ulcerans -positive Ae. notoscriptus , M. ulcerans -positive possum excreta and human Buruli ulcer cases. Metabarcoding analyses of blood-fed Ae. notoscriptus showed in idual mosquitoes had fed both on humans and native possums. Enrichment genome sequencing from PCR-positive mosquitoes confirmed identical M. ulcerans genome single-nucleotide polymorphism (SNP) profiles between insects, possums and clinical human isolates within the same regions. These findings indicate that certain mosquito species likely transmit M. ulcerans in southeastern Australia and highlight mosquito control as a plausible means to control the Buruli ulcer epidemic in our region.
Publisher: Public Library of Science (PLoS)
Date: 13-08-2018
Publisher: American Society for Microbiology
Date: 05-2013
DOI: 10.1128/JCM.03332-12
Abstract: Next-generation sequencing (NGS) of bacterial genomes has recently become more accessible and is now available to the routine diagnostic microbiology laboratory. However, questions remain regarding its feasibility, particularly with respect to data analysis in nonspecialist centers. To test the applicability of NGS to outbreak investigations, Ion Torrent sequencing was used to investigate a putative multidrug-resistant Escherichia coli outbreak in the neonatal unit of the Mercy Hospital for Women, Melbourne, Australia. Four suspected outbreak strains and a comparator strain were sequenced. Genome-wide single nucleotide polymorphism (SNP) analysis demonstrated that the four neonatal intensive care unit (NICU) strains were identical and easily differentiated from the comparator strain. Genome sequence data also determined that the NICU strains belonged to multilocus sequence type 131 and carried the bla CTX-M-15 extended-spectrum beta-lactamase. Comparison of the outbreak strains to all publicly available complete E. coli genome sequences showed that they clustered with neonatal meningitis and uropathogenic isolates. The turnaround time from a positive culture to the completion of sequencing (prior to data analysis) was 5 days, and the cost was approximately $300 per strain (for the reagents only). The main obstacles to a mainstream adoption of NGS technologies in diagnostic microbiology laboratories are currently cost (although this is decreasing), a paucity of user-friendly and clinically focused bioinformatics platforms, and a lack of genomics expertise outside the research environment. Despite these hurdles, NGS technologies provide unparalleled high-resolution genotyping in a short time frame and are likely to be widely implemented in the field of diagnostic microbiology in the next few years, particularly for epidemiological investigations (replacing current typing methods) and the characterization of resistance determinants. Clinical microbiologists need to familiarize themselves with these technologies and their applications.
Publisher: Cold Spring Harbor Laboratory
Date: 26-05-2022
DOI: 10.1101/2022.05.26.493552
Abstract: Realising the promise of genomics to revolutionise routine AMR diagnosis and surveillance has been a long-standing challenge in clinical and public health microbiology. We have directly addressed this issue by creating and validating abritAMR , an ISO-accredited bioinformatics platform for genomics-based bacterial AMR gene detection. abritAMR utilises the NCBI AMRFinderPlus for detection of AMR genes and mutations, with additional features to classify AMR determinants into an antibiotic class. We validated abritAMR by comparing with multiplex PCR or gold-standard reference genomes, together representing 1500 different bacteria across 29 genera and covering 415 antibiotic resistance alleles. We also assessed inference of phenotypic resistance by comparing genomic predictions with agar dilution results for 864 Salmonella spp. Performance of abritAMR was excellent, detecting AMR genes with 99.9% accuracy (95% CI 99.9-99.9%), 97.9% sensitivity (95% CI 97.5-98.4%) and 100% specificity (100-100%). Phenotypic inference of resistance for Salmonella spp. was equally impressive, with 98.9% accuracy (98.7-99.1%). Validation data were submitted to the governing authority and ISO15189 accreditation was achieved. Implementation of abritAMR resulted in streamlined bioinformatics and reporting pathways, and it was readily updated and re-verified with database revisions or changes in reporting requirements. abritAMR is publicly and freely available to assist clinical and public health microbiology laboratories everywhere harness the power of AMR genomics in their professional practice.
Publisher: Oxford University Press (OUP)
Date: 09-06-2016
DOI: 10.1093/CID/CIW359
Publisher: Wiley
Date: 11-02-2019
Publisher: Elsevier BV
Date: 2021
DOI: 10.2139/SSRN.3830366
Publisher: Microbiology Society
Date: 05-2007
DOI: 10.1099/MIC.0.2006/004564-0
Abstract: Screening of the genome sequence of the Mycobacterium ulcerans strain Agy99 from Ghana with tandem repeats finder software revealed 34 novel non-degenerate tandem repeats containing loci suitable for variable number tandem repeats (VNTR) typing. All loci revealed polymorphism within M. ulcerans isolates of geographically erse origins. The results confirm the evolutionary scenario suggested by multi-locus sequence typing in which a progenitor of all M. ulcerans lineages emerged from the environmental species Mycobacterium marinum and subsequently erged into several geographical lineages. For further attempts to develop a VNTR-based genetic fingerprinting tool for M. ulcerans, it is suggested that the focus should rather be on M. marinum than on the African M. ulcerans Agy99 genome sequence as a starting point.
Publisher: Frontiers Media SA
Date: 28-04-2022
DOI: 10.3389/FCIMB.2022.855290
Abstract: Latent HIV-1 provirus in infected in iduals on suppressive therapy does not always remain transcriptionally silent. Both HIV-1 LTR and human gene promoter derived transcriptional events can contribute HIV-1 sequences to the mRNA produced in the cell. In addition, chimeric cellular:HIV mRNA can arise through readthrough transcription and aberrant splicing. Using target enrichment coupled to the Illumina Mi-Seq and PacBio RS II platforms, we show that 3’ LTR activation is frequent in latently infected cells from both the CCL19-induced primary cell model of HIV-1 latency as well as ex vivo s les. In both systems of latent HIV-1 infection, we detected several chimeric species that were generated via activation of a cryptic splice donor site in the 5’ LTR of HIV-1. Aberrant splicing involving the major HIV-1 splice donor sites, SD1 and SD4 disrupts post-transcriptional processing of the gene in which HIV-1 is integrated. In the primary cell model of HIV-1 latency, Tat-encoding sequences are incorporated into the chimeric mRNA transcripts through the use of SD4. Our study unravels clues to the characteristics of HIV-1 integrants that promote formation of chimeric cellular:HIV mRNA and improves the understanding of the HIV-1 RNA footprint in latently infected cells.
Publisher: Springer Science and Business Media LLC
Date: 2008
Publisher: Oxford University Press (OUP)
Date: 23-04-2013
DOI: 10.1093/JAC/DKT130
Abstract: We noted four cases of apparent in vivo emergence of teicoplanin resistance during failed therapy for initially teicoplanin-susceptible vanB vancomycin-resistant Enterococcus faecium (VREfm) infections in solid organ transplant recipients at our institution over a 12 month period. We investigated if in vivo emergence of resistance had occurred, if transplant-related vancomycin-resistant Enterococcus (VRE) infections had occurred and identified clinical predictors of resistance emergence. Whole genome sequencing was performed on nine VREfm isolates for phylogenetic analysis and to identify determinants of teicoplanin resistance. Clinical treatment details were compared with other patients who received teicoplanin for confirmed vanB VRE infections but did not develop resistance during the same year at our institution. A high-resolution, core genome phylogeny was inferred for nine VREfm isolates and confirmed in vivo development of resistance during failed therapy in four cases. Four different non-synonymous single nucleotide polymorphisms (SNPs) were observed in the vanRS genes of teicoplanin-resistant strains compared with the index teicoplanin-susceptible strains, and these SNPs were predicted to confer teicoplanin resistance. VREfm within a cluster of early transplant-related infections were phylogenetically identical at the core genome level, indicating a common source donor. Focus eradication and absence of prosthetic material were characteristics of those patients treated successfully. Clinicians should be cautious of resistance emerging during teicoplanin therapy for vanB VRE, particularly in immunosuppressed patients or where source control is difficult.
Publisher: Elsevier BV
Date: 09-2022
DOI: 10.1016/J.TIM.2022.01.013
Abstract: The human gut is host to a erse range of microorganisms that offer protection against colonization by multidrug-resistant bacteria. Antibiotic use, medications, health conditions, and lifestyle factors can alter the composition of the gut microbiota in such a way that results in loss of colonization resistance and increased susceptibility to invading pathogenic antibiotic-resistant bacteria. Therapeutics aiming to restore a erse and protective microbiome are fast advancing. In this review, we focus on the compositional changes within the gut microbiome that are associated with colonization resistance and discuss their use as potential targets for therapeutics or diagnostics.
Publisher: American Society for Microbiology
Date: 05-2019
DOI: 10.1128/IAI.00163-19
Abstract: Staphylococcus aureus is a major human pathogen of the skin. The global burden of diabetes is high, with S. aureus being a major complication of diabetic wound infections.
Publisher: American Society for Microbiology
Date: 20-02-2020
DOI: 10.1128/IAI.00753-19
Abstract: The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans . There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, R 4 Pam 2 Cys.
Publisher: Microbiology Society
Date: 06-1999
Publisher: American Society for Microbiology
Date: 06-2017
DOI: 10.1128/JCM.00197-17
Abstract: Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera . Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera . We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 erse genera confirmed the in silico -predicted specificity for M. chimaera . Screening 33 water s les from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative s les positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera .
Publisher: Wiley
Date: 28-10-2014
DOI: 10.1111/MMI.12806
Publisher: Oxford University Press (OUP)
Date: 22-11-2018
DOI: 10.1093/JAC/DKX405
Publisher: Oxford University Press (OUP)
Date: 15-08-2016
DOI: 10.1093/JAC/DKW314
Abstract: Enterococcus faecium is a major nosocomial pathogen causing significant morbidity and mortality worldwide. Assessment of E. faecium using MLST to understand the spread of this organism is an important component of hospital infection control measures. Recent studies, however, suggest that MLST might be inadequate for E. faecium surveillance. To use WGS to characterize recently identified vancomycin-resistant E. faecium (VREfm) isolates non-typeable by MLST that appear to be causing a multi-jurisdictional outbreak in Australia. Illumina NextSeq and Pacific Biosciences SMRT sequencing platforms were used to determine the genome sequences of 66 non-typeable E. faecium (NTEfm) isolates. Phylogenetic and bioinformatics analyses were subsequently performed using a number of in silico tools. Sixty-six E. faecium isolates were identified by WGS from multiple health jurisdictions in Australia that could not be typed by MLST due to a missing pstS allele. SMRT sequencing and complete genome assembly revealed a large chromosomal rearrangement in representative strain DMG1500801, which likely facilitated the deletion of the pstS region. Phylogenomic analysis of this population suggests that deletion of pstS within E. faecium has arisen independently on at least three occasions. Importantly, the majority of these isolates displayed a vancomycin-resistant genotype. We have identified NTEfm isolates that appear to be causing a multi-jurisdictional outbreak in Australia. Identification of these isolates has important implications for MLST-based typing activities designed to monitor the spread of VREfm and provides further evidence supporting the use of WGS for hospital surveillance of E. faecium.
Publisher: American Society for Microbiology
Date: 02-2004
DOI: 10.1128/AEM.70.2.1097-1103.2004
Abstract: Mycobacterium ulcerans is the causative agent of Buruli ulcer, one of the most common mycobacterial diseases of humans. Recent studies have implicated aquatic insects in the transmission of this pathogen, but the contributions of other elements of the environment remain largely unknown. We report here that crude extracts from two green algae added to the BACTEC 7H12B culture medium halved the doubling time of M. ulcerans and promoted biofilm formation. Using the 7H12B medium, modified by the addition of the algal extract, and immunomagnetic separation, we also demonstrate that M. ulcerans is associated with aquatic plants in an area of the Ivory Coast where Buruli ulcer is endemic. Genotype analysis showed that plant-associated M. ulcerans had the same profile as isolates recovered in the same region from both aquatic insects and clinical specimens. These observations implicate aquatic plants as a reservoir of M. ulcerans and add a new potential link in the chain of transmission of M. ulcerans to humans.
Publisher: American Society for Microbiology
Date: 2010
DOI: 10.1128/CMR.00042-09
Abstract: The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) over the past decade has provided a challenge to diagnostic microbiologists to detect these strains, clinicians treating patients with infections due to these strains, and researchers attempting to understand the resistance mechanisms. Recent data show that these strains have been detected globally and in many cases are associated with glycopeptide treatment failure however, more rigorous clinical studies are required to clearly define the contribution of hVISA to glycopeptide treatment outcomes. It is now becoming clear that sequential point mutations in key global regulatory genes contribute to the hVISA and VISA phenotypes, which are associated predominately with cell wall thickening and restricted vancomycin access to its site of activity in the ision septum however, the phenotypic features of these strains can vary because the mutations leading to resistance can vary. Interestingly, changes in the staphylococcal surface and expression of agr are likely to impact host-pathogen interactions in hVISA and VISA infections. Given the subtleties of vancomycin susceptibility testing against S. aureus , it is imperative that diagnostic laboratories use well-standardized methods and have a framework for detecting reduced vancomycin susceptibility in S. aureus .
Publisher: American Society for Microbiology
Date: 04-1999
DOI: 10.1128/JCM.37.4.1018-1023.1999
Abstract: Molecular analysis of Mycobacterium ulcerans has revealed two new insertion sequences (ISs), IS 2404 and IS 2606 . IS 2404 was identified by complete sequencing of a previously described repetitive DNA segment from M. ulcerans . This element is 1,274 bp long, contains 12-bp inverted repeats and a single open reading frame (ORF) potentially encoding a protein of 327 amino acids (aa), and apparently generates 7-bp direct repeats upon transposition. Amino acid similarity was found between the putative transposase and those encoded by ISs in other bacterial sequences from Aeromonas salmonicida ( As Is 1 ), Escherichia coli (H repeat element), Vibrio cholerae ( Vc IS 1 ), and Porphyromonas gingivalis ( PG IS 2 ). The second IS, IS 2606 , was discovered by sequence analysis of a Hae III fragment of M. ulcerans genomic DNA containing a repetitive sequence. This element is 1,404 bp long, with 12-bp inverted repeats and a single ORF potentially encoding a protein of 445 aa. Database searches revealed a high degree of amino acid identity (70%) with the putative transposase of IS 1554 from M. tuberculosis . Significant amino acid identity (40%) was also observed with transposases from several other microorganisms, including Rhizobium meliloti (IS Rm3 ), Burkholderia cepacia (IS 1356 ), Corynebacterium diphtheriae , and Yersinia pestis . PCR screening of DNA from 45 other species of mycobacteria with primers for IS 2404 confirm that this element is found only in M. ulcerans . However, by PCR, IS 2606 was also found in Mycobacterium lentiflavum , another slow-growing member of the genus Mycobacterium that is apparently genetically distinct from M. ulcerans . Testing the sensitivity of PCR based on IS 2404 and IS 2606 primers demonstrated the ability to detect 0.1 and 1 M. ulcerans genome equivalents, respectively. The ability to detect small numbers of cells by using two gene targets will be particularly useful for analyzing environmental s les, where there may be low concentrations of M. ulcerans among large numbers of other environmental mycobacteria.
Publisher: Public Library of Science (PLoS)
Date: 31-05-2005
Publisher: Springer Science and Business Media LLC
Date: 09-2013
Abstract: In this report we have explored the genomic and microbiological basis for a sustained increase in bloodstream infections at a major Australian hospital caused by Enterococcus faecium multi-locus sequence type (ST) 203, an outbreak strain that has largely replaced a predecessor ST17 sequence type. To establish a ST203 reference sequence we fully assembled and annotated the genome of Aus0085, a 2009 vancomycin-resistant Enterococcus faecium (VREfm) bloodstream isolate, and the first ex le of a completed ST203 genome. Aus0085 has a 3.2 Mb genome, comprising a 2.9 Mb circular chromosome and six circular plasmids (2 kb–130 kb). Twelve percent of the 3222 coding sequences (CDS) in Aus0085 are not present in ST17 E. faecium Aus0004 and ST18 E. faecium TX16. Extending this comparison to an additional 12 ST17 and 14 ST203 E. faecium hospital isolate genomes revealed only six genomic regions spanning 41 kb that were present in all ST203 and absent from all ST17 genomes. The 40 CDS have predicted functions that include ion transport, riboflavin metabolism and two phosphotransferase systems. Comparison of the vancomycin resistance-conferring Tn 1549 transposon between Aus0004 and Aus0085 revealed differences in transposon length and insertion site, and van locus sequence variation that correlated with a higher vancomycin MIC in Aus0085. Additional phenotype comparisons between ST17 and ST203 isolates showed that while there were no differences in biofilm-formation and killing of Galleria mellonella , ST203 isolates grew significantly faster and out-competed ST17 isolates in growth assays. Here we have fully assembled and annotated the first ST203 genome, and then characterized the genomic differences between ST17 and ST203 E. faecium . We also show that ST203 E. faecium are faster growing and can out-compete ST17 E. faecium . While a causal genetic basis for these phenotype differences is not provided here, this study revealed conserved genetic differences between the two clones, differences that can now be tested to explain the molecular basis for the success and emergence of ST203 E. faecium .
Publisher: PeerJ
Date: 30-10-2016
DOI: 10.7287/PEERJ.PREPRINTS.2562V1
Abstract: From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80kb region of recombination, also gaining Tn 1549 and Tn 916 , transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specialized conditions of hospital and healthcare environments.
Publisher: Cold Spring Harbor Laboratory
Date: 29-03-2023
DOI: 10.1101/2023.03.29.534673
Abstract: Mycobacterium ulcerans (MU) causes Buruli ulcer (BU), a geographically restricted infection that can result in skin loss, contracture, and permanent scarring. Lesion-location maps compiled from more than 640 cases in south eastern Australia suggest biting insects are likely involved in transmission, but it is unclear whether MU is brought by insects to their target or if MU is already on the skin and inoculation is an opportunistic event that need not be insect dependent. We validated a PCR swab detection assay and defined its dynamic range using laboratory cultured M. ulcerans and fresh pigskin. We invited volunteers in Buruli-endemic and non-endemic areas to s le their skin surfaces with self-collected skin swabs tested by IS2404 quantitative PCR. Pigskin validation experiments established a limit-of-detection of 0.06 CFU/cm 2 at a qPCR cycle threshold (Ct) of 35. Fifty-seven volunteers returned their self-collected kits of 4 swabs (bilateral ankles, calves, wrists, forearms), 10 from control areas and 47 from endemic areas. Collection was timed to coincide with the known peak-transmission period of Buruli. All swabs from human volunteers tested negative (Ct ≥35). M. ulcerans was not detected on the skin of humans from highly BU endemic areas. Buruli ulcer (BU) incidence is increasing in temperate south eastern Australia. We have yet to develop public health programs to assist people avoid BU partly because the precise mode of transmission is contentious. Recent research has shown that environmental contamination with M. ulcerans (the cause of BU) is widespread in endemic areas as a result of faecal shedding from infected possums that live close to humans, although direct human-possum contact is rare. We investigated the possibility that the skin of humans in endemic areas could become transiently contaminated with M. ulcerans while outdoors. If this were the case then BU prevention programs could be developed around skin protection and regular washing/showering. To study this possibility, we developed a sensitive skin swab PCR-assay that we tested using a pigskin laboratory model so we could be confident in our results. We asked volunteers to collect their own skin swabs after spending at least 4 hours outside during the known period of peak Buruli transmission. Fifty seven volunteers returned swab sets for testing. Our results were negative. We did not find evidence that humans in our endemic zone have M. ulcerans contamination on their skin.
Publisher: Cold Spring Harbor Laboratory
Date: 31-10-2018
DOI: 10.1101/457838
Abstract: Staphylococcus aureus is a significant human pathogen whose evolution and adaptation has been shaped in part by mobile genetic elements (MGEs), facilitating global spread of extensive antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding MGEs is incomplete, in particular how changes in the structure of multidrug-resistant (MDR) plasmids may influence important staphylococcal phenotypes. Here, we undertook a population-and functional-genomics study of 212 methicillin-resistant S. aureus (MRSA) ST239 isolates collected over 32 years to explore the evolution of the pSK1 family of MDR plasmids, illustrating how these plasmids have co-evolved with and contributed to the successful adaptation of this persistent MRSA lineage. Using complete genomes and temporal phylogenomics we reconstructed the evolution of the pSK1 family lineage from its emergence in the late 1970s, with multiple structural variants arising. Plasmid maintenance and stability was linked to IS 256 - and IS 257 -mediated chromosomal integration and disruption of plasmid replication machinery. Overlaying genomic comparisons with phenotypic susceptibility data for gentamicin and chlorhexidine, it appeared that pSK1 has contributed to enhanced resistance in ST239 MRSA through two mechanisms: (i) acquisition of plasmid-borne resistance mechanisms increasing rates of gentamicin resistance and reduced chlorhexidine susceptibility, and (ii) changes in plasmid configuration linked with further enhancement of chlorhexidine tolerance. While the exact mechanism of enhanced tolerance remains elusive, this research has uncovered a clear evolutionary response of ST239 MRSA to chlorhexidine, one which may contribute to the ongoing persistence and adaptation of this lineage within healthcare institutions.
Publisher: American Society for Microbiology
Date: 30-08-2013
Abstract: Nosocomial outbreaks of vancomycin-resistant Enterococcus faecium (VREfm) are thought to occur by transmission of VREfm between patients, predicting that infection control interventions will limit cross-transmission. Despite implementation of such strategies, the incidence of VREfm infections continues to rise. We aimed to use genomics to better understand the epidemiology of E. faecium within a large hospital and investigate the reasons for failure of infection control strategies. Whole-genome sequencing was performed on 61 E. faecium (36 VREfm) isolates, predominately from blood cultures collected at a single hospital between 1998 and 2009, and on five vanB -positive anaerobic commensal bacteria isolated from human feces. Phylogenomic analysis and precise mapping of the vanB gene, which contains the Tn 1549 transposon, showed that at least 18 of the 36 VREfm isolates had acquired the transposon via independent insertion events, indicating de novo generation of VREfm rather than cross-transmission. Furthermore, Tn 1549 sequences found in 15 of the 36 VREfm isolates were the same as the Tn 1549 sequence from one of the gut anaerobes. National and international comparator E. faecium isolates were phylogenetically interspersed with isolates from our hospital, suggesting that our findings might be globally representative. These data demonstrate that VREfm generation within a patient is common, presumably occurring in the human bowel during antibiotic therapy, and help explain our inability to reduce VREfm infections. A recommendation from our findings is that infection control practices should include screening patients for specific hospital clones of vancomycin-susceptible E. faecium rather than just VREfm. IMPORTANCE Enterococcus faecium is an increasingly important human pathogen causing predominantly antibiotic-resistant infections in hospitalized patients. Large amounts of health care funding are spent trying to control antibiotic-resistant bacteria in hospitals globally, yet in many institutions around the world, vancomycin-resistant E. faecium (VREfm) infections continue to rise. The new findings from this study help explain the failures of our current approaches to controlling vanB VREfm in health care institutions. Given the importance of this bacterium as a cause of hospital-acquired infections and the difficulties faced by infection control units in trying to prevent colonization in their institutions, the novel findings from this study provide evidence that a new approach to controlling VREfm in hospitals is required. In particular, more attention should be given to understanding the epidemiology of hospital-adapted vancomycin-susceptible E. faecium , and patients at higher risk for de novo generation of VREfm need to be identified and optimally managed.
Publisher: Cold Spring Harbor Laboratory
Date: 11-01-2022
DOI: 10.1101/2022.01.10.22269030
Abstract: In recent years reported cases of Buruli ulcer (BU), caused by Mycobacterium ulcerans (MU), have increased substantially in Victoria, Australia, with the epidemic also expanding geographically. To develop an understanding of how MU circulates in the environment and transmits to humans we analyzed environmental s les collected from 115 properties of recent BU cases and from 115 postcode-matched control properties, for the presence of MU. Environmental factors associated with increased odds of MU presence at a property included certain native plant species and native vegetation in general, more alkaline soil, lower altitude, the presence of common ringtail possums ( Pseudocheirus peregrinus ) and overhead powerlines. However, only powerlines and the absence of the native plant Melaleuca lanceolata were associated with BU case properties. S les positive for MU were more likely to be found at case properties and were associated with detections of MU in ringtail possum feces, supporting the hypothesis that MU is zoonotic, with ringtail possums the strongest reservoir host candidate. However, the disparity in environmental risk factors associated with MU positive properties versus case properties indicates a strong human behavioral component or the influence of other environmental factors in disease acquisition that requires further study. Possums, powerlines, and native vegetation are associated with the presence of Mycobacterium ulcerans in residential properties in Victoria, Australia.
Publisher: Springer Science and Business Media LLC
Date: 04-12-2015
DOI: 10.1038/SREP17693
Abstract: Infection of human skin with Mycobacterium ulcerans , the causative agent of Buruli ulcer, is associated with the systemic diffusion of a bacterial macrolide named mycolactone. Patients with progressive disease show alterations in their serum proteome, likely reflecting the inhibition of secreted protein production by mycolactone at the cellular level. Here, we used semi-quantitative metabolomics to characterize metabolic perturbations in serum s les of infected in iduals and human cells exposed to mycolactone. Among the 430 metabolites profiled across 20 patients and 20 healthy endemic controls, there were significant differences in the serum levels of hexoses, steroid hormones, acylcarnitines, purine, heme, bile acids, riboflavin and lysolipids. In parallel, analysis of 292 metabolites in human T cells treated or not with mycolactone showed alterations in hexoses, lysolipids and purine catabolites. Together, these data demonstrate that M. ulcerans infection causes systemic perturbations in the serum metabolome that can be ascribed to mycolactone. Of particular importance to Buruli ulcer pathogenesis is that changes in blood sugar homeostasis in infected patients are mirrored by alterations in hexose metabolism in mycolactone-exposed cells.
Publisher: PeerJ
Date: 06-2016
DOI: 10.7717/PEERJ.2065
Abstract: Identifying the source reservoirs of Mycobacterium ulcerans is key to understanding the mode of transmission of this pathogen and controlling the spread of Buruli ulcer (BU). In Australia, the native possum can harbor M. ulcerans in its gastrointestinal tract and shed high concentrations of the bacteria in its feces. To date, an analogous animal reservoir in Africa has not been identified. Here we tested the hypothesis that common domestic animals in BU endemic villages of Ghana are reservoir species analogous to the Australian possum. Using linear-transects at 10-meter intervals, we performed systematic fecal surveys across four BU endemic villages and one non-endemic village in the Asante Akim North District of Ghana. One hundred and eighty fecal specimens from a single survey event were collected and analyzed by qPCR for the M. ulcerans diagnostic DNA targets IS 2404 and KR-B. Positive and negative controls performed as expected but all 180 test s les were negative. This structured snapshot survey suggests that common domestic animals living in and around humans do not shed M. ulcerans in their feces. We conclude that, unlike the Australian native possum, domestic animals in rural Ghana are unlikely to be major reservoirs of M. ulcerans.
Publisher: American Society for Microbiology
Date: 12-2003
DOI: 10.1128/JB.185.23.6870-6882.2003
Abstract: Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans , we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum . We identified several DNA fragments specific to M. ulcerans , in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans .
Publisher: Cold Spring Harbor Laboratory
Date: 28-02-2023
DOI: 10.1101/2023.02.27.530350
Abstract: Among the 16 two-component systems (TCSs) in the opportunistic human pathogen Staphylococcus aureus , only WalKR is essential. Like orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodelling and is therefore intimately involved in cell ision. However, despite the importance of WalKR in S. aureus , the basis for its essentiality is not understood and the regulon poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including ChIP-seq, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis ( ltaS ) translation (rplK ) DNA compaction ( hup ) initiation of DNA replication ( dnaA, hup ) and purine nucleotide metabolism ( prs ). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by co-ordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Collectively, our findings address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics.
Publisher: Elsevier BV
Date: 11-2021
Publisher: American Society for Microbiology
Date: 15-11-2000
DOI: 10.1128/JB.182.22.6322-6330.2000
Abstract: Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum , respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS 2404 and IS 2606 , have recently been reported in M. ulcerans , and significantly, these elements are not present in M. marinum . Hybridization of the Ase I restriction fragments from M. ulcerans with IS 2404 and IS 2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently erged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis , where species ersity is being driven mainly by the activity of mobile DNA elements.
Publisher: eLife Sciences Publications, Ltd
Date: 08-06-2023
DOI: 10.7554/ELIFE.84778
Abstract: Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic ersity of intracellular bacteria. Using a well-established epithelial cell line model , we have developed a platform called InToxSa ( in tracellular tox icity of S. a ureus ) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S . aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA , encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.
Publisher: Oxford University Press (OUP)
Date: 15-10-2010
DOI: 10.1086/656319
Abstract: A significant increase in the rate of vancomycin-resistant Enterococcus faecium (VREfm) bacteremia at our health service, despite improved infection control, prompted us to investigate the cause. E. faecium bacteremia (including VREfm) over a 12-year period (1998-2009) was investigated using multilocus sequence typing, antibiotic and antiseptic susceptibility profiles, optical mapping, and whole genome sequencing of historical and recent isolates. For 10 years, the rate of bacteremia due to vanB VREfm remained stable and sequence type (ST) 17 was predominant. In 2005, ST203 vancomycin-susceptible E. faecium first appeared at our institution, and from March 2007, coinciding with the appearance of a vanB VREfm ST203, the rate of VRE bacteremia has increased exponentially. Although we found no difference in antiseptic susceptibility or presence of genes encoding putative virulence determinants (esp(Efm), hyl(Efm), and fms genes), comparative genomics revealed almost 500 kb of unique sequence when an ST17 and an ST203 VREfm isolate were compared, suggesting that other genomic factors are responsible for the apparent success of E. faecium. The application of multilocus sequence typing has uncovered the emergence of an epidemic clone of E. faecium ST203 that appears to have acquired the vanB locus and has caused a sustained outbreak of VRE bacteremia.
Publisher: Microbiology Society
Date: 31-01-2017
Publisher: American Society of Tropical Medicine and Hygiene
Date: 04-12-2019
Publisher: Public Library of Science (PLoS)
Date: 10-01-2022
DOI: 10.1371/JOURNAL.PPAT.1010166
Abstract: A hallmark of Listeria (L . ) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L . monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L . monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria -containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L . monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L . monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L . monocytogenes in macrophages is not inevitably tied to bacterial persistence.
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 11-2007
Publisher: Public Library of Science (PLoS)
Date: 10-02-2015
Publisher: American Society for Microbiology
Date: 28-02-2018
Abstract: Increasing antibiotic resistance in the major human pathogen Staphylococcus aureus is threatening the ability to treat patients with these infections. Recent laboratory studies suggest that mutations in the gene commonly associated with rif in resistance may also impact susceptibility to other last-line antibiotics in S. aureus however, the overall frequency and clinical impact of these mutations are unknown. By mining a global collection of clinical S. aureus genomes and by mutagenesis experiments, this work reveals that common rif in-induced rpoB mutations promote phenotypic plasticity that has led to the global emergence of stable, multidrug-resistant S. aureus lineages that are associated with increased risk of therapeutic failure through coresistance to other last-line antimicrobials. We recommend decreasing susceptibility breakpoints for rif in to allow phenotypic detection of critical rpoB mutations conferring low resistance to rif in and reconsidering the appropriate use of rif in to reduce the fixation and spread of these deleterious mutations globally.
Publisher: Elsevier BV
Date: 07-2017
DOI: 10.1016/J.IJANTIMICAG.2017.02.014
Abstract: Antimicrobial resistance in non-typhoidal Salmonella is a critical problem globally, with the emergence of resistance to third-generation cephalosporins (3GCs) a particular concern. The aim of this study was to use whole-genome sequencing (WGS) to characterise recently identified human and non-human isolates of 3GC-resistant Salmonella enterica subsp. enterica serovar Typhimurium from Australia. The Illumina NextSeq sequencing platform was used to determine the genome sequences of 78 S. Typhimurium definitive type 44 isolated in Australia between 1992 and 2016, including 31 3GC-resistant isolates. Phylogenetic and bioinformatics analyses were subsequently performed using a number of in silico tools. We report the emergence of 3GC resistance in locally-acquired Australian S. Typhimurium for the first time. Phenotypically resistant isolates of human and animal origin were geographically restricted and were found by WGS all to be closely related and to carry bla
Publisher: Oxford University Press (OUP)
Date: 09-2008
DOI: 10.1111/J.1574-6968.2008.01264.X
Abstract: In the past few years, a mycolactone-producing subgroup of the Mycobacterium marinum complex has been identified and analyzed. These IS2404-positive species cause pathology in frogs and fish. A recently isolated mycobacterial strain from a fish in Belgium was analyzed using a variety of molecular methods and the results were identical to those obtained from a mycolactone-producing M. marinum from Israel.
Publisher: American Society for Microbiology
Date: 02-2016
DOI: 10.1128/JCM.02344-15
Abstract: Whole-genome sequencing (WGS) has emerged as a powerful tool for comparing bacterial isolates in outbreak detection and investigation. Here we demonstrate that WGS performed prospectively for national epidemiologic surveillance of Listeria monocytogenes has the capacity to be superior to our current approaches using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), binary typing, and serotyping. Initially 423 L. monocytogenes isolates underwent WGS, and comparisons uncovered a erse genetic population structure derived from three distinct lineages. MLST, binary typing, and serotyping results inferred in silico from the WGS data were highly concordant ( %) with laboratory typing performed in parallel. However, WGS was able to identify distinct nested clusters within groups of isolates that were otherwise indistinguishable using our current typing methods. Routine WGS was then used for prospective epidemiologic surveillance on a further 97 L. monocytogenes isolates over a 12-month period, which provided a greater level of discrimination than that of conventional typing for inferring linkage to point source outbreaks. A risk-based alert system based on WGS similarity was used to inform epidemiologists required to act on the data. Our experience shows that WGS can be adopted for prospective L. monocytogenes surveillance and investigated for other pathogens relevant to public health.
Publisher: Springer Science and Business Media LLC
Date: 31-08-2018
Publisher: Elsevier BV
Date: 09-2003
Publisher: Proceedings of the National Academy of Sciences
Date: 16-09-2019
Abstract: Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, h ered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rif icin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type–like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read licon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.
Publisher: Microbiology Society
Date: 08-2017
Publisher: Elsevier BV
Date: 05-2021
Publisher: Springer Science and Business Media LLC
Date: 2007
Start Date: 2013
End Date: 2015
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2014
End Date: 2017
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2012
End Date: 2015
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2011
End Date: 2013
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2012
End Date: 2012
Funder: Australian Research Council
View Funded ActivityStart Date: 2011
End Date: 2013
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2012
End Date: 2015
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2011
End Date: 06-2014
Amount: $480,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 05-2023
End Date: 05-2026
Amount: $690,517.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2015
End Date: 12-2015
Amount: $610,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 03-2012
End Date: 03-2013
Amount: $380,000.00
Funder: Australian Research Council
View Funded Activity