ORCID Profile
0000-0002-4230-6572
Current Organisation
Macquarie University
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Synthetic biology | Microbial genetics | Microbial Genetics | Biochemistry and cell biology | Biomedical Instrumentation | Medicinal and Biomolecular Chemistry | Biochemistry and Cell Biology | Microbiology | Biologically Active Molecules | Microbiology | Genetics | Synthetic Biology | Genomics | Mechanical Engineering | Microelectromechanical Systems (MEMS) | Bioprocessing, Bioproduction and Bioproducts | Analytical Biochemistry | Genomics
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Service Industries Standards and Calibrations | Nutraceuticals and Functional foods | Manufacturing not elsewhere classified | Expanding Knowledge in Technology | Human Pharmaceutical Treatments (e.g. Antibiotics) |
Related Links
Publications
Acinetobacter baumannii Fatty Acid Desaturases Facilitate Survival in Distinct Environments.
Publisher: American Chemical Society (ACS)
Date: 08-06-2021
Portrait of a killer: Uncovering resistance mechanisms and global spread of Acinetobacter baumannii.
Publisher: Public Library of Science (PLoS)
Date: 10-08-2023
DOI: 10.1371/JOURNAL.PPAT.1011520
Abstract: Antibiotic resistance is a growing global concern in the field of medicine as it renders bacterial infections difficult to treat and often more severe. Acinetobacter baumannii is a gram-negative bacterial pathogen causing a wide range of infections, including pneumonia, sepsis, urinary tract infections, and wound infections. A . baumannii has emerged as a significant healthcare-associated pathogen due to its high level of antibiotic resistance. The global spread of antibiotic-resistant strains of A . baumannii has resulted in limited treatment options, leading to increased morbidity and mortality rates, especially in vulnerable populations such as the elderly and immunocompromised in iduals, as well as longer hospital stays and higher healthcare costs. Further complicating the situation, multi- and pan-drug-resistant strains of A . baumannii are becoming increasingly common, and these deadly strains are resistant to all or almost all available antibiotics. A . baumannii employs various clever strategies to develop antibiotic resistance, including horizontal transfer of resistance genes, overexpression of inherent efflux pumps that remove drugs from the cell, intrinsic mutations, combined with natural selection under antibiotic selective pressure leading to emergence of successful resistance clones. The typical multidrug resistance phenotype of A . baumannii is, therefore, an orchestrated collimation of all these mechanisms combined with the worldwide spread of “global clones,” rendering infections caused by this pathogen challenging to control and treat. To address the escalating problem of antibiotic resistance in A . baumannii , there is a need for increased surveillance, strict infection control measures, and the development of new treatment strategies, requiring a concerted effort by healthcare professionals, researchers, and policymakers.
The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries
Publisher: Oxford University Press (OUP)
Date: 21-01-2016
DOI: 10.1093/BIOINFORMATICS/BTW022
Abstract: Summary: Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR lification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology. Availability and implementation: The optimized sequencing protocol is included as supplementary information. The Bio-Tradis analysis pipeline is available under a GPL license at anger-pathogens/Bio-Tradis Contact: parkhill@sanger.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.
The secondary resistome of multidrug-resistant Klebsiella pneumoniae
Publisher: Springer Science and Business Media LLC
Date: 15-02-2017
DOI: 10.1038/SREP42483
Abstract: Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of in idual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of ,000 unique Tn 5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Tra nsposon D irected I nsertion-site S equencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the “secondary resistome”. As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance ( dedA ) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial “helper” drugs that restore the efficacy of existing antimicrobials.
A CRISPR view of genome sequences
Publisher: Springer Science and Business Media LLC
Date: 11-03-2013
DOI: 10.1038/NRMICRO2997
Breaching the Barrier: Genome-Wide Investigation into the Role of a Primary Amine in Promoting E. coli Outer-Membrane Passage and Growth Inhibition by Ampicillin.
Publisher: American Society for Microbiology
Date: 21-12-2022
DOI: 10.1128/SPECTRUM.03593-22
Abstract: Gram-negative bacteria are problematic for antibiotic development due to the low permeability of their cell envelopes. To rationally design new antibiotics capable of breaching this barrier, more information is required about the specific components of the cell envelope that prevent the passage of compounds with different physiochemical properties. Ampicillin and benzylpenicillin are β-lactam antibiotics with identical chemical structures except for a clever synthetic addition of a primary amine group in icillin, which promotes its accumulation in Gram-negatives. Previous work showed that icillin is better able to pass through the outer membrane porin OmpF in Escherichia coli compared to benzylpenicillin. It is not known, however, how the primary amine may affect interaction with other cell envelope components. This study applied TraDIS to identify genes that affect E. coli fitness in the presence of equivalent subinhibitory concentrations of icillin and benzylpenicillin, with a focus on the cell envelope. Insertions that compromised the outer membrane, particularly the lipopolysaccharide layer, were found to decrease fitness under benzylpenicillin exposure, but had less effect on fitness under icillin treatment. These results align with expectations if benzylpenicillin is poorly able to pass through porins. Disruption of genes encoding the AcrAB-TolC efflux system were detrimental to survival under both antibiotics, but particularly icillin. Indeed, insertions in these genes and regulators of
Comment on: Nucleotide sequence of the chromosomal region conferring multidrug resistance (R-type ASSuT) in Salmonella Typhimurium and monophasic Salmonella Typhimurium strains
Publisher: Oxford University Press (OUP)
Date: 06-12-2011
DOI: 10.1093/JAC/DKR525
The Antimicrobial Properties of PdII− and RuII−pyta Complexes**
Publisher: Wiley
Date: 05-09-2023
Abstract: Infections associated with antimicrobial resistance (AMR) are poised to become the leading cause of death in the next few decades, a scenario that can be ascribed to two phenomena: antibiotic over‐prescription and a lack of antibiotic drug development. The crowd‐sourced initiative CO‐ADD has been testing research compounds contributed by researchers around the world to find new antimicrobials to combat AMR, and during this c aign has found that metallodrugs might be a promising, yet untapped source. To this end, we submitted 18 Pd(II) and Ru(II) – pyridyl‐1,2,3‐triazolyl complexes which were developed as catalysts for their antimicrobial properties. It was found that the Pd‐complexes possessed potent antifungal activity, especially Pd1, with MICs between 0.06 – 0.125 µg/mL against C. glabrata. The in vitro studies were extended to in vivo studies in G. mellonella larvae where it was established that the compounds were non‐toxic. Here we effectively demonstrate the potential of Pd(II)‐pyta complexes as antifungal agents.
Systematic analyses identify modes of action of ten clinically relevant biocides and antibiotic antagonism in Acinetobacter baumannii
Publisher: Springer Science and Business Media LLC
Date: 09-10-2023
Complete Genome Sequence of Pseudomonas aeruginosa Reference Strain PAK
Publisher: American Society for Microbiology
Date: 10-10-2019
DOI: 10.1128/MRA.00865-19
Abstract: We report the complete genome of Pseudomonas aeruginosa strain PAK, a strain which has been instrumental in the study of a range of P. aeruginosa virulence and pathogenesis factors and has been used for over 50 years as a laboratory reference strain.
A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa
Publisher: Cold Spring Harbor Laboratory
Date: 19-07-2018
DOI: 10.1101/372805
Abstract: Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a erse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, tra nsposon d irected insertion-site s equencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa . Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa . Additionally, our data suggests a differential effect on twitching motility by flagellum components based upon their cellular location. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.
Using genomics to combat infectious diseases on a global scale
Publisher: Springer Science and Business Media LLC
Date: 19-11-2015
DksA is a conserved master regulator of stress response in Acinetobacter baumannii
Publisher: Oxford University Press (OUP)
Date: 09-05-2023
DOI: 10.1093/NAR/GKAD341
Abstract: Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.
Emergence and Evolution of Multiply Antibiotic-Resistant
Salmonella enterica
Serovar Paratyphi B
d
-Tartrate-Utilizing Strains Contain
Publisher: American Society for Microbiology
Date: 06-2009
DOI: 10.1128/AAC.01532-08
Abstract: The first Australian isolate of Salmonella enterica serovar Paratyphi B d -tartrate-utilizing (dT + ) that is resistant to icillin, chlor henicol, florfenicol, streptomycin, spectinomycin, sulfonamides, and tetracycline (ApCmFlSmSpSuTc) and contains SGI1 was isolated from a patient with gastroenteritis in early 1995. This is the earliest reported isolation globally. The incidence of infections caused by these SGI1-containing multiply antibiotic-resistant S. enterica serovar Paratyphi B dT + strains increased during the next few years and occurred sporadically in all states of Australia. Several molecular criteria were used to show that the early isolates are very closely related to one another and to strains isolated during the following few years and in 2000 and 2003 from home aquariums and their owners. Early isolates from travelers returning from Indonesia shared the same features. Thus, they appear to represent a true clone arising from a single cell that acquired SGI1. Some minor differences in the resistance profiles and molecular profiles also were observed, indicating the ongoing evolution of the clone, and phage type differences were common, indicating that this is not a useful epidemiological marker over time. Three isolates from 1995, 1998, and 1999 contained a complete sul1 gene but were susceptible to sulfamethoxazole due to a point mutation that creates a premature termination codon. This SGI1 type was designated SGI1-R. The loss of resistance genes also was examined. When strains were grown for many generations in the absence of antibiotic selection, the loss of SGI1 was not detected. However, variants SGI1-C (resistance profile SmSpSu) and SGI1-B (resistant to ApSu), which had lost part of the integron, arose spontaneously, presumably via homologous recombination between duplications in the In104 complex integron.
Inhibitors of bacterial RNA polymerase transcription complex
Publisher: Elsevier BV
Date: 2022
DOI: 10.1016/J.BIOORG.2021.105481
Abstract: A series of hybrid compounds that incorporated anthranilic acid with activated 1H-indoles through a glyoxylamide linker were designed to target bacterial RNA polymerase holoenzyme formation using computational docking. Synthesis, in vitro transcription inhibition assays, and biological testing of the hybrids identified a range of potent anti-transcription inhibitors with activity against a range of pathogenic bacteria with MICs as low as 3.1 μM. A structure activity relationship study identified the key structural components necessary for inhibition of both bacterial growth and transcription. Correlation of in vitro transcription inhibition activity with in vivo mechanism of action was established using fluorescence microscopy and resistance passaging using Gram-positive bacteria showed no resistance development over 30 days. Furthermore, no toxicity was observed from the compounds in a wax moth larvae model, establishing a platform for the development of a series of new antibacterial drugs with an established mode of action.
Microfluidic separation and enrichment of Escherichia coli by size using viscoelastic flows
Publisher: American Chemical Society (ACS)
Date: 19-01-2023
Platinum Cyclooctadiene Complexes with Activity against Gram‐positive Bacteria
Publisher: Wiley
Date: 08-07-2021
Abstract: Antimicrobial resistance is a looming health crisis, and it is becoming increasingly clear that organic chemistry alone is not sufficient to continue to provide the world with novel and effective antibiotics. Recently there has been an increased number of reports describing promising antimicrobial properties of metal‐containing compounds. Platinum complexes are well known in the field of inorganic medicinal chemistry for their tremendous success as anticancer agents. Here we report on the promising antibacterial properties of platinum cyclooctadiene (COD) complexes. Amongst the 15 compounds studied, the simplest compounds Pt(COD)X 2 (X=Cl, I, Pt1 and Pt2 ) showed excellent activity against a panel of Gram‐positive bacteria including vancomycin and methicillin resistant Staphylococcus aureus . Additionally, the lead compounds show no toxicity against mammalian cells or haemolytic properties at the highest tested concentrations, indicating that the observed activity is specific against bacteria. Finally, these compounds showed no toxicity against Galleria mellonella at the highest measured concentrations. However, preliminary efficacy studies in the same animal model found no decrease in bacterial load upon treatment with Pt1 and Pt2 . Serum exchange studies suggest that these compounds exhibit high serum binding which reduces their bioavailability in vivo , mandating alternative administration routes such as e. g. topical application.
Approaches to querying bacterial genomes with transposon-insertion sequencing
Publisher: Informa UK Limited
Date: 07-2013
DOI: 10.4161/RNA.24765
High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile
Publisher: American Society for Microbiology
Date: 05-2015
Abstract: Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been h ered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro . We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. IMPORTANCE Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been h ered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection.
Editorial: Secondary Effects of Antibiotic Exposure
Publisher: Frontiers Media SA
Date: 24-08-2021
A Proposed Framework to Identify Dispensable and Essential Functions in Bifidobacteria: Case Study of Bifidobacterium breve UCC2003 as a Prototype of Its Genus
Publisher: Springer US
Date: 29-10-2021
DOI: 10.1007/978-1-0716-1720-5_15
Abstract: Functional genomics of bacteria commonly aims at establishing genotype-phenotype links in microorganisms of industrial, technological and biomedical relevance. In this regard, random transposon mutagenesis coupled to high-throughput next-generation sequencing approaches, termed transposon-insertion sequencing (TIS), has emerged as a robust, genome-wide alternative to perform functional genome analysis. Though these approaches have been used in a large number of studies involving pathogenic and clinically relevant bacteria, they have received little attention in the fields of commensal and potentially beneficial bacteria, including probiotic microorganisms. In this chapter, we describe the implementation of the TIS method Transposon-Directed Insertion Sequencing to describe the set of essential genes in a representative strain of a genus encompassing several commensal and potentially probiotic bacteria and discuss considerations when applying similar methodological approaches to other Bifidobacterium species/strains of interest.
Cross-protection and cross-feeding between Klebsiella pneumoniae and Acinetobacter baumannii promotes their co-existence
Publisher: Springer Science and Business Media LLC
Date: 09-02-2023
DOI: 10.1038/S41467-023-36252-2
Abstract: Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isolated from polymicrobial infections. The infections where these pathogens co-exist can be more severe and recalcitrant to therapy than infections caused by either species alone, however there is a lack of knowledge on their potential synergistic interactions. In this study we characterise the genomes of A. baumannii and K. pneumoniae strains co-isolated from a single human lung infection. We examine various aspects of their interactions through transcriptomic, phenomic and phenotypic assays that form a basis for understanding their effects on antimicrobial resistance and virulence during co-infection. Using co-culturing and analyses of secreted metabolites, we discover the ability of K. pneumoniae to cross-feed A. baumannii by-products of sugar fermentation. Minimum inhibitory concentration testing of mono- and co-cultures reveals the ability for A. baumannii to cross-protect K. pneumoniae against the cephalosporin, cefotaxime. Our study demonstrates distinct syntrophic interactions occur between A. baumannii and K. pneumoniae , helping to elucidate the basis for their co-existence in polymicrobial infections.
Elucidating essential genes in plant-associatedPseudomonas protegensPf-5 using transposon insertion sequencing
Publisher: Cold Spring Harbor Laboratory
Date: 16-07-2020
DOI: 10.1101/2020.07.16.205906
Abstract: Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacteria that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used in this species. Here we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses and transport were under-represented. Comparison of the essential gene sets of Pf-5 and P. aeruginosa PA14, an opportunistic human pathogen, provides insight into the biological processes important for their different lifestyles. Key differences include cytochrome c biogenesis, formation of periplasmic disulfide bonds, lipid biosynthesis, ribonuclease activity, lipopolysaccharides and cell surface structures. Comparison of the Pf-5 in silico predicted and in vitro determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles can improve our understanding of the biological processes important for survival and growth in different environmental niches. Essential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens, but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analysis of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of P. aeruginosa PA14, an opportunistic human pathogen, provides insight into differences in gene essentiality which may be linked to their different lifestyles.
Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii
Publisher: American Society for Microbiology
Date: 02-11-2016
Abstract: Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii . A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii . Furthermore, the method identified a new transcriptional regulator that controls expression of amvA . In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell ision, cell morphology, or aggregation in A. baumannii . IMPORTANCE Transposon-directed insertion sequencing (TraDIS) and related technologies have emerged as powerful methods to identify genes required for bacterial survival or competitive fitness under various selective conditions. We applied fluorescence-activated cell sorting (FACS) to physically enrich for phenotypes of interest within a mutant population prior to TraDIS. To our knowledge, this is the first time that a physical selection method has been applied in parallel with TraDIS rather than a fitness-induced selection. The results demonstrate the feasibility of this combined approach to generate significant results and highlight the major multidrug efflux pumps encoded in an important pathogen. This FACS-based approach, TraDISort, could have a range of future applications, including the characterization of efflux pump inhibitors, the identification of regulatory factors controlling gene or protein expression using fluorescent reporters, and the identification of genes involved in cell replication, morphology, and aggregation.
Splicing factor proline and glutamine rich intron retention, reduced expression and aggregate formation are pathological features of amyotrophic lateral sclerosis
Publisher: Wiley
Date: 02-08-2021
DOI: 10.1111/NAN.12749
Abstract: Splicing factor proline and glutamine rich (SFPQ) is an RNA–DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case–control cohorts. We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA‐sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ‐positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.
Molecular Surveillance Identifies Multiple Transmissions of Typhoid in West Africa
Publisher: Public Library of Science (PLoS)
Date: 22-09-2016
Bacteria separation and enrichment using viscoelastic flows in a straight microchannel
Publisher: Elsevier BV
Date: 09-2023
Clinical and laboratory-induced colistin-resistance mechanisms in Acinetobacter baumannii
Publisher: Microbiology Society
Date: 02-2019
Transposons Related to Tn1696in IncHI2 Plasmids in Multiply Antibiotic ResistantSalmonella entericaSerovar Typhimurium from Australian Animals
Publisher: Mary Ann Liebert Inc
Date: 09-2010
Abstract: Conjugative IncHI2 plasmids carrying tetracycline, trimethoprim, and sulphonamide resistance genes were recovered from two multiply antibiotic resistant Salmonella enterica serovar Typhimurium isolates from Australian food-producing animals. Transposons related to the mercury resistance transposon Tn1696 were identified in both IncHI2 plasmids. These transposons contained an In4-type class 1 integron that carried a dfrA5 trimethoprim resistance gene cassette and the sul1 sulfonamide resistance gene. These integrons were located in the same position as In4 in Tn1696. The integron from one isolate includes a large transposon-like structure containing four IS26 and the strAB, sul2, bla(TEM), and aphA1 genes conferring resistance to streptomycin, sulphonamides, icillin, kanamycin, and neomycin, respectively. This structure is flanked by an 8-bp duplication, but it includes both the aphA1-containing transposon Tn4352 and a transposon, Tn6029, carrying genes derived from RSF1010 and from Tn2. However, Tn4352 and Tn6029 overlap, sharing one IS26 copy. This suggests that they do not move by a standard transpositional mechanism. A circular intermediate, carrying only the region containing the resistance gene(s) and one of the IS26 bounding it, is proposed as an intermediate.
Elucidating Essential Genes in Plant-Associated Pseudomonas protegens Pf-5 Using Transposon Insertion Sequencing
Publisher: American Society for Microbiology
Date: 08-03-2021
DOI: 10.1128/JB.00432-20
Abstract: Essential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens but only a few plant-associated bacteria.
Identification of a Novel LysR Family Transcriptional Regulator Controlling Acquisition of Sulfur Sources in <i>Acinetobacter baumannii</i>
Publisher: S. Karger AG
Date: 2023
DOI: 10.1159/000529038
Abstract: sc l /sc -cysteine biosynthesis from inorganic sulfur represents a major mechanism by which reduced sulfur is incorporated into organic compounds. Cysteine biosynthesis and regulation is well characterized in i Escherichia coli /i . However, the regulation of sulfur metabolism in i Acinetobacter baumannii /i is only partly understood, with the LysR-type regulator, GigC known to control some aspects of sulfur reduction. In this study, we have used transcriptomics and bioinformatic analyses to characterize a novel LysR-type transcriptional regulator encoded by ABUW_1016 ( i cbl /i ), in a highly multidrug resistant and virulent isolate of i A. baumannii. /i We have shown that Cbl is involved in controlling expression of the genes required for uptake and reduction of various sulfur sources in i A. baumannii /i . Collectively, we have identified the global regulon of Cbl and proposed a model of cysteine biosynthesis and its regulation by Cbl and GigC in i A. baumannii /i .
The 3’ UTR ofvigRis required for virulence inStaphylococcus aureusand has expanded through STAR sequence repeat insertions
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.
Length-based separation of Bacillus subtilis bacterial populations by viscoelastic microfluidics
Publisher: Springer Science and Business Media LLC
Date: 19-01-2022
DOI: 10.1038/S41378-021-00333-3
Abstract: In this study, we demonstrated the label-free continuous separation and enrichment of Bacillus subtilis populations based on length using viscoelastic microfluidics. B. subtilis , a gram-positive, rod-shaped bacterium, has been widely used as a model organism and an industrial workhorse. B. subtilis can be arranged in different morphological forms, such as single rods, chains, and clumps, which reflect differences in cell types, phases of growth, genetic variation, and changing environmental factors. The ability to prepare B. subtilis populations with a uniform length is important for basic biological studies and efficient industrial applications. Here, we systematically investigated how flow rate ratio, poly(ethylene oxide) (PEO) concentration, and channel length affected the length-based separation of B. subtilis cells. The lateral positions of B. subtilis cells with varying morphologies in a straight rectangular microchannel were found to be dependent on cell length under the co-flow of viscoelastic and Newtonian fluids. Finally, we evaluated the ability of the viscoelastic microfluidic device to separate the two groups of B. subtilis cells by length (i.e., 1–5 μm and μm) in terms of extraction purity (EP), extraction yield (EY), and enrichment factor (EF) and confirmed that the device could separate heterogeneous populations of bacteria using elasto-inertial effects.
Physical enrichment of transposon mutants from saturation mutant libraries using the TraDISort approach
Publisher: Informa UK Limited
Date: 31-03-2017
Drug Resistance inSalmonella entericaser. Typhimurium Bloodstream Infection, Malawi
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 11-2014
Evolution of IncHI2 plasmids via acquisition of transposons carrying antibiotic resistance determinants
Publisher: Oxford University Press (OUP)
Date: 14-02-2012
DOI: 10.1093/JAC/DKS004
Abstract: To investigate the relationships between IncHI2 plasmids conferring resistance to antibiotics isolated in Australia and those from other countries. PCR, restriction digestion, cloning and DNA sequencing were used to characterize transposons and determine their location in IncHI2 plasmids recovered from Salmonella enterica isolates from Australian animals. Tn10, carrying the tet(B) tetracycline resistance determinant, was found in IncHI2 plasmids pSRC26 and pSRC125 recovered from S. enterica serovar Typhimurium from Australian cattle and in IncHI2 plasmids from serovar Infantis isolates from chickens. Its location was the same as seen in the IncHI2 reference plasmid R478. The location of Tn1696-related mercury and multiple antibiotic resistance transposons was also the same in all of the Australian plasmids, and the mer end was in the same position as the mer module in R478. However, R478 has lost the tnp end (including most of the integron) and some adjacent sequence. The sequence adjacent to the tnp end of the Tn1696-related transposons in the Australian plasmids is in the bla(CMY-8)-carrying plasmid pK29, but only 22 bp from the transposon inverted repeat remains. These plasmids all belong to the same evolutionary lineage. Neither transposon was found in TP116, which represents a separate lineage. Transposon locations are useful markers for lineages of closely related plasmids. The configuration surrounding the Tn1696-like transposons in the Australian IncHI2 plasmids is ancestral to those found in R478 and pK29, each of which has part of the transposon and adjacent sequence replaced by other resistance genes.
Identification of genes required for the fitness of Streptococcus equi subsp. equi in whole equine blood and hydrogen peroxide
Publisher: Microbiology Society
Date: 04-2020
The evolutionary tale of eight novel plasmids in a colistin-resistant environmental Acinetobacter baumannii isolate
Publisher: Microbiology Society
Date: 12-05-2023
Abstract: Acinetobacter baumannii is an important opportunistic pathogen known for its high levels of resistance to many antibiotics, particularly those considered last resorts such as colistin and carbapenems. Plasmids of this organism are increasingly associated with the spread of clinically important antibiotic resistance genes. Although A. baumannii is a ubiquitous organism, to date, most of the focus has been on studying strains recovered from clinical s les ignoring those isolated in the environment (soil, water, food, etc.). Here, we analysed the genetic structures of eight novel plasmids carried by an environmental colistin-resistant A. baumannii (strain E-072658) recovered in a recycled fibre pulp in a paper mill in Finland. It was shown that E-072658 carries a new variant of the mcr-4 colistin resistance gene ( mcr-4.7 ) in a novel Tn 3- family transposon (called Tn 6926 ) carried by a novel plasmid p8E072658. E-072658 is also resistant to sulphonamide compounds consistent with this, the sul2 sulphonamide resistance gene was found in a p dif module. E-072658 also carries six additional plasmids with no antibiotic resistance genes, but they contained several p dif modules shared with plasmids carried by clinical strains. Detailed analysis of the genetic structure of all eight plasmids carried by E-072658 showed a complex evolutionary history revealing genetic exchange events within the genus Acinetobacter beyond the clinical or environmental origin of the strains. This work provides evidence that environmental strains might act as a source for some of the clinically significant antibiotic resistance genes.
Cover Feature: Nontoxic Cobalt(III) Schiff Base Complexes with Broad‐Spectrum Antifungal Activity (Chem. Eur. J. 6/2021)
Publisher: Wiley
Date: 24-11-2020
Nontoxic Cobalt(III) Schiff Base Complexes with Broad‐Spectrum Antifungal Activity
Publisher: Wiley
Date: 24-11-2020
Genomic landscape of extended-spectrum β-lactamase resistance in Escherichia coli from an urban African setting
Publisher: Oxford University Press (OUP)
Date: 04-03-2017
DOI: 10.1093/JAC/DKX058
Identification of Tse8 as a Type VI secretion system toxin from Pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells
Publisher: Springer Science and Business Media LLC
Date: 19-08-2021
Defining the ABC of gene essentiality in streptococci
Publisher: Springer Science and Business Media LLC
Date: 31-05-2017
O-Antigen-Dependent Colicin Insensitivity of Uropathogenic Escherichia coli
Publisher: American Society for Microbiology
Date: 15-02-2019
DOI: 10.1128/JB.00545-18
Abstract: Escherichia coli infections can be a major health burden, especially with the organism becoming increasingly resistant to “last-resort” antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenic E. coli strain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity among E. coli organisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenic E. coli such as uropathogenic E. coli (UPEC).
Shape-based separation of drug-treated Escherichia coli using viscoelastic microfluidics
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D2LC00339B
Abstract: A viscoelastic microfluidic device for shape-based separation of drug-treated Escherichia coli .
Whole genome sequence analysis of Shigella from Malawi identifies fluoroquinolone resistance
Publisher: Cold Spring Harbor Laboratory
Date: 20-11-2020
DOI: 10.1101/2020.11.20.391235
Abstract: Increasing antimicrobial resistance and limited alternative treatments led to fluoroquinolone resistant Shigella strain inclusion on the WHO global priority pathogens list. In this study we characterised multiple Shigella isolates from Malawi with whole genome sequence analysis, identifying the acquirable fluoroquinolone resistance determinant qnrS1 .
The Molecular Basis of Acinetobacter baumannii Cadmium Toxicity and Resistance
Publisher: American Society for Microbiology
Date: 28-10-2021
DOI: 10.1128/AEM.01718-21
Abstract: Cadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, including Acinetobacter baumannii , which is notorious for persisting in harsh environments.
High-throughput transposon mutagenesis in the family Enterobacteriaceae reveals core essential genes and rapid turnover of essentiality
Publisher: Cold Spring Harbor Laboratory
Date: 20-10-2022
DOI: 10.1101/2022.10.20.512852
Abstract: The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the gut commensal and model organism Escherichia coli , as well as several major human pathogens including multiple serovars of Salmonella enterica and Klebsiella pneumoniae . Essential gene sets have been determined for several members of the Enterobacteriaceae, with the E. coli Keio single-gene deletion library often regarded as a gold standard for gene essentiality studies. However, it remains unclear how gene essentiality varies between related strains and species. To investigate this, we have assembled a collection of thirteen sequenced high-density transposon mutant libraries from five genera within the Enterobacteriaceae. We first benchmark a number of gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations we develop a new classifier for gene essentiality. Using this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes, and reconstruct an ancestral essential gene set of 296 genes. Despite the presence of a large cohort of variably essential genes, surprisingly we find an absence of evidence for genus-specific essential genes. A clear ex le of this sporadic essentiality is given by the set of genes regulating the σ E extracytoplasmic stress response, which appears to have independently become essential multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss in obligate insect endosymbionts that have emerged from within the Enterobacteriaceae. This isolates a remarkably small set of genes absolutely required for survival, and uncovers several instances of essential stress responses masked by redundancy in free-living bacteria.
Whole genome sequence analysis of Shigella from Malawi identifies fluoroquinolone resistance
Publisher: Microbiology Society
Date: 04-05-2021
Abstract: Increasing antimicrobial resistance and limited alternative treatments have led to fluoroquinolone-resistant Shigella strain inclusion on the WHO global priority pathogens list. In this study we characterized multiple Shigella isolates from Malawi with whole genome sequence analysis, identifying the acquirable fluoroquinolone resistance determinant qnrS1 .
Recombination: genomic mix 'n' match
Publisher: Springer Science and Business Media LLC
Date: 10-11-2014
DOI: 10.1038/NRMICRO3388
Genomic analysis of Klebsiella pneumoniae isolates from Malawi reveals acquisition of multiple ESBL determinants across diverse lineages
Publisher: Oxford University Press (OUP)
Date: 18-02-2019
DOI: 10.1093/JAC/DKZ032
Abstract: ESBL-producing Klebsiella pneumoniae (KPN) pose a major threat to human health globally. We carried out a WGS study to understand the genetic background of ESBL-producing KPN in Malawi and place them in the context of other global isolates. We sequenced genomes of 72 invasive and carriage KPN isolates collected from patients admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi. We performed phylogenetic and population structure analyses on these and previously published genomes from Kenya (n = 66) and from outside sub-Saharan Africa (n = 67). We screened for presence of antimicrobial resistance (AMR) genetic determinants and carried out association analyses by genomic sequence cluster, AMR phenotype and time. Malawian isolates fit within the global population structure of KPN, clustering into the major lineages of KpI, KpII and KpIII. KpI isolates from Malawi were more related to those from Kenya, with both collections exhibiting more clonality than isolates from the rest of the world. We identified multiple ESBL genes, including blaCTX-M-15, several blaSHV, blaTEM-63 and blaOXA-10, and other AMR genes, across erse lineages of the KPN isolates from Malawi. No carbapenem resistance genes were detected however, we detected IncFII and IncFIB plasmids that were similar to the carbapenem resistance-associated plasmid pNDM-mar. There are multiple ESBL genes across erse KPN lineages in Malawi and plasmids in circulation that are capable of carrying carbapenem resistance. Unless appropriate interventions are rapidly put in place, these may lead to a high burden of locally untreatable infection in vulnerable populations.
Evolution of a multiple antibiotic resistance region in IncHI1 plasmids: reshaping resistance regions in situ
Publisher: Oxford University Press (OUP)
Date: 10-08-2012
DOI: 10.1093/JAC/DKS317
Abstract: To determine the structure of the resistance region in an IncHI1 plasmid conferring resistance to multiple antibiotics, including gentamicin, recovered from a Salmonella enterica serovar Typhimurium isolate from a horse. Plasmids were recovered by conjugation. The plasmid type, resistance genes and their context were identified by PCR, cloning, hybridization and DNA sequencing. The sequence was compared using bioinformatic tools with available resistance region sequences. In isolate SRC27, an IncI1 plasmid, pSRC27-I, conferred streptomycin resistance via the strA and strB genes contained within Tn5393a. An IncHI1 plasmid, pSRC27-H, was found to carry the aacC2 gentamicin resistance gene within a 34.6 kb multiple antibiotic resistance region that included nine further antibiotic resistance genes, aadA2, aphA1, bla(TEM), catA1, dfrA12, strA and strB, sul2 and tetA(B), conferring resistance to streptomycin and spectinomycin, kanamycin and neomycin, icillin, chlor henicol, trimethoprim, streptomycin, sulfamethoxazole and tetracycline, respectively. This complex resistance region has evolved from Tn2670 and Tn10 via loss and gain of DNA segments. It includes Tn6029 and Tn4352, and a new transposon carrying the aacC2 gene. It also contains five copies of IS26, two of IS1 and one each of IS10 and ISCfr1. This region of pSRC27-H is related to ones present at the same position in three sequenced IncHI1 plasmids, pHCM1, pO111_1 and pMAK1, but has acquired new segments carrying antibiotic resistance genes. Evolution via loss and gain of resistance genes has occurred within the large resistance region of pHCM1-type IncHI1 plasmids leading to different resistance phenotypes.
Non-toxic Cobalt(III) Schiff Base Complexes with Broad Spectrum Antifungal Activity
Publisher: American Chemical Society (ACS)
Date: 30-07-2020
DOI: 10.26434/CHEMRXIV.12736505
Abstract: Resistance to currently available antifungal drugs has quietly been on the rise but overshadowed by the alarming spread of antibacterial resistance. There is a striking lack of attention to the threat of drug resistant fungal infections, with only a handful of new drugs currently in development. Given that metal complexes have proven to be useful new chemotypes in the fight against diseases such as cancer, malaria, and bacterial infections, it stands to reason to explore their possible utility in treating fungal infections. Herein we report a series of cobalt(III) Schiff base complexes with broad spectrum antifungal activity. Some of these complexes (1-3) show minimum inhibitory concentrations (MIC) in the low micro- to nanomolar range against a series of Candida and Cryptococcus yeasts. Additionally, we demonstrate that these compounds show no cytotoxicity against both bacterial and human cells. Finally, we report first in vivo toxicity data on these compounds in Galleria mellonella, showing that doses as high as 266 mg/kg are tolerated without adverse effects, paving the way for further in vivo studies of these complexes. br
Transposon Tn5393e Carrying theaphA1-Containing Transposon Tn6023Upstream ofstrABDoes Not Confer Resistance to Streptomycin
Publisher: Mary Ann Liebert Inc
Date: 08-2011
Abstract: The simplest form of transposon Tn5393 carries the strA and strB genes that confer resistance to streptomycin, in addition to its transposition determinants. Tn5393e, made up of Tn5393 and a second transposon, Tn6023, was found in an IncHI2 plasmid, pSRC125, recovered from a multiply antibiotic-resistant Salmonella enterica serovar Typhimurium isolate of bovine origin. Tn6023 is made up of the aphA1b gene flanked by two inversely oriented copies of insertion sequence (IS) IS26 and is flanked by an 8 bp duplication. It is related to several other transposons that carry aphA1b in fragments of differing length, also flanked by copies of IS26. Tn6023 is located in the tnpR gene of Tn5393, which lies upstream of the strA and strB genes, and the combined structure was designated Tn5393e. Although neither strA nor strB contain any mutations that would inactivate them, pSRC125 does not confer resistance to streptomycin, indicating that the strA and strB genes are not expressed. In Tn5393, strA and strB are transcribed from the tnpR promoter, and in Tn5393e neither this transcript nor the transcript from the aphA1b promoter in Tn6023 must reach strAB. Tn5393e was previously found in different locations in Corynebacteria, indicating that it can move and suggesting a wide distribution. The structures of several further variants of Tn5393 found in GenBank were analyzed and assigned variant designations Tn5393f-Tn5393i.
Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in Escherichia coli O157
Publisher: American Society for Microbiology
Date: 05-09-2018
Abstract: Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene ( sspA ) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens. IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication.
Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the last-line antibiotic colistin
Publisher: Springer Science and Business Media LLC
Date: 29-06-2018
DOI: 10.1038/S41598-018-28199-Y
Abstract: Colistin remains one of the few antibiotics effective against multi-drug resistant (MDR) hospital pathogens, such as Klebsiella pneumoniae . Yet resistance to this last-line drug is rapidly increasing. Characterized mechanisms of col R in K . pneumoniae are largely due to chromosomal mutations in two-component regulators, although a plasmid-mediated col R mechanism has recently been uncovered. However, the effects of intrinsic colistin resistance are yet to be characterized on a whole-genome level. Here, we used a genomics-based approach to understand the mechanisms of adaptive col R acquisition in K . pneumoniae . In controlled directed-evolution experiments we observed two distinct paths to colistin resistance acquisition. Whole genome sequencing identified mutations in two colistin resistance genes: in the known col R regulator phoQ which became fixed in the population and resulted in a single amino acid change, and unstable minority variants in the recently described two-component sensor crrB . Through RNAseq and microscopy, we reveal the broad range of effects that colistin exposure has on the cell. This study is the first to use genomics to identify a population of minority variants with mutations in a col R gene in K . pneumoniae .
A global data-driven census ofSalmonellasmall proteins and their potential functions in bacterial virulence
Publisher: Oxford University Press (OUP)
Date: 2020
Abstract: Small proteins are an emerging class of gene products with erse roles in bacterial physiology. However, a full understanding of their importance has been h ered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than Escherichia coli. We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Salmonella Typhimurium. We merged the annotated small proteome of Salmonella with new small proteins predicted with in silico and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (Transposon-directed insertion sequencing), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a Salmonella ΔmgrB mutant and reveal an effect of MgrB in regulating the Salmonella transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available ‘omics’ datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in erse bacteria.
A decade of advances in transposon-insertion sequencing
Publisher: Springer Science and Business Media LLC
Date: 12-06-2020
The Transcriptomic Signature of Tigecycline in Acinetobacter baumannii
Publisher: Frontiers Media SA
Date: 27-10-2020
A high‐resolution genomic analysis of multidrug‐resistant hospital outbreaks of Klebsiella pneumoniae
Publisher: EMBO
Date: 20-02-2015
Abstract: Multidrug‐resistant ( MDR ) Klebsiella pneumoniae has become a leading cause of nosocomial infections worldwide. Despite its prominence, little is known about the genetic ersity of K. pneumoniae in resource‐poor hospital settings. Through whole‐genome sequencing ( WGS ), we reconstructed an outbreak of MDR K. pneumoniae occurring on high‐dependency wards in a hospital in Kathmandu during 2012 with a case‐fatality rate of 75%. The WGS analysis permitted the identification of two MDR K. pneumoniae lineages causing distinct outbreaks within the complex endemic K. pneumoniae . Using phylogenetic reconstruction and lineage‐specific PCR , our data predicted a scenario in which K. pneumoniae , circulating for 6 months before the outbreak, underwent a series of ward‐specific clonal expansions after the acquisition of genes facilitating virulence and MDR . We suggest that the early detection of a specific NDM ‐1 containing lineage in 2011 would have alerted the high‐dependency ward staff to intervene. We argue that some form of real‐time genetic characterisation, alongside clade‐specific PCR during an outbreak, should be factored into future healthcare infection control practices in both high‐ and low‐income settings.
An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria
Publisher: Springer Science and Business Media LLC
Date: 23-06-2020
DOI: 10.1038/S41467-020-16950-X
Abstract: Peptide antibiotics are an abundant and synthetically tractable source of molecular ersity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an hipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina . The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC , a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3–4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the ‘no observable adverse effect level’ (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.
The essential genomic landscape of the commensal Bifidobacterium breve UCC2003
Publisher: Springer Science and Business Media LLC
Date: 17-07-2017
DOI: 10.1038/S41598-017-05795-Y
Abstract: Bifidobacteria are common gut commensals with purported health-promoting effects. This has encouraged scientific research into bifidobacteria, though recalcitrance to genetic manipulation and scarcity of molecular tools has h ered our knowledge on the precise molecular determinants of their health-promoting attributes and gut adaptation. To overcome this problem and facilitate functional genomic analyses in bifidobacteria, we created a large Tn 5 transposon mutant library of the commensal Bifidobacterium breve UCC2003 that was further characterized by means of a Tra nsposon D irected I nsertion S equencing (TraDIS) approach. Statistical analysis of transposon insertion distribution revealed a set of 453 genes that are essential for or markedly contribute to growth of this strain under laboratory conditions. These essential genes encode functions involved in the so-called bifid-shunt, most enzymes related to nucleotide biosynthesis and a range of housekeeping functions. Comparison to the Bifidobacterium and B. breve core genomes highlights a high degree of conservation of essential genes at the species and genus level, while comparison to essential gene datasets from other gut bacteria identified essential genes that appear specific to bifidobacteria. This work establishes a useful molecular tool for scientific discovery of bifidobacteria and identifies targets for further studies aimed at characterizing essential functions not previously examined in bifidobacteria.
A global data-driven census of Salmonella small proteins and their potential functions in bacterial virulence
Publisher: Cold Spring Harbor Laboratory
Date: 27-05-2020
DOI: 10.1101/2020.05.26.116038
Abstract: Small proteins are an emerging class of gene products with erse roles in bacterial physiology. However, a full understanding of their importance has been h ered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than Escherichia coli . We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Salmonella Typhimurium. We merged the annotated small proteome of Salmonella with new small proteins predicted with in silico and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (TraDIS), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a Salmonella Δ mgrB mutant and reveal an effect of MgrB in regulating the Salmonella transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available “omics” datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in erse bacteria.
How antibiotics work together: molecular mechanisms behind combination therapy
Publisher: Elsevier BV
Date: 10-2020
The Murray collection of pre-antibiotic era Enterobacteriacae: a unique research resource
Publisher: Springer Science and Business Media LLC
Date: 28-09-2015
Metal Complexes as Antifungals? from a Crowd-Sourced Compound Library to the First in Vivo Experiments
Publisher: American Chemical Society (ACS)
Date: 23-09-2022
The Role of Zinc Efflux during Acinetobacter baumannii Infection
Publisher: American Chemical Society (ACS)
Date: 28-10-2019
Evolution of IncHI1 plasmids: Two distinct lineages
Publisher: Elsevier BV
Date: 09-2013
DOI: 10.1016/J.PLASMID.2013.03.005
Abstract: The IncHI1 plasmid pSRC27-H from Salmonella enterica serovar Typhimurium carries a region containing several genes that confer resistance to different antibiotics, and this resistance region is in the same position as related resistance regions in a group of sequenced IncHI1 plasmids from various sources that includes pHCM1. Four further additional segments are found in pHCM1 relative to another IncHI1 plasmid, R27. Using PCR or DNA sequencing to detect the presence or absence of each of these additional segments in the same position in the IncHI1 backbone, plasmid pSRC27-H was found to include them. However, in one case the additional segment was smaller in pSRC27-H, lacking a transposon carrying a second resistance region in pHCM1. The sequences of IncHI1 plasmids, pO111_1 and pMAK1, were also examined and found to share the same or closely related additional segments. The structure of the additional material in pHCM1, pO111_1 and pMAK1 was examined, and potential novel transposons were identified. These additional segments define an IncHI1 lineage (pHCM1, pO111_1, pMAK1, pSRC27-H) which we designated type 2 to distinguish it from type 1 (R27, pAKU_1, pP-stx-12). A segment from the Escherichia coli genome and an adjacent copy of IS1 in pHCM1 was defined by comparison to pO111_1 and pMAK1, which lack it. pSRC27-H also lacks it. This structure is present in the same position in R27 and type 1 plasmids, but in the opposite orientation, and appears to have been incorporated via IS1-mediated transposition. The PCRs developed provide a simple means of distinguishing type 1 and type 2 IncHI1 plasmids based on the presence or absence of variable regions.
Non-toxic Cobalt(III) Schiff Base Complexes with Broad Spectrum Antifungal Activity
Publisher: American Chemical Society (ACS)
Date: 30-07-2020
DOI: 10.26434/CHEMRXIV.12736505.V1
Abstract: Resistance to currently available antifungal drugs has quietly been on the rise but overshadowed by the alarming spread of antibacterial resistance. There is a striking lack of attention to the threat of drug resistant fungal infections, with only a handful of new drugs currently in development. Given that metal complexes have proven to be useful new chemotypes in the fight against diseases such as cancer, malaria, and bacterial infections, it stands to reason to explore their possible utility in treating fungal infections. Herein we report a series of cobalt(III) Schiff base complexes with broad spectrum antifungal activity. Some of these complexes (1-3) show minimum inhibitory concentrations (MIC) in the low micro- to nanomolar range against a series of Candida and Cryptococcus yeasts. Additionally, we demonstrate that these compounds show no cytotoxicity against both bacterial and human cells. Finally, we report first in vivo toxicity data on these compounds in Galleria mellonella, showing that doses as high as 266 mg/kg are tolerated without adverse effects, paving the way for further in vivo studies of these complexes.
A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa
Publisher: Microbiology Society
Date: 11-2018
Genomic investigation of a suspected Klebsiella pneumoniae outbreak in a neonatal care unit in sub-Saharan Africa
Publisher: Microbiology Society
Date: 18-11-2021
Abstract: A special-care neonatal unit from a large public hospital in Malawi was noted as having more frequent, difficult-to-treat infections, and a suspected outbreak of multi-drug-resistant Klebsiella pneumoniae was investigated using genomic characterisation. All K. pneumoniae bloodstream infections (BSIs) from patients in the neonatal ward ( n =62), and a subset of K. pneumoniae BSI isolates ( n =38) from other paediatric wards in the hospital, collected over a 4 year period were studied. After whole genome sequencing, the strain sequence types (STs), plasmid types, virulence and resistance genes were identified. One ST340 clone, part of clonal complex 258 (CC258) and an ST that drives hospital outbreaks worldwide, harbouring numerous resistance genes and plasmids, was implicated as the likely cause of the outbreak. This study contributes molecular information necessary for tracking and characterizing this important hospital pathogen in sub-Saharan Africa.
A synthetic peptide mimic killsCandida albicansand synergistically prevents infection
Publisher: Cold Spring Harbor Laboratory
Date: 25-09-2023
Contrasting patterns of longitudinal population dynamics and antimicrobial resistance mechanisms in two priority bacterial pathogens over 7 years in a single center
Publisher: Springer Science and Business Media LLC
Date: 02-09-2019
Related Organisations
Malawi Liverpool Wellcome Trust Clinical Research Programme
Location: Malawi
Wellcome Sanger Institute
Location: United Kingdom of Great Britain and Northern Ireland
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ARC Future Fellowships - Grant ID: FT220100152
Start Date: 2023
End Date: 12-2026
Amount: $935,803.00
Funder: Australian Research Council
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Amount: $674,004.00
Funder: Australian Research Council
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View Funded ActivityDiscovery Projects - Grant ID: DP200102269
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Funder: Australian Research Council
View Funded ActivityDiscovery Early Career Researcher Award - Grant ID: DE180100929
Start Date: 12-2018
End Date: 12-2022
Amount: $365,058.00
Funder: Australian Research Council
View Funded ActivityIndustrial Transformation Training Centres - Grant ID: IC210100040
Start Date: 02-2022
End Date: 02-2027
Amount: $4,997,903.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE200100016
Start Date: 06-2020
End Date: 06-2021
Amount: $400,000.00
Funder: Australian Research Council
View Funded Activity