ORCID Profile
0000-0002-6944-3980
Current Organisation
University of the Sunshine Coast
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Publisher: Springer Science and Business Media LLC
Date: 06-2017
DOI: 10.1038/S41598-017-02096-2
Abstract: Plasmacytoid dendritic cells (pDC) are activators of innate and adaptive immune responses that express HLA-DR, toll-like receptor (TLR) 7, TLR9 and produce type I interferons. The role of human pDC in malaria remains poorly characterised. pDC activation and cytokine production were assessed in 59 malaria-naive volunteers during experimental infection with 150 or 1,800 P. falciparum- parasitized red blood cells. Using RNA sequencing, longitudinal changes in pDC gene expression were examined in five adults before and at peak-infection. pDC responsiveness to TLR7 and TLR9 stimulation was assessed in-vitro . Circulating pDC remained transcriptionally stable with gene expression altered for 8 genes (FDR 0.07). There was no upregulation of co-stimulatory molecules CD86, CD80, CD40, and reduced surface expression of HLA-DR and CD123 (IL-3R-α). pDC loss from the circulation was associated with active caspase-3, suggesting pDC apoptosis during primary infection. pDC remained responsive to TLR stimulation, producing IFN-α and upregulating HLA-DR, CD86, CD123 at peak-infection. In clinical malaria, pDC retained HLA-DR but reduced CD123 expression compared to convalescence. These data demonstrate pDC retain function during a first blood-stage P. falciparum exposure despite sub-microscopic parasitaemia downregulating HLA-DR. The lack of evident pDC activation in both early infection and malaria suggests little response of circulating pDC to infection.
Publisher: Future Medicine Ltd
Date: 09-2018
Abstract: Aim: To develop a probe-based triplex quantitative real-time PCR assay to simultaneously detect the upregulation of the efflux pumps AmrAB–OprA, BpeAB–OprB and BpeEF–OprC in Burkholderia pseudomallei strains exhibiting increased minimum inhibitory concentrations toward meropenem, doxycycline or trimethoprim-sulfamethoxazole. Methods: The triplex assay was developed and subsequently tested on RNA isolated from eight clinical and eight laboratory-generated B. pseudomallei mutants harboring efflux pump regulator mutations. Results: The triplex assay accurately detected efflux pump upregulation in all clinical and laboratory mutants, which corresponded with decreased antibiotic susceptibility or antibiotic resistance. Conclusion: Rapid detection of antibiotic resistance provides clinicians with a tool to identify potential treatment failure in near real time, enabling informed alteration of treatment during an infection and improved patient outcomes.
Publisher: Oxford University Press (OUP)
Date: 31-01-2018
DOI: 10.1093/CID/CIY069
Abstract: Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug meropenem is the current gold standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance toward meropenem has not yet been documented however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure. We examined our 30-year clinical collection of melioidosis cases to identify B. pseudomallei isolates with reduced meropenem susceptibility. Isolates were subjected to minimum inhibitory concentration (MIC) testing toward meropenem. Paired isolates from patients who had evolved decreased susceptibility were subjected to whole-genome sequencing. Select agent-compliant genetic manipulation was carried out to confirm the molecular mechanisms conferring resistance. We identified 11 melioidosis cases where B. pseudomallei isolates developed decreased susceptibility toward meropenem during treatment, including 2 cases not treated with this antibiotic. Meropenem MICs increased from 0.5-0.75 µg/mL to 3-8 µg/mL. Comparative genomics identified multiple mutations affecting multidrug resistance-nodulation- ision (RND) efflux pump regulators, with concomitant overexpression of their corresponding pumps. All cases were refractory to treatment despite aggressive, targeted therapy, and 2 were associated with a fatal outcome. This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. These findings have important ramifications for the diagnosis, treatment, and management of life-threatening melioidosis cases.
Publisher: Cold Spring Harbor Laboratory
Date: 19-12-2021
DOI: 10.1101/2021.12.12.21266792
Abstract: Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug resistant ESKAPE pathogen, Pseudomonas aeruginosa . We aimed to develop two SYBR Green-based mismatch lification mutation assays (SYBR-MAMAs) targeting GyrA T83I ( gyrA 248), and GyrA D87N, D87Y, and D87H ( gyrA 259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa . Following assay validation, the gyrA 248 and gyrA 259 SYBR-MAMAs were tested on 84 clinical P. aeruginosa isolates from Queensland, Australia, 45 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (84%) of isolates with intermediate/full FQ resistance. Importantly, all FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100% concordance with SYBR-MAMA genotypes. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa . An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91%. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.
Publisher: American Society for Microbiology
Date: 02-2016
DOI: 10.1128/AEM.03013-15
Abstract: Melioidosis is a disease of humans and animals that is caused by the saprophytic bacterium Burkholderia pseudomallei . Once thought to be confined to certain locations, the known presence of B. pseudomallei is expanding as more regions of endemicity are uncovered. There is no vaccine for melioidosis, and even with antibiotic administration, the mortality rate is as high as 40% in some regions that are endemic for the infection. Despite high levels of recombination, phylogenetic reconstruction of B. pseudomallei populations using whole-genome sequencing (WGS) has revealed surprisingly robust biogeographic separation between isolates from Australia and Asia. To date, there have been no confirmed autochthonous melioidosis cases in Australia caused by an Asian isolate likewise, no autochthonous cases in Asia have been identified as Australian in origin. Here, we used comparative genomic analysis of 455 B. pseudomallei genomes to confirm the unprecedented presence of an Asian clone, sequence type 562 (ST-562), in Darwin, northern Australia. First observed in Darwin in 2005, the incidence of melioidosis cases attributable to ST-562 infection has steadily risen, and it is now a common strain in Darwin. Intriguingly, the Australian ST-562 appears to be geographically restricted to a single locale and is genetically less erse than other common STs from this region, indicating a recent introduction of this clone into northern Australia. Detailed genomic and epidemiological investigations of new clinical and environmental B. pseudomallei isolates in the Darwin region and ST-562 isolates from Asia will be critical for understanding the origin, distribution, and dissemination of this emerging clone in northern Australia.
Publisher: Cold Spring Harbor Laboratory
Date: 04-02-2022
DOI: 10.1101/2022.02.03.22270419
Abstract: The rise of antimicrobial-resistant (AMR) bacteria is a global health emergency. One critical facet in tackling this epidemic is more rapid AMR diagnosis in serious multi-drug resistant pathogens like Pseudomonas aeruginosa . Here, we designed and then validated two multiplex quantitative real-time PCR (qPCR) assays to simultaneously detect differential expression of the resistance-nodulation- ision efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM, the AmpC β-lactamase, and the porin OprD, which are commonly associated with chromosomally-encoded AMR. Next, qPCRs were tested on 15 sputa from 11 participants with P. aeruginosa respiratory infections to determine AMR profiles in vivo . We confirm multiplex qPCR testing feasibility directly on sputa, representing a key advancement in in vivo AMR diagnosis. Notably, comparison of sputa with their derived isolates grown in Luria-Bertani broth (±2.5% NaCl) or a 5-antibiotic cocktail showed marked expression differences, illustrating the difficulty in replicating in vivo expression profiles in vitro . Cystic fibrosis sputa showed significantly reduced mexE and mexY expression when compared with chronic obstructive pulmonary disease sputa, despite harbouring fluoroquinolone- and aminoglycoside-resistant strains, indicating that these loci are not contributing to AMR in vivo. oprD was also significantly downregulated in cystic fibrosis sputa, even in the absence of contemporaneous carbapenem use, suggesting a common adaptive trait in chronic infections that may affect carbapenem efficacy. Sputum C expression was highest in participants receiving carbapenems (6.7-15x), some of whom were simultaneously receiving cephalosporins, the latter of which would be rendered ineffective by the upregulated C . Our qPCR assays provide valuable insights into the P. aeruginosa resistome, and their use on clinical specimens will permit timely treatment alterations that will improve patient outcomes and antimicrobial stewardship measures.
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 11-2015
Publisher: American Society for Microbiology
Date: 18-05-2021
DOI: 10.1128/AAC.00130-21
Abstract: Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. In addition to identification of isolates to the species level by routine mass spectrometry and antibiotic sensitivity testing, isolates were examined using rapid (∼40-min) real-time PCR assays targeting the most common extended-spectrum β-lactamases (ESBLs bla CTX-M-1 and bla CTX-M-9 groups, plus bla TEM , bla SHV , and an internal 16S rRNA gene control).
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 06-2015
Publisher: Frontiers Media SA
Date: 06-09-2017
Publisher: Cold Spring Harbor Laboratory
Date: 06-12-2017
DOI: 10.1101/229823
Abstract: The melioidosis bacterium, Burkholderia pseudomallei , is increasingly being recognized as a pathogen in patients with cystic fibrosis (CF). We have recently catalogued genome-wide variation of paired, isogenic B. pseudomallei isolates from seven Australasian CF cases, which were collected between four and 55 months apart. Here, we extend this investigation by documenting the transcriptomic changes in B. pseudomallei in five cases. Following growth in an artificial CF sputum medium, four of the five paired isolates exhibited significant differential gene expression (DE) that affected between 32 and 792 genes. The greatest number of DE events was observed between patient CF9 strains, consistent with the hypermutator status of the latter strain, which is deficient in the DNA mismatch repair protein MutS. Two patient isolates harbored duplications that concomitantly increased expression of the β-lactamase gene penA , and a 35kb deletion in another abolished expression of 29 genes. Convergent expression profiles in the chronically-adapted isolates identified two significantly downregulated and 17 significantly upregulated loci, including the antibiotic resistance-nodulation- ision (RND) efflux pump BpeEF-OprC, the quorum-sensing hhqABCDE operon, and a cyanide- and pyocyanin-insensitive cytochrome bd quinol oxidase. These convergent pathoadaptations lead to increased expression of pathways that may suppress competing bacterial and fungal pathogens and that enhance survival in oxygen-restricted environments, the latter of which may render conventional antibiotics less effective in vivo . Treating chronically-adapted B. pseudomallei infections with antibiotics designed to target anaerobic infections, such as the nitroimidazole class of antibiotics, may significantly improve pathogen eradication attempts by exploiting this Achilles heel.
Publisher: Elsevier BV
Date: 07-2008
DOI: 10.1016/J.PLASMID.2008.02.003
Abstract: pPSY is a 12kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10kb. In the present study three different genes were lified by PCR, cloned into pGEM-T Easy and sub-cloned into the EcoRI site of pPSY. The first gene, vioA, is a FAD-dependent l-tryptophan amino acid oxygenase from the high G+C Gram-negative bacterium Chromobacterium violaceum. VioA is involved in the synthesis of the indolocarbazole antitumour antibiotic violacein. It was found that vioA was strongly expressed in Escherichia coli from its native promoter. Two other genes encoding recombinase A (recA) and an amylase (amyA), derived from the high G+C Gram-positive streptomycete, Streptomyces lividans, were also tested. Despite recA lacking its native promoter sequence, it was strongly expressed in E. coli using the lac promoter of pGEM-T Easy. Similar to vioA, S. lividansamyA was strongly expressed in E. coli from its native promoter. Unlike pGEM-T Easy, pPSY stably maintained all three genes without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable licon cloning vector for the expression of heterologous genes in E. coli.
Publisher: Public Library of Science (PLoS)
Date: 21-02-2012
Publisher: Frontiers Media SA
Date: 16-07-2019
Publisher: Springer Science and Business Media LLC
Date: 16-05-2015
Publisher: Microbiology Society
Date: 02-2020
Abstract: Human-to-human transmission of the melioidosis bacterium, Burkholderia pseudomallei , is exceedingly rare, with only a handful of suspected cases documented to date. Here, we used whole-genome sequencing (WGS) to characterize one such unusual B. pseudomallei transmission event, which occurred between a breastfeeding mother with mastitis and her child. Two strains corresponding to multilocus sequence types (STs)-259 and -261 were identified in the mother’s sputum from both the primary culture sweep and in purified colonies, confirming an unusual polyclonal infection in this patient. In contrast, primary culture sweeps of the mother’s breast milk and the child’s cerebrospinal fluid and blood s les contained only ST-259, indicating monoclonal transmission to the child. Analysis of purified ST-259 isolates showed no genetic variation between mother and baby isolates, providing the strongest possible evidence of B. pseudomallei human-to-human transmission, probably via breastfeeding. Next, phylogenomic analysis of all isolates, including the mother’s mixed ST-259/ST-261 sputum s le, was performed to investigate the effects of mixtures on phylogenetic inference. Inclusion of this mixture caused a dramatic reduction in the number of informative SNPs, resulting in branch collapse of ST-259 and ST-261 isolates, and several instances of incorrect topology in a global B. pseudomallei phylogeny, resulting in phylogenetic incongruence. Although phylogenomics can provide clues about the presence of mixtures within WGS datasets, our results demonstrate that this methodology can lead to phylogenetic misinterpretation if mixed genomes are not correctly identified and omitted. Using current bioinformatic tools, we demonstrate a robust method for bacterial mixture identification and strain parsing that avoids these pitfalls.
Publisher: Cold Spring Harbor Laboratory
Date: 17-03-2020
DOI: 10.1101/2020.03.12.20032722
Abstract: The Elizabethkingia genus has gained global attention in recent years as a nosocomial pathogen. Elizabethkingia spp. are intrinsically multidrug resistant, primarily infect immunocompromised in iduals, and are associated with high mortality (∼20-40%). Although Elizabethkingia infections appear sporadically worldwide, gaps remain in our understanding of transmission, global strain relatedness and patterns of antimicrobial resistance. To address these knowledge gaps, 22 clinical isolates collected in Queensland, Australia, over a 16-year period along with six hospital environmental isolates were examined using MALDI-TOF MS (VITEK ® MS) and whole-genome sequencing to compare with a global strain dataset. Phylogenomic reconstruction against all publicly available genomes ( n =100) robustly identified 22 E. anophelis , three E. miricola , two E. meningoseptica and one E. bruuniana from our isolates, most with previously undescribed ersity. Global relationships show Australian E. anophelis isolates are genetically related to those from the USA, England and Asia, suggesting shared ancestry. Genomic examination of clinical and environmental strains identified evidence of nosocomial transmission in patients admitted several months apart, indicating probable infection from a hospital reservoir. Furthermore, broth microdilution of the 22 clinical Elizabethkingia spp. isolates against 39 antimicrobials revealed almost ubiquitous resistance to aminoglycosides, carbapenems, cephalosporins and penicillins, but susceptibility to minocycline, levofloxacin and trimethoprim/sulfamethoxazole. Our study demonstrates important new insights into the genetic ersity, environmental persistence and transmission of Australian Elizabethkingia species. Furthermore, we show that Australian isolates are highly likely to be susceptible to minocycline, levofloxacin and trimethoprim/sulfamethoxazole, suggesting that these antimicrobials may provide effective therapy for Elizabethkingia infections. Elizabethkingia are a genus of environmental Gram-negative, multidrug resistant, opportunistic pathogens. Although an uncommon cause of nosocomial and community-acquired infections, Elizabethkingia spp. are known to infect those with underlying co-morbidities and/or immunosuppression, with high mortality rates of ∼20-40%. Elizabethkingia have a presence in Australian hospitals and patients however, their origin, epidemiology, and antibiotic resistance profile of these strains is poorly understood. Here, we performed phylogenomic analyses of clinical and hospital environmental Australian Elizabethkingia spp., to understand transmission and global relationships. Next, we performed extensive minimum inhibitory concentration testing to determine antimicrobial susceptibility profiles. Our findings identified a highly erse Elizabethkingia population in Australia, with many being genetically related to international strains. A potential transmission source was identified within the hospital environment where two transplant patients were infected and three E. anophelis strains formed a clonal cluster within the phylogeny. Furthermore, near ubiquitous susceptibility to tetracyclines, fluoroquinolones and trimethoprim/sulfamethoxazole was observed in clinical isolates. We provide new insights into the origins, transmission and epidemiology of Elizabethkingia spp., in addition to understanding their intrinsic resistance profiles and potential effective treatment options, which has implications to managing infections and detecting outbreaks globally.
Publisher: Public Library of Science (PLoS)
Date: 11-03-2014
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 05-2018
Publisher: Elsevier BV
Date: 09-2011
DOI: 10.1016/J.PLASMID.2011.07.006
Abstract: Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to 'graze' on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan "plaque" formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.
Publisher: American Society for Microbiology
Date: 15-03-2018
Abstract: The Burkholderia pseudomallei isolate MSHR1435 is a fully virulent environmental sequence type 131 (ST131) isolate that is epidemiologically associated with a 17.5-year chronic melioidosis infection. The completed genome will serve as a reference for studies of environmental ecology, virulence, and chronic B. pseudomallei infections.
Publisher: Public Library of Science (PLoS)
Date: 20-03-2014
Publisher: Public Library of Science (PLoS)
Date: 13-08-2013
Publisher: Elsevier BV
Date: 12-2008
Publisher: Microbiology Society
Date: 04-07-2022
Abstract: Between 2010 and 2015, nocardiosis outbreaks caused by Nocardia seriolae affected many permit farms throughout Vietnam, causing mass fish mortalities. To understand the biology, origin and epidemiology of these outbreaks, 20 N . seriolae strains collected from farms in four provinces in the South Central Coast region of Vietnam, along with two Taiwanese strains, were analysed using genetics and genomics. PFGE identified a single cluster amongst all Vietnamese strains that was distinct from the Taiwanese strains. Like the PFGE findings, phylogenomic and SNP genotyping analyses revealed that all Vietnamese N. seriolae strains belonged to a single, unique clade. Strains fell into two subclades that differed by 103 SNPs, with almost no ersity within clades (0–5 SNPs). There was no association between geographical origin and subclade placement, suggesting frequent N. seriolae transmission between Vietnamese mariculture facilities during the outbreaks. The Vietnamese strains shared a common ancestor with strains from Japan and China, with the closest strain, UTF1 from Japan, differing by just 220 SNPs from the Vietnamese ancestral node. Draft Vietnamese genomes range from 7.55 to 7.96 Mbp in size, have an average G+C content of 68.2 % and encode 7 602–7958 predicted genes. Several putative virulence factors were identified, including genes associated with host cell adhesion, invasion, intracellular survival, antibiotic and toxic compound resistance, and haemolysin biosynthesis. Our findings provide important new insights into the epidemiology and pathogenicity of N. seriolae and will aid future vaccine development and disease management strategies, with the ultimate goal of nocardiosis-free aquaculture.
Publisher: American Society for Microbiology
Date: 17-05-2022
DOI: 10.1128/AAC.00204-22
Abstract: The rise of antimicrobial-resistant (AMR) bacteria is a global health emergency. One critical facet of tackling this epidemic is more rapid AMR diagnosis in serious multidrug-resistant pathogens like Pseudomonas aeruginosa .
Publisher: Cold Spring Harbor Laboratory
Date: 28-12-2021
DOI: 10.1101/2021.12.28.474260
Abstract: Nocardia seriolae has caused significant fish losses in Asia and the Americas in recent decades, including in Vietnam, which has witnessed devastating economic and social impacts due to this bacterial pathogen. Surveillance strategies are urgently needed to mitigate N. seriolae dissemination in Vietnamese aquaculture and mariculture industries. Whole-genome sequencing (WGS) offers the highest level of resolution to discriminate closely related strains and to determine their putative origin and transmission routes. However, WGS is impractical for epidemiological investigations and pathogen surveillance due to its time-consuming and costly nature, putting this technology out-of-reach for many industry end-users. To overcome this issue, we targeted two previously characterised, phylogenetically informative single-nucleotide polymorphisms (SNPs) in N. seriolae that accurately distinguish: i) Vietnamese from non-Vietnamese strains, and ii) the two Vietnamese subclades. Using the mismatch lification mutation assay (MAMA) format, we developed assays that genotype strains based on differences in licon melting temperature (melt-MAMA) and size (agarose-MAMA). Our MAMA assays accurately genotyped strains both from culture and fish tissues at low cost, using either real-time (~AUD$1 er s le) or conventional (~AUD$0.50 er s le) PCR instrumentation. Our novel assays provide a rapid, reproducible, and cost-effective tool for routine genotyping of this pathogen, allowing faster identification and treatment of nocardiosis-effected permit fish within Vietnamese aquaculture/mariculture facilities, an essential step in mitigating N. seriolae -associated losses.
Publisher: Public Library of Science (PLoS)
Date: 05-03-2020
Publisher: American Society for Microbiology
Date: 2014
DOI: 10.1128/AAC.01842-13
Abstract: Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei . Resistance to gentamicin is generally a hallmark of B. pseudomallei , and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei . A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB , encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental s ling of other locations to test for B. pseudomallei endemicity.
Publisher: Oxford University Press (OUP)
Date: 28-11-2012
DOI: 10.1111/LAM.12022
Abstract: A PCR assay was developed to genotypically characterize Francisella tularensis and F. novicida. An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F. tularensis and genotype 14 variable number tandem repeat (VNTR) markers. Assay performance was evaluated with 117 Francisella s les. S le DNA was lified, and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry (ESI-MS). The base compositions of the PCR licons were derived from these high-accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all s les were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among s les. Sequence variants were observed with a number of assay markers, but these did not interfere with s le characterization, and served to increase the genetic ersity detected by the assay.
Publisher: Microbiology Society
Date: 03-2019
Publisher: Microbiology Society
Date: 14-11-2017
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 10-2014
Publisher: Cold Spring Harbor Laboratory
Date: 31-08-2021
DOI: 10.1101/2021.08.30.21262864
Abstract: Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are characterised by increasingly frequent acute pulmonary exacerbations that reduce life quality and length. Human airways are home to a rich polymicrobial environment, including members of the obligately anaerobic genus, Prevotella . Despite their commonness, surprisingly little is known about the prevalence, role, genomic ersity, and antimicrobial resistance (AMR) potential of Prevotella species/strains in healthy and diseased airways. Here, we used comparative genomics to develop a real-time PCR assay to permit rapid Prevotella spp. quantification from cultures and clinical specimens. Assay specificity was validated across a panel of Prevotella and non- Prevotella species, followed by PCR screening of CF and COPD respiratory-derived cultures. Next, 35 PCR-positive isolates were subjected to whole-genome sequencing. Of eight identified species, P. histicola, P. melaninogenica, P. nanceiensis, P. salivae and P. denticola overlapped between participant cohorts. Phylogenomic analysis revealed considerable interhost but limited intrahost ersity, suggesting patient-specific lineages in the lower airways, probably from oral cavity aspirations. Correlation of phenotypic AMR profiles with AMR gene presence identified excellent correlation between tetQ presence and decreased doxycycline susceptibility, and ermF presence and decreased azithromycin susceptibility and clindamycin resistance. AMR rates were higher in the CF isolates, reflecting greater antibiotic use in this cohort. All tested Prevotella isolates were tobramycin-resistant, providing a potential selection method to improve Prevotella culture retrieval rates. Our addition of 35 airway-derived Prevotella genomes to public databases will enhance ongoing efforts to unravel the role of this erse and enigmatic genus in both chronic respiratory diseases and healthy lungs. Thirty-five Prevotella spp . genomes generated in this study are available in the Sequence Read Archive (SRA) and GenBank databases under BioProject accession PRJNA742126.
Publisher: Public Library of Science (PLoS)
Date: 26-07-2019
Publisher: Microbiology Society
Date: 27-10-2022
DOI: 10.1099/JMM.0.001593
Abstract: Background. Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug-resistant ESKAPE pathogen, Pseudomonas aeruginosa . Objectives. We aimed to develop two SYBR Green-based mismatch lification mutation assays (SYBR-MAMAs) targeting GyrA T83I ( gyrA 248) and GyrA D87N, D87Y and D87H ( gyrA 259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa . Methods. Following assay validation, the gyrA 248 and gyrA 259 SYBR-MAMAs were tested on 84 Australian clinical P. aeruginosa isolates, 46 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing. Results. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (83%) of isolates with intermediate/full FQ resistance. All FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100 % concordance with SYBR-MAMA genotypes. Conclusions. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa . An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91 %. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.
Publisher: Cold Spring Harbor Laboratory
Date: 19-04-2019
DOI: 10.1101/614214
Abstract: Non-typeable Haemophilus influenzae (NTHi), an opportunistic pathogen of the upper airways of healthy children, can infect the lower airways, driving chronic lung disease. However, the molecular basis underpinning NTHi transition from a commensal to a pathogen is not clearly understood. Here, we performed comparative genomic and transcriptomic analyses of 12 paired, isogenic NTHi strains, isolated from the nasopharynx (NP) and bronchoalveolar lavage (BAL) of 11 children with chronic lung disease, to identify convergent molecular signatures associated with lung adaptation. Comparative genomic analyses of the 12 NP-BAL pairs demonstrated that five were genetically identical, with the remaining seven differing by only 1 to 3 mutations. Within-patient transcriptomic analyses identified between 2 and 58 differentially expressed genes in 8 of the 12 NP-BAL pairs, including pairs with no observable genomic changes. Whilst no convergence was observed at the gene level, functional enrichment analysis revealed significant under-representation of differentially expressed genes belonging to Coenzyme metabolism, Function unknown, Translation, ribosomal structure and biogenesis C luster of Orthologous Groups categories. In contrast, Carbohydrate transport and metabolism, Cell motility and secretion, Intracellular trafficking and secretion , and Energy production categories were over-represented. This observed trend amongst genetically-unrelated NTHi strains provides evidence of convergent transcriptional adaptation of NTHi to paediatric airways that deserves further exploration. Understanding the pathoadaptative mechanisms that NTHi employs to infect and persist in the lower paediatric airways is essential for devising targeted diagnostics and treatments aimed at minimising disease severity, and ultimately, preventing NTHi lung infections and subsequent chronic lung disease in children.
Publisher: Cold Spring Harbor Laboratory
Date: 20-10-2017
DOI: 10.1101/205070
Abstract: Burkholderia pseudomallei , the causative agent of the high-mortality disease melioidosis, is a Gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug, meropenem, is the current gold-standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance towards meropenem has not yet been documented however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure. Here, we document 11 melioidosis cases in which B. pseudomallei isolates developed decreased susceptibility towards meropenem during treatment, including two cases not treated with this antibiotic. Meropenem minimum inhibitory concentrations increased over time from 0.5-0.75 to 3-8 μg/mL. Using comparative genomics, we identified multiple mutations affecting multidrug resistance-nodulation- ision (RND) efflux pump regulators, leading to over-expression of their corresponding pumps. The most commonly affected pump was AmrAB-OprA, although alterations in the local regulators of BpeEF-OprC or BpeAB-OprB were observed in three cases. This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei . Further, we document two concerning ex les of severe melioidosis where the reduced treatment efficacy of meropenem was associated with a fatal outcome. The bacterium Burkholderia pseudomallei , which causes the often-fatal tropical disease melioidosis, is difficult to eradicate. Due to high levels of intrinsic antibiotic resistance, only a handful of antibiotics are effective against this pathogen. One of these, meropenem, is commonly used in the treatment of melioidosis patients who are unresponsive to other treatments or are critically ill. Here, we describe 11 melioidosis cases whereby patients exhibited prolonged or repeated infections that were associated with the development of decreased meropenem susceptibility. We identified the molecular basis for this decreased susceptibility in latter B. pseudomallei isolates obtained from these patients, and functionally confirmed the mechanism conferring this phenotype. Our findings have important ramifications for the diagnosis, treatment and management of life-threatening melioidosis cases.
Publisher: American Society for Microbiology
Date: 02-11-2016
Abstract: Whole-genome sequence (WGS) data are commonly used to design diagnostic targets for the identification of bacterial pathogens. To do this effectively, genomics databases must be comprehensive to identify the strict core genome that is specific to the target pathogen. As additional genomes are analyzed, the core genome size is reduced and there is erosion of the target-specific regions due to commonality with related species, potentially resulting in the identification of false positives and/or false negatives. IMPORTANCE A comparative analysis of 1,130 Burkholderia genomes identified unique markers for many named species, including the human pathogens B. pseudomallei and B. mallei . Due to core genome reduction and signature erosion, only 38 targets specific to B. pseudomallei /mallei were identified. By using only public genomes, a larger number of markers were identified, due to unders ling, and this larger number represents the potential for false positives. This analysis has implications for the design of diagnostics for other species where the genomic space of the target and/or closely related species is not well defined.
Publisher: American Society for Microbiology
Date: 04-2015
DOI: 10.1128/JCM.03453-14
Abstract: Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillus Burkholderia pseudomallei . Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic. B. pseudomallei is classed as a tier 1 select agent by the Centers for Disease Control and Prevention from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure. B. pseudomallei isolates from the property's groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir of B. pseudomallei . Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens.
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 08-2012
Publisher: Oxford University Press (OUP)
Date: 25-02-2019
DOI: 10.1093/OFID/OFZ091
Publisher: American Society for Microbiology
Date: 27-04-2016
Abstract: Sporadic melioidosis cases have been reported in the African mainland and Indian Ocean islands, but until recently, these regions were not considered areas where B. pseudomallei is endemic. Given the high mortality rate of melioidosis, it is crucial that this disease be recognized and suspected in all regions of endemicity. Previous work has shown that B. pseudomallei originated in Australia, with subsequent introduction into Asia however, the precise origin of B. pseudomallei in other tropical regions remains poorly understood. Using whole-genome sequencing, we characterized B. pseudomallei isolates from Madagascar and Burkina Faso. Next, we compared these strains to a global collection of B. pseudomallei isolates to identify their evolutionary origins. We found that African B. pseudomallei strains likely originated from Asia and were closely related to South American strains, reflecting a relatively recent shared evolutionary history. We also identified substantial genetic ersity among African strains, suggesting long-term B. pseudomallei endemicity in this region.
Publisher: American Society for Microbiology
Date: 03-05-2017
Abstract: Cystic fibrosis (CF) is a genetic disorder characterized by progressive lung function decline. CF patients are at an increased risk of respiratory infections, including those by the environmental bacterium Burkholderia pseudomallei , the causative agent of melioidosis. Here, we compared the genomes of B. pseudomallei isolates collected between ~4 and 55 months apart from seven chronically infected CF patients. Overall, the B. pseudomallei strains showed evolutionary patterns similar to those of other chronic infections, including emergence of antibiotic resistance, genome reduction, and deleterious mutations in genes involved in virulence, metabolism, environmental survival, and cell wall components. We documented the first reported B. pseudomallei hypermutators, which were likely caused by defective MutS. Further, our study identified both known and novel molecular mechanisms conferring resistance to three of the five clinically important antibiotics for melioidosis treatment. Our report highlights the exquisite adaptability of microorganisms to long-term persistence in their environment and the ongoing challenges of antibiotic treatment in eradicating pathogens in the CF lung. Convergent evolution with other CF pathogens hints at a degree of predictability in bacterial evolution in the CF lung and potential targeted eradication of chronic CF infections in the future. IMPORTANCE Burkholderia pseudomallei , the causative agent of melioidosis, is an environmental opportunistic bacterium that typically infects immunocompromised people and those with certain risk factors such as cystic fibrosis (CF). Patients with CF tend to develop chronic melioidosis infections, for reasons that are not well understood. This report is the first to describe B. pseudomallei evolution within the CF lung during chronic infection. We show that the pathways by which B. pseudomallei adapts to the CF lung are similar to those seen in better-studied CF pathogens such as Pseudomonas aeruginosa , Staphylococcus aureus , and Burkholderia cepacia complex species. Adaptations include the accumulation of antibiotic resistance, loss of nonessential genes, metabolic alterations, and virulence factor attenuation. Known and novel mechanisms of resistance to three of the five antibiotics used in melioidosis treatment were identified. Similar pathways of evolution in CF pathogens, including B. pseudomallei , provide exciting avenues for more-targeted treatment of chronic, recalcitrant infections.
Publisher: Microbiology Society
Date: 09-2016
DOI: 10.1099/JMM.0.000312
Abstract: The Burkholderiapseudomallei multilocus sequence typing (MLST) database (pseudomallei/) contains the largest global sequence repository for B. pseudomallei and its closest genetic relatives. Using conventional MLST and in silico MLST data derived from publicly available whole-genome sequences, we first defined the phylogenetic relatedness of B. pseudomallei and its nearest neighbours. Based on this analysis, we propose that the recently described B. pseudomallei complex (Bpc) should be expanded to encompass B. pseudomallei, Burkholderiahumptydooensis (proposed), Burkholderiamallei, Burkholderiaoklahomensis, Burkholderiathailandensis and three unassigned Burkholderia Clades A, B and C (represented by type strains BDU 5, BDU 8 and MSMB0265, respectively). Of note, the MLST narK locus is present in all Bpc species but is missing in all other Burkholderia spp., including all Burkholderiacepacia complex species, with the exception of most Burkholderiaubonensis strains, which contain narK but encode genetically distinct sequences. The presence of narK is thus indicative of a Bpc strain. Next, we revisited in silico the performance of the existing MLST primers, which prompted redesign of primers targeting the gmhD, lepA, lipA, narK and ndh loci to encompass genetic ersity among Bpc strains and to address lification/sequencing issues. We show in silico and in vitro that the redesigned primers yield good-quality lification and sequencing results for the gmhD, lepA, lipA, narK and ndh loci in Bpc species. These primers provide an alternative for lification and sequencing of MLST loci in Bpc species in cases when poor-quality lification or sequencing data are obtained using the original MLST primers.
Publisher: Springer Science and Business Media LLC
Date: 27-08-2015
Publisher: American Society for Microbiology
Date: 06-2012
DOI: 10.1128/JCM.06737-11
Abstract: Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei . In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1- orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia -like fimbrial/ Burkholderia thailandensis -like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin.
Publisher: American Society for Microbiology
Date: 27-11-2014
DOI: 10.1128/CVI.00632-13
Abstract: Nontypeable Haemophilus influenzae (NTHi)-associated disease is a major health problem globally. Whole-genome sequence analysis identified the absence of hpd genes encoding Haemophilus protein D in 3 of 16 phylogenetically distinct NTHi isolates. This novel finding is of potential clinical significance, as protein D and hpd represent important NTHi vaccine antigen and diagnostic targets, respectively.
Publisher: Cold Spring Harbor Laboratory
Date: 23-10-2023
Publisher: Cold Spring Harbor Laboratory
Date: 12-02-2020
DOI: 10.1101/2020.02.11.944942
Abstract: Several members of the Gram-negative environmental bacterial genus, Achromobacter , are associated with serious infections in immunocompromised in iduals, of which Achromobacter xylosoxidans is the most common. Despite their pathogenic potential, comparatively little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management of Achromobacter infections. Here, we performed comparative genomics of 158 Achromobacter spp. genomes to robustly identify species boundaries, to reassign several incorrectly speciated taxa, and to identify genetic sequences specific for the Achromobacter genus and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to lify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ∼110 genome equivalents, and detected down to ∼12 and ∼1 genome equivalent/s, respectively. In silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult CF sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive, and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans , and will facilitate the rapid and accurate diagnosis of this important group of pathogens.
Publisher: Microbiology Society
Date: 10-2019
Publisher: Public Library of Science (PLoS)
Date: 15-01-2010
Publisher: American Society of Tropical Medicine and Hygiene
Date: 06-01-2016
Publisher: Microbiology Society
Date: 03-02-2022
Abstract: Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are characterized by increasingly frequent acute pulmonary exacerbations that reduce life quality and length. Human airways are home to a rich polymicrobial environment, which includes members of the obligately anaerobic genus Prevotella . Despite their commonness, surprisingly little is known about the prevalence, role, genomic ersity and antimicrobial resistance (AMR) potential of Prevotella species and strains in healthy and diseased airways. Here, we used comparative genomics to develop a real-time PCR assay to permit rapid Prevotella species identification and quantification from cultures and clinical specimens. Assay specificity was validated across a panel of Prevotella and non- Prevotella species, followed by PCR screening of CF and COPD respiratory-derived cultures. Next, 35 PCR-positive isolates were subjected to whole-genome sequencing. Of eight identified Prevotella species, P. histicola , P. melaninogenica , P. nanceiensis , P. salivae and P. denticola overlapped between participant cohorts. Phylogenomic analysis revealed considerable interhost but limited intrahost ersity, suggesting patient-specific lineages in the lower airways, probably from oral cavity aspirations. Correlation of phenotypic AMR profiles with AMR genes identified excellent correlation between tetQ presence and decreased doxycycline susceptibility, and ermF presence and decreased azithromycin susceptibility and clindamycin resistance. AMR rates were higher in the CF isolates, reflecting greater antibiotic use in this cohort. All tested Prevotella isolates were tobramycin-resistant, providing a potential selection method to improve Prevotella culture retrieval rates. Our addition of 35 airway-derived Prevotella genomes to public databases will enhance ongoing efforts to unravel the role of this erse and enigmatic genus in both diseased and healthy lungs.
Publisher: Microbiology Society
Date: 13-06-2017
Publisher: Microbiology Society
Date: 09-2013
Abstract: Acinetobacter spp. are a erse group of Gram-negative bacteria frequently implicated in nosocomial infections. Genotypic methods have been instrumental in studying Acinetobacter , but few offer high resolution, rapid turnaround time, technical ease and high inter-laboratory reproducibility, which has h ered understanding of disease incidence, transmission patterns and ersity within this genus. Here, we further evaluated multilocus PCR electrospray ionization/mass spectrometry (PCR/ESI-MS), a method that is simple and robust, and provides both species characterization and strain-level resolution of Acinetobacter spp. on a single platform. We examined 125 Acinetobacter isolates from 21 hospitals, laboratories and medical centres spanning four counties in Arizona, USA, using PCR/ESI-MS. We compared PCR/ESI-MS with an in-house lified fragment length polymorphism (AFLP) genotyping scheme. PCR/ESI-MS demonstrated that Acinetobacter spp. from Arizonan hospitals had similar species and strain distributions to other US civilian hospitals. Furthermore, we showed that the PCR/ESI-MS and AFLP genotypes were highly congruent, with the former having the advantages of robust inter-laboratory reproducibility, rapid turnaround time and simple experimental set-up and data analysis. PCR/ESI-MS is an effective and high-throughput platform for strain typing of Acinetobacter baumannii and for identification of other Acinetobacter spp., including the emerging nosocomial pathogens Acinetobacter pittii and Acinetobacter nosocomialis .
Publisher: Future Medicine Ltd
Date: 06-2017
Abstract: Aim: To design a highly specific and sensitive multiplex real-time PCR assay for the differentiation of the pathogen Haemophilus influenzae from its nonpathogenic near-neighbor Haemophilus haemolyticus. Materials & methods: A comparison of 380 Haemophilus spp. genomes was used to identify loci specific for each species. Novel PCR assays targeting H. haemolyticus (hypD) and H. influenzae (siaT) were designed. Results & discussion: PCR screening across 143 isolates demonstrated 100% specificity for hypD and siaT. These two assays were multiplexed with the recently described fucP assay for further differentiation among H. influenzae. Conclusion: The triplex assay provides rapid, unambiguous, sensitive and highly specific genotyping results for the simultaneous detection of hypD and siaT, including fucose-positive H. influenzae (fucP), in a single PCR.
Publisher: Informa UK Limited
Date: 08-2012
DOI: 10.2147/IDR.S35529
Publisher: American Society for Microbiology
Date: 05-2018
DOI: 10.1128/IAI.00938-17
Abstract: The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of erse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei . A panel of B. pseudomallei dsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically erse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.
Publisher: Cold Spring Harbor Laboratory
Date: 12-2021
DOI: 10.1101/2021.11.30.470482
Abstract: Between 2010 and 2015, nocardiosis outbreaks caused by Nocardia seriolae affected many permit farms throughout Vietnam, causing mass fish mortalities. To understand the biology, origin, and epidemiology of these outbreaks, 20 N. seriolae strains collected from farms in four provinces in the South-Central Coast of Vietnam, along with two Taiwanese strains, were analysed using genetics and genomics. Pulsed-field gel electrophoresis identified a single cluster amongst all Vietnamese strains that was distinct from the Taiwanese strains. Like the PFGE findings, phylogenomic and single-nucleotide polymorphism (SNP) genotyping analyses revealed that all Vietnamese N. seriolae strains belonged to a single, unique clade. Strains fell into two subclades that differed by 103 SNPs, with almost no ersity within clades (0-2 SNPs). There was no association between geographic origin and subclade placement, suggesting frequent N. seriolae transmission between Vietnamese mariculture facilities during the outbreaks. Vietnamese strains shared a common ancestor with strains from Japan and China, with the closest strain, UTF1 from Japan, differing by just 217 SNPs from the Vietnamese ancestral node. Draft Vietnamese genomes range from 7.55-7.96 Mbp in size, have an average G+C content of 68.2%, and encode 7,602-7,958 predicted genes. Several putative virulence factors were identified, including genes associated with host cell adhesion, invasion, intracellular survival, antibiotic and toxic compound resistance, and haemolysin biosynthesis. Our findings provide important new insights into N. seriolae epidemiology and pathogenicity and will aid future vaccine development and disease management strategies, with the ultimate goal of nocardiosis-free aquaculture.
Publisher: Microbiology Society
Date: 08-2018
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1016/J.PLASMID.2009.03.002
Abstract: The broad host range, cloning and expression vector pPSX has been completely sequenced and analysed. pPSX is 14.7kb in length and contains the fusion of two continuous segments of the parental 34kb, IncW plasmid pR388. pPSX appears to have retained at least three sets of gene/s which contribute in different ways to plasmid stability. The first of these parB, is a known participant in the partitioning of low-copy number plasmids. While the adjoining gene, orf35, has high homology with kfrA, a putative plasmid nucleoid organiser that is often associated with the ParAB family of proteins. The second set of genes orfs18, 19, 20, whose exact functions are not clear, have homology to the stability operons of both IncW and IncN plasmids. The third is the resolvase, resP, which may resolve plasmid multimers that can lead to plasmid instability. pPSX is a small, stable cloning vector good for cloning and expression of a wide range of genes, including those from streptomycetes.
Publisher: Elsevier BV
Date: 04-2019
DOI: 10.1016/J.TIM.2019.01.003
Abstract: Bacterial infections are the primary cause of respiratory decline and mortality in cystic fibrosis (CF) patients. In a recent study, Diaz Caballero and colleagues [1] (PLoS Pathog. 2018 :e1007453) catalogued the molecular adaptation of a decade-long Burkholderia multivorans infection in a Canadian CF patient, which evolved to become resistant towards multiple classes of antibiotics.
Publisher: Wiley
Date: 17-10-2011
DOI: 10.1002/MRM.23190
Abstract: The classic paradigm for MRI requires a homogeneous B(0) field in combination with linear encoding gradients. Distortions are produced when the B(0) is not homogeneous, and several postprocessing techniques have been developed to correct them. Field homogeneity is difficult to achieve, particularly for short-bore magnets and higher B(0) fields. Nonlinear magnetic components can also arise from concomitant fields, particularly in low-field imaging, or intentionally used for nonlinear encoding. In any of these situations, the second-order component is key, because it constitutes the first step to approximate higher-order fields. We propose to use the fractional Fourier transform for analyzing and reconstructing the object's magnetization under the presence of quadratic fields. The fractional fourier transform provides a precise theoretical framework for this. We show how it can be used for reconstruction and for gaining a better understanding of the quadratic field-induced distortions, including ex les of reconstruction for simulated and in vivo data. The obtained images have improved quality compared with standard Fourier reconstructions. The fractional fourier transform opens a new paradigm for understanding the MR signal generated by an object under a quadratic main field or nonlinear encoding.
Publisher: Public Library of Science (PLoS)
Date: 14-09-2017
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.PLASMID.2006.11.004
Abstract: A cosmid cloning vector has been constructed that demonstrates high levels of segregational stability in Escherichia coli K12. pPSX is a 14-kilobase vector derived from the IncW plasmid pR388. pPSX is highly stable in E. coli in the absence of antibiotic selection, even while expressing the toxic indolocarbazole antitumor antibiotic violacein. The incorporation of the lambdacos sequence enables construction of cosmid libraries with inserts ranging from 24 to 36kb. The inclusion of a lacZalpha multiple cloning site (MCS) allows blue/white screening. pPSX cosmids can be extracted from the host cell with commercial plasmid extraction kits facilitating downstream analysis, sequencing and sub-cloning. pPSX can be transferred to a variety of heterologous hosts by either electroporation or mobilization from E. coli S17-1. While it is unstable in non-E. coli hosts without antibiotic selection, heterologous host strains such as Rhodobacter sphaeroides and Pseudomonas stutzeri will maintain the plasmid under antibiotic selection to allow screening of expressed inserts. pPSX provides the benefits of large insert sizes with high stability to allow cloning of chemotherapeutic gene clusters in E. coli and a range of other heterologous hosts.
Publisher: Cambridge University Press (CUP)
Date: 06-11-2015
DOI: 10.1017/S0950268815002770
Abstract: Melioidosis is an infectious disease caused by Burkholderia pseudomallei , a bacterium endemic in Southeast Asia and northern Australia. In New Caledonia, sporadic cases were first described in 2005 since then, more cases have been identified. To improve our understanding of melioidosis epidemiology in New Caledonia, we compared the local cases and B. pseudomallei isolates with those from endemic areas. Nineteen melioidosis cases have been diagnosed in New Caledonia since 1999, mostly severe and with frequent bacteraemia, leading to three (16%) fatalities. All but one occurred in the North Province. Besides sporadic cases caused by non-clonal strains, we also identified a hotspot of transmission related to a clonal group of B. pseudomallei that is phylogenetically related to Australian strains.
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 05-2017
Publisher: American Society for Microbiology
Date: 22-12-2020
DOI: 10.1128/MSYSTEMS.00726-20
Abstract: Burkholderia pseudomallei is predominantly a tropical pathogen uncommonly found in the environment of temperate climatic regions. It is unclear if introduction into temperate regions is sporadic and temporary or if B. pseudomallei can persist in such environments. B. pseudomallei was identified in the environment of southwest Western Australia with melioidosis cases between 1966 and 1991. We report a new cluster with 23 animal fatalities in the same region from 2017, with B. pseudomallei again being recovered from the environment. Comparison of the isolates from the first and second clusters using genomics revealed a single sequence type, high clonality, and limited recombination, even though the time of recovery of the isolates spanned 51 years. This is a major contrast to the extensive genomic ersity seen in the tropics. Our data support the suggestion that B. pseudomallei has the ability to persist in nontropical environments, potentially in a latent state, and has the ability to activate following favorable conditions (rainfall) and then infect animals and humans.
Publisher: American Society for Microbiology
Date: 2015
DOI: 10.1128/JCM.02574-14
Abstract: Burkholderia pseudomallei isolates with shared multilocus sequence types (STs) have not been isolated from different continents. We identified two STs shared between Australia and Cambodia. Whole-genome analysis revealed substantial ersity within STs, correctly identified the Asian or Australian origin, and confirmed that these shared STs were due to homoplasy.
Publisher: American Society of Tropical Medicine and Hygiene
Date: 07-08-2013
Publisher: Cold Spring Harbor Laboratory
Date: 17-04-2018
DOI: 10.1101/301960
Abstract: Burkholderia pseudomallei , the causative agent of the deadly tropical disease melioidosis, is intrinsically resistant to many antibiotics, leaving few effective treatment options. Trimethoprim-sulfamethoxazole (SXT), meropenem (MEM) and doxycycline (DOX) are valuable antibiotics for melioidosis treatment due to inherently low or no primary resistance. Although considered rare, upregulation of one or more resistance-nodulation- ision (RND) efflux pumps is now known to lead to acquired resistance towards these drugs in B. pseudomallei. Here, we developed a triplex quantitative PCR assay to detect upregulation of the three clinically relevant RND efflux systems: AmrAB-OprA, BpeB-OprB and BpeEF-OprC. The triplex assay was tested on seven clinically-derived B. pseudomallei isogenic pairs, where the latter strain of each pair had altered regulator activity and exhibited reduced susceptibility to SXT, MEM or DOX. The triplex assay accurately detected efflux pump upregulation between isogenic pairs, which corresponded with decreased antibiotic susceptibility. We further verified assay performance on eight laboratory-generated B. pseudomallei mutants encoding efflux pump regulator mutations. Targeting antibiotic resistance in B. pseudomallei using molecular genotyping provides clinicians with a rapid tool to identify potential treatment failure in near real-time, enabling informed alteration of treatment during an infection and improved patient outcomes. The melioidosis bacterium Burkholderia pseudomallei is intrinsically resistant to many antibiotics, limiting treatment options to a handful of drugs including meropenem, doxycycline and trimethoprim-sulfamethoxazole. Although rare, there have now been several documented melioidosis cases where resistance to these antibiotics has developed during an infection, leading to treatment failure and increased mortality rates. Interestingly, all strains resistant to these drugs exhibit increased efflux pump expression, representing a shared molecular signature that can be exploited for rapid diagnostic purposes. Here, we developed and validated a single-tube real-time qPCR assay to detect clinically relevant efflux pump upregulation in B. pseudomallei , an important first step towards high-level resistance. This triplex assay offers a drastically reduced turn-around-time compared to current methodology, enabling earlier detection of resistance emergence. Implementation of this new diagnostic will aid clinicians in the selection of appropriate therapy, thereby minimizing resistance development and treatment failure for this high-mortality disease.
Publisher: Cold Spring Harbor Laboratory
Date: 23-02-2020
DOI: 10.1101/2020.02.20.958728
Abstract: Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using rapid (∼40 min) real-time PCR assays targeting the most common extended spectrum β-lactamases (ESBLs CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). AmpC CMY β-lactamase prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E . coli and K. pneumoniae isolates , respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one β-lactamase gene, with 144 (50%) encoding bla CTX-M-1 group, 92 (31.9%) bla CTX-M-9 group, 48 (16.7%) bla SHV , 133 (46.2%) bla TEM , and 34 (11.8%) bla CMY . A subset of isolates (n=98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants, and to verify genotyping accuracy. WGS of β-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including bla NDM and bla IMP , and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.
Publisher: Cold Spring Harbor Laboratory
Date: 12-08-2022
DOI: 10.1101/2022.08.11.22278689
Abstract: Antimicrobial resistance (AMR) is an intensifying threat that requires urgent mitigation to avoid a post-antibiotic era. The bacterial pathogen, Pseudomonas aeruginosa , represents one of the greatest AMR concerns due to ever-increasing multi- and pan-drug resistance rates. Shotgun sequencing is gaining traction for AMR profiling due to its unambiguity and data transferability however, existing tools for in silico AMR prediction perform poorly for this pathogen. We curated the most comprehensive database to date of known P. aeruginosa AMR determinants. We then performed comparative genomics and microbial genome-wide association (mGWAS) across a Global isolate Dataset ( n =1391) with paired antimicrobial phenotype and genome data to identify novel AMR determinants. Finally, we assessed the predictive performance of our ARDaP-implemented database, along with AMRFinderPlus and ResFinder, for both the Global Dataset and an additional Validation Dataset of 89 isolates. Our AMR database of 754 chromosomal variants includes 79 newly discovered variants associated with AMR phenotype/s, and 246 variants in AMR-encoding loci that do not confer AMR. Interrogation of the Global Dataset with our AMR database achieved genotype-phenotype concordance (as measured by balanced accuracy [bACC]) of 86%, compared with bACCs of 57% with AMRFinderPlus and 63% with ResFinder. For the Validation Dataset, bACCs of 80%, 54%, and 53%, respectively, were attained. Our pipeline provides the most accurate tool yet for AMR prediction in P. aeruginosa . Implementing our ARDaP-compatible AMR determinant database for routine AMR prediction from P. aeruginosa (meta)genomes will vastly improve AMR identification and treatment modalities, addressing a critical facet in combatting this treatment-refractory pathogen. This work was supported by Advance Queensland (AQIRF0362018 and AQRF13016-17RD2), the Wishlist Sunshine Coast Health Foundation (2019-14), and the National Health and Medical Research Council (GNT455919). The funders had no role in study design, data acquisition, analysis, interpretation, writing, or manuscript submission. Several bioinformatic tools have been developed with the purpose of facilitating rapid and accurate AMR prediction from microbial (meta)genomic data, particularly for superbugs. To this end, we searched PubMed on July 29, 2022 using ((((“pseudomonas aeruginosa”) AND (“antimicrobial resistance”)) AND (“genome”)) AND (“predict*”)) NOT (review) for studies published in all languages. Twenty-four results were obtained, of which 20 described the use of one or more bioinformatic methods to predict AMR determinants (8 x ResFinder, 6 x CARD, 4 x comparative genomics, 4 x machine learning, 2 x transcriptomics, 1 x OpGen internal bioinformatic pipeline). Only 1 study created an AMR determinant database, which demonstrated limited ability to predict AMR across a dataset of 100 P. aeruginosa isolates. No study has yet created a publicly available, open-source tool and associated AMR determinant database for widespread use. Our ARDaP-compatible AMR determinant database, which was built from the largest-ever paired P. aeruginosa AMR phenotype-genome dataset to date ( n =1480 isolates), provides a crucial advancement in the way we use WGS data to rapidly and accurately predict AMR in P. aeruginosa . Using ARDaP, systematic analysis of large volumes of WGS data can be used to develop a more detailed understanding of the specific genetic variants that underpin AMR. AMR prediction tools will play an essential role in the battle against superbugs as they provide precise information on the genetic basis of a pathogen’s resistance profile – information that can be used to inform timely antimicrobial-treatment shifts, reduce the reliance on broad-spectrum antibiotics, and ultimately improve patient outcomes and slow the spread of AMR. Our study demonstrates that predicting AMR phenotype from genotype in P. aeruginosa can rival the performance of phenotypic AMR sensitivity testing such as the VITEK ® 2, which is routinely used in clinical microbiology laboratories worldwide. The rapid detection of emerging AMR and multidrug resistance, and subsequent implementation of personalised treatment regimens, serves as our best chance to extend antibiotic efficacy amidst the AMR crisis.
Publisher: Springer Science and Business Media LLC
Date: 25-02-2016
DOI: 10.1038/NCOMMS10688
Abstract: Chlamydia trachomatis causes sexually transmitted infections and the blinding disease trachoma. Current data on C. trachomatis phylogeny show that there is only a single trachoma-causing clade, which is distinct from the lineages causing urogenital tract (UGT) and lymphogranuloma venerum diseases. Here we report the whole-genome sequences of ocular C. trachomatis isolates obtained from young children with clinical signs of trachoma in a trachoma endemic region of northern Australia. The isolates form two lineages that fall outside the classical trachoma lineage, instead being placed within UGT clades of the C. trachomatis phylogenetic tree. The Australian trachoma isolates appear to be recombinants with UGT C. trachomatis genome backbones, in which loci that encode immunodominant surface proteins ( ompA and pmpEFGH ) have been replaced by those characteristic of classical ocular isolates. This suggests that ocular tropism and association with trachoma are functionally associated with some sequence variants of ompA and pmpEFGH .
Publisher: American Society for Microbiology
Date: 2015
DOI: 10.1128/JCM.02560-14
Abstract: Twelve Burkholderia pseudomallei isolates collected over a 32-month period from a patient with chronic melioidosis demonstrated identical multilocus sequence types (STs). However, whole-genome sequencing suggests a polyclonal infection. This study is the first to report a mixed infection with the same ST.
Publisher: Springer Science and Business Media LLC
Date: 07-07-2017
Publisher: Elsevier BV
Date: 09-2018
Publisher: Cold Spring Harbor Laboratory
Date: 15-07-2019
DOI: 10.1101/702985
Abstract: Stenotrophomonas maltophilia is emerging as an important cause of disease in nosocomial and community-acquired settings, including bloodstream, wound and catheter-associated infections. Cystic fibrosis airways also provide optimal growth conditions for various opportunistic pathogens with high antibiotic tolerance, including S. maltophilia . Currently, there is no rapid, cost-effective, and accurate molecular method for detecting this potentially life-threatening pathogen, particularly in polymicrobial specimens, suggesting that its true prevalence may be underestimated. Here, we used large-scale comparative genomics to identify a specific genetic target for S. maltophilia , with subsequent development and validation of a real-time PCR assay for its detection. Analysis of 165 Stenotrophomonas spp. genomes identified a 4kb region specific to S. maltophilia , which was targeted for Black Hole Quencher assay design. Our assay yielded the positive detection of 89 of 89 (100%) clinical S. maltophilia strains, and no lification of 23 non- S. maltophilia clinical isolates. S. maltophilia was detected in 10/16 CF sputa, demonstrating the utility for direct detection in respiratory specimens. The assay demonstrated good sensitivity, with limits of detection and quantitation on pure culture of ~10 and ~100 genome equivalents, respectively. Our assay provides a highly specific, sensitive, and cost-effective method for the accurate identification of S. maltophilia , and will improve the diagnosis and treatment of this under-recognized pathogen by enabling its accurate and rapid detection from polymicrobial clinical and environmental s les.
Publisher: American Society for Microbiology
Date: 10-2019
DOI: 10.1128/IAI.00528-19
Abstract: Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes.
Publisher: Public Library of Science (PLoS)
Date: 18-05-2012
Publisher: Microbiology Society
Date: 07-2020
Abstract: Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to lify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.
Publisher: Oxford University Press (OUP)
Date: 06-2019
DOI: 10.1093/OFID/OFZ263
Abstract: Community-onset bacteremic Acinetobacter baumannii pneumonia recurred in 3 of 30 (10%) patients followed prospectively, all with ongoing hazardous alcohol intake, 3–56 months after initial pneumonia. Paired isolates underwent whole-genome sequencing. Phylogenetic analysis showed that recurrence strains were all distinct from preceding strains, indicating reinfection in susceptible in iduals rather than relapse.
Publisher: American Society for Microbiology
Date: 30-04-2015
Abstract: We report here five improved high-quality draft genomes of Burkholderia pseudomallei isolated from Australian cystic fibrosis (CF) patients. This pathogen is rarely seen in CF patients. These genomes will be used to better understand chronic carriage of B. pseudomallei in the CF lung and the within-host evolution of longitudinal isolates from these patients.
Publisher: Elsevier BV
Date: 2020
Publisher: American Society for Microbiology
Date: 30-08-2013
Abstract: Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR , an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host. IMPORTANCE Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.
Publisher: American Society for Microbiology
Date: 26-02-2015
Abstract: Here, we present the draft genome sequences of 80 isolates of Burkholderia pseudomallei . The isolates represent clinical cases of melioidosis and environmental isolates from regions in Australia and Papua New Guinea where B. pseudomallei is endemic. The genomes provide further context for the ersity of the pathogen.
Publisher: Springer Science and Business Media LLC
Date: 16-10-2018
Publisher: Cold Spring Harbor Laboratory
Date: 15-10-2019
DOI: 10.1101/804344
Abstract: Human-to-human transmission of the melioidosis bacterium, Burkholderia pseudomallei , is exceedingly rare, with only a handful of suspected cases documented to date. Here, we used whole-genome sequencing (WGS) to characterise one such unusual B. pseudomallei transmission event, which occurred between a breastfeeding mother with mastitis and her child. Two strains corresponding to multilocus sequence types (STs) 259 and 261 were identified in the mother’s sputum from both the primary culture sweep and in purified colonies, confirming an unusual polyclonal infection in this patient. In contrast, primary culture sweeps of the mother’s breast milk and the child’s cerebrospinal fluid and blood s les contained only ST-259, indicating monoclonal transmission to the child. Analysis of purified ST-259 isolates showed no genetic variation between mother and baby isolates, providing the strongest possible evidence of B. pseudomallei transmission, probably via breastfeeding. Next, phylogenomic analysis of all isolates, including the mother’s mixed ST-259/261 sputum s le was performed to investigate the effects of mixtures on phylogenetic inference. Inclusion of this mixture caused a dramatic reduction in the number of informative SNPs, resulting in branch collapse of ST-259 and ST-261 isolates, and several instances of incorrect topology in a global B. pseudomallei phylogeny, resulting in phylogenetic incongruence. Although phylogenomics can provide clues about the presence of mixtures within WGS datasets, our results demonstrate that this methodology can lead to phylogenetic misinterpretation if mixed genomes are not correctly identified and omitted. Using current bioinformatic tools, we demonstrate a robust method for bacterial mixture identification and strain parsing that avoids these pitfalls. Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of high mortality. B. pseudomallei infections occur almost exclusively through contact with contaminated soil and water. Using whole-genome sequencing (WGS), we investigated a rare case of suspected B. pseudomallei transmission from mother to child. The mother’s sputum, breast milk and the baby’s blood and cerebrospinal fluid (CSF) specimens were collected, and DNA was extracted from both pure colonies and primary culture sweeps to capture potential strain mixtures. In-depth analysis of genetic variants identified two strains in the mother’s sputum belonging to multilocus sequence types ST-259 and ST-261, whereas the child was infected with only ST-259. Comparative genomics revealed no genetic differences between mother and child ST-259 isolates, providing the strongest possible evidence of transmission to the child via breast milk. The sputum strain mixture was subsequently used to develop a bioinformatic method for identification and quantification of mixtures from WGS data. Using this method, we found ST-259 and ST-261 at an 87%:13% ratio, respectively. Finally, we demonstrate the negative impact that even a single strain mixture event can have on both within-ST and global phylogenomic inferences. Our findings highlight the need for bioinformatic quality control to avoid unintended consequences of phylogenomic incongruence and branch collapse. Whole-genome sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) and GenBank under BioProject accession number PRJNA559002 . The GenBank accession number for MSHR0643 assembly is VXLH00000000.1. The SRA accession numbers for all raw sequence data are listed in Table 1. All sequencing data generated as part of this study can be found under the NCBI BioProject PRJNA559002 with accession numbers listed in Table 1.
Publisher: Public Library of Science (PLoS)
Date: 05-11-2018
Publisher: Springer Science and Business Media LLC
Date: 08-09-2014
Publisher: American Society for Microbiology
Date: 08-11-2017
Abstract: The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus co-trimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to co-trimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM , encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT , bpeS , and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Co-trimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium. IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium’s resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA , but not folP , and folM , a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.
Publisher: Microbiology Society
Date: 11-07-2016
Publisher: American Society for Microbiology
Date: 06-2014
DOI: 10.1128/AEM.00128-14
Abstract: Burkholderia pseudomallei is a Gram-negative soil bacillus that is the etiological agent of melioidosis and a biothreat agent. Little is known about the biogeography of this bacterium in Australia, despite its hyperendemicity in the northern region of this continent. The population structure of 953 Australian B. pseudomallei strains representing 779 and 174 isolates of clinical and environmental origins, respectively, was analyzed using multilocus sequence typing (MLST). Bayesian population structure and network SplitsTree analyses were performed on concatenated MLST loci, and sequence type (ST) ersity and evenness were examined using Simpson's and Pielou's indices and a multivariate dissimilarity matrix. Bayesian analysis found two B. pseudomallei populations in Australia that were geographically distinct isolates from the Northern Territory were grouped mainly into the first population, whereas the majority of isolates from Queensland were grouped in a second population. Differences in ST evenness were observed between s ling areas, confirming that B. pseudomallei is widespread and established across northern Australia, with a large number of fragmented habitats. ST analysis showed that B. pseudomallei populations ersified as the s ling area increased. This observation was in contrast to smaller s ling areas where a few STs predominated, suggesting that B. pseudomallei populations are ecologically established and not frequently dispersed. Interestingly, there was no identifiable ST bias between clinical and environmental isolates, suggesting the potential for all culturable B. pseudomallei isolates to cause disease. Our findings have important implications for understanding the ecology of B. pseudomallei in Australia and for potential source attribution of this bacterium in the event of unexpected cases of melioidosis.
Publisher: Cold Spring Harbor Laboratory
Date: 31-07-2019
DOI: 10.1101/720607
Abstract: Antimicrobial resistance (AMR) poses a major threat to human health. Whole-genome sequencing holds great potential for AMR identification however, there remain major gaps in comprehensively detecting AMR across the spectrum of AMR-conferring determinants and pathogens. Using 16 wild-type Burkholderia pseudomallei and 25 with acquired AMR, we first assessed the performance of existing AMR software (ARIBA and CARD) for detecting clinically relevant AMR in this pathogen. B. pseudomallei was chosen due to limited treatment options, high fatality rate, and AMR caused exclusively by chromosomal mutation (i.e. single-nucleotide polymorphisms [SNPs], insertions-deletions [indels], copy-number variations [CNVs], and functional gene loss). Due to poor performance with existing tools, we developed ARDaP ( A ntimicrobial R esistance D etection a nd P rediction) to identify the spectrum of AMR-conferring determinants in B. pseudomallei . CARD failed to identify any clinically-relevant AMR in B. pseudomallei , ARIBA cannot differentiate AMR determinants from natural genetic variation, and neither CARD or ARIBA can identify CNV or gene loss determinants. In contrast, ARDaP accurately detected all SNP, indel, CNV, and gene loss AMR determinants described in B. pseudomallei ( n ≈50). Additionally, ARDaP accurately predicted three previously undescribed determinants. In mixed strain data, ARDaP identified AMR to as low as ~5% allelic frequency. We demonstrate that existing AMR software are inadequate for comprehensive AMR detection ARDaP overcomes the shortcomings of existing tools. Further, ARDaP enables AMR prediction from mixed sequence data down to 5% allelic frequency. ARDaP databases can be constructed for any microbial species of interest for comprehensive AMR detection.
Publisher: American Society for Microbiology
Date: 02-2014
DOI: 10.1128/JCM.02239-13
Abstract: The Darwin Prospective Melioidosis Study has documented 785 melioidosis cases over 23 years. Recurrent melioidosis occurred in 39/679 (5.7%) patients surviving initial infection 29 patients suffered relapse of the original infection, and 10 presented with a new Burkholderia pseudomallei infection. With improved therapy, relapse has become rare in recent years.
Publisher: Elsevier BV
Date: 07-2010
Publisher: American Society for Microbiology
Date: 24-08-2020
DOI: 10.1128/JCM.00730-20
Abstract: The Elizabethkingia genus has gained global attention in recent years as containing sporadic, worldwide, nosocomial pathogens. Elizabethkingia spp. are intrinsically multidrug resistant, primarily infect immunocompromised in iduals, and are associated with high mortality (∼20 to 40%). As yet, gaps remain in our understanding of transmission, global strain relatedness, antimicrobial resistance, and effective therapy. Over a 16-year period, 22 clinical and 6 hospital environmental isolates were collected from Queensland, Australia.
Publisher: American Society for Microbiology
Date: 06-04-2017
Abstract: We report here paired isogenic Burkholderia pseudomallei genomes obtained from three patients receiving intravenous meropenem for melioidosis treatment, with post-meropenem isolates developing decreased susceptibility. Two genomes were finished, and four were drafted to improved high-quality standard. These genomes will be used to identify meropenem resistance mechanisms in B. pseudomallei .
Publisher: American Society for Microbiology
Date: 06-2017
DOI: 10.1128/AAC.00268-17
Abstract: The soil-dwelling bacterium Burkholderia pseudomallei is the causative agent of the potentially fatal disease melioidosis. The lack of a vaccine toward B. pseudomallei means that melioidosis treatment relies on prolonged antibiotic therapy, which can last up to 6 months in duration or longer. Due to intrinsic resistance, few antibiotics are effective against B. pseudomallei . The lengthy treatment regimen required increases the likelihood of resistance development, with subsequent potentially fatal relapse. Doxycycline (DOX) has historically played an important role in the eradication phase of melioidosis treatment. Both primary and acquired DOX resistances have been documented in B. pseudomallei however, the molecular mechanisms underpinning DOX resistance have remained elusive. Here, we identify and functionally characterize the molecular mechanisms conferring acquired DOX resistance in an isogenic B. pseudomallei pair. Two synergistic mechanisms were identified. The first mutation occurred in a putative S -adenosyl- l -methionine-dependent methyltransferase (encoded by BPSL3085 ), which we propose leads to altered ribosomal methylation, thereby decreasing DOX binding efficiency. The second mutation altered the function of the efflux pump repressor gene, amrR , resulting in increased expression of the resistance-nodulation- ision efflux pump, AmrAB-OprA. Our findings highlight the erse mechanisms by which B. pseudomallei can become resistant to antibiotics used in melioidosis therapy and the need for resistance monitoring during treatment regimens, especially in patients with prolonged or recrudesced positive cultures for B. pseudomallei .
Start Date: 2017
End Date: 2020
Funder: Advance Queensland
View Funded ActivityStart Date: 2014
End Date: 2016
Funder: Channel 7 Children's Research Foundation
View Funded ActivityStart Date: 2016
End Date: 2020
Funder: National Natural Sciences Foundation China (NSFC)
View Funded ActivityStart Date: 2016
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2013
End Date: 2015
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2016
End Date: 2021
Funder: National Health and Medical Research Council
View Funded Activity