Biology subjects

Williamson, D. A.

Publications and source records attributed to Williamson, D. A..

17 recordsLinked to original sources

bioRxiv2024

Dynamics of antimicrobial resistance and virulence of Salmonella enterica serovar Dublin

Salmonella Dublin is a host-adapted, invasive non-typhoidal Salmonella (iNTS) serovar that causes bloodstream infections in humans and demonstrates increasing prevalence of antimicrobial resistance (AMR). Using a global dataset of 1,303 genomes, coupled with in vitro assays, we examined the evolutionary, resistance, and virulence characteristics of S. Dublin. Our analysis revealed strong geographic associations between AMR profiles and plasmid types, with highly resistant isolates confined predominantly to North America, linked to IncC plasmids co-encoding AMR and heavy metal resistance. By contrast, Australian isolates were largely antimicrobial-susceptible, reflecting differing AMR pressures. We identified two phylogenetically distinct Australian lineages, ST10 and ST74, with a small number of ST10 isolates harbouring a novel hybrid plasmid encoding both AMR and mercuric resistance. Whereas the ST10 lineage remains globally dominant, the ST74 lineage was less prevalent. ST74 exhibited unique genomic features including a larger pan genome compared to ST10 and the absence of key virulence loci including SPI-19 which encodes a type VI secretion system (T6SS). Despite these genomic differences, the ST74 lineage displayed enhanced intracellular replication in human macrophages and induced less pro-inflammatory responses compared with ST10, suggesting alternative virulence strategies that may support systemic dissemination of ST74. The Vi antigen was absent in all ST10 and ST74 genomes, highlighting challenges for serotyping and vaccine development, and has implications for current diagnostic and control strategies for S. Dublin infections. Collectively, this study represents the most comprehensive investigation of S. Dublin to date and importantly, has revealed distinct adaptations of two genotypes within the same serovar, leading to different epidemiological success. The regional emergence and evolution of distinct S. Dublin lineages highlights the need to understand the divergence of intra-serovar virulence mechanisms which may impact the development of effective control measures against this important global pathogen.

microbiology↗

bioRxiv2024

Global diversity and evolution of Salmonella Panama, an understudied serovar causing gastrointestinal and invasive disease worldwide: a genomic epidemiology study

BackgroundNontyphoidal Salmonella (NTS) is a globally important bacterial pathogen, typically associated with foodborne gastrointestinal infection. Some NTS serovars can also colonise normally sterile sites in humans to cause invasive NTS (iNTS) disease. One understudied Salmonella enterica serovar which is responsible for a significant number of cases of iNTS disease is Panama. Despite global dissemination, numerous outbreaks, and a reported association with iNTS disease, S. enterica serovar Panama (S. Panama) has not been investigated in detail. MethodsUsing combined epidemiological and whole genome sequencing data we analysed 836 S. Panama genomes derived from historical collections, national surveillance datasets, and publicly available data. The collection represents all inhabited continents and includes isolates collected between 1931 and 2019. Maximum likelihood and Bayesian phylodynamic approaches were used to determine population structure & evolutionary history, and to infer geo-temporal dissemination. A combination of different bioinformatic approaches utilising short-read and long-read data were used to characterise geographic and clade-specific trends in antimicrobial resistance (AMR), and genetic markers for invasiveness. FindingsWe identified the presence of multiple geographically linked S. Panama clades, and regional trends in antimicrobial resistance profiles. Most isolates were pan-susceptible to antibiotics and belonged to clades circulating in the United States of America, Latin America, and the Caribbean. Multidrug resistant (MDR) isolates in our collection belonged to two phylogenetic clades circulating in Europe and Asia/Oceania, which exhibited the highest invasiveness indices based on the conservation of 196 extra-intestinal predictor genes. InterpretationThis first large-scale phylogenetic analysis of S. Panama revealed important information about population structure, AMR, global ecology, and genetic markers of invasiveness of the identified genomic subtypes. Our findings provide an important baseline for understanding S. Panama infection in the future. The presence of MDR clades with an elevated invasiveness index should be monitored by ongoing surveillance as such clades may pose an increased public health risk. FundingThe project was supported by both a Global Challenges Research Fund (GCRF) data & resources grant BBS/OS/GC/000009D and the BBSRC Core Capability Grant to the Earlham Institute BB/CCG1720/1. JCDH received a Wellcome Trust Investigator award (grant number 222528/Z/21/Z). CVP was supported by the John Lennon Memorial Scholarship from the University of Liverpool and a Fee Bursary Award from the Institute of Integrative Biology at the University of Liverpool. FXW was awarded the Institut Pasteur; Sante publique France; the Fondation Le Roch-Les Mousquetaires; and the French governments Investissement dAvenir programme, Laboratoire dExcellence "Integrative Biology of Emerging Infectious Diseases" (grant number ANR-10-LABX-62-IBEID). MDS was supported by a BBSRC grant (KSB: BB/V009184/1). RJBengtsson was supported by an MRC grant (KSB: MR/R020787/1). RJBennett was supported by a BBSRC DTP (BB/M011186/1). DJI was supported by a National Health and Medical Research Council (NHMRC) Emerging Leadership Fellowship (GNT1195210). BPH was supported by NHMRC Leadership Fellowship (GNT1196103). KSB and EVR are affiliated to the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Gastrointestinal Infections at University of Liverpool in partnership with the United Kingdom Health Security Agency, in collaboration with University of Warwick. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health and Social Care or UKHSA. MAC is affiliated to the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) (NIHR200892) in Genomics and Enabling Data at University of Warwick in partnership with the UK Health Security Agency (UKHSA), in collaboration with Universities of Cambridge and Oxford. MAC is based at UKHSA. The views expressed are those of the author(s) and not necessarily those of the NIHR, the Department of Health and Social Care or the UK Health Security Agency. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. No payment was received by any pharmaceutical company or other agency to write this article. The authors were not precluded from accessing data in the study, and accept responsibility to submit for publication. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSalmonella Panama has consistently been reported as a frequently isolated Salmonella serovar in national surveillance datasets, causing both sporadic cases and larger outbreaks. However, this picture has been masked due to most of the focus being placed on the top two serovars associated with nontyphoidal Salmonella (NTS) and invasive NTS disease: Typhimurium and Enteritidis. Previous works on S. Panama have determined transmission to include human faeces and breast milk, as well as non-human sources such as environmental reservoirs and animals (reptiles and pigs used for food). In contrast to most of the Salmonella serovars causing gastroenteritis, S. Panama has also been associated with a range of systemic infections including septicaemia, meningitis, and osteomyelitis, mostly affecting infants. The patterns of antimicrobial resistance of this pathogen were not well known, with studies reporting a mixture of different antimicrobial resistance profiles. Added value of this studyHere we conducted a large-scale study of 836 globally relevant S. Panama isolates to understand global population structure and disease ecology. The S. Panama genomes used in this study were sourced from a combination of historical collections (including the first ever isolated strain of this serovar dating from 1931), national surveillance datasets, and publicly available data. Some of these isolates had been linked to travel, allowing an inferred location for understanding population structure. Overall, this assembled S. Panama collection spanned a range of 88 years and covered 45 countries and regions of all inhabited continents. We applied bacterial phylodynamic approaches to determine population structure, evolutionary history, and clade-specific trends of invasiveness and antimicrobial resistance correlated with geo-temporal parameters. Implications of all the available evidenceThis study revealed the population structure and global ecology of S. Panama, indicating that multidrug resistant S. Panama circulates in European and Asian regions, and presents an increased number of genetic markers for extra-intestinal invasiveness. The transmission and expansion of antimicrobial-resistant S. Panama strains presented in this study highlight the significance of supporting control and monitoring efforts from an international perspective.

genomics↗

bioRxiv2023

The inter-continental population dynamics of Neisseria gonorrhoeae

Neisseria gonorrhoeae is a globally distributed sexually transmitted bacterial pathogen. Recent studies have revealed that its evolution has been shaped by antibiotic use, while molecular surveillance efforts have demonstrated large changes in lineage composition over relatively short time-spans. However, the global population dynamics of N. gonorrhoeae remain unsatisfyingly characterized. To reconstruct recent large-scale population dynamics, we generated a dated phylogeny from 9,732 N. gonorrhoeae genomes and found the effective population size of the species to have expanded gradually over the past two centuries. While the effective population size of clades with reduced susceptibility to extended-spectrum cephalosporins started declining around 2010, a major clade containing a mosaic mtr operon associated with cephalosporin susceptibility and decreased azithromycin did not display any reduction in population size. Using ancestral trait reconstruction analyses, we delineated transmission lineages, defined as groups of sequences in which all the samples can be traced back to the same import event to a given location. Import, export and local transmission dynamics across two densely sampled locations (Norway and Victoria, Australia) were investigated in detail. Norway exhibited substantially higher rates of strain import and export compared to Victoria, where incidence was to a larger extent fuelled by locally transmitted lineages. Taken together, our work highlights the power of large-scale phylogenomic analyses to uncover the complex dynamics of lineage transmission in N. gonorrhoeae.

microbiology↗

bioRxiv2023

A transmission chain linking Mycobacterium ulcerans with Aedes notoscriptus mosquitoes, possums and human Buruli ulcer cases in southeastern Australia

In temperate southeastern Australia over the past two decades there has been a marked progressive increase in human cases of Buruli ulcer, an infection of subcutaneous tissue caused by Mycobacterium ulcerans. Native possums are the major local environmental reservoir of M. ulcerans as they not only develop Buruli lesions but they also shed M. ulcerans in their excreta. However the way humans acquire M. ulcerans from possums has not been determined. Previous case-control studies, insect field surveys and vector competence studies have suggested a role for mosquitoes in M. ulcerans transmission between possums and humans. To explore these links we conducted an extensive, 4-month structured mosquito field survey and four ad hoc field surveys across an area of 350km2 on the Mornington Peninsula, an area endemic for Buruli ulcer to the south of the major metropolitan city of Melbourne. We then compared spatial and temporal patterns of M. ulcerans-positive mosquito occurrence with M. ulcerans-positive possums (established by previous possum excreta surveys) and human Buruli ulcer cases across the region. We used metabarcoding to assess mosquito blood-feeding host preference and to reconstruct M. ulcerans genomes from positive mosquitoes to test epidemiological inferences. We collected 66,325 mosquitoes spanning 26 different species from 180 repeatedly sampled traps over a 4-month period. Culex molestus and Aedes notoscriptus were the dominant species (42% and 35% of trapped mosquitoes, respectively). PCR screening 25% of trapped mosquitoes revealed a significant association between M. ulcerans and Ae. notoscriptus (p<0.0001) with a maximum likelihood estimate (MLE) of 5.88 M. ulcerans positive mosquitoes per 1,000 tested. Using spatial scanning statistics, we also observed significant overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and human Buruli ulcer cases. Metabarcoding analyses of blood-fed Ae. notoscriptus showed individual mosquitoes had fed both on humans and native possums. Enrichment genome sequencing from PCR-positive mosquitoes confirmed shared M. ulcerans genome single-nucleotide polymorphism (SNP) profiles between mosquitoes, possum excreta and clinical human isolates within the same regions. These findings indicate that Ae. notoscriptus likely transmit M. ulcerans in southeastern Australia and highlight mosquito control as a plausible means to control the Buruli ulcer epidemic in our region.

microbiology↗

bioRxiv2022

Rapid recall and de novo T cell responses during SARS-CoV-2 breakthrough infection

While the protective role of neutralising antibodies against COVID-19 is well-established, questions remain about the relative importance of cellular immunity. Using 6 pMHC-multimers in a cohort with early and frequent sampling we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post-symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable levels of expansion. Strikingly, high levels of SARS-CoV-2-specific CD8+ T cell activation at baseline and peak were strongly correlated with reduced peak SARS-CoV-2 RNA levels in nasal swabs and accelerated clearance of virus. Our study demonstrates rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

immunology↗

bioRxiv2022

Tracking the horizontal transfer of plasmids in Shigella sonnei and Shigella flexneri using phylogenetics

The silent pandemic of antimicrobial resistance (AMR) represents a significant global public health threat. AMR genes in bacteria are often carried on mobile elements, such as plasmids. The horizontal movement of plasmids allows AMR genes and resistance to key therapeutics to disseminate in a population. However, the quantification of the movement of plasmids remains challenging with existing computational approaches. Here, we introduce a novel method that allows us to reconstruct and quantify the movement of plasmids in bacterial populations over time. To do so, we model chromosomal and plasmid DNA co-evolution using a joint coalescent and plasmid transfer process in a Bayesian phylogenetic network approach. This approach reconstructs differences in the evolutionary history of plasmids and chromosomes to reconstruct instances where plasmids likely move between bacterial lineages while accounting for parameter uncertainty. We apply this new approach to a five-year dataset of Shigella, exploring the plasmid transfer rates of five different plasmids with different AMR and virulence profiles. In doing so, we reconstruct the co-evolution of the large Shigella virulence plasmid with the chromosome DNA. We quantify higher plasmid transfer rates of three small plasmids that move between lineages of Shigella sonnei. Finally, we determine the recent dissemination of a multidrug-resistant plasmid between S. sonnei and S. flexneri lineages in multiple independent events and through steady growth in prevalence since 2010. This approach has a strong potential to improve our understanding of the evolutionary dynamics of AMR-carrying plasmids as they are introduced, circulate, and are maintained in bacterial populations.

evolutionary biology↗

bioRxiv2020

Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection

SARS-CoV-2 uses subgenomic (sg)RNA to produce viral proteins for replication and immune evasion. We applied long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA was upregulated earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of ORF1ab containing nsp1 joined to ORF10 and 3UTR was upregulated at 48 hours post infection in human cell lines. We identified double-junction sgRNA containing both TRS-dependent and independent junctions. We found multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA, and that sgRNA modifications are stable across transcript clusters, host cells and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle. Our results are available via an interactive web-app at http://coinlab.mdhs.unimelb.edu.au/.

microbiology↗

bioRxiv2020

Global population structure and genotyping framework for genomic surveillance of the major dysentery pathogen, Shigella sonnei

Shigella sonnei is the most common agent of shigellosis in high-income countries, and causes a significant disease burden in low- and middle-income countries. Antimicrobial resistance is increasingly common in all settings. Whole genome sequencing (WGS) is increasingly utilised for S. sonnei outbreak investigation and surveillance, but comparison of data between studies and labs is challenging. Here, we present a genomic framework and genotyping scheme for S. sonnei to efficiently identify genotype and resistance determinants from WGS data. The scheme is implemented in the software package Mykrobe and tested on thousands of genomes. Applying this approach to analyse >4,000 S. sonnei isolates sequenced in public health labs in three countries identified several common genotypes associated with increased rates of ciprofloxacin resistance and azithromycin resistance, confirming intercontinental spread of highly-resistant S. sonnei clones and demonstrating the genomic framework can facilitate monitoring of the emergence and spread of resistant clones at local and global scales.

microbiology↗

bioRxiv2020

Systematic analysis of key parameters for genomics-based real-time detection and tracking of multidrug-resistant bacteria

BackgroundPairwise single nucleotide polymorphisms (SNPs) are a cornerstone for genomic approaches to multidrug-resistant organisms (MDROs) transmission inference in hospitals. However, the impact of key analysis parameters on these inferences has not been systematically analysed. MethodsWe conducted a multi-hospital 15-month prospective study, sequencing 1537 MDRO genomes for comparison; methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae. We systematically assessed the impact of sample and reference genome diversity, masking of prophage and regions of recombination, cumulative genome analysis compared to a three-month sliding-window, and the comparative effects each of these had when applying a SNP threshold for inferring likely transmission ([≤]15 SNPs for S. aureus, [≤]25 for other species). FindingsAcross the species, using a reference genome of the same sequence type provided a greater degree of pairwise SNP resolution, compared to species and outgroup-reference alignments that typically resulted in inflated SNP distances and the possibility of missed transmission events. Omitting prophage regions had minimal impacts, however, omitting recombination regions a highly variable effect, often inflating the number of closely related pairs. Estimating pairwise SNP distances was more consistent using a sliding-window than a cumulative approach. InterpretationThe use of a closely-related reference genome, without masking of prophage or recombination regions, and a sliding-window for isolate inclusion is best for accurate and consistent MDRO transmission inference. The increased stability and resolution provided by these approaches means SNP thresholds for putative transmission inference can be more reliably applied among diverse MDROs. FundingThis work was supported by the Melbourne Genomics Health Alliance (funded by the State Government of Victoria, Department of Health and Human Services, and the ten member organizations); an National Health and Medical Research Council (Australia) Partnership grant (GNT1149991) and individual grants from National Health and Medical Research Council (Australia) to NLS (GNT1093468), JCK (GNT1008549) and BPH (GNT1105905).

genomics↗

bioRxiv2020

Validation of a Single-step, Single-tube Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2 RNA

2.IntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 L of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65{degrees}C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals. ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD {+/-}7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19. 3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2020

Cellular and structural basis of synthesis of the unique intermediate dehydro-F420-0 in mycobacteria

F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo, substrate, and product bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic pre-complex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.

biochemistry↗

bioRxiv2020

Targeted surveillance strategies for efficient detection of novel antibiotic resistance variants

Genotype-based diagnostics for antibiotic resistance represent a promising alternative to empiric therapy, reducing inappropriate and ineffective antibiotic use. However, because such assays infer resistance phenotypes based on the presence or absence of known genetic markers, their utility will wane in response to the emergence of novel resistance. Maintenance of these diagnostics will therefore require surveillance designed to ensure early detection of novel resistance variants, but efficient strategies to do so remain to be defined. Here, we evaluate the efficiency of targeted sampling approaches informed by patient and pathogen characteristics in detecting genetic variants associated with antibiotic resistance or diagnostic escape in Neisseria gonorrhoeae, focusing on this pathogen because of its high burden of disease, the imminent threat of treatment resistance, and the use and ongoing development of genotype-based diagnostics. We show that incorporating patient characteristics, such as demographics, geographic regions, or anatomical sites of isolate collection, into sampling approaches is not a reliable strategy for increasing variant detection efficiency. In contrast, sampling approaches informed by pathogen characteristics, such as genomic diversity and genomic background, are significantly more efficient than random sampling in identifying genetic variants associated with antibiotic resistance and diagnostic escape.

genomics↗

bioRxiv2020

Increased antibiotic susceptibility in Neisseria gonorrhoeae through adaptation to the cervical environment

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association on the largest collection of N. gonorrhoeae isolates to date (n=4852) to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical isolates relative to urethral isolates (odds ratio (OR) = 3.74, 95% CI [1.98-6.70]). In support of a model in which pump expression incurs a fitness cost in this niche, cervical isolates were also enriched relative to urethral isolates in LOF mutations in the mtrCDE activator mtrA (OR = 8.60, 95% CI [4.96-14.57]) and in farA, a subunit of the FarAB efflux pump (OR = 6.25, 95% CI [3.90-9.83]). In total, approximately 2 in 5 cervical isolates (42.6%) contained a LOF mutation in either the efflux pump components mtrC or farA or the activator mtrA. Our findings extend beyond N. gonorrhoeae to other Neisseria: mtrC LOF mutations are rare (<1%) in the primarily nasopharyngeal-colonizing N. meningitidis in a collection of 14,798 genomes but enriched in a heterosexual urethritis-associated lineage (8.6%, p = 9.90x10-5), indicating that efflux pump downregulation contributes broadly to the adaptation of pathogenic Neisseria to the female urogenital tract. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.

microbiology↗

bioRxiv2019

Complete microbial genomes for public health in Australia and Southwest Pacific

Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health lab activities. Here, we present complete genomes of pathogen strains of regional importance to the Southwest Pacific and Australia. These enrich the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health labs.\n\nAnnouncementWhole-genome sequence (WGS) data is increasingly important in public health microbiology (1-4). The data can be used to replicate many of the basic bacterial sub-typing approaches, as well as support epidemiological investigations, such as surveillance and outbreak investigation (5-7). The appeal of WGS data comes from the promise of a single workflow to process all microbial pathogens that can provide easily portable data that promotes deeper integration of surveillance and investigation efforts across jurisdictions. This promise is leading to a concerted effort to move microbial public health to a primarily genome-based workflow at numerous jurisdictions (8-10), including Australia (11).

genomics↗

bioRxiv2018

Remodelling of pSK1 Family Plasmids and Enhanced Chlorhexidine Tolerance in Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is a significant human pathogen whose evolution and adaptation has been shaped in part by mobile genetic elements (MGEs), facilitating global spread of extensive antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding MGEs is incomplete, in particular how changes in the structure of multidrug-resistant (MDR) plasmids may influence important staphylococcal phenotypes. Here, we undertook a population-and functional-genomics study of 212 methicillin-resistant S. aureus (MRSA) ST239 isolates collected over 32 years to explore the evolution of the pSK1 family of MDR plasmids, illustrating how these plasmids have co-evolved with and contributed to the successful adaptation of this persistent MRSA lineage. Using complete genomes and temporal phylogenomics we reconstructed the evolution of the pSK1 family lineage from its emergence in the late 1970s, with multiple structural variants arising. Plasmid maintenance and stability was linked to IS256- and IS257-mediated chromosomal integration and disruption of plasmid replication machinery. Overlaying genomic comparisons with phenotypic susceptibility data for gentamicin and chlorhexidine, it appeared that pSK1 has contributed to enhanced resistance in ST239 MRSA through two mechanisms: (i) acquisition of plasmid-borne resistance mechanisms increasing rates of gentamicin resistance and reduced chlorhexidine susceptibility, and (ii) changes in plasmid configuration linked with further enhancement of chlorhexidine tolerance. While the exact mechanism of enhanced tolerance remains elusive, this research has uncovered a clear evolutionary response of ST239 MRSA to chlorhexidine, one which may contribute to the ongoing persistence and adaptation of this lineage within healthcare institutions.

evolutionary biology↗

bioRxiv2017

Genome reconstruction and characterisation of extensively drug-resistant bacterial pathogens through direct metagenomic sequencing of human faeces

Whole-genome sequencing of microbial pathogens is revolutionising modern approaches to outbreaks of infectious diseases and is reliant upon organism culture. Culture-independent methods have shown promise in identifying pathogens, but high level reconstruction of microbial genomes from microbiologically complex samples for more in-depth analyses remains a challenge. Here, using metagenomic sequencing of a human faecal sample and analysis by tetranucleotide frequency profiling projected onto emergent self-organising maps, we were able to reconstruct the underlying populations of two extensively-drug resistant pathogens, Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae and vancomycin-resistant Enterococcus faecium. From these genomes, we were able to ascertain molecular typing results, such as MLST, and identify highly discriminatory mutations in the metagenome to distinguish closely related strains. These proof-of-principle results demonstrate the utility of clinical sample metagenomics to recover sequences of important drug-resistant bacteria and application of the approach in outbreak investigations, independent of the need to culture the organisms.

genomics↗

bioRxiv2017

A target-specific assay for rapid and quantitative detection of Mycobacterium chimaera DNA in environmental and clinical specimens

Mycobacterium chimaera is an opportunistic environmental mycobacterium, belonging to the Mycobacterium intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest world-wide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays, specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan qPCR assay for M. chimaera with a detection limit of 10 CFU in whole blood. In vitro screening against DNA extracted from 40 other mycobacteria and 22 bacterial species from 21 diverse genera confirmed in silico predicted specificity for M. chimaera. Screening 33 water samples from heater cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera.

microbiology↗