Microbiology & Immunology - Research Publications
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ItemDrug resistance and genetic profile of bacterial species associated with Buruli ulcer wound infections in two districts of Ghana(BMJ, 2017-02)Background: We identified secondary infection of Buruli ulcer (BU) wounds as a cause of healing delay. In order to contribute to the improvement of wound management and reduction of healing delay, we initiated a study to gain understanding of the possible routes of infection and also characterised the resistant profiles of Gram negative bacteria isolated from the wounds of patients attending two health facilities in Ghana. Methods: Staphylococcus aureus isolates were characterised by the spa gene, mecA and the Pantone Valentine Leukocidin (PVL) toxin followed by spa sequencing and whole genome sequencing of a subset of isolates. Phenotypic antibiotic susceptibility testing of Gram negative clinical isolates was performed and multidrug-resistant Pseudomonas aeruginosa identified. The Enterobacteriaceae were further investigated for ESBL and carbapenem production, and some resistance conferring genes were analysed by PCR. Results: Twenty-four isolates were identified as methicillin-resistant S. aureus (MRSA), and lukFS genes encoding PVL were identified in 67 isolates. Typing and sequencing of the spa gene from 91 isolates identified 29 different spa types with t355 (ST152), t186 (ST88), and t346 dominating. While many distinct strains were isolated from both health centres, genotype clustering was identified within centres pointing to possible health care-associated transmission. Phylogenomic analysis confirmed these clusters. Among the GNB, phenotype screening showed widespread resistance to ampicillin, chloramphenicol, ticarcillin-clavulanic acid, cefuroxime and sulphamethoxazole-trimethoprim. ESBL production was confirmed in 15 isolates phenotypically while 61.5% of screen-positive isolates harboured at least one ESBL-conferring gene. Carbapenem encoding genes were detected in 41% of the isolates. Conclusions: Our findings indicate that the health-care environment likely contributes to superinfection of BU wounds and calls for training in wound management and infection control techniques. The observed frequency of ESBL and carbapenem resistance indicates the need to set up surveillance networks and strictly enforce policies which guide the rational use of antibiotics.
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ItemThe changing landscape of vancomycin-resistant Enterococcus faecium in Australia: a population-level genomic study(OXFORD UNIV PRESS, 2018-12)BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, there has been a recent concerning increase in bacteraemia associated with the vanA genotype, prompting investigation into the genomic epidemiology of VREfm. METHODS: A population-level study of VREfm (10 November-9 December 2015) was conducted. A total of 321 VREfm isolates (from 286 patients) across Victoria State were collected and sequenced with Illumina NextSeq. SNPs were used to assess relatedness. STs and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data. Illumina reads from remaining isolates were then mapped to these assemblies to identify their probable vanA-harbouring plasmid. RESULTS: vanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2%, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm were more closely related within hospitals versus between them [core SNPs 10 (IQR 1-357) versus 356 (179-416), respectively], suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within versus between hospitals, due to widespread dissemination of ST796. Different vanA-harbouring plasmids were found across STs. With the exception of ST78 and ST796, Tn1546 transposons also varied. Phylogenetic analysis revealed Australian strains were often interspersed with those from other countries, suggesting ongoing cross-continental transmission. CONCLUSIONS: Emerging vanA-VREfm in Australia is polyclonal, indicating repeat introductions of vanA-VREfm into hospitals and subsequent dissemination. The close relationship to global strains reinforces the need for ongoing screening and control of VREfm in Australia and abroad.
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ItemBiosynthesis and Ether-Bridge Formation in Nargenicin Macrolides(WILEY-V C H VERLAG GMBH, 2019-03-18)The nargenicin family of antibiotics are macrolides containing a rare ether-bridged cis-decalin motif. Several of these compounds are highly active against multi-drug resistant organisms. Despite the identification of the first members of this family almost 40 years ago, the genetic basis for the production of these molecules and the enzyme responsible for formation of the oxa bridge, remain unknown. Here, the 85 kb nargenicin biosynthetic gene cluster was identified from a human pathogenic Nocardia arthritidis isolate and this locus is solely responsible for nargenicin production. Further investigation of this locus revealed a putative iron-α-ketoglutarate-dependent dioxygenase, which was found to be responsible for the formation of the ether bridge from the newly identified deoxygenated precursor, 8,13-deoxynargenicin. Uncovering the nargenicin biosynthetic locus provides a molecular basis for the rational bioengineering of these interesting antibiotic macrolides.
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ItemSystematic analysis of key parameters for genomics-based real-time detection and tracking of multidrug-resistant bacteria( 2020-09-25)Background: Pairwise single nucleotide polymorphisms (SNPs) are a cornerstone for genomic approaches to multidrug-resistant organisms (MDROs) transmission inference in hospitals. However, the impact of key analysis parameters on these inferences has not been systematically analysed. Methods: We conducted a multi-hospital 15-month prospective study, sequencing 1537 MDRO genomes for comparison; methicillin-resistant Staphylococcus aureus , vancomycin-resistant Enterococcus faecium , and extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae . We systematically assessed the impact of 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). Findings: Across the species, using a reference genome of the same sequence type provided a greater degree of pairwise SNP resolution, compared to species and outgroup-reference alignments that typically resulted in inflated SNP distances and the possibility of missed transmission events. Omitting prophage regions had minimal impacts, however, omitting recombination regions a highly variable effect, often inflating the number of closely related pairs. Estimating pairwise SNP distances was more consistent using a sliding-window than a cumulative approach. Interpretation: The use of a closely-related reference genome, without masking of prophage or recombination regions, and a sliding-window for isolate inclusion is best for accurate and consistent MDRO transmission inference. The increased stability and resolution provided by these approaches means SNP thresholds for putative transmission inference can be more reliably applied among diverse MDROs. Funding: This work was supported by the Melbourne Genomics Health Alliance (funded by the State Government of Victoria, Department of Health and Human Services, and the ten member organizations); an National Health and Medical Research Council (Australia) Partnership grant (GNT1149991) and individual grants from National Health and Medical Research Council (Australia) to NLS (GNT1093468), JCK (GNT1008549) and BPH (GNT1105905).
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ItemTracking the COVID-19 pandemic in Australia using genomics( 2020)BACKGROUND: Whole-genome sequencing of pathogens can improve resolution of outbreak clusters and define possible transmission networks. We applied high-throughput genome sequencing of SARS-CoV-2 to 75% of cases in the State of Victoria (population 6.24 million) in Australia. METHODS: Cases of SARS-CoV-2 infection were detected through active case finding and contact tracing. A dedicated SARS-CoV-2 multidisciplinary genomic response team was formed to enable rapid integration of epidemiological and genomic data. Phylodynamic analysis was performed to assess the putative impact of social restrictions. RESULTS: Between 25 January and 14 April 2020, 1,333 COVID-19 cases were reported in Victoria, with a peak in late March. After applying internal quality control parameters, 903 samples were included in genomic analyses. Sequenced samples from Australia were representative of the global diversity of SARS-CoV-2, consistent with epidemiological findings of multiple importations and limited onward transmission. In total, 76 distinct genomic clusters were identified; these included large clusters associated with social venues, healthcare facilities and cruise ships. Sequencing of sequential samples from 98 patients revealed minimal intra-patient SARS-CoV-2 genomic diversity. Phylodynamic modelling indicated a significant reduction in the effective viral reproductive number (Re) from 1.63 to 0.48 after the implementation of travel restrictions and population-level physical distancing. CONCLUSIONS: Our data provide a comprehensive framework for the use of SARS-CoV-2 genomics in public health responses. The application of genomics to rapidly identify SARS-CoV-2 transmission chains will become critically important as social restrictions ease globally. Public health responses to emergent cases must be swift, highly focused and effective.
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ItemComplete microbial genomes for public health in Australia and the Southwest Pacific(MICROBIOLOGY SOC, 2020-11)Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health laboratory activities. Here, we announce a BioProject (PRJNA556438) dedicated to sharing complete genomes chosen to represent a range of pathogenic bacteria with regional importance to Australia and the Southwest Pacific; enriching the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health microbiology laboratories. In this first step, we present 26 complete high-quality bacterial genomes. Additionally, we describe here a framework for reconstructing complete microbial genomes and highlight some of the challenges and considerations for accurate and reproducible genome reconstruction.
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ItemComprehensive Genomic Investigation of Adaptive Mutations Driving the Low-Level Oxacillin Resistance Phenotype in Staphylococcus aureus(AMER SOC MICROBIOLOGY, 2020-12-08)Antistaphylococcal penicillins such as oxacillin are the key antibiotics in the treatment of invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections; however, mec gene-independent resistance adaptation can cause treatment failure. Despite its clinical relevance, the basis of this phenomenon remains poorly understood. Here, we investigated the genomic adaptation to oxacillin at an unprecedented scale using a large collection of 503 clinical mec-negative isolates and 30 in vitro-adapted isolates from independent oxacillin exposures. By combining comparative genomics, evolutionary convergence, and genome-wide association analysis, we found 21 genetic loci associated with low-level oxacillin resistance, underscoring the polygenic nature of this phenotype. Evidence of adaptation was particularly strong for the c-di-AMP signal transduction pathways (gdpP and dacA) and in the clpXP chaperone-protease complex. The role of mutations in gdpP in conferring low-level oxacillin resistance was confirmed by allele-swapping experiments. We found that resistance to oxacillin emerges at high frequency in vitro (median, 2.9 × 10-6; interquartile range [IQR], 1.9 × 10-6 to 3.9 × 10-6), which is consistent with a recurrent minimum inhibitory concentration (MIC) increase across the global phylogeny of clinical isolates. Nevertheless, adaptation in clinical isolates appears sporadically, with no stably adapted lineages, suggesting a high fitness cost of resistance, confirmed by growth assessment of mutants in rich media. Our data provide a broader understanding of the emergence and dynamics of oxacillin resistance adaptation in S. aureus and a framework for future surveillance of this clinically important phenomenon.IMPORTANCE The majority of Staphylococcus aureus strains causing human disease are methicillin-susceptible (MSSA) and can be treated with antistaphylococcal penicillins (such as oxacillin). While acquisition of the mec gene represents the main resistance mechanism to oxacillin, S. aureus can acquire low-level resistance through adaptive mutations in other genes. In this study, we used genomic approaches to understand the basis of S. aureus adaption to oxacillin and its dynamic at the population level. By combining a genome analysis of clinical isolates from persistent MSSA infections, in vitro selection of oxacillin resistance, and genome-wide association analysis on a large collection of isolates, we identified 21 genes linked to secondary oxacillin resistance. Adaptive mutations in these genes were easy to select when S. aureus was exposed to oxacillin, but they also came at a substantial cost in terms of bacterial fitness, suggesting that this phenotype emerges preferentially in the setting of sustained antibiotic exposure.
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ItemGenomic Exploration of Within-Host Microevolution Reveals a Distinctive Molecular Signature of Persistent Staphylococcus aureus Bacteraemia(BMC, 2018-02-28)Background: Large-scale genomic studies of within-host evolution during Staphylococcus aureus bacteraemia (SAB) are needed to understanding bacterial adaptation underlying persistence and thus refining the role of genomics in management of SAB. However, available comparative genomic studies of sequential SAB isolates have tended to focus on selected cases of unusually prolonged bacteraemia, where secondary antimicrobial resistance has developed. To understand the bacterial genomic evolution during SAB more broadly, we applied whole genome sequencing to a large collection of sequential isolates obtained from patients with persistent or relapsing bacteraemia. Results: We show that, while adapation pathways are heterogenous and episode-specific, isolates from persistent bacteraemia have a distinctive molecular signature, characterised by a low mutation frequency and high proportion of non-silent mutations. By performing an extensive analysis of structural genomic variants in addition to point mutations, we found that these often overlooked genetic events are commonly acquired during SAB. We discovered that IS256 insertion may represent the most effective driver of within-host microevolution in selected lineages, with up to three new insertion events per isolate even in the absence of other mutations. Genetic mechanisms resulting in significant phenotypic changes, such as increases in vancomycin resistance, development of small colony phenotypes, and decreases in cytotoxicity, included mutations in key genes (rpoB, stp, agrA) and an IS256 insertion upstream of the walKR operon. Conclusions: This study provides for the first time a large-scale analysis of within-host evolution during invasive S. aureus infection and describes specific patterns of adaptation that will be informative for both understanding S. aureus pathoadaptation and utilising genomics for management of complicated S. aureus infections.
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ItemWhole-genome sequencing reveals transmission of gonococcal antibiotic resistance among men who have sex with men: an observational study(BMJ PUBLISHING GROUP, 2018-03)OBJECTIVES: Drug-resistant Neisseria gonorrhoeae are now a global public health threat. Direct transmission of antibiotic-resistant gonococci between individuals has been proposed as a driver for the increased transmission of resistance, but direct evidence of such transmission is limited. Whole-genome sequencing (WGS) has superior resolution to investigate outbreaks and disease transmission compared with traditional molecular typing methods such as multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence (NG-MAST). We therefore aimed to systematically investigate the transmission of N. gonorrhoeae between men in sexual partnerships using WGS to compare isolates and their resistance to antibiotics at a genome level. METHODS: 458 couples from a large prospective cohort of men who have sex with men (MSM) tested for gonorrhoea together between 2005 and 2014 were included, and WGS was conducted on all isolates from couples where both men were culture-positive for N. gonorrhoeae. Resistance-determining sequences were identified from genome assemblies, and comparison of isolates between and within individuals was performed by pairwise single nucleotide polymorphism and pangenome comparisons, and in silico predictions of NG-MAST and MLST. RESULTS: For 33 of 34 (97%; 95% CI 85% to 100%) couples where both partners were positive for gonorrhoea, the resistance-determining genes and mutations were identical in isolates from each partner (94 isolates in total). Resistance determinants in isolates from 23 of 23 (100%; 95% CI 86% to 100%) men with multisite infections were also identical within an individual. These partner and within-host isolates were indistinguishable by NG-MAST, MLST and whole genomic comparisons. CONCLUSIONS: These data support the transmission of antibiotic-resistant strains between sexual partners as a key driver of resistance rates in gonorrhoea among MSM. This improved understanding of the transmission dynamics of N. gonorrhoeae between sexual partners will inform treatment and prevention guidelines.