Microbiology & Immunology - Research Publications
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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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ItemNo Preview AvailableGenomics-informed responses in the elimination of COVID-19 in Victoria, Australia: an observational, genomic epidemiological study(ELSEVIER SCI LTD, 2021-08)BACKGROUND: A cornerstone of Australia's ability to control COVID-19 has been effective border control with an extensive supervised quarantine programme. However, a rapid recrudescence of COVID-19 was observed in the state of Victoria in June, 2020. We aim to describe the genomic findings that located the source of this second wave and show the role of genomic epidemiology in the successful elimination of COVID-19 for a second time in Australia. METHODS: In this observational, genomic epidemiological study, we did genomic sequencing of all laboratory-confirmed cases of COVID-19 diagnosed in Victoria, Australia between Jan 25, 2020, and Jan 31, 2021. We did phylogenetic analyses, genomic cluster discovery, and integrated results with epidemiological data (detailed information on demographics, risk factors, and exposure) collected via interview by the Victorian Government Department of Health. Genomic transmission networks were used to group multiple genomic clusters when epidemiological and genomic data suggested they arose from a single importation event and diversified within Victoria. To identify transmission of emergent lineages between Victoria and other states or territories in Australia, all publicly available SARS-CoV-2 sequences uploaded before Feb 11, 2021, were obtained from the national sequence sharing programme AusTrakka, and epidemiological data were obtained from the submitting laboratories. We did phylodynamic analyses to estimate the growth rate, doubling time, and number of days from the first local infection to the collection of the first sequenced genome for the dominant local cluster, and compared our growth estimates to previously published estimates from a similar growth phase of lineage B.1.1.7 (also known as the Alpha variant) in the UK. FINDINGS: Between Jan 25, 2020, and Jan 31, 2021, there were 20 451 laboratory-confirmed cases of COVID-19 in Victoria, Australia, of which 15 431 were submitted for sequencing, and 11 711 met all quality control metrics and were included in our analysis. We identified 595 genomic clusters, with a median of five cases per cluster (IQR 2-11). Overall, samples from 11 503 (98·2%) of 11 711 cases clustered with another sample in Victoria, either within a genomic cluster or transmission network. Genomic analysis revealed that 10 426 cases, including 10 416 (98·4%) of 10 584 locally acquired cases, diagnosed during the second wave (between June and October, 2020) were derived from a single incursion from hotel quarantine, with the outbreak lineage (transmission network G, lineage D.2) rapidly detected in other Australian states and territories. Phylodynamic analyses indicated that the epidemic growth rate of the outbreak lineage in Victoria during the initial growth phase (samples collected between June 4 and July 9, 2020; 47·4 putative transmission events, per branch, per year [1/years; 95% credible interval 26·0-85·0]), was similar to that of other reported variants, such as B.1.1.7 in the UK (mean approximately 71·5 1/years). Strict interventions were implemented, and the outbreak lineage has not been detected in Australia since Oct 29, 2020. Subsequent cases represented independent international or interstate introductions, with limited local spread. INTERPRETATION: Our study highlights how rapid escalation of clonal outbreaks can occur from a single incursion. However, strict quarantine measures and decisive public health responses to emergent cases are effective, even with high epidemic growth rates. Real-time genomic surveillance can alter the way in which public health agencies view and respond to COVID-19 outbreaks. FUNDING: The Victorian Government, the National Health and Medical Research Council Australia, and the Medical Research Future Fund.
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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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ItemKey parameters for genomics-based real-time detection and tracking of multidrug-resistant bacteria: a systematic analysis(ELSEVIER, 2021-11)BACKGROUND: Pairwise single nucleotide polymorphisms (SNPs) are a cornerstone of genomic approaches to the inference of transmission of multidrug-resistant (MDR) organisms in hospitals. However, the impact of many key analytical approaches on these inferences has not yet been systematically assessed. This study aims to make such a systematic assessment. METHODS: We conducted a 15-month prospective study (2-month pilot phase, 13-month implementation phase), across four hospital networks including eight hospitals in Melbourne, VIC, Australia. Patient clinical and screening samples containing one or more isolates of meticillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae were collected and underwent whole genome sequencing. Using the genome data from the top four most numerous sequence types from each species, 16 in total, we systematically assessed the: (1) impact of sample and reference genome diversity through multiple core genome alignments using different data subsets and reference genomes, (2) effect of masking of prophage and regions of recombination in the core genome alignments by assessing SNP distances before and after masking, (3) differences between a cumulative versus a 3-month sliding-window approach to sample genome inclusion in the dataset over time, and (4) the comparative effects each of these approaches had when applying a previously defined SNP threshold for inferring likely transmission. FINDINGS: 2275 samples were collected (397 during the pilot phase from April 4 to June 18, 2017; 1878 during the implementation phase from Oct 30, 2017, to Nov 30, 2018) from 1870 patients. Of these 2275 samples, 1537 were identified as arising from the four most numerous sequence types from each of the four target species of MDR organisms in this dataset (16 sequence types in total: S aureus ST5, ST22, ST45, and ST93; E faecium ST80, ST203, ST1421, and ST1424; K pneumoniae ST15, ST17, ST307, and ST323; and E coli ST38, ST131, ST648, and ST1193). Across the species, using a reference genome of the same sequence type provided a greater degree of pairwise SNP resolution, compared with species and outgroup-reference alignments that mostly resulted in inflated SNP distances and the possibility of missed transmission events. Omitting prophage regions had minimal effect; however, omitting recombination regions had a highly variable effect, often inflating the number of closely related pairs. Estimated SNP distances between isolate pairs over time were more consistent using a sliding-window than a cumulative approach. INTERPRETATION: We propose that the use of a closely related reference genome, without masking of prophage or recombination regions, and of a sliding-window approach for isolate inclusion is best for accurate and consistent MDR organism transmission inference, when using core genome alignments and SNP thresholds. These approaches provide increased stability and resolution, so SNP thresholds can be more reliably applied for putative transmission inference among diverse MDR organisms, reducing the chance of incorrectly inferring the presence or absence of close genetic relatedness and, therefore, transmission. The establishment of a broadly applicable and standardised approach, as proposed here, is necessary to implement widespread prospective genomic surveillance for MDR organism transmission. FUNDING: Melbourne Genomics Health Alliance, and National Health and Medical Research Council of Australia.
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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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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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ItemA phylogenomic framework for assessing the global emergence and evolution of clonal complex 398 methicillin-resistant Staphylococcus aureus(MICROBIOLOGY SOC, 2017-01)Distinct clones of methicillin-resistant Staphylococcus aureus (MRSA) have emerged as important causes of infection in individuals who have exposure to livestock (livestock-associated MRSA; LA-MRSA). Clonal complex 398 (CC398) is the most prevalent LA-MRSA clone, and has been reported from several geographical settings, including Europe, the Americas and Asia. To understand the factors contributing to the global dissemination of this clone, we analysed CC398 MRSA isolates from New Zealand (NZ), a geographically isolated country with an economy strongly dependent on livestock farming. We supplemented the NZ CC398 MRSA collection with global datasets of CC398 MRSA and CC398 methicillin-susceptible S. aureus. Here, we demonstrate multiple sporadic incursions of CC398 MRSA into NZ, as well as recent importation and spread of a swine-associated clade related to the European LA-MRSA lineage. Within a larger global phylogenomic framework, Bayesian modelling suggested that this NZ clade emerged in the late 2000s, with a probable origin in swine from Western Europe. Elucidating the factors responsible for the incursion and spread of LA-MRSA in geographically distant regions, such as NZ, provides important insights into global pathways of S. aureus transmission, and will inform strategies to control importation and spread.
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ItemGenomic Insights into a Sustained National Outbreak of Yersinia pseudotuberculosis(OXFORD UNIV PRESS, 2016-12)In 2014, a sustained outbreak of yersiniosis due to Yersinia pseudotuberculosis occurred across all major cities in New Zealand (NZ), with a total of 220 laboratory-confirmed cases, representing one of the largest ever reported outbreaks of Y. pseudotuberculosis. Here, we performed whole genome sequencing of outbreak-associated isolates to produce the largest population analysis to date of Y. pseudotuberculosis, giving us unprecedented capacity to understand the emergence and evolution of the outbreak clone. Multivariate analysis incorporating our genomic and clinical epidemiological data strongly suggested a single point-source contamination of the food chain, with subsequent nationwide distribution of contaminated produce. We additionally uncovered significant diversity in key determinants of virulence, which we speculate may help explain the high morbidity linked to this outbreak.
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ItemGenomic epidemiology and antimicrobial resistance of Neisseria gonorrhoeae in New Zealand(OXFORD UNIV PRESS, 2018-02)BACKGROUND: Antimicrobial-resistant Neisseria gonorrhoeae is a major threat to public health. No studies to date have examined the genomic epidemiology of gonorrhoea in the Western Pacific Region, where the incidence of gonorrhoea is particularly high. METHODS: A population-level study of N. gonorrhoeae in New Zealand (October 2014 to May 2015). Comprehensive susceptibility testing and WGS data were obtained for 398 isolates. Relatedness was inferred using phylogenetic trees, and pairwise core SNPs. Mutations and genes known to be associated with resistance were identified, and correlated with phenotype. RESULTS: Eleven clusters were identified. In six of these clusters, >25% of isolates were from females, while in eight of them, >15% of isolates were from females. Drug resistance was common; 98%, 32% and 68% of isolates were non-susceptible to penicillin, ciprofloxacin and tetracycline, respectively. Elevated MICs to extended-spectrum cephalosporins (ESCs) were observed in 3.5% of isolates (cefixime MICs ≥ 0.12 mg/L, ceftriaxone MICs ≥ 0.06 mg/L). Only nine isolates had penA XXXIV genotypes, three of which had decreased susceptibility to ESCs (MIC = 0.12 mg/L). Azithromycin non-susceptibility was identified in 43 isolates (10.8%); two of these isolates had 23S mutations (C2611T, 4/4 alleles), while all had mutations in mtrR or its promoter. CONCLUSIONS: The high proportion of females in clusters suggests transmission is not exclusively among MSM in New Zealand; re-assessment of risk factors for transmission may be warranted in this context. As elevated MICs of ESCs and/or azithromycin were found in closely related strains, targeted public health interventions to halt transmission are urgently needed.
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ItemNGMASTER: in silico multi-antigen sequence typing for Neisseria gonorrhoeae(MICROBIOLOGY SOC, 2016-08)Whole-genome sequencing (WGS) provides the highest resolution analysis for comparison of bacterial isolates in public health microbiology. However, although increasingly being used routinely for some pathogens such as Listeria monocytogenes and Salmonella enterica, the use of WGS is still limited for other organisms, such as Neisseria gonorrhoeae. Multi-antigen sequence typing (NG-MAST) is the most widely performed typing method for epidemiological surveillance of gonorrhoea. Here, we present NGMASTER, a command-line software tool for performing in silico NG-MAST on assembled genome data. NGMASTER rapidly and accurately determined the NG-MAST of 630 assembled genomes, facilitating comparisons between WGS and previously published gonorrhoea epidemiological studies. The source code and user documentation are available at https://github.com/MDU-PHL/ngmaster.