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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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.
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ItemNo Preview AvailableGenomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis(NATURE PUBLISHING GROUP, 2013-02)Global spread and limited genetic variation are hallmarks of M. tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology are restricted to East Africa. Here, we sequenced and analyzed the whole genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5× coverage), 454/Roche (13-18× coverage) and/or Illumina DNA sequencing (45-105× coverage). We show that STB isolates are highly recombinogenic and evolutionarily early branching, with larger genome sizes, higher rates of genetic variation, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse infection experiments showed that STB strains are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral STB-like pool of mycobacteria by gain of persistence and virulence mechanisms, and we provide insights into the molecular events involved.
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ItemComparative analysis of the complete genome of an epidemic hospital sequence type 203 clone of vancomycin-resistant Enterococcus faecium(BMC, 2013-09-01)BACKGROUND: In this report we have explored the genomic and microbiological basis for a sustained increase in bloodstream infections at a major Australian hospital caused by Enterococcus faecium multi-locus sequence type (ST) 203, an outbreak strain that has largely replaced a predecessor ST17 sequence type. RESULTS: To establish a ST203 reference sequence we fully assembled and annotated the genome of Aus0085, a 2009 vancomycin-resistant Enterococcus faecium (VREfm) bloodstream isolate, and the first example of a completed ST203 genome. Aus0085 has a 3.2 Mb genome, comprising a 2.9 Mb circular chromosome and six circular plasmids (2 kb-130 kb). Twelve percent of the 3222 coding sequences (CDS) in Aus0085 are not present in ST17 E. faecium Aus0004 and ST18 E. faecium TX16. Extending this comparison to an additional 12 ST17 and 14 ST203 E. faecium hospital isolate genomes revealed only six genomic regions spanning 41 kb that were present in all ST203 and absent from all ST17 genomes. The 40 CDS have predicted functions that include ion transport, riboflavin metabolism and two phosphotransferase systems. Comparison of the vancomycin resistance-conferring Tn1549 transposon between Aus0004 and Aus0085 revealed differences in transposon length and insertion site, and van locus sequence variation that correlated with a higher vancomycin MIC in Aus0085. Additional phenotype comparisons between ST17 and ST203 isolates showed that while there were no differences in biofilm-formation and killing of Galleria mellonella, ST203 isolates grew significantly faster and out-competed ST17 isolates in growth assays. CONCLUSIONS: Here we have fully assembled and annotated the first ST203 genome, and then characterized the genomic differences between ST17 and ST203 E. faecium. We also show that ST203 E. faecium are faster growing and can out-compete ST17 E. faecium. While a causal genetic basis for these phenotype differences is not provided here, this study revealed conserved genetic differences between the two clones, differences that can now be tested to explain the molecular basis for the success and emergence of ST203 E. faecium.
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ItemExploring the Zoonotic Potential of Mycobacterium avium Subspecies paratuberculosis through Comparative Genomics(PUBLIC LIBRARY SCIENCE, 2011-07-22)A comparative genomics approach was utilised to compare the genomes of Mycobacterium avium subspecies paratuberculosis (MAP) isolated from early onset paediatric Crohn's disease (CD) patients as well as Johne's diseased animals. Draft genome sequences were produced for MAP isolates derived from four CD patients, one ulcerative colitis (UC) patient, and two non-inflammatory bowel disease (IBD) control individuals using Illumina sequencing, complemented by comparative genome hybridisation (CGH). MAP isolates derived from two bovine and one ovine host were also subjected to whole genome sequencing and CGH. All seven human derived MAP isolates were highly genetically similar and clustered together with one bovine type isolate following phylogenetic analysis. Three other sequenced isolates (including the reference bovine derived isolate K10) were genetically distinct. The human isolates contained two large tandem duplications, the organisations of which were confirmed by PCR. Designated vGI-17 and vGI-18 these duplications spanned 63 and 109 open reading frames, respectively. PCR screening of over 30 additional MAP isolates (3 human derived, 27 animal derived and one environmental isolate) confirmed that vGI-17 and vGI-18 are common across many isolates. Quantitative real-time PCR of vGI-17 demonstrated that the proportion of cells containing the vGI-17 duplication varied between 0.01 to 15% amongst isolates with human isolates containing a higher proportion of vGI-17 compared to most animal isolates. These findings suggest these duplications are transient genomic rearrangements. We hypothesise that the over-representation of vGI-17 in human derived MAP strains may enhance their ability to infect or persist within a human host by increasing genome redundancy and conferring crude regulation of protein expression across biologically important regions.
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ItemThe Dominant Australian Community-Acquired Methicillin-Resistant Staphylococcus aureus Clone ST93-IV [2B] Is Highly Virulent and Genetically Distinct(PUBLIC LIBRARY SCIENCE, 2011-10-03)Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding α-hemolysin, Panton-Valentine leukocidin (PVL) and α-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a highly conserved accessory genome from a common source.