Doherty Institute - Research Publications
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ItemSingle-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus(BMC, 2015-05-16)BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors. RESULTS: Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage. CONCLUSIONS: The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.
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ItemMinim Typing - A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae(PUBLIC LIBRARY SCIENCE, 2012-03-12)Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.
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ItemWhole genome sequencing to investigate a putative outbreak of the virulent community-associated methicillin-resistant Staphylococcus aureus ST93 clone in a remote Indigenous community(MICROBIOLOGY SOC, 2016-12)We report two cases of severe pneumonia due to clone ST93 methicillin-resistant Staphylococcus aureus (MRSA) presenting from a remote Australian Indigenous community within a 2-week period, and the utilization of whole genome sequences to determine whether these were part of an outbreak. S. aureus was isolated from 12 of 92 nasal swabs collected from 25 community households (including the two index households); one isolate was ST93. Three of five skin lesion S. aureus isolates obtained at the community were ST93. Whole genome sequencing of the ST93 isolates from this study and a further 20 ST93 isolates from the same region suggested that recent transmission and progression to disease had not taken place. The proximity in time and space of the two severe pneumonia cases is probably a reflection of the high burden of disease due to ST93 MRSA in this population where skin infections and household crowding are common.
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ItemMultisite Direct Determination of the Potential for Environmental Contamination of Urine Samples Used for Diagnosis of Sexually Transmitted Infections(OXFORD UNIV PRESS, 2014-09)BACKGROUND: The detection of a sexually transmitted infection (STI) agent in a urine specimen from a young child is regarded as an indicator of sexual contact. False positives may conceivably arise from the transfer of environmental contaminants in clinic toilet or bathroom facilities into urine specimens. METHODS: The potential for contamination of urine specimens with environmental STI nucleic acid was tested empirically in the male and female toilets or bathrooms at 10 Northern Territory (Australia) clinics, on 7 separate occasions at each. At each of the 140 experiments, environmental contamination with Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis nucleic acid contamination was determined by swabbing 10 locations, and urine collection was simulated 5 times, using a (1) synthetic urine surrogate and (2) a standardized finger contamination procedure. RESULTS: The most contaminated toilets and bathrooms were in remote Indigenous communities. No contamination was found in the Northern Territory Government Sexual Assault Referral Centre clinics, and intermediate levels of contamination were found in sexual health clinics and in clinics in regional urban centres. The frequency of surrogate urine sample contamination was low but non-zero. For example, 4 of 558 of the urine surrogate specimens from remote clinics were STI positive. CONCLUSIONS: This is by far the largest study addressing the potential environmental contamination of urine samples with STI agents. Positive STI tests arising from environmental contamination of urine specimens cannot be ruled out. The results emphasize that urine specimens from young children taken for STI testing should be obtained by trained staff in clean environments, and duplicate specimens should be obtained if possible.
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ItemEvolution and Global Transmission of a Multidrug-Resistant, Community-Associated Methicillin-Resistant Staphylococcus aureus Lineage from the Indian Subcontinent(AMER SOC MICROBIOLOGY, 2019-11-26)The evolution and global transmission of antimicrobial resistance have been well documented for Gram-negative bacteria and health care-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. In this study, we traced the recent origins and global spread of a multidrug-resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole-genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data show that the clone emerged on the Indian subcontinent in the early 1960s and disseminated rapidly in the 1990s. Short-term outbreaks in community and health care settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the emergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth, and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional health care-associated clones with the epidemiological transmission of community-associated methicillin-resistant S. aureus (MRSA). Our study demonstrates the importance of whole-genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.IMPORTANCE The Bengal Bay clone (ST772) is a community-associated and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we showed that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally, resulting in small-scale community and health care outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug resistance of health care-associated S. aureus lineages. This study demonstrates the importance of whole-genome sequencing for the surveillance of highly antibiotic-resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.