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We describe the demographics, clinical and molecular epidemiology of extended-spectrum β-lactamase (ESBL) Escherichia coli bloodstream infections (BSI) in Central Australia. All ESBL-producing E. coli bloodstream isolates from January 2018 to December 2020 were retrospectively identified. Demographic and clinical information was extracted by chart review. Whole-genome sequencing was performed for multi-locus sequence typing, antibiotic-resistance genes, and phylogenetic relationships. We identified 41 non-duplicate episodes of ESBL E. coli BSI. Median age was 55 years (IQR 47-63), 78% were female, 93% were Aboriginal, and half came from a remote community. Infections were predominantly urinary (68%, 28/41). In the 12 months prior, 70% (26/37) of identified patients had been hospitalised and 81% (30/37) prescribed antibiotics. Meropenem and piperacillin-tazobactam susceptibility was maintained in 100% and 95% of isolates, respectively. Co-resistance to non-β-lactam antibiotics was 32% to gentamicin, 61% to trimethoprim/sulfamethoxazole, and 68% to ciprofloxacin. For sequenced isolates, 41% (16/35) were sequence type 131 (ST131). Mean acquired antibiotic-resistance genes for each isolate was 12.3 (SD 3.1). Four isolates carried an OXA-1 gene. Only non-ST131 isolates carried AmpC and acquired quinolone-resistance genes. There was some evidence of clustering of closely related strains, but no evidence of community or healthcare admission overlap. ESBL rates are rapidly rising in Central Australia, which is a conducive environment for antibiotic resistance development (e.g., overcrowding, socioeconomic disadvantages, high healthcare exposure and high antibiotic use). Future research is required to explore resistance-transmission dynamics in this unique setting., (Copyright © 2024 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6')-aph(2")) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA., (© 2024. The Author(s).)

Background: The clinical and genomic epidemiology of melioidosis varies across regions., Aim: To describe the clinical and genetic diversity of B. pseudomallei across Queensland, Australia., Methods: Whole genome sequencing of clinical isolates stored at the melioidosis reference lab from 1996-2020 was performed and analysed in conjunction with available clinical data., Results: Isolates from 292 patients were analysed. Bacteraemia was present in 71% and pneumonia in 65%. The case-fatality rate was 25%. Novel sequence types (ST) accounted for 51% of all isolates. No association was identified between the variable virulence factors assessed and patient outcome. Over time, the proportion of First Nation's patients declined from 59% to 26%, and the proportion of patients aged >70 years rose from 13% to 38%., Conclusion: This study describes a genomically diverse and comparatively distinct collection of B. pseudomallei clinical isolates from across Queensland, Australia. An increasing incidence of melioidosis in elderly patients may be an important factor in the persistently high case-fatality in this region and warrants further investigation and directed intervention., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gassiep et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) sequence type 78 (ST78) in a large tertiary Australian hospital. A collection of 63 VREfm ST78 isolates, identified during a routine genomic surveillance program, were subjected to genomic epidemiological analysis based on whole-genome sequencing (WGS) data. The population structure was reconstructed using phylogenetic analysis, and a collection of publicly available VREfm ST78 genomes were used to provide global context. Core genome single nucleotide polymorphism (SNP) distances and available clinical metadata were used to characterize outbreak clusters and reconstruct transmission events. In silico genotyping confirmed that all study isolates were vanB -type VREfm carrying virulence characteristics of the hospital-associated E. faecium. Phylogenetic analysis identified two distinct phylogenetic clades, only one of which was responsible for a hospital outbreak. Four outbreak subtypes could be defined with examples of recent transmissions. Inference on transmission trees suggested complex transmission routes with unknown environmental reservoirs mediating the outbreak. WGS-based cluster analysis with publicly available genomes identified closely related Australian ST78 and ST203 isolates, highlighting the capacity for WGS to resolve complex clonal relationships between the VREfm lineages. Whole genome-based analysis has provided a high-resolution description of an outbreak of vanB -type VREfm ST78 in a Queensland hospital. Combined routine genomic surveillance and epidemiological analysis have facilitated better understanding of the local epidemiology of this endemic strain, providing valuable insight for better targeted control of VREfm. IMPORTANCE Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of health care-associated infections (HAIs) globally. In Australia, the spread of hospital-adapted VREfm is largely driven by a single clonal group (clonal complex [CC]), CC17, to which the lineage ST78 belongs. While implementing a genomic surveillance program in Queensland, we observed increased incidence of ST78 colonizations and infections among patients. Here, we demonstrate the use of real-time genomic surveillance as a tool to support and enhance infection control (IC) practices. Our results show that real-time whole-genome sequencing (WGS) can efficiently disrupt outbreaks by identifying transmission routes that in turn can be targeted using resource-limited interventions. Additionally, we demonstrate that by placing local outbreaks in a global context, high-risk clones can be identified and targeted prior to them becoming established within clinical environments. Finally, the persistence of these organism within the hospital highlights the need for routine genomic surveillance as a management tool to control VRE transmission., Competing Interests: The authors declare no conflict of interest.

Fowl cholera caused by Pasteurella multocida has re-emerged in Australian poultry production since the increasing adoption of free-range production systems. Currently, autogenous killed whole-cell vaccines prepared from the isolates previously obtained from each farm are the main preventative measures used. In this study, we use whole-genome sequencing and phylogenomic analysis to investigate outbreak dynamics, as well as monitoring and comparing the variations in the lipopolysaccharide (LPS) outer core biosynthesis loci of the outbreak and vaccine strains. In total, 73 isolates from two different free-range layer farms were included. Our genomic analysis revealed that all investigated isolates within the two farms (layer A and layer B) carried LPS type L3, albeit with a high degree of genetic diversity between them. Additionally, the isolates belonged to five different sequence types (STs), with isolates belonging to ST9 and ST20 being the most prevalent. The isolates carried ST-specific mutations within their LPS type L3 outer core biosynthesis loci, including frameshift mutations in the outer core heptosyltransferase gene ( htpE ) (ST7 and ST274) or galactosyltransferase gene ( gatG ) (ST20). The ST9 isolates could be separated into three groups based on their LPS outer core biosynthesis loci sequences, with evidence for potential phase variation mechanisms identified. The potential phase variation mechanisms included a tandem repeat insertion in natC and a single base deletion in a homopolymer region of gatG . Importantly, our results demonstrated that two of the three ST9 groups shared identical rep-PCR (repetitive extragenic palindromic PCR) patterns, while carrying differences in their LPS outer core biosynthesis loci region. In addition, we found that ST9 isolates either with or without the natC tandem repeat insertion were both associated with a single outbreak, which would indicate the importance of screening more than one isolate within an outbreak. Our results strongly suggest the need for a metagenomics culture-independent approach, as well as a genetic typing scheme for LPS, to ensure an appropriate vaccine strain with a matching predicted LPS structure is used.

Background: Sepsis contributes significantly to morbidity and mortality globally. In Australia, 20,000 develop sepsis every year, resulting in 5,000 deaths, and more than AUD$846 million in expenditure. Prompt, appropriate antibiotic therapy is effective in improving outcomes in sepsis. Conventional culture-based methods to identify appropriate therapy have limited yield and take days to complete. Recently, nanopore technology has enabled rapid sequencing with real-time analysis of pathogen DNA. We set out to demonstrate the feasibility and diagnostic accuracy of pathogen sequencing direct from clinical samples, and estimate the impact of this approach on time to effective therapy when integrated with personalised software-guided antimicrobial dosing in children and adults on ICU with sepsis., Methods: The DIRECT study is a pilot prospective, non-randomized multicentre trial of an integrated diagnostic and therapeutic algorithm combining rapid direct pathogen sequencing and software-guided, personalised antibiotic dosing in children and adults with sepsis on ICU., Participants and Interventions: DIRECT will collect microbiological and pharmacokinetic samples from approximately 200 children and adults with sepsis admitted to one of four ICUs in Brisbane. In Phase 1, we will evaluate Oxford Nanopore Technologies MinION sequencing direct from blood in 50 blood culture-proven sepsis patients recruited from consecutive patients with suspected sepsis. In Phase 2, a further 50 consecutive patients with suspected sepsis will be recruited in whom MinION sequencing will be combined with Bayesian software-guided (ID-ODS) personalised antimicrobial dosing., Outcome Measures: The primary outcome is time to effective antimicrobial therapy, defined as trough drug concentrations above the MIC of the pathogen. Secondary outcomes are diagnostic accuracy of MinION sequencing from whole blood, time to pathogen identification and susceptibility testing using sequencing direct from whole blood and from positive blood culture broth., Discussion: Rapid pathogen sequencing coupled with antimicrobial dosing software has great potential to overcome the limitations of conventional diagnostics which often result in prolonged inappropriate antimicrobial therapy. Reduced time to optimal antimicrobial therapy may reduce sepsis mortality and ICU length of stay. This pilot study will yield key feasibility data to inform further, urgently needed sepsis studies. Phase 2 of the trial protocol is registered with the ANZCTR (ACTRN12620001122943)., Trial Registration: Registered with the Australia New Zealand Clinical Trials Registry Number ACTRN12620001122943., Competing Interests: AI has received research funding and teaching honoraria from Gilead Sciences inc. unrelated to this work. DP has received research funding from Pfizer, Merck and Shionogi and funding for advisory boards or speaking engagements from Merck, Pfizer, BioMerieux, Sumitomo, Accelerate, QPex and Entasis, unrelated to this work. LC has received research funding from Oxford Nanopore Technologies unrelated to this work, and received travel reimbursement to travel to a conference. JR has consulted for or received grants from The Medicines Company, MSD, Biomerieux, QPEX, Pfizer and Discuva. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Irwin, Coin, Harris, Cotta, Bauer, Buckley, Balch, Kruger, Meyer, Shekar, Brady, Fourie, Sharp, Vlad, Whiley, Beatson, Forde, Paterson, Clark, Hajkowicz, Raman, Bialasiewicz, Lipman, Schlapbach and Roberts.)

Epidemiological surveillance of Shigella spp. in Australia is conducted to inform public health response. Multi-drug resistance has recently emerged as a contributing factor to sustained local transmission of Shigella spp. All data were collected as part of routine public health surveillance, and strains were whole-genome sequenced for further molecular characterisation. 108 patients with an endemic regional Shigella flexneri strain were identified between 2016 and 2019. The S. flexneri phylogroup 3 strain endemic to northern Australia acquired a multi-drug resistance conferring bla DHA plasmid, which has an IncFII plasmid backbone with virulence and resistance elements typically found in IncR plasmids. This is the first report of multi-drug resistance in Shigella sp. in Australia that is not associated with men who have sex with men. This strain caused an outbreak of multi-drug-resistant S. flexneri in northern Australia that disproportionality affects Aboriginal and Torres Strait Islander children. Community controlled public health action is recommended.

Sentinel hospital surveillance was instituted in Australia to detect the presence of pandemic group A Streptococcus strains causing scarlet fever. Genomic and phylogenetic analyses indicated the presence of an Australian GAS emm12 scarlet fever isolate related to United Kingdom outbreak strains. National surveillance to monitor this pandemic is recommended., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5'-CTGC m6 AG-3') in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

Pasteurella multocida is a heterogeneous bacterium, which has the capacity to cause disease in a wide range of host species and is also recognized as an important zoonotic pathogen. Two sequential deaths in captive fur seals occurred at Sea World, Australia during December 2017. A fibrinosuppurative bronchopneumonia in a Subantarctic fur seal (Arctocephalus tropicalis) resulted in death within 24 h of nonspecific signs of illness, whereas a septic peritonitis in a New Zealand fur seal (Arctocephalus forsteri) resulted in death within 12 h of clinical presentation. The cases happened within three days in two different pool locations, although both had previously been housed in the same area. A total of six Pasteurella multocida isolates were obtained from several internal organs at necropsy in both cases and were subjected to whole genome sequencing and phylogenomic analysis. In-silico typing of the isolates revealed that all belonged to Multi-Locus Sequence Type 7 and carried lipopolysaccharide outer core biosynthesis loci Type 3. Phylogenomic analysis of the isolates confirmed that the isolates were near identical at the core genome level, suggesting acquisition from a common source. The results also revealed the presence of within host and across animal diversity of P. multocida isolates for the first time even in a clearly connected outbreak., (Copyright © 2019 Elsevier B.V. All rights reserved.)