Stanley Pang

Background
Tuberculosis is a global public health priority. In Australia, genomic analysis of Mycobacterium tuberculosis (Mtb) occurs at state or territory level, thus multijurisdictional genomics of Mtb remains poorly understood. We present the first national assessment of Mtb using nine years of genomic data from all Australian jurisdictions, as a part of the Australian Pathogen Genomics program.

Methods
Our national Mtb sequence dataset spanned 2015–2023 and represented culture-confirmed cases. AusTrakka was optimised to allow secure, national contribution of Mtb genomes and metadata. Drug resistance was determined using tbtamr, and genomic clusters were identified via hierarchical single linkage clustering (5 SNPs) from pairwise SNP distances. Criteria were established to identify clusters of national public health significance.

Findings
We analysed 6616 Mtb sequences, representing 59% of notified cases nationwide. Lineages 1–4 were detected, with lineage 2 the most prevalent and drug resistant. At the 5-SNP threshold, 373 genomic (17% of sequences) clusters were identified, with 48 (13%) clusters identified as nationally significant. Critically, 31 genomic clusters spanned multiple jurisdictions and persisted for >5 years. Genomic analysis inferred low rates of Mtb drug resistance in Australia.

Interpretation
This first-of-a-kind study on genomic diversity of Mtb in Australia highlighted the benefits of national aggregation and analysis of Mtb sequencing data. It identified multi-jurisdictional clusters which, on average, persisted longer than clusters within jurisdictions. We also established the framework and infrastructure for implementing national coordination, detection, and surveillance of priority clusters to enhance tuberculosis control in Australia.

Funding
Australian National Health and Medical Research Council, Medical Research Futures Fund (FSPGN00049).

The Cepheid Xpert® MRSA/SA Blood Culture test is widely used for rapid molecular identification of Staphylococcus aureus directly from positive blood cultures with suggestive microscopy, and for upfront characterisation of methicillin susceptibility (MSSA) or resistance (MRSA). The assay targets proprietary sequences of the spa gene, a highly conserved genetic element present in virtually all S. aureus. It also incorporates targets for the mecA and staphylococcal cassette chromosome mec (SCCmec) genes, to differentiate MSSA and MRSA.

We describe a false-negative Xpert MRSA/SA blood culture interpreted as neither S. aureus nor MRSA (negative spa, mecA and SCCmec), but S. aureus was ultimately identified by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, 16s-rRNA sequencing, and Illumina sequencing. Whole genome sequencing identified a ∼7000 base pair deletion of the spa gene region, and absent key SCCmec elements and mecA gene. In silico multi-locus sequence typing assigned sequence type 45, a common MRSA lineage, however our isolate was phenotypically penicillin-susceptible and tested negative for blaZ beta-lactamase, and repeated susceptibility testing attempts with the Vitek 2 system failed from termination.

This clinically significant S. aureus demonstrated large-scale spa gene deletions and uncharacteristic susceptibilities for sequence type, highlighting the necessity for continued multimodal identification and susceptibility testing methods given unpredictable species mutability.

Exudative epidermitis (EE) causes substantial morbidity and mortality in piglets. This study investigated the microbial ecology, antimicrobial resistance (AMR), and genomic diversity of Staphylococcus hyicus associated with an EE outbreak in an Australian piggery.

Lesion swabs from 20 affected piglets yielded 160 bacterial isolates (including S. hyicus and cohabiting species). Isolates underwent species identification, antimicrobial susceptibility testing, and whole-genome sequencing (WGS) of S. hyicus for AMR/virulence gene profiling and core-genome SNP analysis to assess genomic relatedness.

S. hyicus predominated among lesion isolates. Phenotypic testing showed varied AMR, with frequent resistance to erythromycin and tetracycline. WGS of 27 S. hyicus isolates identified five distinct genotypic AMR profiles, including combinations spanning multiple drug classes. All S. hyicus carried the exfoliative toxin gene shetA, and 24 also carried exhD.

Core-genome analysis indicated a highly clonal outbreak: 24/27 genomes differed by 0 core SNPs, with the remaining three closely related. Despite this clonality, resistance gene carriage varied across isolates. Consequently, reliance on a single colony to represent an outbreak could understate resistance and overstate treatability.

These findings support routine multi-isolate sampling to capture within-clone AMR variability, bolster antimicrobial selection during EE management, and inform consideration of autogenous vaccines targeting dominant outbreak clones.

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.

Background
Diphtheria caused by toxin-producing Corynebacterium ulcerans is a re-emerging human disease that can cause local and systemic sequelae. In Australia, toxigenic diphtheria is a rare notifiable communicable disease, due to high-vaccination coverage. The public health management of cutaneous cases of toxigenic C. ulcerans varies between jurisdictions, as opposed to the more uniform public health response to toxigenic Corynebacterium diphtheriae presenting as respiratory or laryngeal diphtheria.

Aim
To report a case of zoonotically acquired C. ulcerans, review evidence on the zoonotic reservoir and reported transmission events, and examine public health guidelines for the management of human and animal contacts.

Methods and Results
In this case report, we detail our case investigation, treatment and public health management, including contact tracing and an approach to animal testing. We successfully identified companion canines as probable sources for the human case, with WGS confirming the link. The zoonotic disease link of C. ulcerans to domestic and agricultural animals is established in the literature; however, the management of animal contacts in human cases is inconsistent with jurisdictional or national guidelines.

Conclusions
While a rare disease, a consistent approach to public health management is warranted to systematically elucidate the disease source and improve understanding of transmission.

Background
Food animal AMR surveillance programs assess only small numbers of Escherichia coli (from 100 to 600 per animal class) nationally each year, severely limiting the evaluation of public health risk(s). Here we demonstrate an affordable approach for early detection of emerging resistance on a broad scale that can also accurately characterize spatial and temporal changes in resistance.

Methods
Caecal samples (n = 295) obtained from 10 meat poultry were screened using high-throughput robotics. Initial screening via agar dilution (5310 plates) quantified AMR carriage (cfu/g) for each sample. Ciprofloxacin-resistant isolates (n = 91) proceeded to downstream broth microdilution susceptibility testing. A subset of 28 ciprofloxacin-resistant isolates underwent WGS and phylogenetic analysis.

Results
Intra- and inter-flock carriage of resistance varied with drug class. Ampicillin and tetracycline resistance was ubiquitous to most birds in all flocks with an average carriage rate of 5.8 log10 cfu/g. Gentamicin and ciprofloxacin-resistant E. coli colonized fewer birds, and had an average carriage rate of 1.2 log10 cfu/g and 1.0 log10 cfu/g of faeces, respectively. Resistance to extended-spectrum cephalosporins was absent. ST354 was the dominant ST among the WGS isolates, but they demonstrated markedly lower resistance gene carriage than their international counterparts.

Conclusions
These data amply demonstrate the ineffectiveness of commonly relied-on approaches to AMR surveillance for achieving early detection of emergence, or for measuring spatial and temporal resistance trends. Genetic analysis suggested there has been transnational flow of a ciprofloxacin-resistant strain into Australian poultry flocks, explaining their detection in a nation that prohibits fluoroquinolone use in poultry.

Infection with Pasteurella multocida represents a significant economic threat to Australian pig producers, yet our knowledge of its antimicrobial susceptibilities is lagging, and genomic characterization of P. multocida strains associated with porcine lower respiratory disease is internationally scarce. This study utilized high-throughput robotics to phenotypically and genetically characterize an industry-wide collection of 252 clinical P. multocida isolates that were recovered between 2014 and 2019. Overall, antimicrobial resistance was found to be low, with clinical resistance below 1% for all tested antimicrobials except those from the tetracycline class. Five dominant sequence types, representing 64.8% of all isolates, were identified; they were disseminated across farms and had previously been detected in various animal hosts and countries. P. multocida in Australian farms remain controllable via current antimicrobial therapeutic protocols. The identification of highly dominant, interspecies-infecting strains provides insight into the epidemiology of the opportunistic pathogen, and it highlights a biosecurity threat to the Australian livestock industry.

Vancomycin variable enterococci (VVE) are van-positive enterococci with a vancomycin-susceptible phenotype (VVE-S) that can convert to a resistant phenotype (VVE-R) and be selected for during vancomycin exposure. VVE-R outbreaks have been reported in Canada and Scandinavian countries. The aim of this study was to examine the presence of VVE in whole genome sequenced (WGS) Australian bacteremia Enterococcus faecium (Efm) isolates collected through the Australian Group on Antimicrobial resistance (AGAR) network. Eight potential VVEAus isolates, all identified as Efm ST1421, were selected based on the presence of vanA and a vancomycin-susceptible phenotype. During vancomycin selection, two potential VVE-S harboring intact vanHAX genes, but lacking the prototypic vanRS and vanZ genes, reverted to a resistant phenotype (VVEAus-R). Spontaneous VVEAus-R reversion occurred at a frequency of 4-6 × 10−8 resistant colonies per parent cell in vitro after 48 h and led to high-level vancomycin and teicoplanin resistance. The S to R reversion was associated with a 44-bp deletion in the vanHAX promoter region and an increased vanA plasmid copy number. The deletion in the vanHAX promoter region enables an alternative constitutive promoter for the expression of vanHAX. Acquisition of vancomycin resistance was associated with a low fitness cost compared with the corresponding VVEAus-S isolate. The relative proportion of VVEAus-R vs. VVEAus-S decreased over time in serial passages without vancomycin selection. Efm ST1421 is one of the predominant VanA-Efm multilocus sequence types found across most regions of Australia, and has also been associated with a major prolonged VVE outbreak in Danish hospitals.

Moraxella catarrhalis is an important human respiratory pathogen and a major causative agent of otitis media and chronic obstructive pulmonary disease. Toll-like receptors contribute to, but cannot fully account for, the complexity of the immune response seen in M. catarrhalis infection. Using primary mouse bone marrow-derived macrophages to examine the host response to M. catarrhalis infection, our global transcriptomic and targeted cytokine analyses revealed activation of immune signalling pathways by both membrane-bound and cytosolic pattern-recognition receptors. We show that M. catarrhalis and its outer membrane vesicles or lipooligosaccharide (LOS) can activate the cytosolic innate immune sensor caspase-4/11, gasdermin-D-dependent pyroptosis, and the NLRP3 inflammasome in human and mouse macrophages. This pathway is initiated by type I interferon signalling and guanylate-binding proteins (GBPs). We also show that inflammasomes and GBPs, particularly GBP2, are required for the host defence against M. catarrhalis in mice. Overall, our results reveal an essential role for the interferon-inflammasome axis in cytosolic recognition and immunity against M. catarrhalis, providing new molecular targets that may be used to mitigate pathological inflammation triggered by this pathogen.

Introduction

Enterococcus faecium is an opportunistic pathogen that has become one of the leading causes of hospital acquired infection that are resistant to multiple critically important antimicrobials.

Aim

The objective of the study was to describe the molecular characteristics and relationship between major strains of E. faecium harbouring the van operon and to determine if the strains had increasing virulence and antimicrobial resistance determinants over time.

Methods

E. faecium harbouring the van operon detected using PCR from surveillance rectal swabs of patients that were admitted to high-risk units at a Perth teaching hospital from 2001 to 2015 were retrospectively analysed using a whole genome sequencing and bioinformatics approach.

Results

ST18, ST78, ST80, ST173, ST203 and ST555 were identified as the major STs accounting for 93.7% of E. faecium isolates. Except for ST173, major STs identified at Royal Perth Hospital (RPH) have been reported across Australia and internationally. Isolates from each ST formed independently branched phylogenetic clusters with each harbouring unique virulence and antimicrobial resistance profiles. Depending on the ST, different genes conferring resistance to similar antimicrobial classes were identified. Except for ST80 which harboured the vanA type operon, all major strains harboured the vanB operon conferring only vancomycin resistance.

Conclusion

Major strains of E. faecium isolated over 15-years showed unique virulome and resistome profiles with no indication of increasing virulence or antimicrobial resistance determinants. Strains were distantly related and the acquisition of different genes encoding similar antimicrobial resistances suggest the independent evolution of each strain.