Obstetric and Neonatal Invasive Meningococcal Disease Caused by Neisseria meningitidis Serogroup W, Western Australia, Australia.

Three mother-baby pairs with invasive meningococcal disease occurred over 7 months in Western Australia, Australia, at a time when serogroup W sequence type 11 clonal complex was the predominant local strain. One mother and 2 neonates died, highlighting the role of this strain as a cause of obstetric and early neonatal death.

Novel Human Parechovirus 3 Diversity, Recombination, and Clinical Impact Across 7 Years: An Australian Story.

BACKGROUND: A novel human parechovirus 3 Australian recombinant (HPeV3-AR) strain emerged in 2013 and coincided with biennial outbreaks of sepsis-like illnesses in infants. We evaluated the molecular evolution of the HPeV3-AR strain and its association with severe HPeV infections. METHODS: HPeV3-positive samples collected from hospitalized infants aged 5-252 days in 2 Australian states (2013-2020) and from a community-based birth cohort (2010-2014) were sequenced. Coding regions were used to conduct phylogenetic and evolutionary analyses. A recombinant-specific polymerase chain reaction was designed and utilized to screen all clinical and community HPeV3-positive samples. RESULTS: Complete coding regions of 54 cases were obtained, which showed the HPeV3-AR strain progressively evolving, particularly in the 3' end of the nonstructural genes. The HPeV3-AR strain was not detected in the community birth cohort until the initial outbreak in late 2013. High-throughput screening showed that most (>75%) hospitalized HPeV3 cases involved the AR strain in the first 3 clinical outbreaks, with declining prevalence in the 2019-2020 season. The AR strain was not statistically associated with increased clinical severity among hospitalized infants. CONCLUSIONS: HPeV3-AR was the dominant strain during the study period. Increased hospital admissions may have been from a temporary fitness advantage and/or increased virulence.

Whole-genome sequencing links Clostridium (Clostridioides) difficile in a single hospital to diverse environmental sources in the community.

AIMS: To investigate if Clostridium (Clostridioides) difficile infection (CDI), traditionally thought of as hospital-acquired, can be genomically linked to hospital or community environmental sources, and to define possible importation routes from the community to the hospital. METHODS AND RESULTS: In 2019, C. difficile was isolated from 89/300 (29.7%) floor and 96/300 (32.0%) shoe sole samples at a tertiary hospital in Western Australia. Non-toxigenic C. difficile ribotype (RT) 010 predominated among floor (96.6%) and shoe sole (73.2%) isolates, while toxigenic RT 014/020 was most prevalent among contemporaneous clinical cases (33.0%) at the hospital. Whole-genome sequencing and high-resolution core genome single nucleotide polymorphism (cgSNP) analysis on C. difficile strains from hospital and community sources showed no clinical C. difficile RT 014/020 strains were genetically related, and evidence of frequent long-distance, multi-directional spread between humans, animals and the environment. In addition, cgSNP analysis of environmental RT 010 strains suggested transportation of C. difficile via shoe soles. CONCLUSIONS: While C. difficile RT 014/020 appears to spread via routes outside the healthcare system, RT 010 displayed a pattern of possible importation from the community into the hospital. SIGNIFICANCE AND IMPACT OF STUDY: These findings suggest developing community-based infection prevention and control strategies could significantly lower rates of CDI in the hospital setting.

Flight-Associated Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 Corroborated by Whole-Genome Sequencing.

To investigate potential transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during a domestic flight within Australia, we performed epidemiologic analyses with whole-genome sequencing. Eleven passengers with PCR-confirmed SARS-CoV-2 infection and symptom onset within 48 hours of the flight were considered infectious during travel; 9 had recently disembarked from a cruise ship with a retrospectively identified SARS-CoV-2 outbreak. The virus strain of those on the cruise and the flight was linked (A2-RP) and had not been previously identified in Australia. For 11 passengers, none of whom had traveled on the cruise ship, PCR-confirmed SARS-CoV-2 illness developed between 48 hours and 14 days after the flight. Eight cases were considered flight associated with the distinct SARS-CoV-2 A2-RP strain; the remaining 3 cases (1 with A2-RP) were possibly flight associated. All 11 passengers had been in the same cabin with symptomatic persons who had culture-positive A2-RP virus strain. This investigation provides evidence of flight-associated SARS-CoV-2 transmission.

Genomic epidemiology and population structure of Neisseria gonorrhoeae from remote highly endemic Western Australian populations.

BACKGROUND: Neisseria gonorrhoeae causes gonorrhoea, the second most commonly notified sexually transmitted infection in Australia. One of the highest notification rates of gonorrhoea is found in the remote regions of Western Australia (WA). Unlike isolates from the major Australian population centres, the remote community isolates have low rates of antimicrobial resistance (AMR). Population structure and whole-genome comparison of 59 isolates from the Western Australian N. gonorrhoeae collection were used to investigate relatedness of isolates cultured in the metropolitan and remote areas. Core genome phylogeny, multilocus sequencing typing (MLST), N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) in addition to hierarchical clustering of sequences were used to characterize the isolates. RESULTS: Population structure analysis of the 59 isolates together with 72 isolates from an international collection, revealed six population groups suggesting that N. gonorrhoeae is a weakly clonal species. Two distinct population groups, Aus1 and Aus2, represented 63% of WA isolates and were mostly composed of the remote community isolates that carried no chromosomal AMR genotypes. In contrast, the Western Australian metropolitan isolates were frequently multi-drug resistant and belonged to population groups found in the international database, suggesting international transmission of the isolates. CONCLUSIONS: Our study suggests that the population structure of N. gonorrhoeae is distinct between the communities in remote and metropolitan WA. Given the high rate of AMR in metropolitan regions, ongoing surveillance is essential to ensure the enduring efficacy of the empiric gonorrhoea treatment in remote WA.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis from pooled rectal, pharyngeal and urine specimens in men who have sex with men.

OBJECTIVES: Screening of men who have sex with men (MSM) for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) requires sampling from anorectal and pharyngeal sites in addition to urogenital sampling. Due to the cost of testing multiple anatomical sites individually testing of pooled specimens has potential merit. The Cepheid GeneXpert CT/NG assay (GeneXpert), which also has potential for point-of-care nucleic acid testing in the sexual health clinic, has not been assessed for pooled specimen testing. METHODS: We prospectively compared GeneXpert testing of pooled pharyngeal and rectal swabs with urine samples to standard of care testing of individual specimens from 107 participants using the Roche cobas 4800 CT/NG assay (cobas) for CT and NG in high-risk MSM attending an inner city sexual health clinic. RESULTS: We found testing of pooled pharyngeal, rectal and urine samples by the GeneXpert to have 100% agreement for NG and 94% overall agreement for CT when compared with individual specimen testing by cobas. For CT testing, 14 cases were detected for both tests, 4for cobas only, 2 for GeneXpert only and 89 participants were negative for both tests. CONCLUSIONS: Pooled specimen CT and NG testing by the GeneXpert was accurate when compared with single specimen testing and has potential for screening MSM for CT and NG. The role of pooled specimen testing with the GeneXpert as a point-of-care nucleic acid test in MSM requires further investigation.

Molecular test for chlamydia and gonorrhoea used at point of care in remote primary healthcare settings: a diagnostic test evaluation.

OBJECTIVES: A new molecular test for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) (GeneXpert CT/NG) has been demonstrated to be as accurate as conventional nucleic acid amplification tests (NAAT), but performance has not been evaluated in routine primary care, performed at the point of care by clinicians. We aimed to examine its diagnostic performance when used by clinicians in remote community health services in Australia with high prevalences of CT and NG infection. The trial was registered with the Australian and New Zealand Clinical Trials Registry (#12613000808741) METHODS: At 12 health services, training was provided to 99 clinicians in the use of the GeneXpert CT/NG assay who tested specimens from all patients undergoing STI screening. Specimens were also sent in parallel for conventional laboratory-based NAATs and the concordance of results was evaluated. RESULTS: Clinicians conducted 2486 tests: CT concordance was 99.4% (95% CI 99.1 to 99.7) with a positive concordance of 98.6% (95% CI 95.9 to 99.7) and negative concordance of 99.5% (95% CI 99.1 to 99.8); NG concordance was 99.9% (95% CI 99.7 to 100.0) with a positive concordance of 100.0% (95% CI 97.5 to 100.0) and negative concordance of 99.9% (95% CI 99.7 to 100.0). CONCLUSIONS: In this first study reporting routine point-of-care use of GeneXpert CT/NG by primary care clinicians, we found excellent concordance with conventional NAATs. The use of the GeneXpert CT/NG at the point of care could potentially transform management and control of these infections in many endemic settings, including low/middle-income countries.

Clonal Expansion of New Penicillin-Resistant Clade of Neisseria meningitidis Serogroup W Clonal Complex 11, Australia.

In Western Australia, Neisseria meningitidis serogroup W clonal complex 11 became the predominant cause of invasive meningococcal disease in 2016. We used core-genome analysis to show emergence of a penicillin-resistant clade that had the penA_253 allele. This new penicillin-resistant clade might affect treatment regimens for this disease.

Serum models accurately predict liver-related clinical outcomes in chronic hepatitis C.

BACKGROUND AND AIM: This study developed liver outcome scores in chronic hepatitis C (CHC) that directly predict liver-related death, hepatocellular carcinoma (HCC), and liver decompensation. METHODS: Six hundred seventeen CHC patients were followed up for a mean of 6 years and randomized into a training set (n = 411) and a validation set (n = 206). Clinical outcomes were determined using a population-based data linkage system. RESULTS: In the training set, albumin, gamma-glutamyl transpeptidase, hyaluronic acid, age, and sex were in the final model to predict 5-year liver-related death (area under receiver operating characteristic curve [AUROC] 0.95). Two cut points (4.0 and 5.5) defined three risk groups with an incidence rate for liver-related death of 0.1%, 2%, and 13.2%, respectively (P < 0.001). Albumin, gamma-glutamyl transpeptidase, hyaluronic acid, age, and sex were used to predict 5-year liver decompensation (AUROC 0.90). A cut point of 4.5 gave a sensitivity of 94% and a specificity of 84% to predict 5-year decompensation and defined two groups with an incidence rate for decompensation of 0.2% and 5.8%, respectively (P < 0.001). Alkaline phosphatase, α2-macroglobulin, age, and sex were used to predict 5-year HCC occurrence (AUROC 0.95). A cut-point of eight had a sensitivity of 90% and specificity of 88% to predict 5-year HCC occurrence and defined two groups with an incidence rate for HCC of 0.2% and 5.6%, respectively (P < 0.001). Similar results were obtained using the validation set. CONCLUSIONS: All three liver outcome scores had excellent predictive accuracy and were able to stratify risk into clinical meaningful categories for CHC patients.

Mycobacterium chimaera colonisation of heater-cooler units (HCU) in Western Australia, 2015: investigation of possible iatrogenic infection using whole genome sequencing.

Following the reported link between heater-cooler unit (HCU) colonisation with Mycobacterium chimaera and endocarditis, mycobacterial sampling of all HCUs in use in Western Australia was initiated from August 2015, revealing M. chimaera colonisation in 10 of 15 HCUs. After M. chimaera was isolated from a pleural biopsy from a cardiothoracic patient who may have been exposed to a colonised HCU, a whole genome sequencing investigation was performed involving 65 specimens from 15 HCUs across five hospitals to assess if this infection was related to the HCU. Genetic relatedness was found between the 10 HCU M. chimaera isolates from four hospitals. However the M. chimaera isolate from the cardiothoracic patient was not genetically related to the HCU M. chimaera isolates from that hospital, nor to the other HCU isolates, indicating that the HCUs were not the source of the infection in this patient.

Acute meningoencephalitis associated with echovirus 9 infection in Sri Lanka, 2009.

The aetiology of acute meningoencephalitis in Sri Lankan children and adults is poorly understood. This study was carried out to determine pathogens responsible for meningoencephalitis in Sri Lanka. A hospital-based cross-sectional study was performed using cerebrospinal fluid samples (22 adult and 17 pediatric) collected from August to December 2009 from patients clinically diagnosed with acute meningoencephalitis at two tertiary care hospitals in Sri Lanka. Routine microbiology for bacterial pathogens together with in-house RT-PCR and PCR assays for the detection of dengue viruses, Japanese encephalitis virus, West Nile virus, chikungunya virus, enteroviruses, mumps virus, measles virus, herpes simplex viruses types 1 and 2, and varicella zoster virus were performed. Bacterial pathogens were not isolated from any patient specimens. However, from nine of the paediatric patients aged 1 month to 10 years (mean age 5.2 years) echovirus 9 (E-9; family Picornaviridae, genus Enterovirus,species Enterovirus B ) was detected by RT-PCR. All nine patients presented with fever, six had headache, and seven had vomiting. Neck stiffness indicating meningitis was present in six of the patients. Phylogenetic analysis of partial VP1 and VP4-VP2 genes showed these E-9 strains to be most closely related to E-9 strains detected in CSF from Korea and France in 2005 and 2006. The remaining patients were negative for all other viruses tested. E-9 was the most common cause of acute meningoencephalitis in the tested paediatric population from Sri Lanka in 2009, which likely reflects circulation of this E-9 strain between Europe and Asia over several years.

Molecular epidemiology of Neisseria gonorrhoeae using multi-antigen sequence typing and pulse-field gel electrophoresis in highly endemic Western Australian populations.

BACKGROUND: The remote and indigenous populations of Western Australia (WA) have one of the highest notification rates of gonorrhoea in the world. Despite this, the low rate of antimicrobial resistance in Neisseria gonorrhoeae from these regions permits the use of amoxycillin as empirical therapy. We describe the first molecular epidemiological study of gonococci isolated from this population using two different typing platforms. METHODS: Pulse-field gel electrophoresis (PFGE), Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) and antimicrobial susceptibility tests were performed on 128 consecutive N. gonorrhoeae isolates cultured between January 2011 and December 2013. To highlight clusters isolates were evaluated based on their tbpB sequence types. RESULTS: No predominant NG-MAST or PFGE types were found. A total of 67 distinct PFGE pulsotypes were identified amongst the 128 isolates in this study with 20 PFGE pulsotypes representing 78 isolates. A total of 59 NG-MAST sequence types were found, represented by 45 porB alleles and 28 tbpB alleles with 13 tbpB genomogroups from 45 NG-MAST sequence types. TbpB genomogroup 29, represented by 45 isolates, was by far the most common genomogroup overall. CONCLUSIONS: Results from this study suggest that gonococcal epidemiology in WA is quite different between remote regions and major population centres and, in some cases, geographically restricted. It is likely that isolates originating from endemic regions of WA mostly represent independent, small sexual networks with an infrequent interchange between other communities and regions. Given the high rate of antimicrobial resistance elsewhere in Australia, ongoing surveillance is essential to ensure the enduring efficacy of amoxycillin empiric use in the remote regions of WA.

Real-time PCR assay for detection of blaZ genes in Staphylococcus aureus clinical isolates.

The Clinical and Laboratory Standards Institute recommends consideration of blaZ gene testing for cases of serious Staphylococcus aureus infection. Conventional PCR methods have demonstrated superior sensitivity and specificity to phenotypic tests. To our knowledge, this is the first description of real-time PCR detection of the blaZ gene.

Non-culture Neisseria gonorrhoeae molecular penicillinase production surveillance demonstrates the long-term success of empirical dual therapy and informs gonorrhoea management guidelines in a highly endemic setting.

OBJECTIVES: Unlike most of the world, penicillin resistance in Neisseria gonorrhoeae from remote regions of Western Australia (WA) with high gonorrhoea notification rates has not increased despite many years of empirical oral therapy. With the advent of non-culture molecular diagnosis of gonorrhoea and the consequent decline in culture-based susceptibility, it is imperative to ensure the ongoing reliability of combination oral azithromycin, amoxicillin and probenecid for uncomplicated gonorrhoea in this setting. PCR-based non-culture N. gonorrhoeae antimicrobial resistance surveillance for penicillinase production was therefore employed. METHODS: Genital and non-genital specimens that were PCR-positive for N. gonorrhoeae were assessed for penicillinase production by detection of the N. gonorrhoeae TEM-1 plasmid using specific real-time PCR. RESULTS: In remote regions of WA where gonorrhoea is highly endemic, <5% of N. gonorrhoeae isolates were penicillinase-producing. This contrasts with rates of up to 20% observed in the more densely populated metropolitan and rural regions. CONCLUSIONS: In the era of molecular diagnosis of gonorrhoea, non-culture-based antimicrobial resistance surveillance proved useful when developing evidence-based guidelines for the clinical management of locally acquired gonorrhoea in highly endemic regions in WA. The continued efficacy of combination oral amoxicillin, probenecid and azithromycin therapy despite many years of use in a setting highly endemic for gonorrhoea may explain the low rate of penicillin resistance in these remote regions and supports the concept of adding azithromycin to ß-lactam antibiotics to help delay the emergence of multiresistant N. gonorrhoeae.

Prediction of morbidity and mortality in patients with chronic hepatitis C by non-invasive liver fibrosis models.

BACKGROUND & AIMS: Liver fibrosis is prognostic of outcomes among patients with chronic hepatitis C (CHC). We evaluated the accuracy of non-invasive markers and liver biopsy in predicting morbidity and mortality in CHC patients. METHODS: Compensated CHC patients were evaluated over a 10-year period. Non-invasive markers including Hepascore, FIB-4, APRI and liver biopsy results were retrospectively collated. Follow-up morbidity and mortality data were obtained from the Western Australian Data Linkage System. The prognostic significance of baseline non-invasive markers and biopsy were assessed using Kaplan-Meier analysis. RESULTS: A total of 406 subjects (64% male, mean age 48 ± 11 years) were followed up for 2385 person-years, during which there were 22 (5.4%) deaths including 14 (3.4%) who died from liver disease or required liver transplantation. Sixteen (3.9%) subjects developed liver decompensation. Hepascore and liver biopsy (P < 0.005) but not APRI or FIB-4 were predictive of overall and liver-related mortality as well as liver decompensation. A Hepascore>0.5 was associated with increased overall mortality [Hazard Ratio (95%CI) 6.7 (2.6-17), P < 0.001], liver-related mortality [32.8 (4.3-250), P = 0.001] and risk of future decompensation [11.8 (3.3-41), P < 0.001], whereas a Hepascore ≤0.5 was associated with a 99% probability of not dying from liver-related causes over 10 years. Hepascore had comparable accuracy with liver biopsy in predicting liver-related mortality with AUROC of 0.86 (95%CI 0.80-0.90) and 0.87 (0.79-0.96), respectively. CONCLUSION: Hepascore is predictive of overall and liver-related mortality and morbidity in CHC patients with comparable accuracy to liver biopsy. Hepascore may be a useful prognostic marker in clinical practice.

Neisseria meningitidis porA, fetA and fHbp gene distribution in Western Australia 2000 to 2011.

BACKGROUND: PorA, fetA and fHbp are three antigen encoding genes useful for meningococcal typing and FHbp is an important component of meningococcal B vaccines. METHODS: We performed sequence analysis of meningococcal porA, fetA and fHbp genes on 128 isolates from Western Australia, relating results to age, gender, race and geographic region. RESULTS: We found predominantly PorA subtypes P1.22,14-16 (n = 23) and P1.7-2,4 (n = 19); FetA subtypes F1-5 (n = 41), F3-6 (n = 11), F5-1 (n = 10), F5-2 (n = 9), F5-5 (n = 8), F3-3 (n = 8); and FHbp variant groups 1 (n = 65) and 2 (n = 44). PorA P1.22,14-16 and FHbp variant group 2 were associated with younger age and aboriginality. CONCLUSIONS: FHbp modular groups of the bivalent recombinant FHbp vaccine and the multicomponent 4CMenB vaccine make up 8.3% and 47.7% respectively of the examined serogroup B isolates from 2000-2011, however to estimate vaccine efficacy requires an account of all vaccine antigens and their levels of expression.

Clinical effectiveness and analytical quality of a national point-of-care testing network for sexually transmitted infections integrated into rural and remote primary care clinics in Australia, 2016-2022: an observational program evaluation.

Background: To address inequitable diagnostic access and improve time-to-treatment for First Nations peoples, molecular point-of-care (POC) testing for chlamydia, gonorrhoea and trichomonas was integrated into 49 primary care clinics across Australia. We conducted an observational evaluation to determine clinical effectiveness and analytical quality of POC testing delivered through this national program. Methods: We evaluated (i) implementation by measuring trends in mean monthly POC testing; ii) clinical effectiveness by comparing proportions of positive patients treated by historical control/intervention period and by test type, and calculated infectious days averted; (iii) analytical quality by calculating result concordance by test type, and proportion of unsuccessful POC tests. Findings: Between 2016 and 2022, 46,153 POC tests were performed; an increasing mean monthly testing trend was observed in the first four years (p < 0.0001). A greater proportion of chlamydia/gonorrhoea positives were treated in intervention compared with historical control periods (≤2 days: 37% vs 22% [RR 1.68; 95% CI 1.12, 2.53]; ≤7 days: 48% vs 30% [RR 1.6; 95% CI 1.10, 2.33]; ≤120 days: 79% vs 54% [RR 1.46; 95% CI 1.10, 1.95]); similarly for trichomonas positives and by test type. POC testing for chlamydia, gonorrhoea and trichomonas averted 4930, 5620 and 7075 infectious days, respectively. Results concordance was high [99.0% (chlamydia), 99.3% (gonorrhoea) and 98.9% (trichomonas)]; unsuccessful POC test proportion was 1.8% for chlamydia/gonorrhoea and 2.1% for trichomonas. Interpretation: Molecular POC testing was successfully integrated into primary care settings as part of a routinely implemented program achieving significant clinical benefits with high analytical quality. In addition to the individual health benefits of earlier treatment, fewer infective days could contribute to reduced transmissions in First Nations communities. Funding: This work was supported by an Australian National Health and Medical Research Council Partnership Grant (APP1092503), the Australian Government Department of Health, Western Australia and Queensland Departments of Health.

Genomic Surveillance of Invasive Meningococcal Disease During a National MenW Outbreak in Australia, 2017-2018.

Background: In Australia, invasive meningococcal disease (IMD) incidence rapidly increased between 2014 and 2017 due to rising serogroup W (MenW) and MenY infections. We aimed to better understand the genetic diversity of IMD during 2017 and 2018 using whole genome sequencing data. Methods: Whole genome sequencing data from 440 Australian IMD isolates collected during 2017 and 2018 and 1737 international MenW:CC11 isolates collected in Europe, Africa, Asia, North America, and South America between 1974 and 2020 were used in phylogenetic analyses; genetic relatedness was determined from single-nucleotide polymorphisms. Results: Australian isolates were as follows: 181 MenW (41%), 144 MenB (33%), 88 MenY (20%), 16 MenC (4%), 1 MenW/Y (0.2%), and 10 nongenogroupable (2%). Eighteen clonal complexes (CCs) were identified, and 3 (CC11, CC23, CC41/44) accounted for 78% of isolates (343/440). These CCs were associated with specific serogroups: CC11 (n = 199) predominated among MenW (n = 181) and MenC (n = 15), CC23 (n = 80) among MenY (n = 78), and CC41/44 (n = 64) among MenB (n = 64). MenB isolates were highly diverse, MenY were intermediately diverse, and MenW and MenC isolates demonstrated the least genetic diversity. Thirty serogroup and CC-specific genomic clusters were identified. International CC11 comparison revealed diversification of MenW in Australia. Conclusions: Whole genome sequencing comprehensively characterized Australian IMD isolates, indexed their genetic variability, provided increased within-CC resolution, and elucidated the evolution of CC11 in Australia.

Defining the phylogenetics and resistome of the major Clostridioides difficile ribotypes circulating in Australia.

Clostridioides difficile infection (CDI) remains a significant public health threat globally. New interventions to treat CDI rely on an understanding of the evolution and epidemiology of circulating strains. Here we provide longitudinal genomic data on strain diversity, transmission dynamics and antimicrobial resistance (AMR) of C. difficile ribotypes (RTs) 014/020 (n=169), 002 (n=77) and 056 (n=36), the three most prominent C. difficile strains causing CDI in Australia. Genome scrutiny showed that AMR was uncommon in these lineages, with resistance-conferring alleles present in only 15/169 RT014/020 strains (8.9 %), 1/36 RT056 strains (2.78 %) and none of 77 RT002 strains. Notably, ~90 % of strains were resistant to MLSB agents in vitro, but only ~5.9 % harboured known resistance alleles, highlighting an incongruence between AMR genotype and phenotype. Core genome analyses revealed all three RTs contained genetically heterogeneous strain populations with limited evidence of clonal transmission between CDI cases. The average number of pairwise core genome SNP (cgSNP) differences within each RT group ranged from 23.3 (RT056, ST34, n=36) to 115.6 (RT002, ST8, n=77) and 315.9 (RT014/020, STs 2, 13, 14, 49, n=169). Just 19 clonal groups (encompassing 40 isolates), defined as isolates differing by ≤2 cgSNPs, were identified across all three RTs (RT014/020, n=14; RT002, n=3; RT056, n=2). Of these clonal groups, 63 % (12/19) comprised isolates from the same Australian State and 37 % (7/19) comprised isolates from different States. The low number of plausible transmission events found for these major RTs (and previously documented populations in animal and environmental sources/reservoirs) points to widespread and persistent community sources of diverse C. difficile strains as opposed to ongoing nationwide healthcare outbreaks dominated by a single clone. Together, these data provide new insights into the evolution of major lineages causing CDI in Australia and highlight the urgent need for enhanced surveillance, and for public health interventions to move beyond the healthcare setting and into a One Health paradigm to effectively combat this complex pathogen.