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Realising the promise of genomics to revolutionise routine AMR diagnosis and surveillance has been a long-standing challenge in clinical and public health microbiology. We have directly addressed this issue by creating and validating abritAMR, an ISO-accredited bioinformatics platform for genomics-based bacterial AMR gene detection. abritAMR utilises the NCBI AMRFinderPlus for detection of AMR genes and mutations, with additional features to classify AMR determinants into an antibiotic class. We validated abritAMR by comparing with multiplex PCR or gold-standard reference genomes, together representing 1500 different bacteria across 29 genera and covering 415 antibiotic resistance alleles. We also assessed inference of phenotypic resistance by comparing genomic predictions with agar dilution results for 864 Salmonella spp. Performance of abritAMR was excellent, detecting AMR genes with 99.9% accuracy (95% CI 99.9-99.9%), 97.9% sensitivity (95% CI 97.5-98.4%) and 100% specificity (100-100%). Phenotypic inference of resistance for Salmonella spp. was equally impressive, with 98.9% accuracy (98.7-99.1%). Validation data were submitted to the governing authority and ISO15189 accreditation was achieved. Implementation of abritAMR resulted in streamlined bioinformatics and reporting pathways, and it was readily updated and re-verified with database revisions or changes in reporting requirements. abritAMR is publicly and freely available to assist clinical and public health microbiology laboratories everywhere harness the power of AMR genomics in their professional practice.

Introduction The sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained. Methods VTM was prepared using different methods of sterilization then ‘spiked’ with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR. Results The best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4 °C, and tested within 48 h. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM was held at room temperature. Discussion The manual handling of VTM production, including filling and sterilization, was optimized. SARS-CoV-2 was spiked into VTM to assess different sterilization methods and measure the effects of storage time and temperature upon VTM performance. While most diagnostic protocols will not require replication competent virus, the use of high quality VTM will allow for the next phase of laboratory analysis in the COVID-19 pandemic, including drug and antibody susceptibility analysis of re-isolated SARS-CoV-2, and for the testing of vaccine escape mutants.

Healthcare workers are at increased risk of occupational transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report 2 instances of healthcare workers contracting SARS-CoV-2 despite no known breach of personal protective equipment. Additional specific equipment cleaning was initiated. Viral genomic sequencing supported this transmission hypothesis and our subsequent response.

Genomic sequencing has significant potential to inform public health management for SARS-CoV-2. Here we report high-throughput genomics for SARS-CoV-2, sequencing 80% of cases in Victoria, Australia (population 6.24 million) between 6 January and 14 April 2020 (total 1,333 COVID-19 cases). We integrate epidemiological, genomic and phylodynamic data to identify clusters and impact of interventions. The global diversity of SARS-CoV-2 is represented, consistent with multiple importations. Seventy-six distinct genomic clusters were identified, including large clusters associated with social venues, healthcare and cruise ships. Sequencing sequential samples from 98 patients reveals minimal intra-patient SARS-CoV-2 genomic diversity. Phylodynamic modelling indicates a significant reduction in the effective viral reproductive number (Re) from 1.63 to 0.48 after implementing travel restrictions and physical distancing. Our data provide a concrete framework for the use of SARS-CoV-2 genomics in public health responses, including its use to rapidly identify SARS-CoV-2 transmission chains, increasingly important as social restrictions ease globally., Genome sequencing can be used to infer pathogen transmission dynamics and inform public health responses. Here, the authors sequence >1,200 SARS-CoV-2 samples from Victoria, Australia and find genomic support for the effectiveness of social restrictions in reducing transmission.

The coronavirus disease pandemic has highlighted the utility of pathogen genomics as a key part of comprehensive public health response to emerging infectious diseases threats, however, the ability to generate, analyse, and respond to pathogen genomic data varies around the world. Papua New Guinea (PNG), which has limited in-country capacity for genomics, has experienced significant outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with initial genomics data indicating a large proportion of cases were from lineages that are not well defined within the current nomenclature. Through a partnership between in-country public health agencies and academic organisations, industry, and a public health genomics reference laboratory in Australia a system for routine SARS-CoV-2 genomics from PNG was established. Here we aim to characterise and describe the genomics of PNG’s second wave and examine the sudden expansion of a lineage that is not well defined but very prevalent in the Western Pacific region. We generated 1797 sequences from cases in PNG and performed phylogenetic and phylodynamic analyses to examine the outbreak and characterise the circulating lineages and clusters present. Our results reveal the rapid expansion of the B.1.466.2 and related lineages within PNG, from multiple introductions into the country. We also highlight the difficulties that unstable lineage assignment causes when using genomics to assist with rapid cluster definitions.

BackgroundCOVID-19 has resulted in many infections in healthcare workers (HCWs) globally. We performed state-wide SARS-CoV-2 genomic epidemiological investigations to identify HCW transmission dynamics and provide recommendations to optimise healthcare system preparedness for future outbreaks.MethodsGenome sequencing was attempted on all COVID-19 cases in Victoria, Australia. We combined genomic and epidemiologic data to investigate the source of HCW infections across multiple healthcare facilities (HCFs) in the state. Phylogenetic analysis and fine-scale hierarchical clustering were performed for the entire Victorian dataset including community and healthcare cases. Facilities provided standardised epidemiological data and putative transmission links.FindingsBetween March and October 2020, approximately 1,240 HCW COVID-19 infection cases were identified; 765 are included here. Genomic sequencing was successful for 612 (80%) cases. Thirty-six investigations were undertaken across 12 HCFs. Genomic analysis revealed that multiple introductions of COVID-19 into facilities (31/36) were more common than single introductions (5/36). Major contributors to HCW acquisitions included mobility of staff and patients between wards and facilities, and characteristics and behaviours of individual patients including super-spreading events. Key limitations at the HCF level were identified.InterpretationGenomic epidemiological analyses enhanced understanding of HCW infections, revealing unsuspected clusters and transmission networks. Combined analysis of all HCWs and patients in a HCF should be conducted, supported by high rates of sequencing coverage for all cases in the population. Established systems for integrated genomic epidemiological investigations in healthcare settings will improve HCW safety in future pandemics.FundingThe Victorian Government, the National Health and Medical Research Council Australia, and the Medical Research Future Fund.

Background The World Health Organization (WHO) coordinates the Global Invasive Bacterial Vaccine-Preventable Diseases (IB-VPD) Surveillance Network to support vaccine introduction decisions and use. The network was established to strengthen surveillance and laboratory confirmation of meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Methods Sentinel hospitals report cases of children Results From 2014 through 2019, >137 000 suspected meningitis cases were reported by 58 participating countries, with 44.6% (n = 61 386) reported from countries in the WHO African Region. More than half (56.6%, n = 77 873) were among children Conclusions This multitier, global surveillance network has supported countries in detecting and serotyping the 3 principal invasive bacterial pathogens that cause pediatric meningitis. Streptococcus pneumoniae was the most common bacterial pathogen detected globally despite the growing number of countries that have nationally introduced PCV. The large proportions of deaths due to S. pneumoniae reflect the high proportion of meningitis cases caused by this pathogen. This global network demonstrated a strong correlation between PCV introduction status and reduction in the proportion of pneumococcal meningitis infections caused by vaccine serotypes. Maintaining case-based, active surveillance with laboratory confirmation for prioritized vaccine-preventable diseases remains a critical component of the global agenda in public health. The World Health Organization (WHO)-coordinated Invasive Bacterial Vaccine-Preventable Disease (IB-VPD) Surveillance Network reported data from 2014 to 2019, contributing to the estimates of the disease burden and serotypes of pediatric meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.

We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific immune responses in a patient with lymphoma and recent programmed death 1 (PD-1) inhibitor therapy with late onset of severe coronavirus disease 2019 disease and prolonged SARS-CoV-2 replication, in comparison to age-matched and immunocompromised controls. High levels of HLA-DR+/CD38+ activation, interleukin 6, and interleukin 18 in the absence of B cells and PD-1 expression was observed. SARS-CoV-2–specific antibody responses were absent and SARS-CoV-2–specific T cells were minimally detected. This case highlights challenges in managing immunocompromised hosts who may fail to mount effective virus-specific immune responses., We describe defects in severe acute respiratory syndrome coronavirus 2–specific immune responses and persistent viral replication in a patient with lymphoma. This case highlights challenges in managing immunocompromised hosts who may fail to mount effective virus-specific immune responses.

Chlamydia abortus, the aetiological agent of enzootic abortion of ewes, is a major cause of reproductive loss in small ruminants worldwide, accounting for significant economic losses to the farming industry. Disease can be managed through the use of commercial inactivated or live whole organism-based vaccines, although both have limitations particularly in terms of efficacy, safety and disease-associated outbreaks. Here we report a comparison of two experimental vaccines (chlamydial outer membrane complex (COMC) and octyl glucoside (OG)-COMC) based on detergent extracted outer membrane preparations of C. abortus and delivered as prime-boost immunisations, with the commercial live vaccine Cevac® Chlamydia in a pregnant sheep challenge model. No abortions occurred in either experimental vaccine group, while a single abortion occurred in the commercial vaccine group. Bacterial shedding, as a measure of potential risk of transmission of infection to naïve animals, was lowest in the COMC vaccinated group, with reductions of 87.5%, 86.4% and 74% observed for the COMC, OG-COMC and live commercial vaccine groups, respectively, compared to the unvaccinated challenge control group. The results show that the COMC vaccine performed the best and is a safer efficacious alternative to the commercial vaccines. However, to improve commercial viability, future studies should optimise the antigen dose and number of inoculations required.

Objective To compare the concordance and acceptability of saliva testing with standard‐of‐care oropharyngeal and bilateral deep nasal swab testing for severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) in children and in general practice. Design Prospective multicentre diagnostic validation study. Setting Royal Children’s Hospital, and two general practices (cohealth, West Melbourne; Cirqit Health, Altona North) in Melbourne, July–October 2020. Participants 1050 people who provided paired saliva and oropharyngeal‐nasal swabs for SARS‐CoV‐2 testing. Main outcome measures Numbers of cases in which SARS‐CoV‐2 was detected in either specimen type by real‐time polymerase chain reaction; concordance of results for paired specimens; positive percent agreement (PPA) for virus detection, by specimen type. Results SARS‐CoV‐2 was detected in 54 of 1050 people with assessable specimens (5%), including 19 cases (35%) in which both specimens were positive. The overall PPA was 72% (95% CI, 58–84%) for saliva and 63% (95% CI, 49–76%) for oropharyngeal‐nasal swabs. For the 35 positive specimens from people aged 10 years or more, PPA was 86% (95% CI, 70–95%) for saliva and 63% (95% CI, 45–79%) for oropharyngeal‐nasal swabs. Adding saliva testing to standard‐of‐care oropharyngeal‐nasal swab testing increased overall case detection by 59% (95% CI, 29–95%). Providing saliva was preferred to an oropharyngeal‐nasal swab by most participants (75%), including 141 of 153 children under 10 years of age (92%). Conclusion In children over 10 years of age and adults, saliva testing alone may be suitable for SARS‐CoV‐2 detection, while for children under 10, saliva testing may be suitable as an adjunct to oropharyngeal‐nasal swab testing for increasing case detection.

Chlamydia abortus, the aetiological agent of enzootic abortion of ewes, is a major cause of reproductive loss in small ruminants worldwide, accounting for significant economic losses to the farming industry. Disease can be managed through the use of commercial inactivated or live whole organism-based vaccines, although both have limitations particularly in terms of efficacy, safety and disease-associated outbreaks. Here we report a comparison of two experimental vaccines (chlamydial outer membrane complex (COMC) and octyl glucoside (OG)-COMC) based on detergent extracted outer membrane preparations of C. abortus and delivered as prime-boost immunisations, with the commercial live vaccine Cevac® Chlamydia in a pregnant sheep challenge model. No abortions occurred in either experimental vaccine group, while a single abortion occurred in the commercial vaccine group. Bacterial shedding, as a measure of potential risk of transmission of infection to naïve animals, was lowest in the COMC vaccinated group, with reductions of 87.5%, 86.4% and 74% observed for the COMC, OG-COMC and live commercial vaccine groups, respectively, compared to the unvaccinated challenge control group. The results show that the COMC vaccine performed the best and is a safer efficacious alternative to the commercial vaccines. However, to improve commercial viability, future studies should optimise the antigen dose and number of inoculations required.

Summary: Background: A cornerstone of Australia's ability to control COVID-19 has been effective border control with an extensive supervised quarantine programme. However, a rapid recrudescence of COVID-19 was observed in the state of Victoria in June, 2020. We aim to describe the genomic findings that located the source of this second wave and show the role of genomic epidemiology in the successful elimination of COVID-19 for a second time in Australia. Methods: In this observational, genomic epidemiological study, we did genomic sequencing of all laboratory-confirmed cases of COVID-19 diagnosed in Victoria, Australia between Jan 25, 2020, and Jan 31, 2021. We did phylogenetic analyses, genomic cluster discovery, and integrated results with epidemiological data (detailed information on demographics, risk factors, and exposure) collected via interview by the Victorian Government Department of Health. Genomic transmission networks were used to group multiple genomic clusters when epidemiological and genomic data suggested they arose from a single importation event and diversified within Victoria. To identify transmission of emergent lineages between Victoria and other states or territories in Australia, all publicly available SARS-CoV-2 sequences uploaded before Feb 11, 2021, were obtained from the national sequence sharing programme AusTrakka, and epidemiological data were obtained from the submitting laboratories. We did phylodynamic analyses to estimate the growth rate, doubling time, and number of days from the first local infection to the collection of the first sequenced genome for the dominant local cluster, and compared our growth estimates to previously published estimates from a similar growth phase of lineage B.1.1.7 (also known as the Alpha variant) in the UK. Findings: Between Jan 25, 2020, and Jan 31, 2021, there were 20 451 laboratory-confirmed cases of COVID-19 in Victoria, Australia, of which 15 431 were submitted for sequencing, and 11 711 met all quality control metrics and were included in our analysis. We identified 595 genomic clusters, with a median of five cases per cluster (IQR 2–11). Overall, samples from 11 503 (98·2%) of 11 711 cases clustered with another sample in Victoria, either within a genomic cluster or transmission network. Genomic analysis revealed that 10 426 cases, including 10 416 (98·4%) of 10 584 locally acquired cases, diagnosed during the second wave (between June and October, 2020) were derived from a single incursion from hotel quarantine, with the outbreak lineage (transmission network G, lineage D.2) rapidly detected in other Australian states and territories. Phylodynamic analyses indicated that the epidemic growth rate of the outbreak lineage in Victoria during the initial growth phase (samples collected between June 4 and July 9, 2020; 47·4 putative transmission events, per branch, per year [1/years; 95% credible interval 26·0–85·0]), was similar to that of other reported variants, such as B.1.1.7 in the UK (mean approximately 71·5 1/years). Strict interventions were implemented, and the outbreak lineage has not been detected in Australia since Oct 29, 2020. Subsequent cases represented independent international or interstate introductions, with limited local spread. Interpretation: Our study highlights how rapid escalation of clonal outbreaks can occur from a single incursion. However, strict quarantine measures and decisive public health responses to emergent cases are effective, even with high epidemic growth rates. Real-time genomic surveillance can alter the way in which public health agencies view and respond to COVID-19 outbreaks. Funding: The Victorian Government, the National Health and Medical Research Council Australia, and the Medical Research Future Fund.

Resistance-guided therapy (RGT) for gonorrhoea may reduce unnecessary use of broad-spectrum antibiotics. When reflexed from the Aptima Combo 2 assay, the ResistancePlus GC assay demonstrated 94.8% sensitivity and 100.0% specificity for Neisseria gonorrhoeae detection., Resistance-guided therapy (RGT) for gonorrhea may reduce unnecessary use of broad-spectrum antibiotics. When reflexed from the Aptima Combo 2 assay, the ResistancePlus GC assay demonstrated 94.8% sensitivity and 100.0% specificity for Neisseria gonorrhoeae detection. Of the 379 concordant N. gonorrhoeae-positive samples, 86.8% were found to possess the gyrA S91F mutation, which was highly predictive for ciprofloxacin resistance and stable across 3,144 publicly available N. gonorrhoeae genomes. Our work supports the feasibility of implementing RGT for gonorrhea into routine molecular workflows.

BACKGROUND: A cornerstone of Australia’s ability to control COVID-19 has been effective border control, using an extensive supervised quarantine program. However, a rapid recrudescence in COVID-19 cases was observed in the state of Victoria in June 2020. Here, we describe the genomic findings that located the source of this second wave as a breach in supervised hotel quarantine and demonstrate the successful elimination of COVID-19 for a second time in Australia. METHODS: Genome sequencing was performed on all available SARS-CoV-2-positive samples in Victoria and integrated genomic and epidemiological investigation undertaken. RESULTS: At 31st January 2021, 20,451 COVID-19 cases were reported in Victoria; samples were sequenced from 75% of cases (15,431/20,451). Genomics revealed 98% (10,426/10,646) of locally-acquired cases during the second wave were derived from a single incursion from hotel quarantine, with the outbreak strain rapidly detected in other Australian states and territories. Phylodynamic analyses indicated an epidemic growth rate comparable to emerging variants, such as B.1.1.7 in the United Kingdom. Strict public health interventions resulted in the elimination of the outbreak strain by 29th October 2020. Subsequent cases represented independent international or interstate introductions, with limited local spread. CONCLUSIONS: Rapid escalation of clonal outbreaks can occur from even a single breach of control practices, as revealed through our genomic ‘enhanced outbreak-detection' system. The subsequent elimination and rapid control of new SARS-CoV-2 incursions reinforce that decisive public health responses to emergent cases are effective even with high epidemic growth rates, and “elimination” should be favored in settings where this is achievable. FUNDING STATEMENT: The Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL) and the Victorian Infectious Diseases Reference Laboratory (VIDRL) at The Doherty Institute are funded by the Victorian Government. This work was supported by the National Health and Medical Research Council, Australia (NHMRC); Partnership Grant (APP1149991), Investigator Grant to BPH (APP1196103), Investigator Grant to DAW (APP1174555), Research Fellowship to TPS (APP1105525), MRFF COVID-19 Genomics Grant (MRF9200006). DECLARATION OF INTERESTS: None to declare. ETHICS APPROVAL STATEMENT: Data were collected in accordance with the Victorian Public Health and Wellbeing Act 2008. Ethical approval was received from the University of Melbourne Human Research Ethics Committee (study number 1954615.3).

Background: In Australia, COVID-19 diagnosis relies on RT-PCR testing which is relatively costly and time-consuming. To date, few studies have assessed the performance and implementation of rapid antigen-based SARS-CoV-2 testing in a setting with a low prevalence of COVID-19 infections, such as Australia. Methods: This study recruited participants presenting for COVID-19 testing at three Melbourne metropolitan hospitals during a period of low COVID-19 prevalence. The Abbott PanBioTM COVID-19 Ag point-of-care test was performed alongside RT-PCR. In addition, participants with COVID-19 notified to the Victorian Government were invited to provide additional swabs to aid validation. Implementation challenges were also documented. Findings: The specificity of the Abbott PanBioTM COVID-19 Ag test was 99.96% (95% CI 99.73 - 100%). Sensitivity amongst participants with RT-PCR-confirmed infection was dependent upon the duration of symptoms reported, ranging from 77.3% (duration 1 to 33 days) to 100% in those within seven days of symptom onset. A range of implementation challenges were identified which may inform future COVID-19 testing strategies in a low prevalence setting. Interpretation: Given the high specificity, antigen-based tests may be most useful in rapidly triaging public health and hospital resources while expediting confirmatory RT-PCR testing. Considering the limitations in test sensitivity and the potential for rapid transmission in susceptible populations, particularly in hospital settings, careful consideration is required for implementation of antigen testing in a low prevalence setting. Funding: This work was funded by the Victorian Department of Health and Human Services. The funder was not involved in data analysis or manuscript preparation.

IntroductionThe sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained.MethodsVTM was prepared using different methods of sterilization then ‘spiked’ with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR (qPCR).ResultsThe best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4oC, and tested within 48 hours. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM was held at room temperature (RT).DiscussionThe manual handling of VTM production, including filling and sterilization, was optimized. SARS-CoV-2 was spiked into VTM to assess different sterilization methods and measure the effects of storage time and temperature upon VTM performance. While most diagnostic protocols will not require replication competent virus, the use of high quality VTM will allow for the next phase of laboratory analysis in the COVID-19 pandemic, including drug and antibody susceptibility analysis of re-isolated SARS--CoV-2, and for the testing of vaccine escape mutants.

2.AbstractIntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 μL of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65°C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals.ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD ±7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.3.Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Background Multiresistant organisms (MROs) pose a critical threat to public health. Population-based programs for control of MROs such as carbapenemase-producing Enterobacterales (CPE) have emerged and evaluation is needed. We assessed the feasibility and impact of a statewide CPE surveillance and response program deployed across Victoria, Australia (population 6.5 million). Methods A prospective multimodal intervention including active screening, carrier isolation, centralized case investigation, and comparative pathogen genomics was implemented. We analyzed trends in CPE incidence and clinical presentation, risk factors, and local transmission over the program’s first 3 years (2016–2018). Results CPE case ascertainment increased over the study period to 1.42 cases/100 000 population, linked to increased screening without a concomitant rise in active clinical infections (0.45–0.60 infections/100 000 population, P = .640). KPC-2 infection decreased from 0.29 infections/100 000 population prior to intervention to 0.03 infections/100 000 population in 2018 (P = .003). Comprehensive case investigation identified instances of overseas community acquisition. Median time between isolate referral and genomic and epidemiological assessment for local transmission was 11 days (IQR, 9–14). Prospective surveillance identified numerous small transmission networks (median, 2; range, 1–19 cases), predominantly IMP and KPC, with median pairwise distance of 8 (IQR, 4–13) single nucleotide polymorphisms; low diversity between clusters of the same sequence type suggested genomic cluster definitions alone are insufficient for targeted response. Conclusions We demonstrate the value of centralized CPE control programs to increase case ascertainment, resolve risk factors, and identify local transmission through prospective genomic and epidemiological surveillance; methodologies are transferable to low-prevalence settings and MROs globally., A statewide multimodal intervention for the control of carbapenemase-producing Enterobacterales was associated with a significant increase in case ascertainment, with no rise in clinical infections. Timely prospective epidemiological and genomic surveillance identified numerous small local transmission networks permitting rapid response.

Background: Multi-resistant organisms (MROs) pose a critical threat to public health. Recently, population-based systems for surveillance and control of MROs such as Carbapenemase-producing Enterobacterales (CPE) have emerged globally and evaluation is needed. In this study, we assess the feasibility and impact of a state-wide CPE surveillance and response program deployed in December 2015 across Victoria, Australia (population 6·5 million). Methods: A prospective population-based multi-modal intervention, including active screening, carrier isolation, centralised case investigation and comparative pathogen genomics was implemented. We assess impact by analysing trend in CPE incidence and clinical presentation, risk factors for acquisition and local transmission, using data from the first three years of implementation (January 2016 to December 2018). Findings: CPE case ascertainment increased over the study period to 1·42 cases/100,000 population, linked to increased screening without a concomitant rise in active clinical infections (0·45-0·60 infections/100,000 population, p=0·640). Occurrence of KPC-2 infection decreased from 0·29 infections/100,000 population prior to intervention to 0·03 infections/100,000 population in 2018 (p=0·003). Comprehensive case investigation enabled identification of novel risk factors, including non-healthcare-related overseas acquisition. Median time between isolate referral and initial centralised genomic and epidemiological assessment for local transmission was 11 days (IQR 9-14). Prospective surveillance identified numerous small transmission networks (median 2, range 1-19 cases), predominantly IMP- and KPC-producing organisms. Genomic and epidemiologically defined clusters had a median pairwise distance of 8 (IQR 4-13) single nucleotide polymorphisms, however low diversity between clusters of the same sequence type suggested genomic cluster definitions alone are insufficient for targeted control measures. Interpretation: Our results demonstrate the value of centralised CPE surveillance and control programs to increase case ascertainment, resolve risk factors and identify putative local transmission through combined genomic and epidemiological surveillance; methodologies are transferable to other low-prevalence settings and MROs globally. Funding Statement: National Health and Medical Research Council (NHMRC) partnership grant (GNT1149991) and the Victorian Government. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All data were collected in accordance with the Victorian Public Health and Wellbeing Act 2008. Ethical approval was obtained from the Human Ethics Advisory Group of the School of Biomedical Sciences, University of Melbourne (Ethics ID 1954615.2).

Background: In Australia, COVID-19 diagnosis relies on RT-PCR testing which is relatively costly and time-consuming. To date, no studies have assessed the performance and implementation of rapid antigen-based SARS-CoV-2 testing in a setting with a low prevalence of COVID-19 infections, such as Australia. Methods: This study recruited participants presenting for COVID-19 testing at three Melbourne metropolitan hospitals during a period of low COVID-19 prevalence. The Abbott PanBioTM COVID-19 Ag point-of-care test was performed alongside RT-PCR. In addition, participants with COVID-19 notified to the Victorian Government were invited to provide additional swabs to aid validation. Implementation challenges were also documented. Findings: The specificity of the Abbott PanBioTM COVID-19 Ag test was 99.96% (95% CI 99.73 - 100%). Sensitivity amongst participants with RT-PCR-confirmed infection was dependent upon the duration of symptoms reported, ranging from 78.9% (duration 1 to 33 days) to 100% in those within 7 days of symptom onset. A range of implementation challenges were identified which may inform future COVID-19 testing strategies in a low prevalence setting. Interpretation: Given the high specificity, antigen-based tests may be most useful in rapidly triaging public health and hospital resources while expediting confirmatory RT-PCR testing. Considering the limitations in test sensitivity and the potential for rapid transmission in susceptible populations, particularly in hospital settings, careful consideration is required for implementation of antigen testing in a low prevalence setting. Funding: This work was funded by the Victorian Department of Health and Human Services. The funder was not involved in data analysis or manuscript preparation. Declaration of Interests: All authors: no conflicts. Ethics Approval Statement: Ethics review and study approval was provided by Monash Health Human Research and Ethics Committee (RES-20-0000-678A) and local Governance approval was provided by Melbourne Health and Austin Health Offices for Research.

The shedding patterns of Salmonella spp. and MLVA profiles of Salmonella enterica subspecies enterica (I) serotype 1,4,[5],12:i:- were monitored in a 12-month longitudinal observational study of five pig herds to inform management; provide indications of potential hazard load at slaughter; and assist evaluation of MLVA for use by animal and public health practitioners. Twenty pooled faecal samples, stratified by age group, were collected quarterly. When Salmonella was cultured, multiple colonies were characterized by serotyping and where S. Typhimurium-like serovars were confirmed, isolates were further characterized by phage typing and multiple locus variable number tandem repeat analysis (MLVA). Salmonella was detected in 43% of samples. Salmonella 1,4,[5],12:i- was one of several serovars that persisted within the herds and was found among colonies from each production stage. Virtually all Salmonella 1,4,[5],12:i:- isolates were phage type 193, but exhibited 12 different, closely-related MLVA profiles. Salmonella 1,4,[5],12:i:- diversity within herds was low and MLVA profiles were stable indicating colonization throughout the herds and suggesting each farm had an endemic strain. High prevalence of S. 1,4,[5],12:i:- specific shedding among terminal animals indicated high hazard load at slaughter, suggesting that primary production may be an important pathway of S. 1,4,[5],12:i:- into the human food chain, this has implications for on-farm management and the application and targeting control measures and further evidence of the need for effective process control procedures to be in place during slaughter and in pork boning rooms. These findings have implications for animal health and food safety risk mitigation and risk management.

Carbapenemase-producing Enterobacterales (CPE) are being increasingly reported in Australia, and integrated clinical and genomic surveillance is critical to effectively manage this threat. We sought to systematically characterize CPE in Victoria, Australia, from 2012 to 2016., Carbapenemase-producing Enterobacterales (CPE) are being increasingly reported in Australia, and integrated clinical and genomic surveillance is critical to effectively manage this threat. We sought to systematically characterize CPE in Victoria, Australia, from 2012 to 2016. Suspected CPE were referred to the state public health laboratory in Victoria, Australia, from 2012 to 2016 and examined using phenotypic, multiplex PCR and whole-genome sequencing (WGS) methods and compared with epidemiological metadata. Carbapenemase genes were detected in 361 isolates from 291 patients (30.8% of suspected CPE isolates), mostly from urine (42.1%) or screening samples (34.8%). IMP-4 (28.0% of patients), KPC-2 (25.3%), NDM (24.1%), and OXA carbapenemases (22.0%) were most common. Klebsiella pneumoniae (48.8% of patients) and Escherichia coli (26.1%) were the dominant species. Carbapenemase-inactivation method (CIM) testing reliably detected carbapenemase-positive isolates (100% sensitivity, 96.9% specificity), identifying an additional five CPE among 159 PCR-negative isolates (IMI and SME carbapenemases). When epidemiologic investigations were performed, all pairs of patients designated “highly likely” or “possible” local transmission had ≤23 pairwise single-nucleotide polymorphisms (SNPs) by genomic transmission analysis; conversely, all patient pairs designated “highly unlikely” local transmission had ≥26 pairwise SNPs. Using this proposed threshold, possible local transmission was identified involving a further 16 patients for whom epidemiologic data were unavailable. Systematic application of genomics has uncovered the emergence of polyclonal CPE as a significant threat in Australia, providing important insights to inform local public health guidelines and interventions. Using our workflow, pairwise SNP distances between CPE isolates of ≤23 SNPs suggest local transmission.

Improved knowledge of factors that promote outbreaks of enteric pathogens among men who have sex with men (MSM) could enable targeted public health interventions. We detected enteric pathogens in 57 of 519 (11%) asymptomatic MSM, and we found that enteric pathogen detection was associated with both oroanal sex (rimming) and group sex.

Invasive Group A Streptococcus (iGAS) disease can cause permanent disability and death. The incidence of iGAS has increased in many developed countries since the 1980s. iGAS disease is not nationally notifiable in Australia or at the state level in Victoria. The Victorian Hospital Pathogen Surveillance Scheme (VHPSS) is a voluntary laboratory-based surveillance system established in 1988. We assessed the trends and molecular epidemiology of iGAS disease in Victoria from 2007-2017.A case of iGAS was defined as an individual for whom Group A Streptococcus (GAS) was isolated from a normally sterile body site. Data on all iGAS cases, as reported to the VHPSS, between 1 January 2007 and 31 December 2017 were examined.A total of 1,311 iGAS cases had associated isolates, and M Protein Gene (emm) typing was performed for 91.6%. The mean annual incidence was 2.1 (95% CI: 1.8-2.5) per 100,000 population per year, increasing 2.7-fold over the study period. In total, 140 different iGAS emm-types were observed, with the ten most prevalent types comprising 63.1% of the sample.Despite limitations in this surveillance data, we observed increasing rates of iGAS disease in Victoria. iGAS incidence exceeded the mean annual incidence for invasive meningococcal disease, calculated using Victorian data from the National Notifiable Diseases Surveillance System (2.1 vs. 0.6 cases per 100,000 population per year, respectively). Mandatory case notification could enhance disease control and prevention. Further, the diversity in emm-types emphasises the importance of effective secondary chemoprophylaxis in prevention, alongside GAS vaccine development.

Background Invasive Group A Streptococcus (iGAS) disease can cause permanent disability and death. The incidence of iGAS has increased in many developed countries since the 1980s. iGAS disease is not nationally notifiable in Australia or at the state level in Victoria. The Victorian Hospital Pathogen Surveillance Scheme (VHPSS) is a voluntary laboratory-based surveillance system established in 1988. We assessed the trends and molecular epidemiology of iGAS disease in Victoria from 2007-2017. Methods A case of iGAS was defined as an individual for whom Group A Streptococcus (GAS) was isolated from a normally sterile body site. Data on all iGAS cases, as reported to the VHPSS, between 1 January 2007 and 31 December 2017 were examined. Results A total of 1,311 iGAS cases had associated isolates, and M Protein Gene (emm) typing was performed for 91.6%. The mean annual incidence was 2.1 (95% CI: 1.8-2.5) per 100,000 population per year, increasing 2.7-fold over the study period. In total, 140 different iGAS emm-types were observed, with the ten most prevalent types comprising 63.1% of the sample. Conclusions Despite limitations in this surveillance data, we observed increasing rates of iGAS disease in Victoria. iGAS incidence exceeded the mean annual incidence for invasive meningococcal disease, calculated using Victorian data from the National Notifiable Diseases Surveillance System (2.1 vs. 0.6 cases per 100,000 population per year, respectively). Mandatory case notification could enhance disease control and prevention. Further, the diversity in emm-types emphasises the importance of effective secondary chemoprophylaxis in prevention, alongside GAS vaccine development.

Background The live, temperature-attenuated vaccine strain 1B of Chlamydia abortus, the aetiological agent of ovine enzootic abortion (OEA), has been implicated in cases of vaccine breakdown. The aim of this study was to understand the nature of this attenuation through sequencing of the vaccine parent strain (AB7) and the derived mutant strains 1B and 1H, as well as to clarify the role of the vaccine strain in causing disease through comparative whole genome analysis. Methods Whole genome sequencing was performed on: vaccine parent strain AB7; N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-induced temperature attenuated mutant strain 1B grown from the commercial live vaccines Cevac Chlamydia and Enzovax; strain 1H a reverted NTG mutant; and 5 strains isolated from cases of OEA originating from animals from the original vaccine safety trial (2 strains) or from vaccinated ewes or ewes exposed to vaccinated animals (3 strains). Results We confirmed that AB7 is in a different lineage from the reference strain S26/3. The genome of vaccine strain 1B contains ten single nucleotide polymorphisms (SNPs) created by the NTG treatment, which are identical to those found in strain 1H. The strains from OEA cases also cluster phylogenetically very tightly with these vaccine strains. Conclusions The results show that C. abortus vaccine strain 1B has an identical genome sequence to the non-attenuated “reverted mutant” strain 1H. Thus, the protection of the 1B vaccine is unlikely to be due to the NTG induced SNPs and is more likely caused by the administration of high doses of C. abortus elementary bodies that stimulate protective immunity. Vaccine-identical strains were also isolated from cases of disease, as well as strains which had acquired 1–3 SNPs, including an animal that had not been vaccinated with either of the commercial live OEA vaccines, indicating that the 1B vaccine strain may be circulating and causing disease.

In Australia, the incidence of Salmonella Typhimurium has increased dramatically over the past decade. Whole-genome sequencing (WGS) is transforming public health microbiology, but poses challenges for surveillance. To compare WGS-based approaches with conventional typing for Salmonella surveillance, we performed concurrent WGS and multilocus variable-number tandem-repeat analysis (MLVA) of Salmonella Typhimurium isolates from the Australian Capital Territory (ACT) for a period of 5 months. We exchanged data via a central shared virtual machine and performed comparative genomic analyses. Epidemiological evidence was integrated with WGS-derived data to identify related isolates and sources of infection, and we compared WGS data for surveillance with findings from MLVA typing. We found that WGS data combined with epidemiological data linked an additional 9% of isolates to at least one other isolate in the study in contrast to MLVA and epidemiological data, and 19% more isolates than epidemiological data alone. Analysis of risk factors showed that in one WGS-defined cluster, human cases had higher odds of purchasing a single egg brand. While WGS was more sensitive and specific than conventional typing methods, we identified barriers to uptake of genomic surveillance around complexity of reporting of WGS results, timeliness, acceptability, and stability. In conclusion, WGS offers higher resolution of Salmonella Typhimurium laboratory surveillance than existing methods and can provide further evidence on sources of infection in case and outbreak investigations for public health action. However, there are several challenges that need to be addressed for effective implementation of genomic surveillance in Australia.

BackgroundUntil recently,Klebsiella pneumoniaecarbapenemase (KPC)-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.MethodsAll KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids.ResultsInitial investigations indicated that the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates (K. pneumoniae92%) identified from 35 different locations across metropolitan and rural Victoria between 2012 and 2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host diversity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation.ConclusionA real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase-producing Enterobacteriaceae surveillance and management.

BackgroundUntil recently, KPC-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.Methods and FindingsAll KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing (WGS) was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids. Initial investigations indicated the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates (K. pneumoniae92%) identified from 35 different locations across metropolitan and rural Victoria between 2012-2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host diversity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation.ConclusionsA real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase producing Enterobacteriaceae management.

Background Chlamydia abortus (formerly Chlamydophila abortus) is an economically important livestock pathogen, causing ovine enzootic abortion (OEA), and can also cause zoonotic infections in humans affecting pregnancy outcome. Large-scale genomic studies on other chlamydial species are giving insights into the biology of these organisms but have not yet been performed on C. abortus. Our aim was to investigate a broad collection of European isolates of C. abortus, using next generation sequencing methods, looking at diversity, geographic distribution and genome dynamics. Results Whole genome sequencing was performed on our collection of 57 C. abortus isolates originating primarily from the UK, Germany, France and Greece, but also from Tunisia, Namibia and the USA. Phylogenetic analysis of a total of 64 genomes shows a deep structural division within the C. abortus species with a major clade displaying limited diversity, in addition to a branch carrying two more distantly related Greek isolates, LLG and POS. Within the major clade, seven further phylogenetic groups can be identified, demonstrating geographical associations. The number of variable nucleotide positions across the sampled isolates is significantly lower than those published for C. trachomatis and C. psittaci. No recombination was identified within C. abortus, and no plasmid was found. Analysis of pseudogenes showed lineage specific loss of some functions, notably with several Pmp and TMH/Inc proteins predicted to be inactivated in many of the isolates studied. Conclusions The diversity within C. abortus appears to be much lower compared to other species within the genus. There are strong geographical signatures within the phylogeny, indicating clonal expansion within areas of limited livestock transport. No recombination has been identified within this species, showing that different species of Chlamydia may demonstrate different evolutionary dynamics, and that the genome of C. abortus is highly stable. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3657-y) contains supplementary material, which is available to authorized users.

This study investigated the pathogenesis of two variant strains (LLG and POS) of Chlamydia abortus, in comparison to a typical wild-type strain (S26/3) which is known to be responsible for late term abortion in small ruminants. Challenge with the three strains at mid-gestation resulted in similar pregnancy outcomes, with abortion occurring in approximately 50–60% of ewes with the mean gestational lengths also being similar. However, differences were observed in the severity of placental pathology, with infection appearing milder for strain LLG, which was reflected in the lower number of organisms shed in vaginal swabs post-partum and less gross pathology and organisms present in placental smears. Results for strain POS were somewhat different than LLG with a more focal restriction of infection observed. Post-abortion antibody responses revealed prominent differences in seropositivity to the major outer membrane protein (MOMP) present in elementary body (EB) preparations under denaturing conditions, most notably with anti-LLG and anti-POS convalescent sera where there was no or reduced detection of MOMP present in EBs derived from the three strains. These results and additional analysis of whole EB and chlamydial outer membrane complex preparations suggest that there are conformational differences in MOMP for the three strains. Overall, the results suggest that gross placental pathology and clinical outcome is not indicative of bacterial colonization and the severity of infection. The results also highlight potential conformational differences in MOMP epitopes that perhaps impact on disease diagnosis and the development of new vaccines.

Porcine proliferative enteropathy (PPE) caused by the bacterium Lawsonia intracellularis causes considerable economic loss to the pig industry. The objective of this study was to evaluate the seroprevalence of L. intracellularis exposure in different age groups of pigs (growers to finishers) within English farms and to identify potential risk factors. Samples were obtained in a cross-sectional study of 147 farms between 2008 and 2009. Twelve samples (six growers and six finishers) from each farm were tested for L. intracellularis by antibody ELISA. At animal level there was a significant positive linear trend between seroprevalence and age in weeks (r(2)=2.65, P0.001), with seroprevalence lowest (24.73%) at 11 weeks and highest (93.33%) at 24 weeks. At farm level, seroprevalence was significantly lower in growers than finishers (56.80% vs. 94.26%, P0.001). Farms reporting minor Salmonella problems and those that brought boars onto the farm had higher odds of testing positive in growers (OR 5.69 and 4.31, respectively. On the other hand, farms where producers considered temperature as an important stress factor (OR=0.3) and which had more than two sites on which pigs are kept (OR=0.16) were less likely to test positive in growers. The current study confirmed the high prevalence of L. intracellularis in English pig farms. The potential risk factors and further information of the disease impact on the farm productivity will aid the development of appropriate control strategies through better understanding of the disease.

Members of the familyMicroviridaehave been identified in a number of chlamydial species infecting humans (phage CPAR39 inChlamydophila pneumoniae), other mammals (φCPG1 inChlamydophila caviae, Chp2 inChlamydophila abortusand Chp3 inChlamydophila pecorum) and birds (Chp1 inChlamydophila psittaci). This study describes the identification and genome sequencing of Chp4, an icosahedral, 4530 bp, ssDNA phage inC. abortus. Chp4 is predicted to contain eight ORFs, six of which could be assigned putative functions based on sequence similarity to characterized bacteriophage. Gene order and content were highly conserved amongst chlamydiaphage, with the highest sequence variability occurring in the IN5 and INS variable regions of the VP1 major coat protein, which has been associated with host cell recognition and binding. Phylogenetic analysis of VP1 indicated that Chp4 is a member of theChlamydiamicrovirus, and is most closely related to phage φCPG1 and CPAR39.

The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria , the Cyanobacteria , and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N 2 fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

Only one isolate each of the class “ Spartobacteria ” (subdivision 2 of the phylum Verrucomicrobia ) and of subdivision 3 of Verrucomicrobia have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class “ Spartobacteria ” and were related to “ Chthoniobacter flavus .” Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class “ Spartobacteria ” were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class “ Spartobacteria ” were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class “ Spartobacteria ” was inhibited by other culturable bacteria.

The culturability of microorganisms in a 10 cm core of an Australian pasture soil was investigated using a minimal agar medium with xylan as the growth substrate. Culturability decreased with increasing depth, from a maximum of 19% of the total microscopically countable cells in the 0-2 cm section to 2.4% in the 8-10 cm section. Seventy-one isolates from the core were identified by comparative 16S rRNA gene sequence analysis. Many of these isolates belong to groups of globally distributed soil bacteria, including well-characterized families of the classes Alphaproteobacteria and Betaproteobacteria, and of the subclass Actinobacteridae. Other isolates belong to groups with few or no cultivated representatives: 10 isolates in two subdivisions of the phylum Acidobacteria, five isolates in a new order and nine isolates in a new family of the class Alphaproteobacteria, two isolates in a new order of the class Gammaproteobacteria, three isolates in two new families of the subclass Actinobacteridae, and two isolates in the subclass Rubrobacteridae. These new isolates represent the first laboratory cultures able to be assigned to some of these groups and greatly increase the number of cultivated strains known for others. This demonstrates that a minimal change in cultivation strategy (using a polymeric growth substrate and longer incubation times) can result in the isolation of globally distributed but previously uncultured phylogenetically novel soil bacteria.

Lawsonia intracellularis is the etiological agent of proliferative enteropathy (PE), causing mild or acute hemorrhagic diarrhea in infected animals. Here we report the genome sequence of strain N343, isolated from a sow that died of hemorrhagic PE. N343 contains 24 single nucleotide polymorphisms and 90 indels compared to the reference strain PHE/MN1-00.