Search

The development of antimicrobial resistance in foodborne pathogens is a growing public health concern. This study was undertaken to determine the antimicrobial susceptibility of Salmonella enterica subspecies enterica isolated from the Australian commercial egg layer industry. S. enterica subspecies enterica (n=307) isolated from Australian commercial layer flock environments (2015-2018) were obtained from reference, research and State Government laboratories from six Australian states. All Salmonella isolates were serotyped. Antimicrobial susceptibility testing (AST) for 16 antimicrobial agents was performed by broth microdilution. Antimicrobial resistance genes and sequence types (STs) were identified in significant isolates by whole genome sequencing (WGS). Three main serotypes were detected, S. Typhimurium (n=61, 19.9%), S. Senftenburg (n=45, 14.7%) and S. Agona (n=37, 12.1%). AST showed 293/307 (95.4%) isolates were susceptible to all tested antimicrobial agents and all isolates were susceptible to amoxicillin-clavulanate, azithromycin, ceftiofur, ceftriaxone, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin and trimethoprim-sulfamethoxazole. Low levels of non-susceptibility were observed to streptomycin (2.3%, n=7), sulfisoxazole (2.0%, n=6), chloramphenicol (1.3%, n=4) and tetracycline (1.0%, n=3). Very low levels of non-susceptibility were observed to ampicillin (2/307; 0.7%) and cefoxitin (2/307; 0.7%). Two isolates (S. Havana and S. Montevideo), exhibited multidrug-resistant phenotypes to streptomycin, sulfisoxazole and tetracycline and possessed corresponding antimicrobial resistance genes (aadA4, aac(6')-Iaa, sul1, tetB). One S. Typhimurium isolate was resistant to ampicillin and tetracycline, and possessed both tetA and blaTEM-1B. WGS also identified these isolates as belonging to ST4 (S. Montevideo), ST578 (S. Havana) and ST19 (S. Typhimurium). The absence of resistance to highest priority critically important antimicrobials as well as the e

The ability to detect SARS-CoV-2 is critical to implementing evidence-based strategies to address the COVID-19 global pandemic. Expanding SARS-CoV-2 diagnostic ability beyond well-equipped laboratories widens the opportunity for surveillance and control efforts. However, such advances are predicated on the availability of rapid, scalable, accessible, yet high-performance diagnostic platforms. Methods to detect viral RNA using reverse transcription loop-mediated isothermal amplification (RT-LAMP) show promise as rapid and field-deployable tests; however, the per-unit costs of the required diagnostic hardware can be a barrier for scaled deployment. Here, we describe a diagnostic hardware configuration for LAMP technology, named the FABL-8, that can be built for approximately US$380 per machine and provide results in under 30 min. Benchmarking showed that FABL-8 has a similar performance to a high-end commercial instrument for detecting fluorescence-based LAMP reactions. Performance testing of the instrument with RNA extracted from a SARS-CoV-2 virus dilution series revealed an analytical detection sensitivity of 50 virus copies per microliter - a detection threshold suitable to detect patient viral load in the first few days following symptom onset. In addition to the detection of SARS-CoV-2, we show that the system can be used to detect the presence of two bacterial pathogens, demonstrating the versatility of the platform for the detection of other pathogens. This cost-effective and scalable hardware alternative allows democratization of the instrumentation required for high-performance molecular diagnostics, such that it could be available to laboratories anywhere - supporting infectious diseases surveillance and research activities in resource-limited settings. Copyright © 2021 The Authors. Published by American Chemical Society.

The COVID-19 pandemic has placed unprecedented global demand on laboratory supplies required for testing. Sample pooling has been investigated by laboratories as a strategy to preserve testing capacity. We evaluate the performance of Cepheid Xpert Xpress SARS-CoV-2 RT-PCR assay for testing samples in pools of 4 and 6. Clinical samples containing SARS-CoV-2, and confirmed negative clinical samples were used to create sample pools. Clinical samples had 'neat' Xpert E gene cycle threshold values ranging between 20 and 28 and all were detected qualitatively when contained in pools of 4 or 6 samples. For these samples, pooling had a median change in cycle threshold value of 2.0 in pools of 4, and of 2.9 in pools of 6. With the use of Cepheid Xpert Xpress SARS-CoV-2 RT-PCR assay, pooling of 4 or 6 samples may be an effective strategy to increase testing capacity.Copyright © 2020 The Authors

Emerging testing technologies for detection of SARS-CoV-2 include those that are rapid and can be used at point-of-care (POC), and those facilitating high throughput laboratory-based testing. Tests designed to be performed at POC (such as antigen tests and molecular assays) have the potential to expedite isolation of infectious patients and their contacts, but most are less sensitive than standard-of-care reverse transcription polymerase chain reaction (RT-PCR). Data on clinical performance of the majority of emerging assays are limited with most evaluations performed on contrived or stored laboratory samples. Further evaluations of these assays are required, particularly when performed at POC on symptomatic and asymptomatic patients and at various time-points after symptom onset. A few studies have so far shown several of these assays have high specificity. However, large prospective evaluations are needed to confirm specificity, particularly before the assays are implemented in low prevalence settings or asymptomatic populations. High throughput laboratory-based testing includes the use of new sample types (e.g., saliva to increase acceptability) or innovative uses of existing technology (e.g., sample pooling). Information detailing population-wide testing strategies for SARS-COV-2 is largely missing from peer-reviewed literature. Logistics and supply chains are key considerations in any plan to 'scale up' testing in the Australian context. The strategic use of novel assays will help strike the balance between achieving adequate test numbers without overwhelming laboratory capacity. To protect testing of high-risk populations, the aims of testing with respect to the phase of the pandemic must be considered.Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.

Multidrug-resistant Staphylococcus and vancomycin-resistant Enterococcus (VRE) are important human pathogens that are resistant to most clinical antibiotics. Treatment options are limited and often require the use of 'last-line' antimicrobials such as linezolid, daptomycin, and in the case of Staphylococcus, also vancomycin. The emergence of resistance to these last-line antimicrobial agents is therefore of considerable clinical concern. This mini-review provides an overview of resistance to last-line antimicrobial agents in Staphylococcus and VRE, with a particular focus on how genomics has provided critical insights into the emergence of resistant clones, the molecular mechanisms of resistance, and the importance of mobile genetic elements in the global spread of resistance to linezolid.© Copyright © 2021 Turner, Lee, Gorrie, Howden and Carter.

Serological assays for SARS-CoV-2 infection are now widely available for use in diagnostic laboratories. Limited data are available on the performance characteristics in different settings, and at time periods remote from the initial infection. Validation of the Abbott (Architect SARS-CoV-2 IgG), DiaSorin (Liaison SARS-CoV-2 S1/S2 IgG) and Roche (Cobas Elecsys Anti-SARS-CoV-2) assays was undertaken utilising 217 serum samples from 131 participants up to 7 months following COVID-19 infection. The Abbott and DiaSorin assays were implemented into routine laboratory workflow, with outcomes reported for 2764 clinical specimens. Sensitivity and specificity were concordant with the range reported by the manufacturers for all assays. Sensitivity across the convalescent period was highest for the Roche at 95.2-100% (95% CI 81.0-100%), then the DiaSorin at 88.1-100% (95% CI 76.0-100%), followed by the Abbott 68.2-100% (95% CI 53.4-100%). Sensitivity of the Abbott assay fell from approximately 5 months; on this assay paired serum samples for 45 participants showed a significant drop in the signal-to-cut-off ratio and 10 sero-reversion events. When used in clinical practice, all samples testing positive by both DiaSorin and Abbott assays were confirmed as true positive results. In this low prevalence setting, despite high laboratory specificity, the positive predictive value of a single positive assay was low. Comprehensive validation of serological assays is necessary to determine the optimal assay for each diagnostic setting. In this low prevalence setting we found implementation of two assays with different antibody targets maximised sensitivity and specificity, with confirmatory testing necessary for any sample which was positive in only one assay.Copyright © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

The COVID-19 pandemic has exposed the dependence of diagnostic laboratories on a handful of large corporations with market monopolies on the worldwide supply of reagents, consumables, and hardware for molecular diagnostics. Global shortages of key consumables for RT-qPCR detection of SARS-CoV-2 RNA have impaired the ability to run essential, routine diagnostic services. Here, we describe a workflow for rapid detection of SARS-CoV-2 RNA in upper respiratory samples including nasal swabs and saliva, utilizing low-cost equipment and readily accessible reagents. Using repurposedCreality3D Ender-3three-dimensional (3D) printers, we built a semiautomated paramagnetic bead RNA extraction platform. The hardware for the system was built for $300 USD, and the material cost per reaction was $1 USD. Named the Ender VX500, instrument performance when paired with RT-qPCR for SARS-CoV-2 detection in nasal and saliva specimens was two virus copies per microliter. There was a high-performance agreement (assessed using 458 COVID-19 nasal swab specimens) with the Aptima SARS-CoV-2 assay run on the Hologic Panther, a commercial automated RNA extraction and detection platform. Inter- and intrainstrument precision was excellent (coefficients of variation (CoV) of 1.10 and 0.66-1.32%, respectively) across four instruments. The platform is scalable with throughput ranging from 23 specimens on a single instrument run by one user in 50 min to 364 specimens on four instruments run by four users in 190 min. Step-by-step instructions and protocols for building and running theEnder VX500have been made available without restriction.Copyright © 2021 The Authors. Published by American Chemical Society

Production of the Panton-Valentine leukocidin (PVL) by Staphylococcus aureus is mediated via the genes lukS-PV and lukF-PV which are carried on bacteriophage ϕSa2. PVL is associated with S. aureus strains that cause serious infections and clones of community-associated methicillin-resistant S. aureus (CA-MRSA) that have additionally disseminated widely. In Western Australia (WA) the original CA-MRSA were PVL negative however, between 2005 and 2008, following the introduction of eight international PVL-positive CA-MRSA, PVL-positive WA CA-MRSA were found. There was concern that PVL bacteriophages from the international clones were transferring into the local clones, therefore a comparative study of PVL-carrying ϕSa2 prophage genomes from historic WA PVL-positive S. aureus and representatives of all PVL-positive CA-MRSA isolated in WA between 2005 and 2008 was performed. The prophages were classified into two genera and three PVL bacteriophage groups and had undergone many recombination events during their evolution. Comparative analysis of mosaic regions of selected bacteriophages using the Alignments of bacteriophage genomes (Alpha) aligner revealed novel recombinations and modules. There was heterogeneity in the chromosomal integration sites, the lysogeny regulation regions, the defence and DNA processing modules, the structural and packaging modules and the lukSF-PV genes. One WA CA-MRSA (WA518751) and one international clone (Korean Clone) have probably acquired PVL-carrying ϕSa2 in WA, however these clones did not disseminate in the community. Genetic heterogeneity made it impossible to trace the source of the PVL prophages in the other WA clones. Against this background of PVL prophage diversity, the sequence of one group, the ϕSa2USA/ϕSa2wa-st93 group, was remarkably stable over at least 20 years and associated with the highly virulent USA300 and ST93-IVa CA-MRSA lineages that have disseminated globally.

Importance Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is associated with mortality of more than 20%. Combining standard therapy with a β-lactam antibiotic has been associated with reduced mortality, although adequately powered randomized clinical trials of this intervention have not been conducted. Objective To determine whether combining an antistaphylococcal β-lactam with standard therapy is more effective than standard therapy alone in patients with MRSA bacteremia. Design, Setting, and Participants Open-label, randomized clinical trial conducted at 27 hospital sites in 4 countries from August 2015 to July 2018 among 352 hospitalized adults with MRSA bacteremia. Follow-up was complete on October 23, 2018. Interventions Participants were randomized to standard therapy (intravenous vancomycin or daptomycin) plus an antistaphylococcal β-lactam (intravenous flucloxacillin, cloxacillin, or cefazolin) (n = 174) or standard therapy alone (n = 178). Total duration of therapy was determined by treating clinicians and the β-lactam was administered for 7 days. Main Outcomes and Measures The primary end point was a 90-day composite of mortality, persistent bacteremia at day 5, microbiological relapse, and microbiological treatment failure. Secondary outcomes included mortality at days 14, 42, and 90; persistent bacteremia at days 2 and 5; acute kidney injury (AKI); microbiological relapse; microbiological treatment failure; and duration of intravenous antibiotics. Results The data and safety monitoring board recommended early termination of the study prior to enrollment of 440 patients because of safety. Among 352 patients randomized (mean age, 62.2 [SD, 17.7] years; 121 women [34.4%]), 345 (98%) completed the trial. The primary end point was met by 59 (35%) with combination therapy and 68 (39%) with standard therapy (absolute difference, −4.2%; 95% CI, −14.3% to 6.0%). Seven of 9 prespecified secondary end points showed no significant difference. For the

Objectives: To describe the first isolation and sequencing of SARS-CoV-2 in Australia and rapid sharing of the isolate. Setting(s): SARS-CoV-2 was isolated from a 58-year-old man from Wuhan, China who arrived in Melbourne on 19 January 2020 and was admitted to the Monash Medical Centre, Melbourne from the emergency department on 24 January 2020 with fever, cough, and progressive dyspnoea. Major outcomes: Clinical course and laboratory features of the first reported case of COVID-19 (the illness caused by SARS-CoV-2) in Australia; isolation, whole genome sequencing, imaging, and rapid sharing of virus from the patient. Result(s): A nasopharyngeal swab and sputum collected when the patient presented to hospital were each positive for SARS-CoV-2 (reverse transcription polymerase chain reaction). Inoculation of Vero/hSLAM cells with material from the nasopharyngeal swab led to the isolation of SARS-CoV-2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles with morphology characteristic of viruses of the family Coronaviridae. Whole genome sequencing of the viral isolate and phylogenetic analysis indicated the isolate exhibited greater than 99.99% sequence identity with other publicly available SARS-CoV-2 genomes. Within 24 hours of isolation, the first Australian SARS-CoV-2 isolate was shared with local and overseas reference laboratories and major North American and European culture collections. Conclusion(s): The ability to rapidly identify, propagate, and internationally share our SARS-CoV-2 isolate is an important step in collaborative scientific efforts to deal effectively with this international public health emergency by developing better diagnostic procedures, vaccine candidates, and antiviral agents.Copyright © 2020 AMPCo Pty Ltd

Objectives: To conduct a pilot study implementing combined genomic and epidemiologic surveillance for hospital-acquired multidrug-resistant organisms (MDROs) to predict transmission between patients and to estimate the local burden of MDRO transmission. Design(s): Pilot prospective multicenter surveillance study. Setting(s): The study was conducted in 8 university hospitals (2,800 beds total) in Melbourne, Australia (population 4.8 million), including 4 acute-care, 1 specialist cancer care, and 3 subacute-care hospitals. Method(s): All clinical and screening isolates from hospital inpatients (April 24 to June 18, 2017) were collected for 6 MDROs: VanA VRE, MRSA, ESBL Escherichia coli (ESBL-Ec) and Klebsiella pneumoniae (ESBL-Kp), and carbapenem-resistant Pseudomonas aeruginosa (CRPa) and Acinetobacter baumannii (CRAb). Isolates were analyzed and reported as routine by hospital laboratories, underwent whole-genome sequencing at the central laboratory, and were analyzed using open-source bioinformatic tools. MDRO burden and transmission were assessed using combined genomic and epidemiologic data. Result(s): In total, 408 isolates were collected from 358 patients; 47.5% were screening isolates. ESBL-Ec was most common (52.5%), then MRSA (21.6%), vanA VRE (15.7%), and ESBL-Kp (7.6%). Most MDROs (88.3%) were isolated from patients with recent healthcare exposure. Combining genomics and epidemiology identified that at least 27.1% of MDROs were likely acquired in a hospital; most of these transmission events would not have been detected without genomics. The highest proportion of transmission occurred with vanA VRE (88.4% of patients). Conclusion(s): Genomic and epidemiologic data from multiple institutions can feasibly be combined prospectively, providing substantial insights into the burden and distribution of MDROs, including in-hospital transmission. This analysis enables infection control teams to target interventions more effectively. Copyright © 2020 by The Society

BACKGROUND: Multi-resistant 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 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). METHOD(S): A prospective multi-modal intervention including active screening, carrier isolation, centralised case investigation and comparative pathogen genomics was implemented. We analyze trend in CPE incidence and clinical presentation, risk factors and local transmission over the program's first three years (January 2016 to December 2018). RESULT(S): 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). 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 identified putative overseas community acquisition. Median time between isolate referral and initial 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. CONCLUSION(S): We demonstrate the value of centralised CPE control programs to increase case ascertainment, resolve risk factors and identify putative local transmission through prospective genomic and epidemiological surveillance; methodologies are transferable to low-prevalence settings and MROs globa

Importance: Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is associated with mortality of more than 20%. Combining standard therapy with a beta-lactam antibiotic has been associated with reduced mortality, although adequately powered randomized clinical trials of this intervention have not been conducted. Objective(s): To determine whether combining an antistaphylococcal beta-lactam with standard therapy is more effective than standard therapy alone in patients with MRSA bacteremia. Design, Setting, and Participant(s): Open-label, randomized clinical trial conducted at 27 hospital sites in 4 countries from August 2015 to July 2018 among 352 hospitalized adults with MRSA bacteremia. Follow-up was complete on October 23, 2018. Intervention(s): Participants were randomized to standard therapy (intravenous vancomycin or daptomycin) plus an antistaphylococcal beta-lactam (intravenous flucloxacillin, cloxacillin, or cefazolin) (n = 174) or standard therapy alone (n = 178). Total duration of therapy was determined by treating clinicians and the beta-lactam was administered for 7 days. Main Outcomes and Measures: The primary end point was a 90-day composite of mortality, persistent bacteremia at day 5, microbiological relapse, and microbiological treatment failure. Secondary outcomes included mortality at days 14, 42, and 90; persistent bacteremia at days 2 and 5; acute kidney injury (AKI); microbiological relapse; microbiological treatment failure; and duration of intravenous antibiotics. Result(s): The data and safety monitoring board recommended early termination of the study prior to enrollment of 440 patients because of safety. Among 352 patients randomized (mean age, 62.2 [SD, 17.7] years; 121 women [34.4%]), 345 (98%) completed the trial. The primary end point was met by 59 (35%) with combination therapy and 68 (39%) with standard therapy (absolute difference, -4.2%; 95% CI, -14.3% to 6.0%). Seven of 9 prespecified secondary end points showed no signi

Healthcare workers are at increased risk of occupational transmission of SARS-CoV-2. We report two 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.Copyright © The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Introduction. The 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. Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. Methodology. We used a commercial RT-LAMP mastermix from OptiGene 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 1 microl 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 degreeC for 30 min and measure fluorescence in the FAM channel at 1 min intervals. Results. Assay 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 min (sd+/-7 min). 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. Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and r

Objective: To describe an outbreak of bacteremia caused by vancomycin-sensitive Enterococcus faecalis (VSEfe). Design(s): An investigation by retrospective case control and molecular typing by whole-genome sequencing (WGS). Setting(s): A tertiary-care neonatal unit in Melbourne, Australia. Method(s): Risk factors for 30 consecutive neonates with VSEfe bacteremia from June 2011 to December 2014 were analyzed using a case control study. Controls were neonates matched for gestational age, birth weight, and year of birth. Isolates were typed using WGS, and multilocus sequence typing (MLST) was determined. Result(s): Bacteremia for case patients occurred at a median time after delivery of 23.5 days (interquartile range, 14.9-35.8). Previous described risk factors for nosocomial bacteremia did not contribute to excess risk for VSEfe. WGS typing results designated 43% ST179 as well as 14 other sequence types, indicating a polyclonal outbreak. A multimodal intervention that included education, insertion checklists, guidelines on maintenance and access of central lines, adjustments to the late onset sepsis antibiotic treatment, and the introduction of diaper bags for disposal of soiled diapers after being handled inside the bed, led to termination of the outbreak. Conclusion(s): Typing using WGS identified this outbreak as predominately nonclonal and therefore not due to cross transmission. A multimodal approach was then sought to reduce the incidence of VSEfe bacteremia.Copyright © 2019 by The Society for Healthcare Epidemiology of America. All rights reserved.

Objective: To describe an outbreak of bacteremia caused by vancomycin-sensitive Enterococcus faecalis (VSEfe). Design: An investigation by retrospective case control and molecular typing by whole-genome sequencing (WGS). Setting: A tertiary-care neonatal unit in Melbourne, Australia. Methods: Risk factors for 30 consecutive neonates with VSEfe bacteremia from June 2011 to December 2014 were analyzed using a case control study. Controls were neonates matched for gestational age, birth weight, and year of birth. Isolates were typed using WGS, and multilocus sequence typing (MLST) was determined. Results: Bacteremia for case patients occurred at a median time after delivery of 23.5 days (interquartile range, 14.9–35.8). Previous described risk factors for nosocomial bacteremia did not contribute to excess risk for VSEfe. WGS typing results designated 43% ST179 as well as 14 other sequence types, indicating a polyclonal outbreak. A multimodal intervention that included education, insertion checklists, guidelines on maintenance and access of central lines, adjustments to the late onset sepsis antibiotic treatment, and the introduction of diaper bags for disposal of soiled diapers after being handled inside the bed, led to termination of the outbreak. Conclusions: Typing using WGS identified this outbreak as predominately nonclonal and therefore not due to cross transmission. A multimodal approach was then sought to reduce the incidence of VSEfe bacteremia.

Background: Various studies have identified numerous factors associated with poor clinical outcomes in patients with Staphylococcus aureus bacteraemia (SAB). A new study was created to provide deeper insight into in-hospital complications and risk factors for treatment failure. Methods: Adult patients hospitalised with Staphylococcus aureus bacteraemia (SAB) were recruited prospectively into a multi-centre cohort. The primary outcome was treatment failure at 30 days (composite of all-cause mortality, persistent bacteraemia, or recurrent bacteraemia), and secondary measures included in-hospital complications and mortality at 6- and 12-months. Data were available for 222 patients recruited from February 2011 to December 2012. Results: Treatment failure at 30-days was recorded in 14.4% of patients (30-day mortality 9.5%). Multivariable analysis predictors of treatment failure included age > 70 years, Pitt bacteraemia score ≥ 2, CRP at onset of SAB > 250 mg/L, and persistent fevers after SAB onset; serum albumin at onset of SAB, receipt of appropriate empiric treatment, recent healthcare attendance, and performing echocardiography were protective. 6-month and 12-month mortality were 19.1% and 24.2% respectively. 45% experienced at least one in-hospital complication, including nephrotoxicity in 19.5%. Conclusions: This study demonstrates significant improvements in 30-day outcomes in SAB in Australia. However, we have identified important areas to improve outcomes from SAB, particularly reducing renal dysfunction and in-hospital treatment-related complications.

Alcohol-based disinfectants and particularly hand rubs are a key way to control hospital infections worldwide. Such disinfectants restrict transmission of pathogens, such as multidrug-resistant Staphylococcus aureus and Enterococcus faecium. Despite this success, health care infections caused by E. faecium are increasing. We tested alcohol tolerance of 139 hospital isolates of E. faecium obtained between 1997 and 2015 and found that E. faecium isolates after 2010 were 10-fold more tolerant to killing by alcohol than were older isolates. Using a mouse gut colonization model of E. faecium transmission, we showed that alcohol-tolerant E. faecium resisted standard 70% isopropanol surface disinfection, resulting in greater mouse gut colonization compared to alcohol-sensitive E. faecium. We next looked for bacterial genomic signatures of adaptation. Alcohol-tolerant E. faecium accumulated mutations in genes involved in carbohydrate uptake and metabolism. Mutagenesis confirmed the roles of these genes in the tolerance of E. faecium to isopropanol. These findings suggest that bacterial adaptation is complicating infection control recommendations, necessitating additional procedures to prevent E. faecium from spreading in hospital settings.Copyright © 2018 The Authors, some rights reserved;.

Background: Various studies have identified numerous factors associated with poor clinical outcomes in patients with Staphylococcus aureus bacteraemia (SAB). A new study was created to provide deeper insight into in-hospital complications and risk factors for treatment failure. Method(s): Adult patients hospitalised with Staphylococcus aureus bacteraemia (SAB) were recruited prospectively into a multi-centre cohort. The primary outcome was treatment failure at 30 days (composite of all-cause mortality, persistent bacteraemia, or recurrent bacteraemia), and secondary measures included in-hospital complications and mortality at 6- and 12-months. Data were available for 222 patients recruited from February 2011 to December 2012. Result(s): Treatment failure at 30-days was recorded in 14.4% of patients (30-day mortality 9.5%). Multivariable analysis predictors of treatment failure included age > 70 years, Pitt bacteraemia score >= 2, CRP at onset of SAB > 250 mg/L, and persistent fevers after SAB onset; serum albumin at onset of SAB, receipt of appropriate empiric treatment, recent healthcare attendance, and performing echocardiography were protective. 6-month and 12-month mortality were 19.1% and 24.2% respectively. 45% experienced at least one in-hospital complication, including nephrotoxicity in 19.5%. Conclusion(s): This study demonstrates significant improvements in 30-day outcomes in SAB in Australia. However, we have identified important areas to improve outcomes from SAB, particularly reducing renal dysfunction and in-hospital treatment-related complications.Copyright © 2018 The Author(s).

Background: Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of hospital-acquired infections. New, presumably better-adapted strains of VRE appear unpredictably; it is uncertain how they spread despite improved infection control. We aimed to investigate the relatedness of a novel sequence type (ST) of vanB E. faecium - ST796 - very near its time of origin from hospitals in three Australian states and New Zealand. Method(s): Following near-simultaneous outbreaks of ST796 in multiple institutions, we gathered then tested colonization and bloodstream infection isolates' antimicrobial resistance (AMR) phenotypes, and phylogenomic relationships using whole genome sequencing (WGS). Patient meta-data was explored to trace the spread of ST796. Result(s): A novel clone of vanB E. faecium (ST796) was first detected at one Australian hospital in late 2011, then in two New Zealand hospitals linked by inter-hospital transfers from separate Melbourne hospitals. ST796 also appeared in hospitals in South Australia and New South Wales and was responsible for at least one major colonization outbreak in a Neonatal Intensive Care Unit without identifiable links between centers. No exceptional AMR was detected in the isolates. While WGS analysis showed very limited diversity at the core genome, consistent with recent emergence of the clone, clustering by institution was observed. Conclusion(s): Evolution of new E. faecium clones, followed by recognized or unrecognized movement of colonized individuals then rapid intra-institutional cross-transmission best explain the multi-center, multistate and international outbreak we observed.Copyright © 2018 The Author(s).

Alcohol-based disinfectants and particularly hand rubs are a key way to control hospital infections worldwide. Such disinfectants restrict transmission of pathogens, such as multidrug-resistant Staphylococcus aureus and Enterococcus faecium. Despite this success, health care infections caused by E. faecium are increasing. We tested alcohol tolerance of 139 hospital isolates of E. faecium obtained between 1997 and 2015 and found that E. faecium isolates after 2010 were 10-fold more tolerant to killing by alcohol than were older isolates. Using a mouse gut colonization model of E. faecium transmission, we showed that alcohol-tolerant E. faecium resisted standard 70% isopropanol surface disinfection, resulting in greater mouse gut colonization compared to alcohol-sensitive E. faecium. We next looked for bacterial genomic signatures of adaptation. Alcohol-tolerant E. faecium accumulated mutations in genes involved in carbohydrate uptake and metabolism. Mutagenesis confirmed the roles of these genes in the tolerance of E. faecium to isopropanol. These findings suggest that bacterial adaptation is complicating infection control recommendations, necessitating additional procedures to prevent E. faecium from spreading in hospital settings.

Background: Enterococcus faecium is an important nosocomial pathogen. It has a high propensity for horizontal gene transfer, which has resulted in the emergence of MDR strains that are difficult to treat. The most notorious of these, vancomycin-resistant E. faecium, are usually treated with linezolid or daptomycin. Resistance has, however, been reported, meaning that new therapeutics are urgently needed. The 1,2,4-oxadiazoles are a recently discovered family of antimicrobials that are active against Gram-positive pathogens and therefore have therapeutic potential for treating E. faecium. However, only limited data are available on the activity of these antimicrobials against E. faecium. Objectives: To determine whether the 1,2,4-oxadiazole antimicrobials are active against MDR and daptomycinnon- susceptible E. faecium. Methods: The activity of the 1,2,4-oxadiazole antimicrobials against vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium was determined using susceptibility testing, time-kill assays and synergy assays. Toxicity was also evaluated against human cells by XTT and haemolysis assays. Results: The 1,2,4-oxadiazoles are active against a range of MDR E. faecium, including isolates that display nonsusceptibility to vancomycin and daptomycin. This class of antimicrobial displays rapid bactericidal activity and demonstrates superior killing of E. faecium compared with daptomycin. Finally, the 1,2,4-oxadiazoles act synergistically with daptomycin against E. faecium, with subinhibitory concentrations reducing the MIC of daptomycin for non-susceptible isolates to a level below the clinical breakpoint. Conclusions: The 1,2,4-oxadiazoles are active against MDR and daptomycin-non-susceptible E. faecium and hold great promise as future therapeutics for treating infections caused by these difficult-to-treat isolates.

Background: Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of hospital-acquired infections. New, presumably better-adapted strains of VRE appear unpredictably; it is uncertain how they spread despite improved infection control. We aimed to investigate the relatedness of a novel sequence type (ST) of vanB E. faecium - ST796 - very near its time of origin from hospitals in three Australian states and New Zealand. Methods: Following near-simultaneous outbreaks of ST796 in multiple institutions, we gathered then tested colonization and bloodstream infection isolates' antimicrobial resistance (AMR) phenotypes, and phylogenomic relationships using whole genome sequencing (WGS). Patient meta-data was explored to trace the spread of ST796. Results: A novel clone of vanB E. faecium (ST796) was first detected at one Australian hospital in late 2011, then in two New Zealand hospitals linked by inter-hospital transfers from separate Melbourne hospitals. ST796 also appeared in hospitals in South Australia and New South Wales and was responsible for at least one major colonization outbreak in a Neonatal Intensive Care Unit without identifiable links between centers. No exceptional AMR was detected in the isolates. While WGS analysis showed very limited diversity at the core genome, consistent with recent emergence of the clone, clustering by institution was observed. Conclusions: Evolution of new E. faecium clones, followed by recognized or unrecognized movement of colonized individuals then rapid intra-institutional cross-transmission best explain the multi-center, multistate and international outbreak we observed.

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555- like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.

Objectives: To investigate the genetic context associated with the emergence of vanA VRE in Australia. Methods: The whole genomes of 18 randomly selected vanA-positive Enterococcus faecium patient isolates, collected between 2011 and 2013 from hospitals in four Australian capitals, were sequenced and analysed. Results: In silico typing and transposon/plasmid assembly revealed that the sequenced isolates represented (in most cases) different hospital-adapted STs and were associated with a variety of different Tn1546 variants and plasmid backbone structures. Conclusions: The recent emergence of vanA VRE in Australia was polyclonal and not associatedwith the dissemination of a single 'dominant' ST or vanA-encoding plasmid. Interestingly, the factors contributing to this epidemiological change are not known and future studies may need to consider investigation of potential community sources.

Objectives: To investigate the genetic context associated with the emergence of vanA VRE in Australia. Method(s): The whole genomes of 18 randomly selected vanA-positive Enterococcus faecium patient isolates, collected between 2011 and 2013 from hospitals in four Australian capitals, were sequenced and analysed. Result(s): In silico typing and transposon/plasmid assembly revealed that the sequenced isolates represented (in most cases) different hospital-adapted STs and were associated with a variety of different Tn1546 variants and plasmid backbone structures. Conclusion(s): The recent emergence of vanA VRE in Australia was polyclonal and not associatedwith the dissemination of a single 'dominant' ST or vanA-encoding plasmid. Interestingly, the factors contributing to this epidemiological change are not known and future studies may need to consider investigation of potential community sources.Copyright © The Author 2016.

Background Methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia is a serious infection resulting in 20–50 % 90-day mortality. The limitations of vancomycin, the current standard therapy for MRSA, make treatment difficult. The only other approved drug for treatment of MRSA bacteraemia, daptomycin, has not been shown to be superior to vancomycin. Surprisingly, there has been consistent in-vitro and in-vivo laboratory data demonstrating synergy between vancomycin or daptomycin and an anti-staphylococcal β-lactam antibiotic. There is also growing clinical data to support such combinations, including a recent pilot randomised controlled trial (RCT) that demonstrated a trend towards a reduction in the duration of bacteraemia in patients treated with vancomycin plus flucloxacillin compared to vancomycin alone. Our aim is to determine whether the addition of an anti-staphylococcal penicillin to standard therapy results in improved clinical outcomes in MRSA bacteraemia. Methods/Design We will perform an open-label, parallel-group, randomised (1:1) controlled trial at 29 sites in Australia, New Zealand, Singapore, and Israel. Adults (aged 18 years or older) with MRSA grown from at least one blood culture and able to be randomised within 72 hours of the index blood culture collection will be eligible for inclusion. Participants will be randomised to vancomycin or daptomycin (standard therapy) given intravenously or to standard therapy plus 7 days of an anti-staphylococcal β-lactam (flucloxacillin, cloxacillin, or cefazolin). The primary endpoint will be a composite outcome at 90 days of (1) all-cause mortality, (2) persistent bacteraemia at day 5 or beyond, (3) microbiological relapse, or (4) microbiological treatment failure. The recruitment target of 440 patients is based on an expected failure rate for the primary outcome of 30 % in the control arm and the ability to detect a clinically meaningful absolute decrease of 12.5 %, with a two-sided alpha of 0.05, a powe

From 1 January to 31 December 2014, 27 institutions around Australia participated in the Australian Staphylococcal Sepsis Outcome Programme (ASSOP). The aim of ASSOP 2014 was to determine the proportion of Staphylococcus aureus bacteraemia (SAB) isolates in Australia that are antimicrobial resistant, with particular emphasis on susceptibility to methicillin and to characterise the molecular epidemiology of the isolates. Overall, 18.8% of the 2,206 SAB episodes were methicillin resistant, which was significantly higher than that reported in most European countries. The 30-day all-cause mortality associated with methicillin-resistant SAB was 23.4%, which was significantly higher than the 14.4% mortality associated with methicillin-sensitive SAB (P <0.0001). With the exception of the beta-lactams and erythromycin, antimicrobial resistance in methicillin-sensitive S. aureus remains rare. However in addition to the beta-lactams, approximately 50‰ of methicillin-resistant S. aureus (MRSA) were resistant to erythromycin and ciprofloxacin and approximately 15% were resistant to co-trimoxazole, tetracycline and gentamicin. When applying the European Committee on Antimicrobial Susceptibility Testing breakpoints, teicoplanin resistance was detected in 2 S. aureus isolates. Resistance was not detected for vancomycin or linezolid. Resistance to non-beta-lactam antimicrobials was largely attributable to 2 healthcare-associated MRSA clones; ST22-IV [2B] (EMRSA-15) and ST239-III [3A] (Aus-2/3 EMRSA). ST22-IV [2B] (EMRSA-15) has become the predominant healthcare associated clone in Australia. Sixty per cent of methicillin-resistant SAB were due to community-associated (CA) clones. Although polyclonal, almost 44% of community-associated clones were characterised as ST93-IV [2B] (Queensland CA-MRSA) and ST1-IV [2B] (WA1). CA-MRSA, in particular the ST45-V [5C2&5] (WA84) clone, has acquired multiple antimicrobial resistance determinants including ciprofloxacin, erythromycin, clindamyci

In the last decade, Clostridium difficile infection (CDI) has reached an epidemic state with increasing incidence and severity in both health care and community settings. Vancomycin is an important first-line therapy for CDI, and the emergence of resistance would have significant clinical consequences. In this study, we describe for the first time a vanB2 vancomycin resistance operon in C. difficile, isolated from an Australian veal calf at slaughter. The operon was carried on an ~42-kb element showing significant homology and synteny to Tn1549, a conjugative transposon linked with the emergence and global dissemination of vancomycin-resistant enterococci (VRE). Notably, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly as a result of an aberrant vanRB gene. As observed for other anaerobic species of the animal gut microbiota, C. difficile may be a reservoir of clinically important vancomycin resistance genes.

Background. In vitro laboratory and animal studies demonstrate a synergistic role for the combination of vancomycin and antistaphylococcal β-lactams for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Prospective clinical data are lacking. Methods. In this open-label, multicenter, clinical trial, adults with MRSA bacteremia received vancomycin 1.5 g intravenously twice daily and were randomly assigned (1:1) to receive intravenous flucloxacillin 2 g every 6 hours for 7 days (combination group) or no additional therapy (standard therapy group). Participants were stratified by hospital and randomized in permuted blocks of variable size. Randomization codes were kept in sealed, sequentially numbered, opaque envelopes. The primary outcome was the duration of MRSA bacteremia in days. Results. We randomly assigned 60 patients to receive vancomycin (n = 29), or vancomycin plus flucloxacillin (n = 31). The mean duration of bacteremia was 3.00 days in the standard therapy group and 1.94 days in the combination group. According to a negative binomial model, the mean time to resolution of bacteremia in the combination group was 65% (95% confidence interval, 41%–102%; P = .06) that in the standard therapy group. There was no difference in the secondary end points of 28- and 90-day mortality, metastatic infection, nephrotoxicity, or hepatotoxicity. Conclusions. Combining an antistaphylococcal β-lactam with vancomycin may shorten the duration of MRSA bacteremia. Further trials with a larger sample size and objective clinically relevant end points are warranted.

Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only detected in recent strains.Copyright © 2015 Elsevier Ltd.

Objective To describe successful termination of an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) colonization within a neonatal service. Setting Multisite neonatal intensive care unit and special care nurseries within a single health care service. Participants Forty-four cases of VREfm-colonized neonatal inpatients-including 2 clinical isolates (eye swab and catheter-urine specimen) and 42 screening isolates. Interventions Active surveillance cultures, patient isolation, contact precautions, enhanced environment cleaning, and staff and parent education. Whole genome sequencing and multilocus sequence typing were used to characterize the outbreak and refine infection control procedures. Results Peak prevalence of VREfm colonization across all sites was 31% upon discovery of the outbreak. Subsequent to the intervention, transmission was halted within 8 weeks and no further isolates of the outbreak strain have been detected as of 12 months following outbreak cessation. Environmental swabs revealed VREfm colonization of baby-weighing scales, a baby bath, and a pharmacy refrigerator within the neonatal intensive care unit. All isolates were of a single multilocus sequence type (sequence type 796) and highly clonal at the core genome level. Conclusions Bundled infection control interventions were effective in rapidly terminating a clonal outbreak of sequence type 796 VREfm colonization within a neonatal inpatient service. Strain-typing and active surveillance cultures were critical in guiding the management of this outbreak. The closed environment of a neonatal unit likely facilitated eradication of the patient and environment reservoirs of VREfm colonization.Copyright © 2015 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only detected in recent strains.

Infections caused by highly successful clones of hospital-associated methicillin-resistant Staphylococcus aureus (HAMRSA) are a major public health burden. The globally dominant sequence type 239 (ST239) HA-MRSA clone has persisted in the health care setting for decades, but the basis of its success has not been identified. Taking a collection of 123 ST239 isolates spanning 32 years, we have used population-based functional genomics to investigate the evolution of this highly persistent and successful clone. Phylogenetic reconstruction and population modeling uncovered a previously unrecognized distinct clade of ST239 that was introduced into Australia from Asia and has perpetuated the epidemic in this region. Functional analysis demonstrated attenuated virulence and enhanced resistance to last-line antimicrobials, the result of two different phenomena, adaptive evolution within the original Australian ST239 clade and the introduction of a new clade displaying shifts in both phenotypes. The genetic diversity between the clades allowed us to employ genome-wide association testing and identify mutations in other essential regulatory systems, including walKR, that significantly associate with and may explain these key phenotypes. The phenotypic convergence of two independently evolving ST239 clades highlights the very strong selective pressures acting on HA-MRSA, showing that hospital environments have favored the accumulation of mutations in essentialMRSAgenes that increase resistance to antimicrobials, attenuate virulence, and promote persistence in the health care environment. Combinations of comparative genomics and careful phenotypic measurements of longitudinal collections of clinical isolates are giving us the knowledge to intelligently address the impact of current and future antibiotic usage policies and practices on hospital pathogens globally. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for innumerable drug-resistant health c