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Enterococci are a major cause of health care-associated infections and account for approximately 10% of all bacteremias globally. The aim of this study was to determine the proportion of enterococcal bacteremia isolates in Australia that are antimicrobial resistant, with particular emphasis on susceptibility to ampicillin and the glycopeptides, and to characterize the molecular epidemiology of the Enterococcus faecalis and Enterococcus faecium isolates. From 1 January to 31 December 2011, 1,079 unique episodes of bacteremia were investigated, of which 95.8% were caused by either E. faecalis (61.0%) or E. faecium (34.8%). The majority of bacteremias were health care associated, and approximately one-third were polymicrobial. Ampicillin resistance was detected in 90.4% of E. faecium isolates but was not detected in E. faecalis isolates. Vancomycin nonsusceptibility was reported in 0.6% and 36.5% of E. faecalis and E. faecium isolates, respectively. Unlike Europe and the United States, where vancomycin resistance in E. faecium is predominately due to the acquisition of the vanA operon, 98.4% of E. faecium isolates harboring van genes carried the vanB operon, and 16.1% of the vanB E. faecium isolates had vancomycin MICs at or below the susceptible breakpoint of the CLSI. Although molecular typing identified 126 E. faecalis pulsed-field gel electrophoresis pulsotypes, >50% belonged to two pulsotypes that were isolated across Australia. E. faecium consisted of 73 pulsotypes from which 43 multilocus sequence types were identified. Almost 90% of the E. faecium isolates were identified as CC17 clones, of which approximately half were characterized as ST203, which was isolated Australia-wide. In conclusion, the Australian Enterococcal Sepsis Outcome Programme (AESOP) study has shown that although they are polyclonal, enterococcal bacteremias in Australia are frequently caused by ampicillin-resistant vanB E. faecium.

Community-acquired Staphylococcus aureus infections are a public health concern, yet little is known about infections that do not present to hospital. We identified community-onset S. aureus infections via specimens submitted to a community-based pathology service. Referring doctors confirmed eligibility and described infection site, severity and treatment. Isolates were characterized on antibiotic resistance, PFGE, MLST/SCCmec, and Panton-Valentine leukocidin (PVL), representing 106 community-onset infections; 34 non-multiresistant methicillin-resistant S. aureus (nmMRSA) (resistant to <3 non-β-lactam antibiotics), 15 multiply antibiotic-resistant MRSA (mMRSA) and 57 methicillin-sensitive S. aureus (MSSA). Most (93%) were skin and soft tissue infections. PVL genes were carried by 42% of nmMRSA isolates [95% confidence interval (CI) 26-61] and 15% of MSSA (95% CI 8-28). PVL was associated with infections of the trunk, head or neck (56·4% vs. 24·3%, P=0·005) in younger patients (23 vs. 52 years, P<0·001), and with boils or abscesses (OR 8·67, 95% CI 2·9-26·2), suggesting underlying differences in exposure and/or pathogenesis.

Background: The community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined., Results: Here, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular α-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of α-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus., Conclusions: These data demonstrate that hyperexpression of α-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular α-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA.

Background: The PVL-positive ST772-MRSA-V is an emerging community-associated (CA-) MRSA clone that has been named Bengal Bay Clone since most patients have epidemiological connections to the Indian subcontinent. It is found increasingly common in other areas of the world., Methods: One isolate of ST772-MRSA-V was sequenced using the Illumina Genome Analyzer System. After initial assembling the multiple sequence contigs were analysed using different in-house annotation scripts. Results were compared to microarray hybridisation results of clinical isolates of ST772-MRSA-V, of related strains and to another ST772-MRSA-V genome sequence., Results: According to MLST e-burst analysis, ST772-MRSA-V belongs to Clonal Complex (CC)1, differing from ST1 only in one MLST allele (pta-22). However, there are several additional differences including agr alleles (group II rather than III), capsule type (5 rather than 8), the presence of the egc enterotoxin gene cluster and of the enterotoxin homologue ORF CM14 as well as the absence of the enterotoxin H gene seh. Enterotoxin genes sec and sel are present. ST772-MRSA-V harbours the genes encoding enterotoxin A (sea) and PVL (lukS/F-PV). Both are located on the same prophage., Conclusions: ST772-MRSA-V may have emerged from the same lineage as globally spread CC1 and CC5 strains. It has acquired a variety of virulence factors, and for a CA-MRSA strain it has an unusually high number of genes associated with antibiotic resistance.

The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric Gram-negative pathogens. The 2010 survey focussed on community-onset infections, examining isolates from urinary tract infections from patients presenting to outpatient clinics, emergency departments or to community practitioners. Two thousand and ninety-two Escherichia coli, 578 Klebsiella species and 268 Enterobacter species were tested using a commercial automated method (Vitek 2, BioMérieux) and results were analysed using Clinical and Laboratory Standards Institute breakpoints from January 2012. Of the key resistances, non-susceptibility to the third-generation cephalosporin, ceftriaxone, was found in 3.2% of E. coli and 3.2%-4.0% of Klebsiella spp. Non-susceptibility rates to ciprofloxacin were 5.4% for E. coli, 1.0%-2.3% for Klebsiella spp., and 2.5%-6.6% in Enterobacter spp, and resistance rates to piperacillin-tazobactam were 2.8%, 3.2%-6.9%, and 16.8%-18.0% for the same 3 groups respectively. Only 3 strains, 2 Klebsiella spp. and 1 Enterobacter spp, were shown to harbour a carbapenemase (IMP-4)., (This work is copyright. Apart from any use as permitted under the Copyright Act 1968, no part may be reproduced by any process without prior written permission from the Commonwealth. Requests and inquiries concerning reproduction and rights should be addressed to the Commonwealth Copyright Administration, Attorney General's Department, Robert Garran Offices, National Circuit, Barton ACT 2600 or posted at http://www.ag.gov.au/cca.)

In 2011, the Australian Group on Antimicrobial Resistance (AGAR) conducted a period-prevalence survey of clinical Staphylococcus aureus isolated from hospital inpatients. Twenty-nine microbiology laboratories from all states and mainland territories participated. Specimens were collected more than 48 hours post-admission. Isolates were tested by Vitek2® antimicrobial susceptibility card (AST-P612 card). Nationally, the proportion of S. aureus that were methicillin-resistant S. aureus (MRSA) was 30.3%; ranging from 19.9% in Western Australia to 36.8% in New South Wales/Australian Capital Territory. Resistance to the non-ß-lactam antimicrobials was common except for rifampicin, fusidic acid, high-level mupirocin and daptomycin. No resistance was detected for vancomycin, teicoplanin or linezolid. Antibiotic resistance in methicillin susceptible S. aureus (MSSA) was rare apart from erythromycin (13.2%) and there was no resistance to vancomycin, teicoplanin or linezolid. Inducible clindamycin resistance was the norm for erythromycin resistant, clindamycin intermediate/susceptible S. aureus in Australia with 90.6% of MRSA and 83.1% of MSSA with this phenotype having a positive double disc diffusion test (D-test). The proportion of S. aureus characterised as being healthcare-associated MRSA (HA-MRSA) was 18.2%, ranging from 4.5% in Western Australia to 28.0% in New South Wales/Australian Capital Territory. Four HA-MRSA clones were characterised and 98.8% of HA-MRSA isolates were classified as either ST22-IV [2B] (EMRSA-15) or ST239-III [3A] (Aus-2/3 EMRSA). Multiclonal community-associated MRSA (CA-MRSA) accounted for 11.7% of all S. aureus. In Australia, regional variation in resistance is due to the differential distribution of MRSA clones between regions, particularly for the major HA-MRSA clone, ST239-III [3A] (Aus-2/3 EMRSA), which is resistant to multiple non-ß-lactam antimicrobials., (This work is copyright. Apart from any use as permitted under the Copyright Act 1968, no part may be reproduced by any process without prior written permission from the Commonwealth. Requests and inquiries concerning reproduction and rights should be addressed to the Commonwealth Copyright Administration, Attorney General's Department, Robert Garran Offices, National Circuit, Barton ACT 2600 or posted at http://www.ag.gov.au/cca.)

In 2010, 15 institutions around Australia conducted a period prevalence study of key resistances in isolates of Enterococcus species associated with a range of clinical disease amongst in- and outpatients. Each institution collected up to 100 consecutive isolates and tested these for susceptibility to commonly used antimicrobials using standardised methods. Vancomycin-resistant Enterococcus faecium and Enterococcus faecalis were characterised by pulsed-field gel electrophoresis. Multilocus sequence typing was performed on representative pulsotypes of E. faecium. Susceptibility results were compared with similar surveys conducted in 1995, 1999, 2003, 2005, 2007 and 2009. In the 2010 survey, E. faecalis (1,201 isolates) and E. faecium (170 isolates) made up 98.9% of the 1,386 isolates tested. Ampicillin resistance was very common (85.3%) in E. faecium and absent in E. faecalis. Non-susceptibility to vancomycin was 36.5% in E. faecium (similar to the 35.2% in 2009 but up from 15.4% in the 2007 survey) and 0.5% in E. faecalis. There were significant differences in the proportion of vancomycin-resistant E. faecium between the states ranging from 0% in Western Australia to 54.4% in South Australia. The vanB gene was detected in 62 E. faecium and 3 E. faecalis isolates. The vanA gene was detected in 1 E. faecium isolate. All vancomycin-resistant E. faecium belonged to clonal complex 17. The most common sequence type (ST) was ST203, which was found in all regions that had reports of vancomycin resistant enterococci. ST341 was detected only in New South Wales/Australian Capital Territory and ST414 only in South Australia and Victoria. High-level resistance to gentamicin was 34.1% in E. faecalis and 66.1% in E. faecium. A subset of isolates was tested against high-level streptomycin, linezolid and quinupristin/dalfopristin. High-level streptomycin resistance was found in 8.2% of E. faecalis isolates and 43.8% of E. faecium isolates. Linezolid non-susceptibility was more common in E. faecalis (5.8%) than E. faecium (0.9%). Overall 9.4% of E. faecium were resistant to quinupristin/dalfopristin (E. faecalis is intrinsically resistant)., (This work is copyright. Apart from any use as permitted under the Copyright Act 1968, no part may be reproduced by any process without prior written permission from the Commonwealth. Requests and inquiries concerning reproduction and rights should be addressed to the Commonwealth Copyright Administration, Attorney General's Department, Robert Garran Offices, National Circuit, Barton ACT 2600 or posted at http://www.ag.gov.au/cca.)

The Australian Group on Antimicrobial Resistance (AGAR) performs regular multicentre period prevalence studies to monitor changes in antimicrobial resistance. In 2011, 29 laboratories in Australia participated in the national surveillance of Staphylococcus aureus resistance. The survey only included unique isolates from clinical specimens collected ≥48h after hospital admission. MRSA accounted for 30.3% of S. aureus isolates. MRSA resistance to ciprofloxacin, erythromycin, tetracycline, trimethoprim/sulfamethoxazole, gentamicin and clindamycin (constitutive resistance) varied considerably between regions. Resistance to non-β-lactam antimicrobials was uncommon in MSSA, with the exception of erythromycin. Regional variation in resistance was due to the differential distribution of MRSA clones between regions. The proportion of S. aureus genetically characterised as healthcare-associated MRSA (HA-MRSA) was significantly lower in this survey (18.2%) compared with the 2005 survey (24.2%) (P<0.0001). Although four HA-MRSA clones were characterised, 98.8% of HA-MRSA were classified as either ST22-MRSA-IV [2B] (EMRSA-15) or ST239-MRSA-III [3A] (Aus-2/3 EMRSA). Multiclonal community-associated MRSA (CA-MRSA) increased markedly from 6.5% in 2005 to 11.7% of all S. aureus in 2011 (P<0.0001). Although the proportion of MRSA resistant to non-β-lactam antimicrobials has decreased nationally, the proportion of S. aureus that are MRSA has remained stable. This is primarily due to non-multiresistant CA-MRSA becoming more common in Australian hospitals at the expense of the long-established multiresistant ST239-MRSA-III [3A] (Aus-2/3 EMRSA). Given hospital outbreaks of CA-MRSA are thought to be extremely rare, it is most likely that patients colonised at admission with CA-MRSA have become infected with the colonising strain during their hospital stay., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Nosocomial outbreaks of vancomycin-resistant Enterococcus faecium (VREfm) are thought to occur by transmission of VREfm between patients, predicting that infection control interventions will limit cross-transmission. Despite implementation of such strategies, the incidence of VREfm infections continues to rise. We aimed to use genomics to better understand the epidemiology of E. faecium within a large hospital and investigate the reasons for failure of infection control strategies. Whole-genome sequencing was performed on 61 E. faecium (36 VREfm) isolates, predominately from blood cultures collected at a single hospital between 1998 and 2009, and on five vanB-positive anaerobic commensal bacteria isolated from human feces. Phylogenomic analysis and precise mapping of the vanB gene, which contains the Tn1549 transposon, showed that at least 18 of the 36 VREfm isolates had acquired the transposon via independent insertion events, indicating de novo generation of VREfm rather than cross-transmission. Furthermore, Tn1549 sequences found in 15 of the 36 VREfm isolates were the same as the Tn1549 sequence from one of the gut anaerobes. National and international comparator E. faecium isolates were phylogenetically interspersed with isolates from our hospital, suggesting that our findings might be globally representative. These data demonstrate that VREfm generation within a patient is common, presumably occurring in the human bowel during antibiotic therapy, and help explain our inability to reduce VREfm infections. A recommendation from our findings is that infection control practices should include screening patients for specific hospital clones of vancomycin-susceptible E. faecium rather than just VREfm., Importance: Enterococcus faecium is an increasingly important human pathogen causing predominantly antibiotic-resistant infections in hospitalized patients. Large amounts of health care funding are spent trying to control antibiotic-resistant bacteria in hospitals globally, yet in many institutions around the world, vancomycin-resistant E. faecium (VREfm) infections continue to rise. The new findings from this study help explain the failures of our current approaches to controlling vanB VREfm in health care institutions. Given the importance of this bacterium as a cause of hospital-acquired infections and the difficulties faced by infection control units in trying to prevent colonization in their institutions, the novel findings from this study provide evidence that a new approach to controlling VREfm in hospitals is required. In particular, more attention should be given to understanding the epidemiology of hospital-adapted vancomycin-susceptible E. faecium, and patients at higher risk for de novo generation of VREfm need to be identified and optimally managed.

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.

The predominant community-associated MRSA strains vary between geographic settings, with ST8-IV USA300 being the commonest clone in North America, and the ST30-IV Southwest Pacific clone established as the dominant clone in New Zealand for the past two decades. Moreover, distinct epidemiological risk factors have been described for colonisation and/or infection with CA-MRSA strains, although these associations have not previously been characterized in New Zealand. Based on data from the annual New Zealand MRSA survey, we sought to describe the clinical and molecular epidemiology of MRSA in New Zealand. All non-duplicate clinical MRSA isolates from New Zealand diagnostic laboratories collected as part of the annual MRSA survey were included. Demographic data was collected for all patients, including age, gender, ethnicity, social deprivation index and hospitalization history. MRSA was isolated from clinical specimens from 3,323 patients during the 2005 to 2011 annual surveys. There were marked ethnic differences, with MRSA isolation rates significantly higher in Māori and Pacific Peoples. Over the study period, there was a significant increase in CA-MRSA, and a previously unidentified PVL-negative ST5-IV spa t002 clone replaced the PVL-positive ST30-IV Southwest Pacific clone as the dominant CA-MRSA clone. Of particular concern was the finding of several successful and virulent MRSA clones from other geographic settings, including ST93-IV (Queensland CA-MRSA), ST8-IV (USA300) and ST772-V (Bengal Bay MRSA). Ongoing molecular surveillance is essential to prevent these MRSA strains becoming endemic in the New Zealand healthcare setting.

To determine the impact of infectious diseases consultation (IDC) in Staphylococcus aureus bacteraemia. All MRSA bacteraemia and a random subset of MSSA bacteraemia were retrospectively analysed. Out of 599 SAB episodes, 162 (27%) were followed by an IDC. Patients with IDC were younger and more frequently intravenous drug users, but fewer resided in a long-term care facility or were indigenous. Hospital length of stay was longer (29.5 vs 17 days, p < 0.001), and endocarditis (19.1% vs 7.3%, p < 0.001) and metastatic seeding (22.2% vs 10.1%, p < 0.001) were more frequent in the IDC group; however, SAPS II scores were lower in the IDC group (27 vs 37, p < 0.001). ICU admission rates in the two groups were similar. The isolate tested susceptible to empirical therapy more frequently in the IDC group (88.9% vs 78.0%, p = 0.003). Seven-day (3.1 vs 16.5%), 30-day (8.0% vs 27.0%) and 1-year mortality (22.2% vs 44.9%) were all lower in the IDC group (all p < 0.001). Multivariate analysis showed that effective initial therapy was the only variable associated with the protective effect of IDC. In patients with SAB, all-cause mortality was significantly lower in patients who had an IDC, because of the higher proportion of patients receiving effective initial antibiotics.

Background: It is uncertain whether particular clones causing invasive community-onset methicillin-resistant and methicillin-sensitive Staphylococcus aureus (cMRSA/cMSSA) infection differ in virulence., Methods: Invasive cMRSA and cMSSA cases were prospectively identified. Principal component analysis was used to derive an illness severity score (ISS) from clinical data, including 30-day mortality, requirement for intensive hospital support, the presence of bloodstream infection, and hospital length of stay. The mean ISS for each S. aureus clone (based on MLST) was compared with its DNA microarray-based genotype., Results: Fifty-seven cMRSA and 50 cMSSA infections were analyzed. Ten clones caused 82 (77%) of these infections and had an ISS calculated. The enterotoxin gene cluster (egc) and the collagen adhesin (cna) gene were found in 4 of the 5 highest-ranked clones (ST47-MSSA, ST30-MRSA-IV[2B], ST45-MSSA, and ST22-MRSA-IV[2B]) compared with none and 1 of the lowest 5 ranked clones, respectively. cMSSA clones caused more severe infection than cMRSA clones. The lukF/lukS Panton-Valentine leukocidin (PVL) genes did not directly correlate with the ISS, being present in the second, fourth, and 10th most virulent clones., Conclusions: The clinical severity of invasive cMRSA and cMSSA infection is likely to be attributable to the isolates' entire genotype rather than a single putative virulence determinant such as PVL.

We report the molecular epidemiology of 27 clinical multidrug-resistant Staphylococcus epidermidis (MDRSE) isolates collected between 2003 and 2007 in an Australian teaching hospital. The dominant genotype (sequence type 2 [ST2]) accounted for 85% of the isolates tested and was indistinguishable from an MDRSE genotype identified in European hospitals, which may indicate that highly adaptable health care-associated genotypes of S. epidermidis have emerged and disseminated worldwide in the health care setting.

Due to a longstanding comprehensive "search and destroy policy", methicillin-resistant Staphylococcus aureus (MRSA) is not endemic in Western Australian (WA) acute care hospitals. As the prevalence of MRSA in the community has increased, healthcare workers (HCW) are at risk of importing MRSA into hospitals. We aimed to determine the prevalence of and risk factors for nasal MRSA colonization in our HCW population. A period prevalence study was conducted at an 850-bed tertiary hospital. Basic demographics and a nasal swab were obtained. A total of 1,542 HCWs employed in our centre were screened for MRSA, of whom 3.4% (n = 52) were colonized. MRSA colonization was more common in patient care assistants (6.8%) and nurses (5.2%) than in allied health professionals (1.7%) and doctors (0.7%) (p < 0.01). Working in "high-risk" wards that cared for MRSA colonized/infected patients was the strongest risk factor for HCW MRSA colonization (p < 0.001). ST1-IV and ST78-IV (the most common community clones in the region) were the most frequently identified clones. In conclusion, MRSA colonization of HCWs occurs primarily in HCWs caring for patients colonized or infected with MRSA. Surveillance screening of HCWs should be regularly performed on wards with patients with high MRSA colonization prevalence to prevent further spread in the hospital.

The recently described phenol-soluble modulin PSM-mec was detected in Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus fleuretti, Staphylococcus hominis, Staphylococcus pseudintermedius, Staphylococcus saprophyticus, Staphylococcus simulans and Staphylococcus vitulinus from different hosts (humans, goats, dogs, cats, pigs, cattle and turkeys). It was identified in isolates harbouring SCCmec types II, IIA, IIB, IID, III, VIII and in some irregular or truncated elements., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In Australia the PVL-positive ST93-IV [2B], colloquially known as "Queensland CA-MRSA" has become the dominant CA-MRSA clone. First described in the early 2000s, ST93-IV [2B] is associated with skin and severe invasive infections including necrotizing pneumonia. A singleton by multilocus sequence typing (MLST) eBURST analysis ST93 is distinct from other S. aureus clones. To determine if the increased prevalence of ST93-IV [2B] is due to the widespread transmission of a single strain of ST93-IV [2B] the genetic relatedness of 58 S. aureus ST93 isolated throughout Australia over an extended period were studied in detail using a variety of molecular methods including pulsed-field gel electrophoresis, spa typing, MLST, microarray DNA, SCCmec typing and dru typing. Identification of the phage harbouring the lukS-PV/lukF-PV Panton Valentine leucocidin genes, detection of allelic variations in lukS-PV/lukF-PV, and quantification of LukF-PV expression was also performed. Although ST93-IV [2B] is known to have an apparent enhanced clinical virulence, the isolates harboured few known virulence determinants. All PVL-positive isolates carried the PVL-encoding phage ΦSa2USA and the lukS-PV/lukF-PV genes had the same R variant SNP profile. The isolates produced similar expression levels of LukF-PV. Although multiple rearrangements of the spa sequence have occurred, the core genome in ST93 is very stable. The emergence of ST93-MRSA is due to independent acquisitions of different dru-defined type IV and type V SCCmec elements in several spa-defined ST93-MSSA backgrounds. Rearrangement of the spa sequence in ST93-MRSA has subsequently occurred in some of these strains. Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVL-positive ST93-IVa [2B]-t202-dt10 predominant across Australia. Whether ST93-IVa [2B] t202-dt10 arose from one PVL-positive ST93-MSSA-t202, or by independent acquisitions of SCCmec-IVa [2B]-dt10 into multiple PVL-positive ST93-MSSA-t202 strains is not known.

The Sequenom MassARRAY iPLEX single-nucleotide polymorphism (SNP) typing platform uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with single-base extension PCR for high-throughput multiplex SNP detection. In this study, we investigated the use of iPLEX MassARRAY technology for methicillin-resistant Staphylococcus aureus (MRSA) genotyping. A 16-plex MassARRAY iPLEX GOLD assay (MRSA-iPLEX) was developed that targets a set of informative SNPs and binary genes for MRSA characterization. The method was evaluated with 147 MRSA isolates, and the results were compared with those of an established SYBR Green-based real-time PCR system utilizing the same SNP-binary markers. A total of 2352 markers belonging to 44 SNP-binary profiles were analysed by both real-time PCR and MRSA-iPLEX. With real-time PCR as the reference standard, MRSA-iPLEX correctly assigned 2298 of the 2352 (97.7%) markers. Sequence variation in the MRSA-iPLEX primer targets accounted for the majority of MRSA-iPLEX erroneous results, highlighting the importance of primer target selection. MRSA-iPLEX provided optimal throughput for MRSA genotyping, and was, on a reagent basis, more cost-effective than the real-time PCR methods. The 16-plex MRSA-iPLEX is a suitable alternative to SYBR Green-based real-time PCR typing of major sequence types and clonal complexes of MRSA., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Background: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was first reported in remote regions of Western Australia and is now the predominant MRSA isolated in the state. The objective of this study is to determine the genetic relatedness of Western Australian CA-MRSA clones within different multilocus sequence type (MLST) clonal clusters providing an insight into the frequency of S. aureus SCCmec acquisition within a region., Results: The CA-MRSA population in Western Australia is genetically diverse consisting of 83 unique pulsed-field gel electrophoresis strains from which 46 MLSTs have been characterised. Forty five of these sequence types are from 18 MLST clonal clusters and two singletons. While SCCmec IV and V are the predominant SCCmec elements, SCCmec VIII and several novel and composite SCCmec elements are present. The emergence of MRSA in diverse S. aureus clonal clusters suggests horizontal transmission of the SCCmec element has occurred on multiple occasions. Furthermore DNA microarray and spa typing suggests horizontal transfer of SCCmec elements has also occurred within the same CC. For many single and double locus variant CA-MRSA clones only a few isolates have been detected., Conclusions: Although multiple CA-MRSA clones have evolved in the Western Australian community only three clones have successfully adapted to the Western Australian community environment. These data suggest the successful evolution of a CA-MRSA clone may not only depend on the mobility of the SCCmec element but also on other genetic determinants.

In 2009, the Australian Group on Antimicrobial Resistance (AGAR) conducted a period-prevalence survey of clinical Staphylococcus aureus isolated from hospital inpatients. Thirty medical microbiology laboratories from each state and mainland territory participated. Specimens were collected more than 48 hours post-admission. Isolates were tested by Vitek2 (AST-P579 card) and by Etest for daptomycin. Nationally, the proportion of S. aureus that were MRSA was 33.6%, ranging from 27.3% in South Australia to 41.4% in New South Wales/Australian Capital Territory. Resistance to the non-beta-lactam antimicrobials was common except for rifampicin, fusidic acid, daptomycin and high-level mupirocin. No resistance was detected for vancomycin, teicoplanin, quinupristin-dalfopristin or linezolid. Resistance in the methicillin susceptible S. aureus (MSSA) was rare apart from erythromycin (12%) and absent for vancomycin, teicoplanin, daptomycin, quinupristin-dalfopristin and linezolid. The proportion of methicillin resistant S. aureus (MRSA) has remained stable since the first AGAR inpatient survey in 2005 yet during the same time frame resistance to many antimicrobials, in particular tetracycline, trimethoprim-sulphamethoxazole and gentamicin, has significantly decreased. This suggests that non-multi-resistant community-associated MRSA (CA-MRSA) clones are becoming more common in the hospital setting and replacing the long-established multi-resistant clones such as ST239-III (Aus 2/3 EMRSA). Given hospital outbreaks of CA-MRSA are thought to be extremely rare it is most likely that patients colonised at admission with CA-MRSA have become infected with the colonising strain during their hospital stay.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding α-hemolysin, Panton-Valentine leukocidin (PVL) and α-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a highly conserved accessory genome from a common source.

We designed a single-step, closed-tube, real-time polymerase chain reaction and high-resolution melting assay to simultaneously detect the presence of the Panton-Valentine leukocidin gene and discriminate histidine and arginine isoforms. Of 223 Staphylococcus aureus isolates from northern Australia, isoforms clustered by clonal complex (CC). All CC93 isolates harbored the arginine isoform., (Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.)

Differences between the features of invasive community-onset methicillin-resistant Staphylococcus aureus (cMRSA) and methicillin-susceptible S. aureus (cMSSA) infections are incompletely understood. Fifty-seven patients with invasive cMRSA infection were prospectively identified at two teaching hospitals; for each cMRSA case, two cases of invasive cMSSA infection acted as controls. The primary outcome was 30-day all-cause mortality. Patients with invasive cMRSA infection were more likely to be Aboriginal (25% vs. 14%, age-adjusted odds ratio [OR] 2.5, p = 0.037), reside in a long-term care facility and/or have been hospitalised in the previous year (51% vs. 34%, p = 0.04) and less likely to have endocarditis (2% vs. 12%, p = 0.02) or require admission to an intensive care unit or high-dependency area (7% vs. 21%, p = 0.02). All-cause mortality at 30 days was similar in the cMRSA and cMSSA groups (9% vs. 7%, p = 0.68). Panton-Valentine leukocidin (PVL) genes were detected in a similar proportion of cMRSA and cMSSA isolates (32% vs. 27%, p = 0.49) and the presence of PVL genes was associated with younger age (35 years vs. 55 years, p < 0.001), Aboriginal ethnicity (38% vs. 10%, p < 0.001), skin and soft-tissue infection (54% vs. 19%, p < 0.001), lower illness severity at presentation (SAPS II score 9 vs. 21, p = 0.001) and shorter hospitalisation (9 days vs. 24 days, p < 0.001). Patients with "PVL-positive" and "PVL-negative" S. aureus infection had similar 30-day all-cause mortality (4% vs. 9%, p = 0.28). Few clinical features differentiated patients with invasive cMRSA infection from those with infection caused by cMSSA. Invasive "PVL-positive" S. aureus infection was associated with less morbidity but similar mortality to "PVL-negative" infection.

Clonal complex 59 (CC59) community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains were characterized using pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, diagnostic DNA microarrays, and PCRs targeting staphylococcal cassette chromosome mec (SCCmec) elements and Panton-Valentine leukocidin (PVL). Six distinct groups within CC59 were characterized. At least seven different variants of SCCmec elements were identified (IVa [2B], IVb [2B], IVd [2B], IV variant [2B], IVa [2B&5], V variant [5C2], and V [5C2&5]). (The structural type is indicated by a Roman numeral, with a lowercase letter indicating the subtype, and the ccr complex and the mec complex are indicated by an Arabic numeral and an uppercase letter, respectively. Where there is an extra ccr element, this is indicated by "&" and an Arabic numeral designating the ccr type.) The first group is similar to the American sequence type 59 (ST59) MRSA-IV CA-MRSA strain USA1000. The second group includes a PVL-negative ST87 strain with an SCCmec element of subtype IVb (2B). The third group comprises PVL-variable ST59 MRSA-IV strains harboring multiple SCCmec IV subtypes. PVL-negative ST59 MRSA strains with multiple or composite SCCmec elements (IVa [2B&5]) form the fourth group. Group 5 corresponds to the internationally known "Taiwan clone," a PVL-positive strain with a variant SCCmec element (V [5C2&5]). This strain proved to be the most common CC59 MRSA strain isolated in Western Australia. Finally, group 6 encompasses the ST59 MRSA-V variant (5C2). The differentiation of CC59 into groups and strains indicates a rapid evolution and spread of SCCmec elements. Observed differences between groups of strains as well as intrastrain variability within a group facilitate the tracing of their spread.

Background: Bacterial pneumonia is a well described complication of influenza. In recent years, community-onset methicillin-resistant Staphylococcus aureus (cMRSA) infection has emerged as a contributor to morbidity and mortality in patients with influenza. Since the emergence and rapid dissemination of pandemic A(H1N1)2009 influenzavirus in April 2009, initial descriptions of the clinical features of patients hospitalized with pneumonia have contained few details of patients with bacterial co-infection., Methodology/principal Findings: Patients with community-acquired pneumonia (CAP) caused by co-infection with pandemic A(H1N1)2009 influenzavirus and cMRSA were prospectively identified at two tertiary hospitals in one Australian city during July to September 2009, the period of intense influenza activity in our region. Detailed characterization of the cMRSA isolates was performed. 252 patients with pandemic A(H1N1)2009 influenzavirus infection were admitted at the two sites during the period of study. Three cases of CAP due to pandemic A(H1N1)2009/cMRSA co-infection were identified. The clinical features of these patients were typical of those with S. aureus co-infection or sequential infection following influenza. The 3 patients received appropriate empiric therapy for influenza, but inappropriate empiric therapy for cMRSA infection; all 3 survived. In addition, 2 fatal cases of CAP caused by pandemic A(H1N1)2009/cMRSA co-infection were identified on post-mortem examination. The cMRSA infections were caused by three different cMRSA clones, only one of which contained genes for Panton-Valentine Leukocidin (PVL)., Conclusions/significance: Clinicians managing patients with pandemic A(H1N1)2009 influenzavirus infection should be alert to the possibility of co-infection or sequential infection with virulent, antimicrobial-resistant bacterial pathogens such as cMRSA. PVL toxin is not necessary for the development of cMRSA pneumonia in the setting of pandemic A( H1N1) 2009 influenzavirus co-infection.

Objective: To document the types of, and mortality from, Staphylococcus aureus bacteraemia in Australia and New Zealand, and determine factors associated with mortality., Design and Setting: Prospective observational study in 27 independent or hospital pathology laboratories in Australia (24) and New Zealand (3), employing a web-based database to prospectively record demographic features, selected risk factors, principal antibiotic treatment and mortality data on all patients with positive blood cultures for S. aureus from June 2007 to May 2008., Main Outcome Measure: 30-day all-cause mortality., Results: 1994 episodes of S. aureus bacteraemia were identified, and complete 30-day follow-up data were available for 1865. Most episodes had their onset in the community (60.8%; 95% CI, 58.7%-63.0%). Methicillin-resistant S. aureus (MRSA) caused 450 episodes (24.1%; 95% CI, 22.2%-25.9%), and 123 of these (27.3%) had a susceptibility profile consistent with community-associated MRSA. All-cause mortality at 30 days was 20.6% (95% CI, 18.8%-22.5%). On univariate analysis, increased mortality was significantly associated with older age, European ethnicity, MRSA infection, infections not originating from a medical device, sepsis syndrome, pneumonia/empyema, and treatment with a glycopeptide or other non-beta-lactam antibiotic. On multivariable analysis, independent predictors of mortality were age, sepsis syndrome, pneumonia/empyema, device-associated infection with a secondary focus, left-sided endocarditis, and treatment with a glycopeptide such as vancomycin, but not MRSA infection., Conclusions: S. aureus bacteraemia is a common infection in both the community and hospitals in Australia and New Zealand, and is associated with appreciable mortality. Invasive MRSA infection may be more life-threatening, partly because of the inferior efficacy of the standard treatment, vancomycin. National web-based surveillance of S. aureus bacteraemia and its outcomes is not only important but also easily achievable.

Objectives: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was first reported in remote regions of Western Australia (WA) in 1992 and is now the predominant MRSA isolated in the State. To gain insights into the emergence of CA-MRSA, 2146 people living in 11 remote WA communities were screened for colonization with S. aureus., Methods: Antibiogram analysis, contour-clamped homogeneous electric field electrophoresis, multilocus sequence typing, Panton-Valentine leucocidin determinant detection and accessory genetic regulator typing were performed to characterize the isolates. MRSA was further characterized by staphylococcal cassette chromosome mec typing., Results: The S. aureus population consisted of 13 clonal complexes and two Singleton lineages together with 56 sporadic isolates. Five lineages contained MRSA; however, these were not the predominant methicillin-susceptible S. aureus (MSSA) lineages. There was greater diversity amongst the MSSA while the MRSA appeared to have emerged clonally following acquisition of the staphylococcal cassette chromosome mec. Three MRSA lineages were considered to have been endemic in the communities and have subsequently become predominant lineages of CA-MRSA in the wider WA community. People colonized with MSSA tended to harbour clones of a different genetic lineage at each anatomical site while people colonized with MRSA tended to harbour clones of the same lineage at each site. Overall, the isolates were resistant to few antimicrobials., Conclusions: Although the evidence suggests that in WA CA-MRSA strains arose in remote communities and have now disseminated into the wider community, there is no evidence that they arose from the predominant MSSA clones in these communities.

The objective was to compare the epidemiology and outcome of healthcare- (HA-) and community-associated (CA-) MRSA bacteraemia. A 10-year retrospective study of MRSA bacteraemia was carried out. Episodes were classified according to established criteria. Molecular typing was performed on a subset of isolates. Of 197 MRSA bacteraemia episodes, 178 (90.4%) were classified as HA-MRSA and 19 (9.6%) as CA-MRSA. All-cause 7- and 30-day mortality rates were similar in the HA and CA-MRSA bacteraemia groups; however, 1-year mortality was higher in the HA-MRSA bacteraemia group (48.3% vs 21.1% [p = 0.023]). Thirty-day all-cause mortality was significantly lower if empiric antimicrobial therapy included agent(s) to which the isolate tested susceptible, compared with patients receiving "inactive" therapy (19% vs 35.1% [p = 0.011]). The majority of MRSA bacteraemia episodes were caused by clones known to circulate in the community. All-cause mortality is as high in HA- as in CA-MRSA bacteraemia. Thirty-day mortality was significantly reduced if the patient received an antibiotic with activity against the MRSA isolate.

Biennial community-based Staphylococcus aureus antimicrobial surveillance programs have been performed by the Australian Group for Antimicrobial Resistance (AGAR) since 2000. Over this time the percentage of S. aureus identified as methicillin resistant has increased significantly from 10.3% in 2000 to 16% in 2006. This increase has occurred throughout Australia and has been due to the emergence of community-associated MRSA (CA-MRSA) clones. However, healthcare associated MRSA were still predominant in New South Wales/Australian Capital Territory and Victoria/Tasmania. In the 2006 survey CA-MRSA accounted for 8.8% of community-onset S. aureus infections. Although multiple CA-MRSA clones were characterised, the predominate clone identified was Queensland (Qld) MRSA (ST93-MRSA-IV) a Panton-Valentine leukocidin (PVL) positive MRSA that was first reported in Queensland and northern New South Wales in 2003 but has now spread throughout Australia. Several international PVL-positive CA-MRSA clones were also identified including USA300 MRSA (ST8-MRSA-IV). In addition, PVL was detected in an EMRSA-15 (ST22-MRSA-IV) isolate; a hospital associated MRSA clone that is known to be highly transmissible in the healthcare setting. With the introduction of the international clones and the transmission of Qld MRSA throughout the country, over 50% of CA-MRSA in Australia are now PVL positive. This change in the epidemiology of CA-MRSA in the Australian community will potentially result in an increase in skin and soft tissue infections in young Australians. As infections caused by these strains frequently results in hospitalisation their emergence is a major health concern.

Objective: To describe an outbreak of invasive methicillin-resistant Staphylococcus aureus (MRSA) infection after percutaneous needle procedures (acupuncture and joint injection) performed by a single medical practitioner., Setting: A medical practitioner's office and 4 hospitals in Perth, Western Australia., Patients: Eight individuals who developed invasive MRSA infection after acupuncture or joint injection performed by the medical practitioner., Methods: We performed a prospective and retrospective outbreak investigation, including MRSA colonization surveillance, environmental sampling for MRSA, and detailed molecular typing of MRSA isolates. We performed an infection control audit of the medical practitioner's premises and practices and administered MRSA decolonization therapy to the medical practitioner., Results: Eight cases of invasive MRSA infection were identified. Seven cases occurred as a cluster in May 2004; another case (identified retrospectively) occurred approximately 15 months earlier in February 2003. The primary sites of infection were the neck, shoulder, lower back, and hip: 5 patients had septic arthritis and bursitis, and 3 had pyomyositis; 3 patients had bacteremia, including 1 patient with possible endocarditis. The medical practitioner was found to be colonized with the same MRSA clone [ST22-MRSA-IV (EMRSA-15)] at 2 time points: shortly after the first case of infection in March 2003 and again in May 2004. After the medical practitioner's premises and practices were audited and he himself received MRSA decolonization therapy, no further cases were identified., Conclusions: This outbreak most likely resulted from a breakdown in sterile technique during percutaneous needle procedures, resulting in the transmission of MRSA from the medical practitioner to the patients. This report demonstrates the importance of surveillance and molecular typing in the identification and control of outbreaks of MRSA infection.

A series of epidemics of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have occurred in Australia, starting in Western Australia in the early 1990s, in the Northern Territory soon thereafter and in eastern states in the mid 1990s. The Western Australian epidemic has been due mainly to Panton-Valentine leukocidin (PVL)-negative clones, whilst PVL-positive clones have predominated in the east. More recently, the major epidemic clones have spread throughout the country, whilst multiple new minor clones have emerged, mainly in Western Australia. A total of 45 clones of CA-MRSA have been detected in Australia to date: 30 of these carry SCCmec IV, 6 carry SCCmec V and 9 carry novel SCCmec types. Overall, CA-MRSA clones have been associated predominantly with skin and soft-tissue infections. PVL-positive clones have been associated with furunculosis, necrotising pneumonia and osteomyelitis and have caused fatalities in otherwise healthy children and young adults. Initial treatment of these infections remains problematic, as it is frequently inappropriate. Of particular concern, healthcare-associated acquisition of CA-MRSA clones is now increasing, although major hospital outbreaks have not occurred yet.

The Australian Group for Antimicrobial Resistance conducted a survey of the prevalence of antimicrobial resistance in unique clinical isolates of Staphylococcus aureus from patients admitted to hospital for more than 48 hours. Thirty-two laboratories from all states and territories collected 2,908 isolates from 1 May 2005, of which 31.9% were methicillin-resistant Staphylococcus aureus (MRSA). The regional prevalence of MRSA varied significantly (P < 0.0001) from 22.5% in Western Australia to 43.4% in New South Wales/Australian Capital Territory. Prevalence of MRSA from individual laboratories varied even more from 4% to 58%. This variation was explained in part by distribution of age with the risk of MRSA significantly (P < 0.0001) increasing with age. Other unmeasured factors including hospital activity and infection control practices in the individual institution may have also contributed. Further investigation is warranted as reductions in prevalence would reduce morbidity, mortality and healthcare costs.

Objective: To describe the control of an outbreak of infection and colonization with the New York/Japan methicillin-resistant Staphylococcus aureus (MRSA) clone in multiple healthcare facilities, and to demonstrate the importance of making an MRSA management policy involving molecular typing of MRSA into a statewide public health responsibility., Setting: A range of healthcare facilities, including 2 metropolitan teaching hospitals and a regional hospital, as well as several community hospitals and long-term care facilities in a nonmetropolitan healthcare region., Interventions: A comprehensive, statewide MRSA epidemiological investigation and management policy., Results: In May 2005, there were 3 isolates referred to the Western Australian Gram-Positive Bacteria Typing and Research Unit that were identified as the New York/Japan MRSA clone, a pandemic MRSA clone with the ability to spread and replace existing clones in a region. Subsequent investigation identified 28 additional cases of infection and/or colonization dating from 2002 onward, including 1 involving a colonized healthcare worker (HCW) who had previously been hospitalized overseas. Of the 31 isolates detected, 25 were linked epidemiologically and via molecular typing to the isolate recovered from the colonized HCW. Four isolates appeared to have been introduced separately from overseas. Although the isolate from the single remaining case patient was genetically indistinct from the isolates that spread within Western Australia, no specific epidemiological link could be established. The application of standard outbreak management strategies reduced further spread., Conclusions: The elimination of the New/York Japan MRSA clone in a healthcare region demonstrates the importance of incorporating MRSA management policy into statewide public health programs. The mainstays of such programs should include a comprehensive and effective outbreak identification and management policy (including pre-employment screening of HCWs, where applicable) and MRSA clone identification by multilocus sequence typing.

Objectives: Methicillin-resistant staphylococci (MRS) isolates from healthy and diseased cats and dogs were characterized by staphylococcal cassette chromosome mec (SCCmec), multilocus sequence typing (MLST) and cassette chromosome recombinase gene (ccrAB) sequencing., Methods: PCR-directed SCCmec typing was carried out for all MRS isolates and two Staphylococcus aureus and two Staphylococcus epidermidis strains were analysed by MLST. Strains belonging to SCCmec type III and IV were sequenced for their ccrAB gene of allotypes 3 and 2, respectively., Results: Five types of SCCmec, types I, III, IV, IV (paediatric) and V SCCmec, were found. The S. aureus strains belonged to sequence type (ST) 239 and the two S. epidermidis belonged to ST43 and ST60 respectively. High sequence conservation was observed for the ccrAB gene of allotypes 2 and 3., Conclusions: MRS isolates from cats and dogs demonstrate a similar diversity of SCCmec types to those found in human staphylococci and ST239-MRSA-III, a widely dispersed strain in human hospitals, was identified in diseased dogs.

Aims: To describe clinical features and molecular epidemiology of non-multiresistant methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia., Methods: Patients with non-multiresistant MRSA isolated from blood at South Western Area Pathology Service from 1 January 1999 to 31 December 2001 were enrolled. Pulsed field gel electrophoresis, phage typing, and (selected instances) multilocus sequence and staphylococcal cassette chromosome typing was performed. PCR was used to detect Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin-1 (TSST-1), and enterotoxin genes., Results: Sixteen patients were detected: eight with UK EMRSA-15 (ST22-MRSA-IV), three with Oceania (South-West Pacific/Western Samoan phage pattern) (ST30-MRSA-IV), two with WA MRSA-5 (ST8-MRSA-IV), and one each with WA MRSA-1 (ST1-MRSA-IV), Queensland strain (ST93-MRSA-IV), and WA MRSA-15 (ST59-MRSA-IV). Prior hospital admissions occurred with six of the eight patients with UK EMRSA-15, none of the three with Oceania, and three of the five with other strains. Thirteen of 16 patients had underlying disease. Three of the three patients with Oceania strain bacteraemia were Polynesians; 11 of 13 of the others were Caucasians. PVL genes were detected in four of 16 isolates (all Oceania and Queensland strains). entC was detected in two EMRSA-15 strains; entA in one Oceania, two WA MRSA-5 and the WA MRSA-1 strain, with entA and entB in the WA MRSA-15 strain. tst was not detected., Conclusions: Multiple epidemic strains cause non-multiresistant MRSA bacteraemia. Most patients had risk factors. Oceania and Queensland strains possess the PVL gene.

Objective: To describe antimicrobial resistance and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated in community settings in Australia., Design and Setting: Survey of S. aureus isolates collected prospectively Australia-wide between July 2004 and February 2005; results were compared with those of similar surveys conducted in 2000 and 2002., Main Outcome Measures: Up to 100 consecutive, unique clinical isolates of S. aureus from outpatient settings were collected at each of 22 teaching hospital and five private laboratories from cities in all Australian states and territories. They were characterised by antimicrobial susceptibilities (by agar dilution methods), coagulase gene typing, pulsed-field gel electrophoresis, multilocus sequence typing, SCCmec typing and polymerase chain reaction tests for Panton-Valentine leukocidin (PVL) gene., Results: 2652 S. aureus isolates were collected, of which 395 (14.9%) were MRSA. The number of community-associated MRSA (CA-MRSA) isolates rose from 4.7% (118/2498) of S. aureus isolates in 2000 to 7.3% (194/2652) in 2004 (P = 0.001). Of the three major CA-MRSA strains, WA-1 constituted 45/257 (18%) of MRSA in 2000 and 64/395 (16%) in 2004 (P = 0.89), while the Queensland (QLD) strain increased from 13/257 (5%) to 58/395 (15%) (P = 0.0004), and the south-west Pacific (SWP) strain decreased from 33/257 (13%) to 26/395 (7%) (P = 0.01). PVL genes were detected in 90/195 (46%) of CA-MRSA strains, including 5/64 (8%) of WA-1, 56/58 (97%) of QLD, and 25/26 (96%) of SWP strains. Among health care-associated MRSA strains, all AUS-2 and AUS-3 isolates were multidrug-resistant, and UK EMRSA-15 isolates were resistant to ciprofloxacin and erythromycin (50%) or to ciprofloxacin alone (44%). Almost all (98%) of CA-MRSA strains were non-multiresistant., Conclusions: Community-onset MRSA continues to spread throughout Australia. The hypervirulence determinant PVL is often found in two of the most common CA-MRSA strains. The rapid changes in prevalence emphasise the importance of ongoing surveillance.

Community-associated methicillin-resistant Staphylococcus aureus (MRSA) was first reported in Western Australia in the early 1990s from indigenous peoples living in remote areas. Although a statewide policy of screening all hospital patients and staff who have lived outside the state for MRSA has prevented the establishment of multidrug-resistant epidemic MRSA, the policy has not prevented SCCmec type IV and type V MRSA clones from becoming established. Of the 4,099 MRSA isolates analyzed (referred to the Gram-positive Bacteria Typing and Research Unit) from July 2003 to December 2004, 77.5% were community-associated MRSA (CA-MRSA). Using multilocus sequence/staphylococcal chromosome cassette mec typing, 22 CA-MRSA clones were characterized. Of these isolates, 55.5% were resistant to >1 non-beta-lactam antimicrobial drug. Five Panton-Valentine leukocidin (PVL)-positive CA-MRSA clones were identified. The emergence of multidrug-resistant CA-MRSA clones and the detection of PVL toxin genes in clones previously reported as PVL negative is a major public health concern.

Twenty Australian community staphylococci harboring the type V staphylococcal cassette chromosome mec (SCCmec) were found to belong to eight multilocus sequence types. Five were previously unreported novel type V SCCmec elements. The mec complexes were of two types, based on the polymorphisms in the IS431 transposase genes. Five isolates were multiresistant.

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a notable cause of hospital-acquired infections. A statewide screening and control policy was implemented in Western Australia (WA) after an outbreak of epidemic MRSA in a Perth hospital in 1982. We report on statutory notifications from 1998 to 2002 and review the 20-year period from 1983 to 2002. The rate of reporting of community-associated Western Australia MRSA (WAMRSA) escalated from 1998 to 2002 but may have peaked in 2001. Several outbreaks were halted, but they resulted in an increase in reports as a result of screening. A notable increase in ciprofloxacin resistance during the study period was observed as a result of more United Kingdom epidemic MRSA (EMRSA) -15 and -16. WA has seen a persistently low incidence of multidrug-resistant MRSA because of the screening and decolonization program. Non-multidrug-resistant, community-associated WAMRSA strains have not established in WA hospitals.

Seventy-one percent of 76 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from two medical centers in El Paso, Texas, represent three similar pulsed-field gel electrophoresis types. Overall, six pulsed-field types were identified represented by multilocus sequence/staphylococcal chromosomal cassette DNA mec (SCCmec) types: ST5-MRSA-II; ST36-MRSA-II; ST8 (untypeable SCCmec); and a newly described clonal cluster 8 strain, ST507-MRSA-IV. This study demonstrates the presence of multiple-antibiotic-resistant epidemic MRSA clones in El Paso.

Increasing reports of the appearance of novel nonmultiresistant methicillin-resistant Staphylococcus aureus MRSA (MRSA) strains in the community and of the spread of hospital MRSA strains into the community are cause for public health concern. We conducted two national surveys of unique isolates of S. aureus from clinical specimens collected from nonhospitalized patients commencing in 2000 and 2002, respectively. A total of 11.7% of 2,498 isolates from 2000 and 15.4% of 2,486 isolates from 2002 were MRSA. Approximately 54% of the MRSA isolates were nonmultiresistant (resistant to less than three of nine antibiotics) in both surveys. The majority of multiresistant MRSA isolates in both surveys belonged to two strains (strains AUS-2 and AUS-3), as determined by pulsed-field gel electrophoresis (PFGE) and resistogram typing. The 3 AUS-2 isolates and 10 of the 11 AUS-3 isolates selected for multilocus sequence typing (MLST) and staphylococcal chromosomal cassette mec (SCCmec) analysis were ST239-MRSA-III (where ST is the sequence type) and thus belonged to the same clone as the eastern Australian MRSA strain of the 1980s, which spread internationally. Four predominant clones of novel nonmultiresistant MRSA were identified by PFGE, MLST, and SCCmec analysis: ST22-MRSA-IV (strain EMRSA-15), ST1-MRSA-IV (strain WA-1), ST30-MRSA-IV (strain SWP), and ST93-MRSA-IV (strain Queensland). The last three clones are associated with community acquisition. A total of 14 STs were identified in the surveys, including six unique clones of novel nonmultiresistant MRSA, namely, STs 73, 93, 129, 75, and 80slv and a new ST. SCCmec types IV and V were present in diverse genetic backgrounds. These findings provide support for the acquisition of SCCmec by multiple lineages of S. aureus. They also confirm that both hospital and community strains of MRSA are now common in nonhospitalized patients throughout Australia.

Community methicillin-resistant Staphylococcus aureus (CMRSA) strains are being isolated with increasing frequency around the world. In Western Australia CMRSA are endemic in geographically remote communities and have been found to belong to five different contour-clamped homogeneous electric field (CHEF) electrophoretic patterns. Representatives of each of these CHEF patterns have been compared to CMRSA representative of CHEF patterns from other Australian states and New Zealand. With one exception, all of the isolates were nonmultiresistant and were not resistant to many antimicrobial agents other than the beta-lactams. With one exception, which is not believed to be a CMRSA, all of the isolates harbored a beta-lactamase plasmid. Erythromycin resistance was associated with a 2-kb plasmid. One of the beta-lactamase plasmids was found to be able to acquire additional resistance determinants to become a multiple resistance plasmid. There were 10 multilocus sequence types belonging to eight distantly related clonal complexes of S. aureus. One new sequence type was found. Although most of the CMRSA harbored the type IVa SCCmec, a type IV structural variant was found and two new SCCmec types were identified. Protein A gene (spa) typing revealed two new spa types and, with two exceptions, corresponded to multilocus sequence typing. In contrast to other reports on CMRSA, most of the CMRSA strains studied here did not contain the Panton-Valentine leukocidin genes. The results also demonstrate that nonmultiresistant hospital strains such as UK EMRSA-15 may be able to circulate in the community and could be mistaken for CMRSA based on their resistance profiles.

Objective: To demonstrate that nosocomial transmission of vancomycin-resistant enterococci (VRE) can be terminated and endemicity prevented despite widespread dissemination of an epidemic strain in a large tertiary-care referral hospital., Interventions: Two months after the index case was detected in the intensive care unit, 68 patients became either infected or colonized with an epidemic strain of vanB vancomycin-resistant Enterococcus faecium despite standard infection control procedures. The following additional interventions were then introduced to control the outbreak: (1) formation of a VRE executive group; (2) rapid laboratory identification (30 to 48 hours) using culture and polymerase chain reaction detection of vanA and vanB resistance genes; (3) mass screening of all hospitalized patients with isolation of carriers and cohorting of contacts; (4) environmental screening and increased cleaning; (5) electronic flagging of medical records of contacts; and (6) antibiotic restrictions (third-generation cephalosporins and vancomycin)., Results: A total of 19,658 patient and 24,396 environmental swabs were processed between July and December 2001. One hundred sixty-nine patients in 23 wards were colonized with a single strain of vanB vancomycin-resistant E. faecium. Introducing additional control measures rapidly brought the outbreak under control. Hospital-wide screening found 39 previously unidentified colonized patients, with only 7 more nonsegregated patients being detected in the next 2 months. The outbreak was terminated within 3 months at a cost of dollar 2.7 million (Australian dollars)., Conclusion: Despite widespread dissemination of VRE in a large acute care facility, eradication was achievable by a well-resourced, coordinated, multifaceted approach and was in accordance with good clinical governance.

Objectives: To investigate the incidence and epidemiology of non-multiresistant methicillin-resistant Staphylococcus aureus (nmMRSA) infection in south-east Queensland, Australia., Study Design: A retrospective survey was done of hospital records of all patients who had non-multiresistant MRSA isolated at Ipswich Hospital (a 250-bed general hospital, 40 km south-west of Brisbane, Queensland, Australia) between March 2000 and June 2001. Laboratory typing of these isolates was done with antibiogram, pulsed-field gel electrophoresis, bacteriophage typing and coagulase gene typing., Results: There were 44 infections caused by nmMRSA. Seventeen infections (39%) occurred in patients from the south-west Pacific Islands (predominantly Samoa, Tonga and New Zealand). Laboratory typing showed that the isolates in Pacific Islanders were Pacific Island strains, and 16/17 of these infections were community acquired. Twenty-three infections (52%) occurred in Caucasians. Eleven of the isolates from Caucasians (48%) were a new predominantly community-acquired strain that we have termed the 'R' pulsotype, nine (39%) were Pacific Island strains, and three (13%) were health care institution-associated strains. Four infections occurred in patients who were not Caucasians or Pacific Islanders. Overall, 34 of all 44 infections (77%) were community acquired., Conclusions: Non-multiresistant MRSA infection, relatively frequently observed in Pacific Islanders in south-east Queensland, is now a risk for Caucasians as well, and is usually community acquired. Clinicians should consider taking microbiological specimens for culture and antimicrobial susceptibility testing in patients with suspected staphylococcal infections who are not responding to empirical therapy with beta-lactam antibiotics.

We developed a standardized DNA sequence-based approach for the accurate and timely identification of medically important fungi by sequencing polymerase chain reaction (PCR) products with a rapid automated capillary electrophoresis system. A simple DNA extraction method and PCR amplification using universal fungal primers was used to amplify ribosomal DNA from a range of clinical isolates and reference strains. The entire internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA region was sequenced using automated dye termination sequencing for 89 clinical isolates. These had previously been identified by traditional methods and included 12 ascomycetous yeast species, three basidiomycetous yeast species, eight dermatophyte species and two thermally dimorphic fungi, Scedosporium prolificans and S. apiospermum. Furthermore, 21 reference strains representing 19 different Candida species, Geotrichum candidum and Malassezia furfur were also sequenced as part of this study and were used either as standards for sequence-based comparisons, or as assay controls. Sequence-based identification was compared to traditional identification in a blinded manner. Of the clinical isolates tested, 88/89 had DNA sequences that were highly homologous to those of reference strains accessioned in GenBank, and 87/89 gave a sequence-based identification result that correlated with the traditional identification. In contrast to relatively slow conventional methods of identification, a sequence-based identification from a pure culture can be obtained within 24 h of a DNA extraction carried out after a minimal period of culture growth. We conclude that this approach is rapid, and may be a more accurate cost-effective alternative than most phenotypic methods for identification of many medically important fungi frequently encountered in a routine diagnostic microbiology laboratory.