The Australian community methicillin resistant Staphylococcus aureus endemic : clonal spread or multiple evolutionary events

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was first reported in remote indigenous communities living in the sparsely populated Kimberley region of Western Australia (WA). Between 1989 and 1995 five Panton Valentine leucocidin (PVL) negative clones were isolated from these communities: ST1-MRSA-IVa [2B] (WA-MRSA-1), ST78-MRSA-IVa [2B] (WA-MRSA-2), ST5- MRSA-IVa [2B] (WA-MRSA-3), ST45-MRSA-V [5C2] (WA-MRSA-4), and ST8- MRSA-IVa [2B] (WA-MRSA-5).Between 1995 and 2003, S. aureus screening of the indigenous populations living in 11 of these remote communities showed the S. aureus population consisted of 13 multilocus sequence type clonal complexes (CCs) and two Singleton lineages. Although five lineages contained MRSA, the MRSA lineages 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 (SCCmec) element. The emergence of CA-MRSA clones in different CCs indicates horizontal transmission of the SCCmec element into S. aureus had occurred on at least six occasions: SCCmec IVa [2B] into CC1 (ST1), CC5 (ST5), CC8 (ST8), CC45 (ST45), CC88 (ST78) and SCCmec V [5C2] into CC45 (ST45). Based upon the spa type and the DNA microarray profile six evolutionary events have subsequently occurred on at least three occasions from these clones (i.e. vertical transmission of the SCCmec element): twice from WA-MRSA-1, WA-MRSA-3, and WA-MRSA-5. Vertical transmission of the SCCmec element has not been identified for WA-MRSA-4 or WA-MRSA-2. The most prevalent MSSA lineage in the communities was the PVLpositive Singleton ST93 clone. As ST93-MRSA-IVa [2B], colloquially known as Queensland CA-MRSA, has become the most prevalent CA-MRSA in Australia, it was surprising in an environment of high β-lactam use and frequent horizontal transmission of SCCmec IVa a methicillin-resistant variant of ST93-MSSA was not found.Within these indigenous communities people colonised with MSSA tended to harbour clones of a different genetic lineage at each anatomical site while people colonised with MRSA tended to harbour clones of the same lineage at each site. Although the anterior nares is the preferred screening site for population studies, in this study many isolates of S. aureus would have been missed if throat and skin lesions had not also been swabbed. Three MRSA clones (WA-MRSA-1, WAMRSA- 2, and WA-MRSA-3) considered to be endemic in these communities have subsequently become predominant clones in the wider Australian community.Although WA-MRSA-1, WA-MRSA-2, WA-MRSA-3 and Queensland CA-MRSA predominate, the CA-MRSA population in Australia is genetically diverse. In WA, between 2003 and 2010, 83 unique pulsed-field gel electrophoresis (PFGE) strains were described from which 46 multilocus sequence types have been characterised. Forty five of these sequence types (STs) were from 18 CCs and two Singletons. While SCCmec IV and V were the predominant SCCmec elements, SCCmec VIII and several novel and composite SCCmec elements were present. The emergence of MRSA in diverse S. aureus CCs suggest horizontal transmission of the SCCmec elements has occurred on multiple occasions. Furthermore, DNA microarray and spa typing suggest 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 were detected. This suggests 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.As WA CA-MRSA, colloquially known as “WA-MRSA” are typically PVL negative many of the MRSA infections in WA have been superficial skin infections. However with the recent introduction of PVL-positive CA-MRSA more severe skin and soft tissues infections accompanied with a significant decrease in the age of patients have been observed.In 2010, 22% of CA-MRSA isolated in WA were PVL positive, with Queensland CA-MRSA being the predominant PVL-positive clone. The emergence of Queensland CA-MRSA (ST93-MRSA-IVa [2B]) has been 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 identified, PVL-positive ST93-MRSA-IVa [2B]-t202-dt10 was the predominant strain. Whether this strain arose from one PVL-positive ST93- MSSA-t202 or by independent acquisition of SCCmec-IVa [2B]-dt10 into multiple PVL-positive ST93-MSSA-t202 strains is yet to be determined.Several international PVL-positive clones have been introduced into WA, including the CC59 strain ST59-MRSA-VT [5C2&5] (Taiwan CA-MRSA clone), and the CC8 strain ST8-MRSA-IV [2B] (USA300). Genetic analysis of these strains indicated they are distinct from WA CA-MRSA clones.Although ST59-MRSA-VT [5C2&5] (Taiwan CA-MRSA clone) was found to be the most prevalent CC59 clone isolated in WA, independent evolution of PVL-negative CC59 CA-MRSA has occurred. Using a variety of molecular techniques, six distinct groups of CC59 were differentiated. Within these groups at least seven different variants of SCCmec elements were distinguished; (IVa [2B], IVb [2B], IVd [2B], IVa [2B]&5, IVv [2B], Vv [5C2], and V [5C2&5]. This suggests rapid evolution and/or multiple transfer events of SCCmec have occurred within this CC. Although some CC59 isolates in WA have overseas origins (eg Taiwan CA-MRSA clone and possibly USA1000), PVL-negative CC59 lineages unique to WA have acquired various SCCmec types on multiple occasions.The PVL-positive ST8-MRSA-IV [2B] strain isolated in WA was found to be closely related to USA300, with most isolates unable to be distinguished from USA300- TC1516. Some isolates however varied in their carriage of resistance and virulence determinants and therefore USA300 in Australia cannot be regarded as being genetically homogeneous. Altogether 16 variants were identified. Notably some isolates did not harbour the ACME locus, which is intriguing because this locus is assumed to be involved in facilitating the spread of USA300 by skin contact.In conclusion, this thesis has shown “WA-MRSA” arose in remote indigeneous communities located in WA, and three of these clones have subsequently become the most prevalent MRSA clones in Australia. However “WA-MRSA” did not arise from the predominant MSSA clones isolated from these remote communities. Although the vertical and horizontal transmission of SCCmec elements into S. aureus has occurred on multiple occasions in the WA community only three “WA-MRSA” clones have found an ecological niche. These three PVL negative clones harbour few additional resistance and virulence genes which paradoxically may contribute to their success. PVL-positive CA-MRSA infections have become more prevalent in young Australians. Although primarily due to Queensland CA-MRSA, international PVL-positive CA-MRSA clones are present in Australia

Community-onset Staphylococcus aureus infections presenting to general practices in South-eastern Australia

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

Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids in Staphylococcus aureus

Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. A significant proportion of antimicrobial-resistance genes are plasmid-borne, but only a minority of S. aureus plasmids encode proteins required for conjugative transfer or Mob relaxase proteins required for mobilisation. The pWBG749 family of S. aureus conjugative plasmids can facilitate the horizontal transfer of diverse antimicrobial-resistance plasmids that lack Mob genes. Here we reveal that these mobilisable plasmids carry copies of the pWBG749 origin-of-transfer (oriT) sequence and that these oriT sequences facilitate mobilisation by pWBG749. Sequences resembling the pWBG749 oriT were identified on half of all sequenced S. aureus plasmids, including the most prevalent large antimicrobial-resistance/virulence-gene plasmids, pIB485, pMW2 and pUSA300HOUMR. oriT sequences formed five subfamilies with distinct inverted-repeat-2 (IR2) sequences. pWBG749-family plasmids encoding each IR2 were identified and pWBG749 mobilisation was found to be specific for plasmids carrying matching IR2 sequences. Specificity of mobilisation was conferred by a putative ribbon-helix-helix-protein gene smpO. Several plasmids carried 2–3 oriT variants and pWBG749-mediated recombination occurred between distinct oriT sites during mobilisation. These observations suggest this relaxase-in trans mechanism of mobilisation by pWBG749-family plasmids is a common mechanism of plasmid dissemination in S. aureus

The Molecular Epidemiology of the Highly Virulent ST93 Australian Community Staphylococcus aureus Strain

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 WSa2USA 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 PVLpositive 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

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolated from Australian veterinarians

This work investigated the molecular epidemiology and antimicrobial resistance of methicillinresistant Staphylococcus aureus (MRSA) isolated from veterinarians in Australia in 2009. The collection (n = 44) was subjected to extensive molecular typing (MLST, spa, SCCmec, dru, PFGE, virulence and antimicrobial resistance genotyping) and antimicrobial resistance phenotyping by disk diffusion. MRSA was isolated from Australian veterinarians representing various occupational emphases. The isolate collection was dominated by MRSA strains belonging to clonal complex (CC) 8 and multilocus sequence type (ST) 22. CC8 MRSA (ST8-IV [2B], spa t064; and ST612-IV [2B] , spa variable,) were strongly associated with equine practice veterinarians (OR = 17.5, 95% CI = 3.3-92.5, P &lt; 0.001) and were often resistant to gentamicin and rifampicin. ST22-IV [2B], spa variable, were strongly associated with companion animal practice veterinarians (OR = 52.5, 95% CI = 5.2-532.7, P &lt; 0.001) and were resistant to ciprofloxacin. A single pig practice veterinarian carried ST398-V [5C2], spa t1451. Equine practice and companion animal practice veterinarians frequently carried multiresistant-CC8 and ST22 MRSA, respectively, whereas only a single swine specialist carried MRSA ST398. The presence of these strains in veterinarians may be associated with specific antimicrobial administration practices in each animal species

Molecular characterization of endocarditis-associated Staphylococcus aureus

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

Evolution and diversity of community-associated methicillin-resistant Staphylococcus aureus in a geographical region

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

Reversible vancomycin susceptibility within emerging ST1421 Enterococcus faecium strains is associated with rearranged vanA-gene clusters and increased vanA plasmid copy number

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