Evolution of a 72-Kilobase Cointegrant, Conjugative Multiresistance Plasmid in Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates from the Early 1990s.

Horizontal transfer of plasmids encoding antimicrobial resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s, the first CA-MRSA strain isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline, and penicillin resistance genes on plasmid pWBG753 (∼30 kb). WA-5 and pWBG753 appeared only briefly in WA; however, fusidic acid resistance plasmids related to pWBG753 were also present in the first European CA-MRSA isolates at the time. Here, we characterize a 72-kb conjugative plasmid, pWBG731, present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749 family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium, and penicillin resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS257, IS257-dependent miniature inverted-repeat transposable elements (MITEs), and the BinL resolution system of the ß-lactamase transposon Tn552 An evolutionarily intermediate ∼42-kb nonconjugative plasmid, pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline resistance plasmid pT181. IS257 likely facilitated the replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized nonconjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous community-associated methicillin-sensitive S. aureus (CA-MSSA) isolates. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA.

An updated view of plasmid conjugation and mobilization in Staphylococcus.

The horizontal gene transfer facilitated by mobile genetic elements impacts almost all areas of bacterial evolution, including the accretion and dissemination of antimicrobial-resistance genes in the human and animal pathogen Staphylococcus aureus. Genome surveys of staphylococcal plasmids have revealed an unexpected paucity of conjugation and mobilization loci, perhaps suggesting that conjugation plays only a minor role in the evolution of this genus. In this letter we present the DNA sequences of historically documented staphylococcal conjugative plasmids and highlight that at least 3 distinct and widely distributed families of conjugative plasmids currently contribute to the dissemination of antimicrobial resistance in Staphylococcus. We also review the recently documented "relaxase-in trans" mechanism of conjugative mobilization facilitated by conjugative plasmids pWBG749 and pSK41, and discuss how this may facilitate the horizontal transmission of around 90% of plasmids that were previously considered non-mobilizable. Finally, we enumerate unique sequenced S. aureus plasmids with a potential mechanism of mobilization and predict that at least 80% of all non-conjugative S. aureus plasmids are mobilizable by at least one mechanism. We suggest that a greater research focus on the molecular biology of conjugation is essential if we are to recognize gene-transfer mechanisms from our increasingly in silico analyses.

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.

Evolutionary dynamics of methicillin-resistant Staphylococcus aureus within a healthcare system.

BACKGROUND: In the past decade, several countries have seen gradual replacement of endemic multi-resistant healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) with clones that are more susceptible to antibiotic treatment. One example is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe. RESULTS: We investigated the population structure of MRSA in Singaporean hospitals spanning three decades, using whole genome sequencing. Applying Bayesian phylogenetic methods we report that prior to the introduction of ST22, the ST239 MRSA population in Singapore originated from multiple introductions from the surrounding region; it was frequently transferred within the healthcare system resulting in a heterogeneous hospital population. Following the introduction of ST22 around the beginning of the millennium, this clone spread rapidly through Singaporean hospitals, supplanting the endemic ST239 population. Coalescent analysis revealed that although the genetic diversity of ST239 initially decreased as ST22 became more dominant, from 2007 onwards the genetic diversity of ST239 began to increase once more, which was not associated with the emergence of a sub-clone of ST239. Comparative genomic analysis of the accessory genome of the extant ST239 population identified that the Arginine Catabolic Mobile Element arose multiple times, thereby introducing genes associated with enhanced skin colonization into this population. CONCLUSIONS: Our results clearly demonstrate that, alongside clinical practice and antibiotic usage, competition between clones also has an important role in driving the evolution of nosocomial pathogen populations.

Methicillin-resistant Staphylococcus aureus clones, Western Australia.

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.

Contour-clamped homogeneous electric field electrophoresis of Staphylococcus aureus.

Contour-clamped homogeneous electric field (CHEF) electrophoresis is a technique of pulsed-field gel electrophoresis that enables the resolution of large fragments of DNA that cannot be resolved by conventional gel electrophoresis. The procedure involves the application of controlled electric fields that change direction at a predetermined angle to samples of DNA that have been embedded in an agarose gel matrix and digested with a restriction endonuclease. Adjustment of the electrophoresis conditions enables the separation of DNA fragments with lengths from 10 kilobases up to 9 megabases in a size-dependent manner in agarose gels. The banding patterns can be used for epidemiological typing, the separated DNA can be immobilized onto a membrane and used for genetic mapping, or individual fragments can be extracted and used for downstream genetic manipulations. The protocol requires specialized equipment and can be completed in a maximum of 7 days.

Methicillin-resistant Staphylococcus aureus, Western Australia.

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.

Community-onset methicillin-resistant Staphylococcus aureus bacteremia in Northern Australia.

BACKGROUND: Community-onset infections caused by methicillin-resistant Staphylococcus aureus (COMRSA) are being increasingly reported worldwide. METHODS: A retrospective study was performed of 14 patients with 15 episodes of COMRSA bacteremia (COMRSAB) admitted to the Royal Darwin Hospital, Northern Territory, Australia from 1998 to 2001. Isolates from COMRSAB episodes underwent extended susceptibility testing and molecular typing by pulsed field gel electrophoresis and allotyping of the staphylococcal cassette chromosome mec (SCCmec) region by polymerase chain reaction. RESULTS: The proportion of community-onset S. aureus bacteremia episodes that were due to COMRSA increased from 9% in 1998 to 20% in 2001. The clinical features of COMRSAB were similar to those seen with methicillin-susceptible strains, including sepsis, endocarditis and metastatic infection. Ineffective empiric antimicrobial therapy was administered in the majority (80%) of episodes. All COMRSAB isolates tested contained allotype IV SCCmec, which is commonly found in community isolates of MRSA and rarely found in isolates from healthcare-associated MRSA infection. CONCLUSION: The increasing incidence of COMRSAB in our region has resulted in the addition of vancomycin to standard empiric therapy in certain patients with suspected S. aureus bacteremia acquired in the community.

Are all community methicillin-resistant Staphylococcus aureus related? A comparison of their mec regions.

AIMS: To compare the relationship of community-acquired, methicillin-resistant Staphylococcus aureus (CMRSA) from five Australian States and New Zealand. METHODS: Contour-clamped homogeneous electric field (CHEF) electrophoresis and analysis of the mec complex and ccr gene complex by PCR were used to compare 22 CMRSA isolates from Western Australia (WA), South Australia (SA), Victoria (VIC), New South Wales (NSW) and New Zealand (NZ) and three hospital-acquired epidemic MRSA (EMRSA). RESULTS: Sixteen community isolates were found to carry Class B mec complex and Type 2 ccr gene complex. Two WA isolates carried the Class B1 mec complex and three VIC and one SA isolate carried a previously unreported mec complex, which has been labelled E. The ccr gene type of the Class B1 and Class E isolates could not be determined. These isolates may carry previously unreported ccr gene complexes. The relatedness of the CHEF patterns of the CMRSA was dependent on their geographical origin. A similar CHEF pattern was found in some WA MRSA, VIC and SA isolates. NSW and NZ CMRSA had the same CHEF patterns and were similar to three VIC isolates and EMRSA-16. Two SA CMRSA isolates had CHEF patterns similar to the English EMRSA-15 strain. A multiply resistant, nosocomial EMRSA from Australia had a class A mec complex, and a CHEF pattern, which was unrelated to any of the CMRSA. CONCLUSION: Most of the CMRSA isolated from five Australian states and New Zealand had unrelated CHEF patterns. However, the majority of them carried the Type IV SCCmec cassette (Class B mec and Type 2 ccr gene complexes), which indicates that they may have acquired their mec complex from the same source or that they have evolved from the same progenitor. Some of the CMRSA had a previously undescribed SCCmec cassette.

Alterations in phage-typing patterns in vancomycin-intermediate Staphylococcus aureus.

The ability of phage-typing and SmaI chromosomal RFLPs to conclude appropriate strain relatedness between a collection of 12 well-characterized in vitro-selected vancomycin-intermediate Staphylococcus aureus (VISA) strains and their seven vancomycin-susceptible parent strains is reported. Generally, no SmaI RFLP alterations were observed in VISA strains when they were compared with their respective parent strains, and clonal relationships between isogenic strains were clearly evident. Unlike the SmaI RFLP results, parent strains and VISA derivatives generally did not share similar phage-typing profiles. Depending on the phage set investigated, some VISA strains even became untypable by this method. Loss of phage infectivity is probably due to cell wall (phage receptor) alterations that are expressed by the VISA strains investigated. Collectively, these findings indicate that inappropriate relationships between VISA and vancomycin-susceptible parents might be drawn if only phage-typing and antibiotic susceptibility are utilized to determine epidemiological relationships.

Characterization of drug-resistant Staphylococcus aureus isolated from poultry processing plants in Western Australia.

Poultry processing plants can provide a favourable environment for the survival and transmission of Staphylococcus aureus. It is known that infections due to antibiotic-resistant strains of S. aureus are an increasingly serious problem clinically and, since antibiotic exposure in food-animal species may lead to antibiotic-resistant bacteria, it is possible that processed poultry may constitute a reservoir for disseminating antibiotic-resistance into the community. The present study was undertaken to determine the prevalence of antimicrobial-resistant S. aureus in two poultry processing plants, and to characterize the isolates by antimicrobial susceptibility and chromosomal and plasmid DNA analysis. One hundred and twenty-six S. aureus were isolated from two poultry processing plants in Western Australia. All were sensitive to 14 of the 26 antimicrobials tested and all isolates were resistant to at least one antibiotic and one chemical marker, the prominent resistance combination being to penicillin and cadmium (89%). Forty-six (36.5%) of the isolates were resistant to six or more of the antimicrobial agents tested. Overall there were no consistent resistance patterns for the isolates and no consistent patterns were found between and within the two processing plants. There were 24 epidemiologically unrelated Sma1 contour-clamped homogeneous electric field (CHEF) groups and 17 different plasmid profiles detected among the isolates. All isolates were found to harbour from between one to seven plasmids. The majority of isolates carried at least one large plasmid (22-48 Kb), and one or more small plasmids (1-3 Kb). Some isolates with epidemiologically related CHEF patterns had similar plasmid profiles and resistance patterns.

Genetic organization of mecA and mecA-regulatory genes in epidemic methicillin-resistant Staphylococcus aureus from Australia and England.

The mecA gene that encodes methicillin resistance in Staphylococcus aureus may be regulated by the mecR1 and mecI genes, and this region has been referred to as the mec complex. An analysis of these regulatory genes in 35 epidemic methicillin-resistant S. aureus (MRSA) isolated in England and Australia has found that they contain three classes of mec complex. Firstly, the Class A mec complex with complete mecR1 and mecI genes. Secondly, a new variant of Class A, the Class A1 mec complex, with a 166 bp deletion in the membrane-spanning domain of mecR1 and a complete mecI gene. Thirdly, the Class B mec complex, in which the penicillin-binding domain of mecR1 and the whole mecI gene are deleted by the insertion of a partial sequence of IS1272. Seven MRSA isolated in England and Australia over different time periods had the Class A mec complex. However, the isolates did not have closely related pulsed-field gel electrophoresis (PFGE) patterns. The Class A1 mec complex was found in 12 Australian isolates and the English epidemic MRSA, EMRSA-1. All these organisms were isolated in the early 1980s and had closely related PFGE patterns. The Class B mec complex region was found in nine EMRSA and seven Australian MRSA isolated over the period from the 1970s to 2000. These isolates had related PFGE patterns. The mecA region was also compared in the isolates and all but two of the isolates had an XbaI restriction site. These results support the global spread of epidemic clones and confirm the close relationship between the Australian and English MRSA strains.

Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community.

Multiple methicillin-resistant Staphylococcus aureus (MRSA) clones carrying type IV staphylococcal cassette chromosome mec were identified in the community-acquired MRSA strains of both the United States and Australia. They multiplied much faster than health-care-associated MRSA and were resistant to fewer non-beta-lactam antibiotics. They seem to have been derived from more diverse S. aureus populations than health-care-associated MRSA strains.

Genetic characterization of the fusidic acid and cadmium resistance determinants of Staphylococcus aureus plasmid pUB101.

We report the cloning of the fusidic acid and cadmium resistance determinants from Staphylococcus aureus plasmid pUB101. The pUB101 fusidic acid resistance determinant was located on a 2.9 kb HindIII fragment. Sequencing of this fragment revealed three putative open reading frames (ORFs) of 213 (far1), 152 (orf152) and 170 amino acids (orf170), which are flanked by the right-hand end of insertion sequence IS431/257 (IS431/257RH) and a partial ORF. Far1 and Orf152 demonstrated homology with a chromosomally encoded fibronectin-binding protein of Listeria monocytogenes and the putative protein YosT, found on the SP beta c2 prophage of Bacillus subtilis, respectively. Transformation of S. aureus with a construct containing a 949 bp far1-specific amplicon led to the isolation of a fusidic acid-resistant transformant, thereby identifying the pUB101 fusidic acid resistance structural gene. Between orf152 and far1 we identified a unique 113 bp symmetrical element and other repeat elements that may be involved with the control of orf152 and/or far1 expression. Hybridization of Southern blots revealed that far1 was not located on the chromosome or plasmid content of a limited number of Australian, UK and Hong Kong fusidic acid-resistant isolates. The pUB101 cadmium resistance determinant was located on a 3.6 kb HindIII fragment that carried a cadDX operon, remnants of two putative plasmid replication protein genes and IS431/257RH. Sequence analysis also demonstrated the presence of a single-stranded origin of replication, normally found on rolling circle replicating plasmids, within the putative promoter region of the cadDX operon.

Proteome analysis of highly immunoreactive proteins of Helicobacter pylori.

BACKGROUND: Identification of the immunoreactive proteins of Helicobacter pylori is important for the development of both diagnostic tests and vaccines relating to the organism. Our aim was to determine whether there are significant differences between human IgG and IgA reactivities to individual H. pylori proteins, and whether patterns of immunoreactivity are sustained across different strains of H. pylori. METHOD: The total complement of protein from seven strains of H. pylori was resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins were transferred electrophoretically onto polyvinylene difluoride (PVDF) membranes, which were probed with sera pooled either from H. pylori-infected patients, or noninfected (control) patients. Highly immunoreactive proteins were detected using chromogenic enzyme-antibody conjugates recognising either serum IgG or IgA. These proteins were then characterised by tryptic peptide-mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Highly immunoreactive proteins were detected which were common to all seven strains, and recognised by both immunoglobulin subclasses. The proteins appear to be localised in five groups. Protein analysis established that these groups encompass multiple isoforms of chaperonin HspB (two subgroups); urease beta-subunit UreB; elongation factor EF-Tu; and flagellin FlaA. The pattern of highly immunoreactive proteins was strongly conserved across the seven strains. CONCLUSION: These results suggest that within a tightly defined region on the H. pylori proteome map there are five groups of proteins that are highly reactive to both IgG and IgA. Our analysis suggests it is unlikely that the highly immunoreactive clusters harbour any significant proteins other than isoforms of HspB, UreB, EF-Tu and FlaA, and that, with the partial exception of FlaA, these clusters are strongly conserved across all seven strains.