Characterization of a novel staphylococcal cassette chromosome composite island from community-associated MRSA isolated in aged care facilities in Western Australia.

BACKGROUND: In Western Australia (WA), clonal complex 5, ST835, community-associated (CA) MRSA is isolated almost exclusively from aged care facilities. In WA four different staphylococcal cassette chromosome (SCC) mec (SCCmec) elements have been identified in this ST, indicating high genetic activity in the SCCmec region. OBJECTIVES: To investigate the SCC region of ST835 CA-MRSA WA MRSA-40 and determine the distribution of an SCCsorbitol element found within the region. RESULTS: The SCC region contained a composite island, SCCmecWA MRSA-40-CI, that was composed of three elements, ΨSCCpls, SCCsorbitol and SCCmecVT (5C2&5). This is the first time that a sorbitol operon has been reported in an SCC element. CONCLUSIONS: Generation of SCCmecWA MRSA-40-CI has involved multiple genetic events and recombination with CoNS has occurred during evolution of the SCC elements. While Staphylococcus aureus is renowned for its ability to utilize mobile genetic elements to disseminate antimicrobial resistance, the SCC region of WA MRSA-40 shows that this clone has also utilized SCC elements to acquire extra virulence and possibly adapt to a niche environment.

Staphylococcus aureus plasmids without mobilization genes are mobilized by a novel conjugative plasmid from community isolates.

OBJECTIVES: To describe a family of conjugative plasmids isolated from colonizing community Staphylococcus aureus and determine their ability to mobilize unrelated antimicrobial resistance/virulence plasmids, not encoding mobilization functions. METHODS: Plasmid pWBG749 was labelled with Tn551 (pWBG749e) to enable laboratory manipulation. Plasmid pWBG749e was conjugated into S. aureus of seven different lineages that harboured unrelated plasmids and mobilization experiments were performed. Plasmids were screened by EcoRI restriction and hybridization with probes prepared from unique pWBG749 conjugation genes. RESULTS: Conjugative plasmids pWBG745, pWBG748 and pWBG749 belong to the same conjugative-plasmid family as the vancomycin resistance plasmid pBRZ01. Plasmid pWBG749e mobilized five unrelated plasmids. Mobilized plasmid pWBG744 is a pIB485-family plasmid that was also found in international S. aureus. CONCLUSIONS: Plasmid pWBG749e can mobilize unrelated S. aureus plasmids whose means of dissemination have not previously been understood.

Population dynamics of methicillin-susceptible and -resistant Staphylococcus aureus in remote communities.

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.

Genetic lineages of community-associated methicillin-resistant Staphylococcus aureus in Kuwait hospitals.

Twenty-six community-associated methicillin-resistant Staphylococcus aureus (CAMSRA) isolates were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and screened for accessory gene regulator (agr), capsular polysaccharide (cap), and Panton-Valentine leucocidin (PVL) genes. They exhibited five PFGE patterns (types A to E). The majority were PFGE type A (12 isolates) or type B (8 isolates). MLST showed that PFGE type A isolates belonged to sequence type 80 (ST80), while the PFGE type B isolates were ST30. The ST80 and ST30 clones contained agr allotype 3, cap type 8, and PVL. The results showed that two internationally recognized CAMRSA clones are dominant in Kuwait hospitals.

Type V staphylococcal cassette chromosome mec in community staphylococci from Australia.

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.

Diversity among community isolates of methicillin-resistant Staphylococcus aureus in 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.

Thermal inactivation of antimicrobial-resistant Gram-positive cocci in chicken meat: D and Z value determinations.

Antimicrobial-resistance in Gram-positive bacteria is reported with increasing frequency in strains isolated from food animals. Their isolation from commercial poultry carcasses and meat products constitute a potential risk that resistant strains or resistance genes might spread to humans via the food chain. As bacterial inactivation by thermal process is a critical control point in the safe preparation of many ready-to-eat foods, it is important to determine the thermal resistance of these organisms. The present study was undertaken to investigate the thermal tolerance (D and Z values) of antimicrobial-resistant, Gram-positive cocci in ground chicken meat. The antimicrobial-resistant, Gram-positive cocci for this study were isolated from two poultry processing plants in Western Australia. D and Z value data indicate that these isolates do not exhibit enhanced thermal resistant characteristics. The estimated lethal effect of the cooking process for chicken meat indicates that an internal temperature of 70 degrees C for 2.1 min would provide a 7-log reduction of all cell suspensions tested.

Persistence of a clone of methicillin-resistant Staphylococcus aureus in a burns unit.

A total of 128 MRSA isolates from a burns unit in 1992 and 1997 was studied by resistotyping, plasmid analysis and pulsed-field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA to ascertain whether a clone of MRSA had persisted in the unit or whether different clones had been introduced at different times. All the MRSA isolates produced beta-lactamase and had high MICs to methicillin (>256 mg/L). All were resistant to tetracycline, kanamycin, cadmium acetate and mercuric chloride. Most were resistant to gentamicin, neomycin, erythromycin, chloramphenicol, trimethoprim, ciprofloxacin, propamidine isethionate and ethidium bromide, and were susceptible to minocycline, vancomycin and teicoplanin. None of the 1992 isolates was resistant to mupirocin, but 56% and 19% of the 1997 isolates expressed high- and low-level mupirocin resistance, respectively. Many of the 1997 isolates had acquired a 38-kb plasmid encoding high-level mupirocin resistance. The 1992 isolates had two main PFGE patterns; 82% of them belonged to PFGE pattern 1. The 1997 isolates had PFGE pattern 1, the same as the majority of the 1992 isolates. All MRSA isolates from both years carried the mecA gene in the same SmaI fragment. These findings demonstrated that a clone of MRSA that was prevalentin the burns unit in 1992 had persisted and became the predominant clone in 1997.

New Staphylococcus aureus incompatibility group 1 plasmids encoding penicillinase production and resistance to different antibacterial agents.

Eleven Staphylococcus aureus plasmids encoding penicillinase production and resistance to different antibacterial agents were transferred to laboratory recipient strains in mixed-culture transfer and transduction experiments and characterized by restriction endonuclease analysis and incompatibility. The plasmids were differentiated into four types (types A-D) on the basis of their resistance phenotypes and restriction endonuclease patterns. One type encoded resistance to cadmium and arsenate. The second type encoded resistance to cadmium, mercuric compounds and nucleic acid-binding compounds. The third type encoded resistance to cadmium, kanamycin, neomycin and streptomycin while the fourth type encoded resistance to kanamycin, neomycin and ethidium bromide. Plasmids within the same class were structurally related or similar and were different from those in the other classes. Three plasmids, pWBG626, pWBG628, and pWBG663, representing three of the four plasmid types, belonged to incompatibility group 1. These new plasmids add to the number of known incompatibility group 1 plasmids and have resistance phenotypes which should be useful for studying incompatibility of new S. aureus plasmids.

Effects of salicylate and related compounds on fusidic acid MICs in Staphylococcus aureus.

Salicylate, acetyl-salicylate, benzoate and ibuprofen increased fusidic acid MICs for fusidic acid-resistant and -susceptible strains of Staphylococcus aureus representing six genetic lineages. The effects of these substances on fusidic acid resistance levels occurred in a strain-dependent manner. The weak acid acetate, and acetaminophen did not alter fusidic acid resistance levels, while the addition of saligenin, the alcohol of salicylate, reduced gradient plate MICs for all strains studied. These findings indicate that a benzoic acid structure is required for the induction of increased intrinsic fusidic acid resistance levels. When 2 mM salicylate was added to media used in population analyses, the number of cells able to survive on high concentrations of fusidic acid increased. This increase in cell survival was observed in two unrelated fusidic acid-resistant strains, with chromosomal (WBG8287) or plasmid (WBG1576) mediated resistance determinants and two unrelated susceptible strains. The salicylate-induced increase in fusidic acid resistance was phenotypic at low fusidic acid concentrations (relative to resistance phenotype) for WBG8287 and a fusidic acid-susceptible strain. On media containing salicylate and high fusidic acid concentrations, the mutation frequency to higher fusidic acid resistance levels was greater for WBG8287, compared with unsupplemented fusidic acid-containing media. These experiments provide evidence for a novel salicylate inducible fusidic acid resistance mechanism in S. aureus.

Community strain of methicillin-resistant Staphylococcus aureus involved in a hospital outbreak.

Western Australia (WA) has been able to prevent methicillin-resistant Staphylococcus aureus (MRSA) strains from outside of the state from becoming established in its hospitals. Recently, a single-strain outbreak of MRSA occurred in a WA metropolitan teaching hospital following admission of an infected patient from a remote community. The strain responsible for the outbreak was unrelated to any imported strains and spread rapidly in the hospital. Screening of two remote communities in the region from which the index case came revealed that 42% of the people in one community and 24% in the other carried MRSA. Isolates were typed by resistance pattern, plasmid analysis, contour-clamped homogeneous electric field electrophoresis, bacteriophage pattern, and coagulase gene restriction fragment length polymorphism. It was found that of the people carrying MRSA, 39% in the former community and 17% in the latter community were carrying an MRSA strain which was indistinguishable from the strain that caused the hospital outbreak.

Growth in the presence of salicylate increases fluoroquinolone resistance in Staphylococcus aureus.

Salicylate and acetylsalicylate slightly increased fluoroquinolone resistance in ciprofloxacin-susceptible and -resistant Staphylococcus aureus. Salicylate allowed a greater number of cells from ciprofloxacin-susceptible and -resistant strains to survive on high fluoroquinolone concentrations. Salicylate also increased the frequency with which a susceptible strain mutated to become more resistant to ciprofloxacin.

Heterogeneous expression of fusidic acid resistance in Staphylococcus aureus with plasmid or chromosomally encoded fusidic acid resistance genes.

Fusidic acid resistance expression in a methicillin susceptible Staphylococcus aureus strain (WBG1576), which carries fusidic acid resistance on plasmid pUB101, and a prevalent Western Australian methicillin-fusidic acid resistant strain (WBG8287) were compared. WBG8287 carries fusidic acid resistance on the chromosome and its plasmid content has no effect on the levels of this resistance. WBG1576 and WBG8287 exhibited similar heterogeneous populations in respect to fusidic acid resistance levels in population analyses. A high-level fusidic acid resistant mutant of WBG1576 (BE8) had alterations in Smal chromosomal profiles, but not in plasmid size or resistance expression. Mutations causing increased fusidic acid resistance in WBG1576 are chromosomally located. A high-level fusidic acid resistant mutant of WBG8287 (BE3) had no alterations in Smal chromosomal profiles, or plasmid content and resistances. Comparison of resistance levels to kanamycin and spectinomycin, between high-level resistant colonies of WBG8287 and WBG8287, indicate that mutations in the chromosomal gene fusA, which encodes elongation factor-G, are probably the cause of the increased resistance levels observed in these mutant strains.

Molecular and phage typing of Staphylococcus aureus harbouring cryptic conjugative plasmids.

The spread of antibiotic resistant-bacterial pathogens in a hospital could be due to the spread of a resistant strain or the spread of a resistance plasmid among unrelated strains. In this study the relatedness of Staphylococcus aureus isolates carrying identical cryptic conjugative plasmids was determined by a combination of resistance profiles, plasmid patterns, pulsed field gel electrophoresis of SmaI digested chromosomal DNA and phage typing. Results of the different typing techniques were in agreement to one another and demonstrated that the isolates were of three different types. The results suggested that a cryptic conjugative plasmid had spread to different S. aureus isolates in the hospital. This is an example of plasmid spread as opposed to strain spread.

A phage-mediated transfer of chromosomally integrated tetracycline resistance plasmid in Staphylococcus aureus.

The ability of a Staphylococcus aureus isolate WBG7416 to transfer its resistance determinants was studied in conjugation and mixed-culture transfer experiments. It carried plasmid-borne resistance to kanamycin, trimethoprim, chloramphenicol, cadmium, propamidine isethionate, and chromosomal resistance to methicillin, gentamicin, streptomycin, erythromycin, clindamycin, tetracycline, and minocycline. It transferred tetracycline resistance in mixed-culture transfer but not in conjugation experiments. DNA-DNA hybridization of genomic DNA from the tetracycline-resistant transferrants against a labeled tetracycline resistance plasmid, pWBG3, probe revealed the presence of an integrated plasmid in their chromosomes. In contrast, no homology to the probe was detected in the chromosome of a tetracycline-resistant mutant of the recipient strain. The results established a role for a bacteriophage in the transfer of chromosomal tetracycline resistance in WBG7416 besides demonstrating the presence of an integrated tetracycline resistance plasmid in the transferrants. It also offered an insight into the nature of the integrated plasmid.

Detection of an integrated tetracycline-resistance plasmid in Staphylococcus aureus from a Nigerian hospital.

The genetics of tetracycline-resistance determinants was studied in eight methicillin-resistant and two methicillin-sensitive Staphylococcus aureus isolated from a Nigerian hospital. Curing and transfer experiments demonstrated that one methicillin-sensitive S. aureus isolate WBG4762, had a 4.4 kb extrachromosomal plasmid- as well as a chromosomally-mediated tetracycline resistance. All others had chromosomal tetracycline resistance and were resistant to either tetracycline and minocycline or tetracycline only. The two methicillin-susceptible isolates were resistant to both tetracycline and minocycline. Chromosomal DNA from all the resistant isolates hybridized with a digoxigenin-11-dUTP labeled 4.4 kb tetracycline-resistance plasmid probe indicating that they contained tetracycline-resistance plasmids similar to the probe integrated into their chromosomes. The results demonstrated the presence of integrated tetracycline-resistance plasmid in both methicillin-resistant and methicillin-susceptible S. aureus resistant to tetracycline and minocycline as well as those resistant only to tetracycline. This is the first demonstration of an integrated tetracycline-resistance plasmid in methicillin-sensitive S. aureus and suggests that the integrated tetracycline-resistance plasmid may be widespread in S. aureus.

Transfer of plasmid-borne resistance from a multiply-resistant Staphylococcus aureus isolate, WBG1022.

Staphylococcus aureus isolate, WBG1022, was resistant to penicillin, kanamycin, neomycin, streptomycin, chloramphenicol, trimethoprim, cadmium, and ethidium bromide and harbored plasmids of 34.5, 24.5, 4.4, 3.2, and 2.6 kilobases. The plasmids were transferred in mixed-culture transfer and conjugation experiments. No resistance phenotype was associated with the 2.6-kb plasmid. The 3.2-kb and 4.4-kb plasmids encoded chloramphenicol and streptomycin resistance respectively. The 24.5-kb plasmid, pWBG626, encoded joint resistance to penicillin, kanamycin, neomycin, and ethidium bromide. Resistance to trimethoprim and cadmium were chromosomal. The 34.5-kb plasmid, pWBG661, had no resistance phenotype but was found to be conjugative. It also mobilized the 4.4-kb and 24.5-kb plasmids in WBG1022. Restriction endonuclease analysis of pWBG661 with EcoRI, ClaI, PvuII, and BglII restriction enzymes demonstrated that pWBG661 was identical to two previously isolated S. aureus conjugative plasmids, pWBG620 and pWBG637, that also lack resistance phenotypes.

Murine strongyloidiasis: the effects of cyclosporin A and thiabendazole administered singly and in combination.

A single Cyclosporin A (CsA) dose of 30 mg kg-1 given orally at day 4 post-infection (p.i.) to Sprague-Dawley rats infected with Strongyloides ratti, reduced the faecal larval count by 46.8 +/- 1.2%. CsA was equally effective when the same dose rate was administered subcutaneously at day 4 p.i., reducing the faecal larval count by 41.6 +/- 8.6%. Thiabendazole (TBZ) given orally at 5 or 10 mg kg-1 (single dose at day 4 p.i.) reduced the faecal larval counts by 57.1 +/- 4.1% and 69.0 +/- 9.6%, respectively. Orally administered CsA was less effective than 5 mg TBZ kg-1 (at day 4 p.i.) Co-administration of 5 mg TBZ kg-1 and CsA did not elicit synergy or additive efficacy, indicating that CsA did not antagonise the anti-strongyloides activity of TBZ. The data suggests that for patients with current, historical or serological evidence of strongyloidiasis, CsA may be used where immunosuppressive therapy is required for other concurrent reasons or when TBZ is contraindicated.