Genetic Diversity of Arginine Catabolic Mobile Element in Staphylococcus epidermidis

BACKGROUND:The methicillin-resistant Staphylococcus aureus clone USA300 contains a novel mobile genetic element, arginine catabolic mobile element (ACME), that contributes to its enhanced capacity to grow and survive within the host. Although ACME appears to have been transferred into USA300 from S. epidermidis, the genetic diversity of ACME in the latter species remains poorly characterized. METHODOLOGY/PRINCIPAL FINDINGS:To assess the prevalence and genetic diversity of ACME, 127 geographically diverse S. epidermidis isolates representing 86 different multilocus sequence types (STs) were characterized. ACME was found in 51% (65/127) of S. epidermidis isolates. The vast majority (57/65) of ACME-containing isolates belonged to the predominant S. epidermidis clonal complex CC2. ACME was often found in association with different allotypes of staphylococcal chromosome cassette mec (SCCmec) which also encodes the recombinase function that facilities mobilization ACME from the S. epidermidis chromosome. Restriction fragment length polymorphism, PCR scanning and DNA sequencing allowed for identification of 39 distinct ACME genetic variants that differ from one another in gene content, thereby revealing a hitherto uncharacterized genetic diversity within ACME. All but one ACME variants were represented by a single S. epidermidis isolate; the singular variant, termed ACME-I.02, was found in 27 isolates, all of which belonged to the CC2 lineage. An evolutionary model constructed based on the eBURST algorithm revealed that ACME-I.02 was acquired at least on 15 different occasions by strains belonging to the CC2 lineage. CONCLUSIONS/SIGNIFICANCE:ACME-I.02 in diverse S. epidermidis isolates were nearly identical in sequence to the prototypical ACME found in USA300 MRSA clone, providing further evidence for the interspecies transfer of ACME from S. epidermidis into USA300

Intermediate Vancomycin Susceptibility in a Community-associated MRSA Clone

We describe a case of treatment failure caused by a strain of USA300 community-associated methicillin-resistant Staphylococcus aureus (MRSA) with intermediate susceptibility to vancomycin and reduced susceptibility to daptomycin. The strain was isolated from the bone of a 56-year-old man with lumbar osteomyelitis after a 6-week treatment course of vancomycin for catheter-associated septic thrombophlebitis

Clonal Characterization of Staphylococcus aureus by Multilocus Restriction Fragment Typing, a Rapid Screening Approach for Molecular Epidemiology

We have developed a rapid and simplified approach for the strain characterization of Staphylococcus aureus on the basis of multilocus sequence typing (MLST) in which sequence variations in the MLST housekeeping gene loci are detected by restriction fragment pattern analysis rather than sequencing; we refer to this approach as multilocus restriction fragment typing (MLRFT). Briefly, MLRFT for S. aureus involves the PCR amplification of each of the seven MLST housekeeping gene loci by using the same primer pairs used in MLST. The amplicons are then digested directly with one or two restriction enzymes and the restriction fragments are resolved by agarose gel electrophoresis. Projection from published MLST data shows that MLRFT captures about 95% of the genetic diversity detected by MLST. The MLRFT approach was validated with a set of 59 methicillin-susceptible and 44 methicillin-resistant S. aureus isolates from community-acquired and nosocomial sources which had previously been characterized by pulsed-field gel electrophoresis (PFGE). MLRFT resolved the 103 isolates into 15 restriction fragment types, giving a discrimination index of 89.0%. Clonal groupings established by MLRFT correlated well with those established by PFGE. In short, MLRFT provides a convenient alternative to MLST and PFGE because it requires minimal laboratory facilities and is relatively simple and inexpensive to perform

Vancomycin Treatment Failure Associated with Heterogeneous Vancomycin-Intermediate Staphylococcus aureus in a Patient with Endocarditis and in the Rabbit Model of Endocarditis

Heterogeneous resistance to vancomycin is thought to precede emergence of intermediate susceptibility to vancomycin in Staphylococcus aureus, but the clinical significance of heterogeneous resistance is unknown. Paired S. aureus isolates from a patient with endocarditis who relapsed after vancomycin treatment were tested for heterogeneous resistance to vancomycin. The pretreatment and the relapse clinical isolates (strains SF1 and SF2, respectively) were genotyped by pulsed-field gel electrophoresis. Susceptibility to vancomycin was assessed by the broth dilution method, population analysis, and time-kill studies and in the rabbit model of endocarditis. Strains SF1 and SF2 had similar genotypes, and the vancomycin MICs for the strains were ≤2 μg/ml. SF2 exhibited heterogeneous resistance to vancomycin. Vancomycin eradicated SF1 in the rabbit model of endocarditis, while SF2 persisted at pretreatment levels. Vancomycin treatment failure in this patient with endocarditis was attributable to heterogeneous resistance to vancomycin

Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation.

Biofilm formation is thought to result from the concerted action of primary attachment to a specific surface and accumulation in multilayered cell clusters. Here we describe the isolation and characterization of transposon (Tn917) mutants of Staphylococcus epidermidis O-47 which were biofilm negative in the polystyrene microtiter plate assay. Among 5,000 Tn917 insertion mutants, 4 biofilm-negative mutants were isolated. Each mutant carried one copy of Tn917. The mutants were divided into two phenotypic classes: class A (mut1 and mut1a) and class B (mut2 and mut2a). Mutants of phenotypic class A lacked four cell surface proteins, were less hydrophobic, and were affected in primary attachment to polystyrene, but were still able to form multilayered cell clusters. They were able to form a biofilm on a glass surface, a trait that was even more pronounced than in the wild-type stain O-47. Loss of several surface proteins might have led to the reduced surface hydrophilic structures, thus favoring primary attachment to a glass surface and leading to subsequent biofilm formation. Mutants of phenotype class B were able to attach to polystyrene but were unable to form multilayered cell clusters, had unchanged cell surface proteins and hydrophobicity, and were unable to form a biofilm on a glass surface, mut1 and mut2 could be complemented by wild-type DNA fragments containing the Tn917 insertion sites of mut1 and mut2, respectively. The complemented biofilm-positive clone mut1 (pRC20) produced a 60-kDa protein which is postulated to function as the adhesin for binding to plastic. The traits of binding to polystyrene and the ability to form multilayered cell clusters are phenotypically and genetically distinct