Evolutionary relationships between sporadic and epidemic strains of healthcare-associated methicillin-resistant Staphylococcus aureus

National surveillance of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates by pulsed-field gel electrophoresis (PFGE) typing allowed identification of rarely occurring 'sporadic' isolates with patterns significantly distinct from those of major epidemic clones of methicillin-resistant S. aureus (MRSA) circulating in Belgian hospitals. The aim of the present study was to compare the genetic background, antibiotic susceptibility profile and in vitro growth rates of 36 MRSA isolates with either 'epidemic' or 'sporadic' PFGE profiles to identify factors that could be involved in the epidemic behaviour of S. aureus . Sequence analysis of seven housekeeping genes (multilocus sequence typing) and seven surface-associated genes, combined with staphylococcal cassette chromosome mec (SCC mec ) typing and spa typing results, segregated sporadic isolates into four groups: (1) isolates phylogenetically distant from epidemic HA-MRSA clones that possessed several properties of community-acquired MRSA strains; (2) isolates derived from the same methicillin-susceptible S. aureus ancestor as epidemic isolates but possessing a distinct type of SCC mec ; and (3) and (4) isolates that were closely related to epidemic strains, either as recent descendants of these or as intermediate evolutionary steps between epidemic HA-MRSA strains and their putative ancestors. Sporadic isolates did not show slower growth in vitro than epidemic isolates. These findings suggest that the SCC mec type and insertion/deletion of other mobile genetic elements may be involved in modulating the epidemic behaviour of MRSA strains of similar genetic background, independently of fitness cost.     

Evolution and molecular characterization of methicillin-resistant Staphylococcus aureus epidemic and sporadic clones in Cordoba, Argentina

Since 1999, a new, epidemic, methicillin-resistant Staphylococcus aureus (MRSA) strain, named the "Cordobes clone," has emerged in Argentina and coexists with the pandemic Brazilian clone. The purpose of this study was to determine the stability over time of the new clone and to investigate its evolutionary relationship with epidemic international MRSA lineages and with other MRSA and methicillin-susceptible S. aureus (MSSA) major clones distributed in this region. One hundred three MRSA isolates recovered in 2001 from Cordoba, Argentina, hospitals and 31 MSSA strains collected from 1999 to 2002 were analyzed by their antibiotic resistance patterns, phage typing, and pulsed-field gel electrophoresis. Additionally, representative members of most MRSA defined genotypes (A, B, C, E, K, and I) were characterized by multilocus sequence typing (MLST) and spaA and SCCmec typing. The most prevalent MSSA pulsotypes were also analyzed by MLST. Our results support the displacement of the Brazilian clone (sequence type [ST] 239, spaA type WGKAOMQ, SCCmec type IIIA) by the Cordobes clone (ST5, spaA type TIMEMDMGMGMK, SCCmec type I) in the hospital environment. MRSA and MSSA isolates shared only ST5. The data support the origin of the Cordobes clone as a member of a lineage that includes the pediatric and New York/Japan international clones and that is genetically related to the British EMRSA-3 strain. Interestingly, the pediatric clone, isolated from most community-acquired infections in Cordoba, was characterized by ST100, a single-locus variant of ST5 and a new variant of SCCmec type related to SCCmec type IVc.     

Staff carriage of epidemic methicillin-resistant Staphylococcus aureus.

Twenty-six nurses were repeatedly screened for carriage of epidemic methicillin-resistant Staphylococcus aureus (EMRSA) immediately before and after duty periods in which they solely attended six patients widely colonized with two EMRSA strains distinguishable by plasmid analysis. EMRSA carriage was detected in 13 nurses. Three EMRSA carriage patterns emerged: transient carriage in 12 nurses, when the EMRSA was isolated from noses or fingers of nurses after duty but was gone before their next day's duty; short-term nasal carriage, seen on occasion in 4 of these 12 nurses, when EMRSA carriage was detected on two consecutive screens; and persistent nasal carriage, seen in 1 nurse only, when the EMRSA was seen on more than two consecutive occasions. All but one of these incidents of carriage could be explained by close patient, rather than environmental, exposure and occurred despite an intensive control programme. Transient or short-term carriage in nurses probably resulted in transfer of the EMRSA between patients. Staff decontamination should be considered following a period of cohort nursing of EMRSA patients, especially if staff members are shortly to nurse unaffected patients. Our findings may explain some of the difficulties in controlling EMRSA.     

Molecular characterization of epidemic ciprofloxacin- and methicillin-resistant Staphylococcus aureus strains colonizing patients in an intensive care unit.

Eighteen methicillin-resistant Staphylococcus aureus (MRSA) samples isolated from patients and the environment in an intensive care unit (ICU) during a routine surveillance were tested for antimicrobial resistance and typed by pulsed-field gel electrophoresis. Three pulsed-field patterns were observed. Sixteen were ciprofloxacin resistant and had identical pulsed-field patterns. The results suggested that a ciprofloxacin-resistant MRSA clone had contaminated the environment and spread among patients. This study demonstrates the application of infection control surveillance combined with strain typing in detecting MRSA colonization in the ICU where it was not known to exist.     

Lysogenicity of methicillin-resistant strains of Staphylococcus aureus

The lysogenic status of 23 strains of methicillin-resistant Staphylococcus aureus, isolated at the Royal Prince Alfred Hospital, Sydney, since 1980, was studied. Twenty strains, belonging to the four predominant phage types isolated in this hospital, carried the same lysogenic phage which we have designated C. Three other phages were isolated from five strains belonging to phage type 84/85/90. The presence of phage C had little effect on the phage-typing pattern of the strains. Similarly, lysogenization with the other three phages did not result in a significant change in phage-typing patterns. However, when strain 1489, isolated in 1969, was lysogenized with these three phages, there was a change in phage-typing pattern. Lysogenization of this strain with phage 47T resulted in a marked loss of sensitivity to both group-I and group-III phages. The lysogenic status of these methicillin-resistant strains of S. aureus was compared with that of strains isolated between 1967 and 1970. There was no evidence that the strains isolated recently were either related to, or derived from, the earlier ones.     

Phenotypic prediction rule for community-associated methicillin-resistant Staphylococcus aureus

Recent studies have suggested that community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are encroaching upon nosocomial settings. We assessed the performance characteristics of a rule using the antimicrobial phenotype to predict genotype. This rule could be applied for epidemiologic purposes to describe the trend in CA-MRSA infections over time.     

Methicillin-resistant Staphylococcus aureus: phylogenetic relatedness between European epidemic clones and Swiss sporadic strains

We have compared the phylogenetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) strains from Switzerland and their phylogenetic relationships with European epidemic clones, using multiprimer random amplification polymorphic DNA (RAPD). Strains included 24 European epidemic clones (59 strains), 66 sporadic strains isolated in Switzerland in 1996-1997, and 15 reference strains of five other Staphylococcus species. Similarity and clustering analysis with the Jaccard's coefficient showed that the maximum genetic distance between MRSA strains was 0.43, whereas the minimum genetic distance between the six Staphylococcus species was 0.97, indicating that the method permits phylogenetic hierarchization. The 24 MRSA clones reported to be epidemic in European countries during the 1990s were distributed into seven different genetic clusters with a maximum distance of 0.29 among them. This clustering pattern was confirmed by the analysis of a subset of MRSA strains by multilocus enzyme electrophoresis at 12 loci. Most of the sporadic Swiss strains were distributed into these seven different genetic clusters, together with the epidemic MRSA clones. This suggests that there is no phylogenetic cluster specific to epidemic clones of MRSA.     

Differential growth of epidemic methicillin-resistant Staphylococcus aureus in vancomycin

This study examines the ability of isolates representing the 17 epidemic methicillin-resistant strains of Staphylococcus aureus to grow in increasing levels of vancomycin. Only EMRSAs 1, 2, 8, 11, 12 and 15 showed any growth and were designated EMRSAs 1A, 2A, 8A, 11A, 12A and 15A. On population analysis, these strains all produced clones that grew on 32 microg/mL vancomycin, while EMRSA 12A and 15A grew at 128 microg/mL. This was associated with increased resistance to lysostaphin and teicoplanin, a loss of phage sensitivity and an increase in cell wall diameter. Typing by pulsed-field gel electrophoresis following SmaI digestion showed no change in EMRSA 8A and 15A, while the other EMRSAs all lost or gained at least one band. EMRSAs 1A, 2A and 15A became more resistant to methicillin, and EMRSAs 8A, 11A and 12A became less resistant to methicillin. These results suggest that more than one mechanism is responsible for this phenomenon.     

DNA polymorphisms in methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus.

Restriction fragment length polymorphisms in methicillin-susceptible and methicillin-resistant (MRSA) strains of Staphylococcus aureus isolated in the same hospital over a 4-month period were studied by using SmaI and ApaI digestion of genomic DNA and pulsed-field gel electrophoresis. Each of the 20 methicillin-susceptible strains had a unique SmaI pattern, but the 27 MRSA strains showed only seven SmaI patterns. More than half of the SmaI fragments in all of these seven patterns were identical, as were those in the patterns from two unrelated MRSA strains. Digestion with ApaI, which cuts staphylococcus DNA into at least twice as many fragments, confirmed the results obtained with SmaI. Lastly, the plasmid contents of MRSA strains showing identical SmaI and ApaI electrophoretic patterns were not identical. These results are interpreted as supporting the hypothesis that all MRSA strains arose from a single clone and emphasize the need to use several methods in epidemiological investigations of MRSA outbreaks.     

Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism.

The X region of the protein A gene of Staphylococcus aureus contains a highly polymorphic sequence which is composed of repeats of 24 bp. We used amplification by PCR to investigate whether this region could be used to discriminate between epidemic and nonepidemic methicillin-resistant S. aureus (MRSA) strains. Most epidemic MRSA strains (24 of 33) harbored more than seven repeats, while most nonepidemic MRSA strains (10 of 14) contained seven or fewer repeats. It is conceivable that a longer X region results in a better exposition of the Fc-binding region of protein A, thereby facilitating colonization of host surfaces and contributing to the epidemic phenotype.     

Comparative genomics of Canadian epidemic lineages of methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that has disseminated throughout Canadian hospitals and communities. Pulsed-field gel electrophoresis of over 9,300 MRSA isolates obtained from the Canadian Nosocomial Infection Surveillance Program has identified 10 epidemic strain types in Canada (CMRSA1 to CMRSA10). In an attempt to determine specific genetic factors that have contributed to their high prevalence in community and/or hospital settings, the genomic content of representative isolates for each of the 10 Canadian epidemic types was compared using comparative genomic hybridizations. Comparison of the community-associated Canadian epidemic isolates (CMRSA7 and CMRSA10) with the hospital-associated Canadian epidemic isolates revealed one open reading frame (ORF) (SACOL0046) encoding a putative protein belonging to a metallo-beta-lactamase family, which was present only in the community-associated Canadian epidemic isolates. A more restricted comparison involving only the most common hospital-associated Canadian epidemic isolates (CMRSA1 and CMRSA2) with the community-associated Canadian epidemic isolates did reveal additional factors that might be contributing to their prevalence in the community and hospital settings, which included ORFs encoding potential virulence factors involved in capsular biosynthesis, serine proteases, epidermin, adhesion factors, regulatory functions, leukotoxins, and exotoxins.     

Undecatungstophosphate sensitized strains of methicillin-resistant Staphylococcus aureus to beta-lactams

Previously, a factor (Factor T) was found in aged mixtures of tungstate and phosphate, which greatly sensitizes strains of methicillin-resistant Staphylococcus aureus to beta-lactams. Factor T was purified and identified as undecatungstophosphate([PW11O39]7-). Undecatungstosilicate([SiW11O39]8-), a compound closely related to undecatungstophosphate, showed a similar enhancing effect. Chemically, these compounds are classified as "polyoxotungstates", and it is expected that these tungsten compounds will be useful as a "tool" in laboratory tests: i.e., in screening media for highly resistant MRSA strains. They may be also useful to investigate the resistant mechanism of the bacterial cells.     

Phenotypic distinctiveness of Staphylococcus aureus strains associated with toxic shock syndrome.

Twenty Staphylococcus aureus strains from patients with toxic shock syndrome (TSS) and 20 from control patients (non-TSS) with infection but no clinical evidence of TSS were compared phenotypically in a collaborative, blinded, randomized study. TSS strains were significantly (P less than 0.05) more likely than non-TSS strains to produce various previously described but related toxic shock-associated proteins (pyrogenic exotoxin C, enterotoxin F, and TSS marker protein), as well as differing in other distinctive phenotypic characteristics, such as hemolysis, bacteriocin susceptibility, arsenate resistance, pigment production, and casein proteolysis. TSS strains were significantly less likely to carry plasmids than control strains. A combination of two variables--proteolysis and toxic shock-associated protein production--statistically accounted for all other phenotypic variations between TSS and non-TSS strains. Only proteolysis covaried with all other significant variables, suggesting a primary role in the phenotypic distinctiveness of TSS S. aureus strains and possibly in the pathogenesis of TSS.     

Countrywide molecular survey of methicillin-resistant Staphylococcus aureus strains in Poland

The present investigation was undertaken to assess the proportion of methicillin-resistant Staphylococcus aureus (MRSA) strains among hospital-acquired isolates and to determine the clones of MRSA currently circulating in Poland by using a number of molecular techniques. Between January and May 2005, methicillin resistance was investigated among a total of 915 S. aureus isolates collected from 39 hospitals. A total of 208 (22.7%) isolates were positive for the mecA gene by PCR. The molecular characterization of MRSA isolates was carried out by the multiple-locus variable-number tandem repeat fingerprinting, pulsed-field gel electrophoresis, multilocus sequence typing, and staphylococcal chromosomal cassette mec (SCCmec) typing methods. The Hungarian (PFGE B; ST239, SCCmec type III [ST239-III]), Iberian (ST247-I), and Berlin (ST45-IV) clones were predominant, representing approximately 52.9, 11.5, and 10.0% of the MRSA isolates, respectively. A decline in the proportion of earlier MRSA clones, such as ST5-IV (a Pediatric clone), ST80-IV) (a Mediterranean clone), ST239-III (a Polish and Brazilian clone), and ST30-IV (a southwest Pacific clone) was observed. Additionally, the emergence of an MRSA clone with SCCmec type V, possibly representing a community-acquired strain, was observed in two hospitals during this study.     

Multiple-locus variable-number tandem-repeat assay analysis of methicillin-resistant Staphylococcus aureus strains

Our objective was to determine if a multiple-locus variable-number tandem-repeat assay (MLVA) for Staphylococcus aureus could predict pulsed-field gel electrophoresis (PFGE) types (i.e., USA types), thus allowing us to replace PFGE with a simpler and more rapid typing method. One hundred three well-characterized isolates representing 13 major lineages of S. aureus were tested by both PFGE and MLVA. MLVA was performed using a rapid DNA extraction technique and PCR primers for sdrCDE, clfA, clfB, sspA, and spa. PFGE was performed with genomic DNA fragments generated using SmaI, as per CDC protocols. Banding patterns were analyzed both visually and with BioNumerics software. All isolates were typeable with MLVA and PFGE. MLVA patterns were highly reproducible. PFGE separated the isolates into 13 types with 42 subtypes. Using any band difference to designate a novel MLVA type, MLVA produced 45 types, including 9 clusters containing multiple isolates. Using BioNumerics and a cutoff of >75% relatedness, MLVA produced 28 types, 11 of which contained >1 isolate. Epidemiologically related outbreak isolates of USA300-0114 from five states clustered in one MLVA pattern. USA100 isolates were present in several unrelated (<40%) MLVA types. A cutoff of >80% separated outbreak strains of USA300-0114 into three distinct MLVA types. MLVA did not differentiate community methicillin-resistant S. aureus (MRSA) lineages (USA300, USA400, USA1000, and USA1100) from health care MRSA lineages (USA100, USA200, or USA500). The ability of MLVA to differentiate among strains that are indistinguishable by PFGE may be of epidemiologic value and warrants further study.     

Molecular characterization of methicillin-resistant Staphylococcus aureus isolates in Korea

We used several molecular typing methods to analyze 196 methicillin-resistant Staphylococcus aureus (MRSA) and 139 methicillin-susceptible S. aureus (MSSA) isolates collected between 1996 and 2005. The sequence type 72 MRSA has increased in frequency in the community in the Republic of Korea and in hospitals in recent years.