Genetic diversity among community methicillin-resistant Staphylococcus aureus strains causing outpatient infections in Australia

Increasing reports of the appearance of novel nonmultiresistant methicillin-resistant Staphylococcus aureus MRSA (MRSA) strains in the community and of the spread of hospital MRSA strains into the community are cause for public health concern. We conducted two national surveys of unique isolates of S. aureus from clinical specimens collected from nonhospitalized patients commencing in 2000 and 2002, respectively. A total of 11.7% of 2,498 isolates from 2000 and 15.4% of 2,486 isolates from 2002 were MRSA. Approximately 54% of the MRSA isolates were nonmultiresistant (resistant to less than three of nine antibiotics) in both surveys. The majority of multiresistant MRSA isolates in both surveys belonged to two strains (strains AUS-2 and AUS-3), as determined by pulsed-field gel electrophoresis (PFGE) and resistogram typing. The 3 AUS-2 isolates and 10 of the 11 AUS-3 isolates selected for multilocus sequence typing (MLST) and staphylococcal chromosomal cassette mec (SCCmec) analysis were ST239-MRSA-III (where ST is the sequence type) and thus belonged to the same clone as the eastern Australian MRSA strain of the 1980s, which spread internationally. Four predominant clones of novel nonmultiresistant MRSA were identified by PFGE, MLST, and SCCmec analysis: ST22-MRSA-IV (strain EMRSA-15), ST1-MRSA-IV (strain WA-1), ST30-MRSA-IV (strain SWP), and ST93-MRSA-IV (strain Queensland). The last three clones are associated with community acquisition. A total of 14 STs were identified in the surveys, including six unique clones of novel nonmultiresistant MRSA, namely, STs 73, 93, 129, 75, and 80slv and a new ST. SCCmec types IV and V were present in diverse genetic backgrounds. These findings provide support for the acquisition of SCCmec by multiple lineages of S. aureus. They also confirm that both hospital and community strains of MRSA are now common in nonhospitalized patients throughout Australia.     

Delineation of the endemic and sporadic methicillin-resistant Staphylococcus aureus clones in a Czech hospital

The aim of this study was to define the endemic clones of methicillin-resistant Staphylococcus aureus (MRSA) among strains collected between September, 2001, and February, 2003, at the regional hospital of Novy Jicín, Czech Republic. The isolates were characterized by susceptibility tests, HindIII ribotyping, and pulsed-field gel electrophoresis. Representatives of each clonal type were analyzed by multilocus sequence typing and staphylococcal cassette chromosome mec (SCCmec) typing. The prevalence of the most important macrolide (ermA, ermB, ermC, and msrA) and aminoglycoside (aac6'-aph2", aph3', and ant4') resistance genes was evaluated as well. Our results document the existence of two international MRSA clones: (1) the Iberian clone (ST247:SCCmec IA:PFGE A:ribotype H2), endemic in the hospital and associated to a single multiresistant phenotype; and (2) clone EMRSA-15 (ST22:SCCmec IV:PFGE H-ribotype H7), appearing in the beginning of 2002 and associated with three phenotypes. These two clones could be distinguished by antibiogram, distribution of macrolide and aminoglycoside resistance genes (ermA, aac6'-aph2", ant4' versus ermC and msrA in a few isolates), production of beta-lactamase, and presence of enterotoxin A (in the Iberian clone).     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Spain: a multicentre prevalence study (2002)

A point-prevalence study, performed in 2002 in 143 Spanish hospitals, collected 439 isolates of Staphylococcus aureus. Of these, 134 (30.5%) were resistant to methicillin (i.e., MRSA). Susceptibility testing was performed by a microdilution method, and mecA was detected by PCR. The isolates were characterised by phage typing, pulsed-field gel electrophoresis (PFGE) after SmaI digestion, and SCCmec typing. The 134 MRSA isolates showed resistance to ciprofloxacin (93.3%), tobramycin (88.8%), erythromycin (67.9%), clindamycin (59.7%), gentamicin (42.5%), mupirocin (17.9%), rifampicin (5.2%) and trimethoprim-sulphamethoxazole (5.2%). All of the isolates were susceptible to glycopeptides. Twenty-five resistance patterns were found, of which four accounted for 66% of the isolates. Phage group III was the most frequent (41.1%). PFGE revealed 31 different patterns, with ten major clones (including two predominant clones with variable antibiotypes that accounted for 43.3% of the MRSA isolates) and 21 sporadic patterns. Two isolates belonged to two variants of the Iberian clone (ST247-MRSA-I), one to the Brazilian clone (ST239-MRSA-III), and seven to the EMRSA-16 clone (ST36-MRSA-II). SCCmecIV accounted for 70.2% of the isolates (73.9% were type IVA), while SCCmecI, SCCmecII and SCCmecIII accounted for 22.1%, 6.9% and 0.8% of isolates, respectively, with three non-typeable isolates. Isolates of SCCmecIV and SCCmecIVA were predominantly nosocomial (95.8% and 97.1%, respectively). None of the isolates produced Panton-Valentine leukocidin. Thus, two clones carrying SCCmecIV and SCCmecIVA, respectively, were predominant among nosocomial MRSA isolates throughout Spain.     

Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period

A total of 629 nonduplicate methicillin-resistant Staphylococcus aureus MRSA isolates were recovered between June and November 2006 from 11 hospitals located in different areas of Portugal. Selected isolates (n = 271, 43%) were typed by pulsed-field gel electrophoresis (PFGE), representatives of which were additionally characterized by spa typing, multilocus sequence typing, staphylococcal cassette chromosome mec (SCCmec) typing, and the presence of Panton-Valentine leukocidin (PVL). The 271 isolates were classified into 13 different clonal types. Three pandemic clones included the majority (n = 241, 88%) of the isolates and were observed in several hospitals: (i) EMRSA-15 (54%)-PFGE type A, ST22, spa type t022, SCCmec IV-was found in the 11 hospitals studied and was identified as the major clone in seven of them; (ii) the New York/Japan clone (17%)-PFGE B, ST5, spa type t067, SCCmec II-was identified in nine hospitals and represented the major clone in four; and (iii) the Brazilian MRSA (17%)-PFGE C, ST239, spa type t037, SCCmec IIIA-was also detected in nine hospitals but never as the main clone. All isolates tested were PVL negative. Clone EMRSA-15 is currently the predominant MRSA clonal type circulating in Portuguese hospitals, but a new wave of MRSA has emerged in the country with the recent introduction and spread of the New York/Japan clone. The Brazilian MRSA that was the leading clone in Portugal in the late 1990s is declining and being progressively replaced by the two former clones. We report the first isolate SCCmec type V (ST45) in Portugal.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Majorcan hospitals: high prevalence of the epidemic clone EMRSA-15

Clinical isolates (n = 389) of methicillin-resistant Staphylococcus aureus (MRSA) recovered from 371 patients between January 2003 and June 2004 at the three major public hospitals on the island of Majorca, Spain were studied. The clonal relatedness of MRSA isolates was determined by pulsed-field gel electrophoresis (PFGE) after digestion with SmaI. During the study period, MRSA was found in 31% of patients with S. aureus-positive cultures. PFGE analysis identified three predominant clones, affecting 94% of the patients. The three clones had been detected since 1999 in one hospital, and were designated as clones A, B and C. Whereas clones A and B (multidrug-resistant) were related to the two most prevalent clones in Spain at this time, clone C was identical to EMRSA-15, currently one of the most common MRSA clones in UK hospitals and also detected in other countries, but rarely in Spanish hospitals. This imported epidemic clone was detected in c. 10% of patients admitted to one of the three hospitals in 2002, but its prevalence has increased significantly (32% of the patients investigated in the three hospitals in the present study), and this clone also accounted for 44% of the isolates from non-hospitalised patients. Even though EMRSA-15 showed the least multidrug resistance of the three major clones, it was apparently more virulent, since it was associated significantly (p 0.001) with bacteraemia, and positive blood cultures were documented for 21% of the patients infected by this clone, compared with only 10% and 7% of patients infected with clones A and B, respectively.     

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.     

Characterization of isolates of methicillin-resistant Staphylococcus aureus from Hong Kong by phage typing, pulsed-field gel electrophoresis, and fluorescent amplified-fragment length polymorphism analysis

The genetic relatedness of 127 methicillin-resistant Staphylococcus aureus (MRSA) isolates, belonging to five major types as identified by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiles, was examined further using phage typing and fluorescent amplified fragment length polymorphism (FAFLP). The MRSA isolates were recovered from patients at the Prince of Wales Hospital (PWH), Hong Kong, over a 13-year period, 1988 to 2000. These strains were also compared with representatives of the well-described MRSA international clones and with epidemic MRSA strains (eMRSA) 1 to 16 from the United Kingdom. Phage typing distinguished two major "clones" at this hospital: all of the phage type 1 (PT1) isolates belonged to PFGE types A, C, D, and E, while most of the PT2 isolates were associated with PFGE type B, which exhibited a unique antibiotic resistance profile. MRSA isolates belonging to PFGE subtype A2 were indistinguishable from the British eMRSA-1, while isolates of PFGE type B were closely related to eMRSA-9 by PFGE. Based on FAFLP, all five predominant PFGE types at the PWH belonged to one group and fell into the same cluster as eMRSA-1, -4, -7, -9, and -11 isolates. Multilocus sequence typing and staphylococcal cassette chromosome mec typing classified representatives of our MRSA isolates as members of the same clone (ST239-MRSA-III). Thus, the predominant MRSA isolates frin the PWH in the last decade are closely related to early United Kingdom eMRSA clones 1, 4, and 11 and are members of a lineage that includes the Brazilian MRSA clone.     

Replacement of methicillin-resistant Staphylococcus aureus clones in Hungary over time: a 10-year surveillance study

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Hungary has been increasing and is now close to 20% among invasive isolates of S. aureus . In order to understand the evolution of MRSA in Hungary, two collections of isolates were studied: 22 representatives of a collection of 238 MRSA isolates recovered between 1994 and 1998, and a collection of 299 MRSA isolates recovered between 2001 and 2004. The isolates were first characterised by pulsed-field gel electrophoresis (PFGE) and were distributed into 19 different PFGE patterns. Representatives of each pattern were further characterised by spa typing, multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCC mec ) typing. The Hungarian clone that was predominant in 1994–1998 (PFGE E, ST239-III) had almost disappeared in 2003–2004, being replaced by the Southern German clone (PFGE B, ST228-I) and the New York/Japan epidemic clone (PFGE A, ST5-II), which represented c.  85% of the 2001–2004 isolates. Thus, this study describes, for the first time, the co-dominance and extensive spread of the New York/Japan clone in a European country.     

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.     

Prevalence of the ST239 clone of methicillin-resistant Staphylococcus aureus and differences in antimicrobial susceptibilities of ST239 and ST5 clones identified in a Korean hospital

A total of 188 nonduplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained between 2001 and 2004 in a university hospital in Daegu, Korea, were analyzed for their clonal types by molecular typing techniques, including multilocus sequence typing, spaA typing, staphylococcal chromosomal cassette mec (SCCmec) typing, and pulsed-field gel electrophoresis (PFGE). They were examined for their antimicrobial susceptibilities. The majority (87%) of MRSA isolates belonged to sequence type 239 (ST239; n = 100; 53%) and ST5 (n = 63, 34%) on the basis of sequence typing. MRSA isolates belonging to ST239 were genotypically homogeneous, while those belonging to ST5 showed variations in spaA type, SCCmec type, and PFGE patterns. The rates of resistance of the MRSA isolates belonging to ST239 to trimethoprim, sulfamethoxazole, tobramycin, gentamicin, erythromycin, and tetracycline were significantly higher than those of the isolates belonging to ST5 (P < 0.05). This study demonstrated that the ST239 clone, while rarely detected in Korea, was prevalent and that the antimicrobial susceptibility of the ST239 clone was significantly different from that of the ST5 clone.     

Evolution, antimicrobial susceptibility and assignment to international clones of methicillin-resistant Staphylococcus aureus isolated over a 9-year period in two Spanish hospitals

Antimicrobial resistance profiles, restriction fragment length polymorphism of the coagulase gene and repetitive element sequence-based PCR were used to classify 210 methicillin-resistant Staphylococcus aureus isolates recovered between 1997 and 2005 in two hospitals in Vigo, north-west Spain. Representative isolates belonging to the epidemic clones were analysed by spa typing and multilocus sequence typing, and the staphylococcal chromosomal cassette (SCC)mec type was determined for all isolates. The New York/Japan clone (t002-ST5-II) was detected in Spain for the first time. However, the New York/Japan and the Brazilian (t037-ST239-IIIA) clones were replaced by EMRSA-16 (t018-ST36-II), which at present is the predominant clone.     

Emergence of clonal complex 5 (CC5) methicillin-resistant Staphylococcus aureus (MRSA) isolates susceptible to trimethoprim-sulfamethoxazole in a Brazilian hospital

In this study, genotyping techniques including staphylococcal chromosomal cassette mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and restriction-modification tests were used to compare the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered at two times within a 10-year interval (1998 and 2008) from a tertiary Brazilian hospital. In addition, the antimicrobial susceptibility profiles were analyzed. All 48 MRSA isolates from 1998 and 85.7% from 2008 (48/56 isolates) displayed multidrug-resistance phenotypes and SCCmec III. All but one of the 13 representative SCCmec III isolates belonged to CC8 and had PFGE patterns similar to that of the BMB9393 strain (Brazilian epidemic clone of MRSA; BEC). In 2008, we found an increased susceptibility to rifampicin and chloramphenicol among the SCCmec III isolates. In addition, we detected the entrance of diverse international MRSA lineages susceptible to trimethoprim-sulfamethoxazole (SXT), almost all belonging to CC5. These non-SCCmec III isolates were related to the USA300 (ST8-SCCmec IV; PFGE-type B), USA800 (ST5-SCCmec IV; subtype D₁), USA100 (ST5-SCCmec II; subtype D₂), and EMRSA-3/Cordobes (ST5-SCCmec I, type C) clones. To the best of our knowledge, this is the first report of the emergence of isolates genetically related to the EMRSA-3/Cordobes clone in southeast Brazil. In this regard, these isolates were the most common non-SCCmec III MRSA in our institution, accounting for 8.9% of all isolates recovered in 2008. Thus, despite the supremacy of BEC isolates in our country, significant changes may occur in local MRSA epidemiology, with possible consequences for the rationality of MRSA empiric therapy.     

Clonal and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus (MRSA) from a Portuguese hospital over time

Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from a general hospital in Oporto, Portugal, during two periods (1992-1993 and 1996-2000) were characterized by pulsed-field gel electrophoresis (PFGE) of SmaI fragments, and by hybridization of ClaI digests with mecA and Tn554 probes, discriminating the isolates in mecA::Tn554::PFGE genotypes. In addition, a representative sample of the defined genotypes was characterized by multilocus sequence typing (MLST) and SCCmec (staphylococcal cassette chromosome mec) typing, generating the corresponding ST-SCCmec types. In 1992-1993, 77% of MRSA belonged to the Iberian clone (genotype I::E::A or ST247-IA). In 1996-2000, the frequency of this clone decreased to 19% and the majority (69%) of the isolates belonged to another international clone, the Brazilian MRSA (genotype XI::B::B or ST239-IIIA). Trimethoprim/sulfamethoxazole (SXT) was confirmed to be an important phenotypic marker to distinguish the Iberian (SXT-susceptible) and the Brazilian (SXT-resistant) clones in MRSA isolates from Portugal. Our observations document major shifts in the dominant MRSA clonal types that occurred in this hospital since 1992, suggesting a selective advantage of the Brazilian relatively to the Iberian clone. In addition to these two MRSA clones that are the most frequent in Portuguese hospitals since the early 1990s, sporadic MRSA clones (representing 14% of the total) were identified and characterized.     

Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus

A multilocus sequence typing (MLST) scheme has been developed for Staphylococcus aureus. The sequences of internal fragments of seven housekeeping genes were obtained for 155 S. aureus isolates from patients with community-acquired and hospital-acquired invasive disease in the Oxford, United Kingdom, area. Fifty-three different allelic profiles were identified, and 17 of these were represented by at least two isolates. The MLST scheme was highly discriminatory and was validated by showing that pairs of isolates with the same allelic profile produced very similar SmaI restriction fragment patterns by pulsed-field gel electrophoresis. All 22 isolates with the most prevalent allelic profile were methicillin-resistant S. aureus (MRSA) isolates and had allelic profiles identical to that of a reference strain of the epidemic MRSA clone 16 (EMRSA-16). Four MRSA isolates that were identical in allelic profile to the other major epidemic MRSA clone prevalent in British hospitals (clone EMRSA-15) were also identified. The majority of isolates (81%) were methicillin-susceptible S. aureus (MSSA) isolates, and seven MSSA clones included five or more isolates. Three of the MSSA clones included at least five isolates from patients with community-acquired invasive disease and may represent virulent clones with an increased ability to cause disease in otherwise healthy individuals. The most prevalent MSSA clone (17 isolates) was very closely related to EMRSA-16, and the success of the latter clone at causing disease in hospitals may be due to its emergence from a virulent MSSA clone that was already a major cause of invasive disease in both the community and hospital settings. MLST provides an unambiguous method for assigning MRSA and MSSA isolates to known clones or assigning them as novel clones via the Internet.     

Clonal types and multidrug resistance patterns of methicillin-resistant Staphylococcus aureus (MRSA) recovered in Italy during the 1990s

A large number (272) of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from Italian hospitals during the early and late 1990s were characterized for multidrug resistance pattern and clonal type using a combination of genotyping methods, including pulsed-field gel electrophoresis (PFGE), spaA typing, multilocus sequence typing (MLST), determination of SCC mec type, and hybridization pattern with Tn 554. The majority of MRSA belonged to four genetic lineages: the pandemic Iberian and Brazilian clones, and two unique clonal types-the "Italian" and "Rome" clones of MRSA. The Italian clone carried the SCC mec type I in the genetic background of ST228, which is a double-locus variant of the sequence type of the multidrug-resistant New York/Japanese clone (ST5). The properties of the Rome clone showed several striking similarities to those of the Archaic clone of MRSA that was dominant among MRSA isolates in the mid-1960s to 1970s, but has not been detected since then in recent global surveillance studies.     

Tracking methicillin-resistant Staphylococcus aureus clones during a 5-year period (1998 to 2002) in a Spanish hospital

Three hundred seventy-five consecutive methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates recovered between 1998 and 2002 at the Nuestra Señora de Candelaria University Hospital in Tenerife, Spain, were analyzed by molecular fingerprinting techniques to determine MRSA clonal types and their prevalence over time. After determining antibiotic susceptibility, we used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize MRSA isolates and to establish PFGE types. Additionally, several selected isolates representative of each major PFGE type were tested by multilocus sequence typing (MLST) and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec), generating the corresponding sequence type (ST)-SCCmec types. Results of PFGE, supported by those of MLST and SCCmec typing, allowed us to identify six MRSA clones within the five major PFGE types and document temporal shifts in the prevalence of these MRSA clones from 1998 to 2002. Four of the clones were the pandemic “Iberian” (designated ST247-MRSA-IA), EMRSA-15 (ST22-MRSA-IV), EMRSA-16 (ST36-MRSA-II), and the so-called pediatric (ST5-MRSA-IV) clones, while the other two (ST125-MRSA-IVA and ST146-MRSA-IVA) clones could be derived from the pediatric one. The most striking temporal shift in the dominance of MRSA clones was the replacement of the multidrug-resistant and highly epidemic Iberian clone by the so-called British EMRSA-16 clone during the 5-year surveillance period. Our results are in accordance with previously stated findings showing the worldwide hospital dominance of relatively few pandemic and presumably virulent MRSA clones. We report for the first time the detection in Spain of the British EMRSA-15 and pediatric clones, as well as the abrupt replacement of the Iberian by the EMRSA-16 as the major MRSA clone.Staphylococcus aureus is the causal agent of most staphylococcal pathologies and is currently a versatile microbial pathogen that has evolved resistance to all antibiotic classes. It is associated with serious community-acquired and nosocomial diseases, although most life-threatening S. aureus infections are hospital acquired (4, 8). Its high level of adaptation to hospital environments has been deeply facilitated by the acquisition of methicillin resistance, an evolutionary step that converted S. aureus to methicillin-resistant S. aureus (MRSA), one of the most common nosocomial pathogens nowadays (19). MRSA emerged with the introduction of an exogenous DNA element into its genome, the staphylococcal cassette chromosome mec (SCCmec), which carries the methicillin resistance mecA gene (16). Recent data shows that acquisition of SCCmec has occurred on multiple occasions and that at least five different methicillin-sensitive S. aureus phylogenetic lineages acquired the element (29). Four main structural types of SCCmec, which differ in size and composition, have been described for S. aureus (14, 15, 20).Genetic studies using molecular typing methods have shown that most hospital-acquired MRSA infections worldwide are due to any of the so-called epidemic MRSA (EMRSA). These EMRSA clones present great fitness to hospital environments and, consequently, are established in many hospitals and have spread internationally (7). This situation highlights the importance of monitoring the distribution and routes of dissemination of such EMRSA clones at both levels, within hospitals and between distant locations (24). With this purpose, several molecular techniques and an international common nomenclature have been applied to track EMRSA (10, 27). Pulsed-field gel electrophoresis (PFGE) is considered the “gold standard” for establishing clonal relationships at the local level, but its detection capacity seems to make it also too discriminative for global comparisons (5, 31). By contrast, multilocus sequence typing (MLST) has been verified as an adequate method for long-term and global epidemiological studies (11, 48). Combination of MLST with SCCmec typing permits the unambiguous assignment of collections of MRSA isolates to known or new MRSA clones (10).The aim of this study was to identify MRSA clones circulating in the Nuestra Señora de Candelaria University Hospital (HUNSC), Tenerife, Spain, and to track shifts in their prevalence during a 5-year period (1998 to 2002). For this purpose, we used a combination of different molecular typing methods, including PFGE, MLST, and SCCmec typing.     

Epidemiology of emerging methicillin-resistant Staphylococcus aureus (MRSA) in Denmark: a nationwide study in a country with low prevalence of MRSA infection

Strict infection control measures introduced during the 1970s have kept the incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections extremely low in Denmark. Nevertheless, similarly to other countries, MRSA infections began to appear in the community in the late 1990s. A nationwide surveillance program has collected and stored all MRSA isolates since 1988 and, since 1999, clinical information has been also recorded. We used this information and isolates in a detailed epidemiological and molecular analysis of the 81 MRSA infections identified in Denmark in 2001. MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and SCCmec typing. Comparison of the 45 community-onset MRSA (CO-MRSA) infections with the 36 hospital-acquired MRSA (HA-MRSA) infections showed several striking contrasts. Most CO-MRSA were recovered from skin and soft tissue infections caused by isolates carrying the Panton-Valentine leucocidin toxin genes, and the majority (84%) of isolates belonged to a single clonal type, ST80-IV, which has been found in the community in other European countries. Clone ST80-IV could be traced in Denmark back to 1993. ST80-IV was rarely found in HA-MRSA infections, which belonged to a large number of clonal types, including some pandemic MRSA clones. The low number of HA-MRSA infections and the diversity of MRSA clones in Danish hospitals may be the result of successful infection control measures that prevent spread of clones in hospitals. The mechanism of spread of the ST80-IV clone in the Danish community is not known, and new control measures are needed to control further spread of this and other CA-MRSA clones.     

Comparison of genetic backgrounds of methicillin-resistant and -susceptible Staphylococcus aureus isolates from Portuguese hospitals and the community

In order to understand the origins of the dominant methicillin-resistant Staphylococcus aureus (MRSA) clones in Portuguese hospitals, we compared the genetic backgrounds of nosocomial MRSA with methicillin-susceptible S. aureus (MSSA) isolates from the same hospitals (n = 155) and from the community (n = 157) where they were located. Pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, and agr type analysis revealed that the genetic backgrounds correspondent to the dominant MRSA clones in Portuguese hospitals during the last 15 years (Iberian ST247, Brazilian ST239, and EMRSA-15 ST22) were scarcely or not found among the present MSSA collection. The four major MSSA clones encountered (A-ST30, B-ST34, C-ST5, and H-ST45) correspond, or are very similar, to the background of other international MRSA pandemic clones, i.e., EMRSA-16, New York/Japan, Pediatric, and Berlin clones. However, with the exception of the Pediatric clone, none of these MRSA clones has been detected in Portugal. Our findings suggest the three major MRSA clones identified in Portuguese hospitals have not originated from the introduction of SCCmec into dominant MSSA backgrounds present in the Portuguese nosocomial or community environment but were probably imported from abroad. In contrast, the MRSA Pediatric clone might have originated in our country by the acquisition of SCCmec type IV into MSSA clone C. Furthermore, we provide evidence that the introduction of SCCmec into sensitive clones is most likely a relatively infrequent event that seems to depend not exclusively on the presence of a successful MSSA lineage.     

MRSA in Austria--an overview.

The aim of this study was to provide an overview of predominant and sporadic methicillin-resistant Staphylococcus aureus (MRSA) strains in large regions of Austria, and to compare the results with those from other European countries. In total, 1439 MRSA isolates, collected routinely between January 1996 and June 2006 from five Austrian federal provinces, were investigated. The isolates were confirmed as MRSA using mecA/femA multiplex PCR assays. Genes encoding Panton-Valentine leukocidin (PVL), which are characteristic of community-acquired MRSA, were also detected by PCR. Subtyping was performed using SmaI macrorestriction digestion of genomic DNA, followed by pulsed-field gel electrophoresis (PFGE) and cluster analysis. Isolates that could not be assigned to clusters were further analysed by spa typing and/or multilocus sequence typing. The predominant clones detected in Austria were ST228 (southern German epidemic clone), ST5 (Rhine-Hessen MRSA), the ST8 Austrian clone and CC8/ST8. Whereas the frequencies of lineages corresponding to ST247, ST45 and ST22 remained comparably low, an increase in the frequency of lineages corresponding to ST5 and to ST228 was recorded. Overall, 20 different MRSA types and 321 subtypes were recognised according to PFGE analysis. The prevalence of different strains varied considerably in the different Austrian regions. When compared to other European countries, the situation in Austria was most similar to that found in Germany.