Methicillin-Resistant Staphylococcus aureus Clonal Types in the Czech Republic

Molecular surveillance studies have documented the extensive spread of methicillin-resistant Staphylococcus aureus (MRSA) clones. Studies carried out by Centro de Epidemiologia Molecular-Network for Tracking Gram-Positive Pathogenic Bacteria (CEM/NET) led to the identification of two international multidrug-resistant strains, which were designated as the Iberian and Brazilian MRSA clones and which were defined by multiple genomic typing methods; these included ClaI restriction digests hybridized with mecA- and Tn554-specific DNA probes and pulsed-field gel electrophoresis (PFGE). The genotypic characteristics of these clones are distinct: the Iberian clone is defined as mecA type I, Tn554 type E (or its variants), and PFGE pattern A (I:E:A), whereas the Brazilian clone is defined as mecA type XI (or its variants), Tn554 type B, and PFGE pattern B (XI:B:B). In this study, we characterized 59 single-patient isolates of MRSA collected during 1996 and 1997 at seven hospitals located in Prague and five other cities in the Czech Republic by using the methodologies mentioned above and by using ribotyping of EcoRI and HindIII digests hybridized with a 16S-23S DNA probe. The Brazilian MRSA clone (XI:B:B) was the major clone (80%) spread in two hospitals located in Prague and one located in Brno; the Iberian MRSA clone (I:E:A or its variant I:DD:A), although less representative (12%), was detected in two hospitals, one in Prague and the other in Plzen. Almost all the strains belonging to clone XI:B:B (45 of 47) corresponded to a unique ribotype, E1H1, whereas most strains of the I:E:A and I:DD:A clonal types (6 of 7) corresponded to ribotype E2H2.     

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.     

Similarity of antibiotic resistance patterns and molecular typing properties of methicillin-resistant Staphylococcus aureus isolates widely spread in hospitals in New York City and in a hospital in Tokyo, Japan

One hundred and forty-three single-patient methicillin-resistant Staphylococcus aureus (MRSA) isolates collected during April-June, 1997, and February, 1998, in a hospital in Tokyo, Japan, were characterized by molecular typing techniques that involved hybridization of ClaI restriction digests with the mecA- and Tn554-specific DNA probes and determination of macrorestriction patterns of SmaI-digested chromosomal DNA by pulsed-field gel electrophoresis (PFGE). A large proportion (76%) of the isolates carried the mecA polymorph I, Tn554 pattern A, and PFGE pattern A (clonal type I:A:A), which was the same as the clonal type of an MRSA widely spread in hospitals in New York City and hospitals in neighboring New Jersey, Connecticut, and Pennsylvania. Also similarly to the New York clone, most of the MRSA isolates from the Japanese hospital were resistant to penicillin, ciprofloxacin, erythromycin, tetracycline, and high concentrations (500 microg/ml) of spectinomycin, but were susceptible to chloramphenicol, sulfamethoxazole-trimethoprim, and rifampin. All of the 143 MRSA isolates had vancomycin MICs < or = 2 mg/L.     

Detection of an Archaic Clone of Staphylococcus aureus with Low-Level Resistance to Methicillin in a Pediatric Hospital in Portugal and in International Samples: Relics of a Formerly Widely Disseminated Strain?

Close to half of the 878 methicillin-resistant Staphylococcus aureus (MRSA) strains recovered between 1992 and 1997 from the pediatric hospital in Lisbon were bacteria in which antibiotic resistance was limited to beta-lactam antibiotics. The other half were multidrug resistant. The coexistence of MRSA with such unequal antibiotic resistance profiles prompted us to use molecular typing techniques for the characterization of the MRSA strains. Fifty-three strains chosen randomly were typed by a combination of genotypic methods. Over 90% of the MRSA strains belonged to two clones: the most frequent one, designated the "pediatric clone," was reminiscent of historically "early" MRSA: most isolates of this clone were only resistant to beta-lactam antimicrobials and remained susceptible to macrolides, quinolones, clindamycin, spectinomycin, and tetracycline. They showed heterogeneous and low-level resistance to methicillin (MIC, 1.5 to 6 microg/ml), carried the ClaI-mecA polymorph II, were free of the transposon Tn554, and showed macrorestriction pattern D (clonal type II::NH::D). The second major clone was the internationally spread and multiresistant "Iberian" MRSA with homogeneous and high-level resistance to methicillin (MIC, >200 microg/ml) and clonal type I::E::A. Surprisingly, the multidrug-resistant and highly epidemic Iberian MRSA did not replace the much less resistant pediatric clone during the 6 years of surveillance. The pediatric clone was also identified among contemporary MRSA isolates from Poland, Argentina, The United States, and Colombia, and the overwhelming majority of these were also associated with pediatric settings. We propose that the pediatric MRSA strain represents a formerly widely spread archaic clone which survived in some epidemiological settings with relatively limited antimicrobial pressure.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Colombian hospitals: dominance of a single unique multidrug-resistant clone

The first study on the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolates from Colombia was performed as part of a global surveillance established by the CEM/NET Initiative, under Project RESIST. Seventy-six MRSA isolates recovered from five hospitals during 1996-1998 were analyzed by the hybridization of ClaI restriction digests with mecA- and Tn554-specific probes, and by pulsed-field gel electrophoresis (PFGE) of chromosomal SmaI digests. All MRSA isolates, with one exception, belonged to a single clonal type II::NH::D. This clone, which was previously described among MRSA isolates recovered in the early 1990s in European and New York and South American hospitals, showed resistance to beta-lactam antibiotics only and appeared to be associated almost exclusively with pediatric infections ("Pediatric clone" of MRSA). While sharing identical molecular typing properties with the Pediatric clone, the Colombian isolates differed by extensive multidrug resistance and were recovered from patients of all ages. It is also noteworthy that the Brazilian clone of MRSA (XI::B::B), another multidrug-resistant international clone currently widely spread in Brazil, Argentina, Uruguay, Chile, and also in several European countries, was completely absent from this set of isolates from Colombia.     

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.     

Wide geographic distribution of a unique methicillin-resistant Staphylococcus aureus clone in Hungarian hospitals

Objective: To determine the genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from six provincial hospitals in Hungary between 1993 and 1994.
Method: Molecular fingerprinting methods were used: hybridization with a mecA -specific DNA probe after Cla I restriction; hybridization with a probe for Tn 554 ; and pulsed-field gel electrophoresis after Sma I digestion of chromosomal DNA.
Results: All strains were resistant to penicillin, oxacillin, erythromycin, gentamicin, tetracycline, imipenem, and cephalosporins, and variably resistant to ofloxacin, clindamycin and tobramycin; all isolates were susceptible to vancomycin. Forty-eight of the 51 isolates carried the mecA gene as determined by Southern hybridization, using a mecA -specific DNA probe, indicating that the methodology used for initial identification may have been in error in three of the cases. Forty-seven of the 48 mecA -positive isolates showed very similar genetic backgrounds as defined by pulsed-field gel electrophoresis (PFGE) patterns after Sma I digestion of chromosomal DNAs: a unique PFGE pattern was seen in 32 isolates and minor variants of it in 15 additional isolates. All the 47 isolates carried the same mecA polymorph ( Cla I type III), as determined by DNA hybridization after Cla I digestion of chromosomal DNA. Only one of the MRSA isolates had a completely different PFGE pattern and a novel mecA polymorph.
Conclusions: The findings demonstrate the existence of a unique, epidemic MRSA clone, in both invasive and colonizing strains, which is widely dispersed in Hungarian hospitals hundreds of kilometers apart.     

Geographic spread of epidemic multiresistant Staphylococcus aureus clone in Brazil.

Staphylococcus aureus isolates from five large teaching hospitals and one medium-size community hospital located in geographically distant parts of Brazil, in the south and southeast (Rio de Janeiro, Niteroi, Sao Paulo, Porto Alegre) and in the north (Manaus), were tested for their antibiotic resistance patterns and genetic backgrounds. Eighty-five of the 152 isolates were identified as methicillin-resistant S. aureus (MRSA) by using a combination of an agar dilution screen and a mecA gene-specific DNA probe. All MRSA isolates were resistant to penicillin, erythromycin, gentamicin, oxacillin, and cephalothin, and the majority of isolates (74%) were also resistant to chloramphenicol, sulfamethoxazole-trimethoprim, ciprofloxacin, and clindamycin as well and were susceptible only to vancomycin. Isolates obtained from hospitals in Sao Paulo, Rio de Janeiro, Niteroi, and Porto Alegre (1,600 km from one another) and Manaus (3,700 km from Rio de Janeiro) were examined by a variety of molecular fingerprinting techniques: the nature of the mecA polymorph and Tn554 attachment sites and restriction fragment length polymorphism of genomic DNAs after SmaI restriction and separation of the digested DNA by pulsed-field gel electrophoresis. The overwhelming majority of the isolates shared a common pulsed-field gel electrophoresis pattern and carried mecA polymorph III in combination with Tn554 pattern B, indicating the presence of a single, epidemic MRSA clone spread over large geographic distances of Brazil.     

Unusually large number of methicillin-resistant Staphylococcus aureus clones in a Portuguese hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hospital de Santa Maria, a 1,300-bed teaching hospital in Lisbon, Portugal, since the mid-1980s with a prevalence of 30% in 1993. A total of 54 MRSA and 93 methicillin-susceptible S. aureus (MSSA) isolates recovered during the first 3 months of 1993 were analyzed for the particular mecA polymorphs and Tn554 attachment sites (in the case of MRSA) and for pulsed-field gel electrophoretic patterns. While all MRSA isolates shared a very similar multidrug resistance antibiogram, molecular methods allowed the identification of an unusually large number of genetic backgrounds (24 different pulsed-field gel electrophoresis patterns in 54 isolates) and three different mecA polymorphs among the MRSA strains. Similar large variation in the genetic backgrounds of MSSA was observed. The most frequent mecA polymorph (mecA type I) was found in association with three different Tn554 patterns. Among the MRSA strains of Hospital Santa Maria, we found two clonal types previously described in Portugal: one corresponding to the dominant clone in an MRSA outbreak at the pediatric ward of the Lisbon Hospital Dona Estefânia and another one identical to the Iberian epidemic clone identified in several Portuguese hospitals and in MRSA outbreaks in Barcelona and Madrid. This suggests that MRSA clones of Hospital de Santa Maria may have been a reservoir for staphylococcal strains over the past decade.     

Three-year assessment of methicillin-resistant Staphylococcus aureus clones in Latin America from 1996 to 1998

Four hundred ninety-nine methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from 1996 to 1998 from 22 hospitals in five countries of Latin America-Argentina, Brazil, Chile, Uruguay and Mexico-were examined for antimicrobial susceptibility and clonal type in order to define the endemic clones in those hospitals. The hybridization of ClaI restriction digests with the mecA- and Tn554-specific DNA probes combined with pulsed-field gel electrophoresis of chromosomal SmaI digests (ClaI-mecA::ClaI-Tn554::PFGE clonal types) documented not only the predominance and persistence of the Brazilian clone (XI::B::B) in Brazil (97%) and Argentina (86%) but also its massive dissemination to Uruguay (100%). Moreover, a close relative of the Brazilian clone (XI::kappa::B) was highly represented in Chile (53%) together with a novel clone (47%) (II::E'::F) resistant to pencillin, oxacillin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, and gentamicin. A unique clonal type (I::NH::M) was detected in Mexico among pediatric isolates and was resistant to penicillin, oxacillin, and gentamicin only. This study clearly documented the very large capacity for geographic expansion and the persistence of the Brazilian clone, contributing not only to the increasing uniformity of the MRSA in South America but worldwide as well.     

Analysis of different molecular methods for typing methicillin-resistant Staphylococcus aureus isolates belonging to the Brazilian epidemic clone

The extensive geographic spread of MRSA isolates belonging to the Brazilian epidemic clone (BEC) limited the value of pulsed-field gel electrophoresis (PFGE) in epidemiological studies of outbreaks caused by these strains. Thus, the discriminatory power of eight different molecular methods was evaluated in an attempt to establish a methodology for genotyping BEC isolates involved in intra-hospital outbreaks. BEC isolates from five hospitals in Teresina City, Piaui State were genotyped by conventional electrophoresis or PFGE of Cla I- or Sma I-digested genomic DNA hybridised with specific labelled mecA, Tn554, IS257 and IS256 probes. The combination of PFGE with Cla I/mecA, Cla I/Tn554, Cla I/IS257, Sma I/mecA and Sma I/IS257 probe-fingerprinting techniques provided a very poor discriminatory power for BEC strains. Although Cla I/IS256 fingerprinting discriminated 17 different polymorphisms among the isolates displaying PFGE A1 pattern, this strategy was not reproducible. In contrast, the combination of PFGE and Sma I/IS256 polymorphisms differentiated BEC isolates into nine stable polymorphisms. Thus combination of PFGE and hybridisation with IS256 probe may be recommended as a useful means of typing BEC strains involved in intra-hospital infections.     

Clonal structure of the methicillin-resistant Staphylococcus aureus (MRSA) population in Poland: revision and update

The clonal structure of the methicilin-resistant Staphylococcus aureus (MRSA) population in Poland has been analyzed in several reports since the mid-1990s. The present study was performed on 253 MRSA isolates (146 archival and 107 new isolates) recovered in 26 hospitals between 1990 and 2001. Whereas all isolates were typed by pulsed-field gel electrophoresis (PFGE) and the analysis of the ClaI::mecA and ClaI::Tn554 RFLP polymorphism, selected isolates were also subjected to multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) comparisons. Based on the PFGE data, 15 MRSA clones were discerned, seven of which were observed in multiple hospitals. Five of these were related to the pandemic Hungarian (MLST clonal complex, CC8), Iberian (CC8), Pediatric (CC5), Mexican (CC30), and Brazilian clones (CC8). MLST confirmed the earlier reports on the similarity of the Hungarian and Brazilian clones, and it revealed that one of the two remaining epidemic clones was related to the Hungarian/Brazilian, and the other--to the Berlin clones. A local strain from the Northeastern part of the country was found to be similar to a minor Greek clone. The MRSA clonal structure and the increasing complexity of the relationships between the genetic and phenotypic traits of this micro-organism in Poland has now been firmly established.     

Archaic strains of methicillin-resistant Staphylococcus aureus: molecular and microbiological properties of isolates from the 1960s in Denmark

Methicillin-resistant Staphylococcus aureus (MRSA) isolated in the 1960s in Denmark are among the historically earliest samples of these bacteria. We determined microbiological and molecular properties of 46 such isolates. They showed remarkably uniform properties, which included: (i) low methicillin MIC value (6-25 microg/ml); (ii) heterogeneous expression of resistance; (iii) the presence of a single, common, mecA polymorph II; (iv) lack of the regulatory gene mecI; (v) frequent lack of Tn554; and (vi) a common pulsed-field gel electrophoresis (PFGE) type. These properties, together with the chronological dates of isolation, and recovery of the strains from 18 hospitals scattered over Denmark, suggest that they represent a lineage close in time to the evolutionary origin of European strains of MRSA.     

Two international methicillin-resistant Staphylococcus aureus clones endemic in a university hospital in Patras,Greece

Pulsed-field gel electrophoresis (PFGE) of SmaI macrofragments and hybridization of ClaI digests with the mecA- and Tn554-specific DNA probes were used to define the endemic clones of methicillin-resistant Staphylococcus aureus (MRSA) among strains collected in 1993 and 1998 to 2000 at the University Hospital of Patras, Patras, Greece. Representatives of each clonal type were analyzed by spaA typing, multilocus sequence typing (MLST), and staphylococcal chromosomal cassette mec (SCCmec) typing. The results indicated the existence of two successive international MRSA clones: (i) a clonal type with PFGE type A, sequence type (ST) 30 (ST30), and SCCmec type IV, which was very similar to a clone widely spread in the United Kingdom, Mexico, and Finland, and (ii) a clonal type with PFGE type B, ST239, and SCCmec III, which was related to the Brazilian clone. Both clones seem to be widespread in Greece as well. A novel MRSA clone is also described and is characterized by a new MLST type (ST80) associated with SCCmec type IV and with the presence of Panton-Valentine leukocidin genes.     

Molecular characterisation of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone in a Mexican hospital (1999–2003)

Methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 216), collected between January 1999 and May 2003 in a tertiary-care university hospital in Guadalajara, Mexico, were characterised by antibiotype, pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments, and hybridisation of ClaI digests with mecA- and Tn554-specific DNA probes. Representatives of the single clonal type found were analysed by spa typing, multilocus sequence typing and staphylococcal chromosomal cassette mec (SCCmec) typing, and were tested for the presence of 22 virulence determinants and agr type. A single PFGE pattern was identified, with minor variations over time, with spa type 2, sequence type 5, SCCmec type II, agr type 2 and the presence of the enterotoxin genes seg and sei, the gamma-haemolysin variant gene hlg-v and the leukocidin lukE-lukD genes. In addition, the isolates showed antimicrobial resistance to beta-lactams, macrolides, chloramphenicol and imipenem, and susceptibility to gentamicin, rifampicin, trimethoprim-sulphamethoxazole and vancomycin. Following its appearance in 1997, this clone spread within the hospital, and is now present in most of the hospital units and wards.     

Distribution of methicillin-resistant Staphylococcus aureus clones among health care facilities in Connecticut, New Jersey, and Pennsylvania.

A previous surveillance study conducted in 12 hospitals in New York City in 1996 identified a unique multidrug-resistant genetic lineage of methicillin-resistant Staphylococcus aureus (MRSA) that was widespread and accounted for as much as 42% of all the MRSA isolates. The purpose of the study described here was to determine possible geographic spread of this New York clone of MRSA to neighboring states. Single-patient MRSA isolates (258) from 29 health care facilities in Connecticut (CT), New Jersey (NJ), and Pennsylvania (PA) were collected during the calendar year 1998. DNA typing, consisting of fingerprinting of chromosomal macrorestriction patterns generated by SmaI digestion followed by pulsed-field gel electrophoresis (PFGE), identified 22 patterns. PFGE type A, closely related to the PFGE type of the previously identified New York clone, accounted for 154 (60%) of 258 isolates. The clone was detected in all facilities, was predominant in 19 of the 29 health care centers, and accounted for 92% of the MRSA isolates collected in PA. The overwhelming majority of MRSA with PFGE type A was also resistant to erythromycin, ciprofloxacin, and clindamycin. One of the two most common PFGE subtypes detected in the three states sampled (PFGE subtype A1) had an identical PFGE pattern to that of the previously described vancomycin-resistant strain of S. aureus (VISA) recently detected in a hospital in Westchester, NY. The second most frequent MRSA clone with PFGE type E and accounting for 26% (68/258 isolates), also described earlier in the 12 New York City hospitals, was resistant not only to erythromycin, ciprofloxacin, and clindamycin, but also to gentamicin and sulfamethoxazole-trimethoprim as well. The unique multidrug resistance pattern of this second clone and its geographic distribution accounted for the differences observed in the frequency of multidrug resistance among MRSA isolates recovered in the three states. The pandemic Iberian clone recently detected in New York City was not detected among the 258 MRSA isolates recovered in CT, NJ, and PA.     

Identification of a variant "Rome clone" of methicillin-resistant Staphylococcus aureus with decreased susceptibility to vancomycin, responsible for an outbreak in an intensive care unit

We describe the identification of a variant of the "Rome clone" of methicillin-resistant Staphylococcus aureus (MRSA), responsible for an outbreak involving 5 patients in a Cardiac Surgery Intensive Care Unit (CS-ICU) of a tertiary-care University Hospital in Rome. All strains isolated from patients and from nasal swabs obtained from four members of the CS-ICU personnel, belonged to the same identified clone. The characteristics of this clone were: (1) resistance to ampicillin, oxacillin, gentamicin, ciprofloxacin, erythromycin, clindamycin, rifampin, spectinomycin, and tetracycline; (2) vancomycin and teicoplanin MICs respectively of 2 and 4 mg/L; (3) heteroresistant subpopulations in the presence of 4 and 6 mg/L of vancomycin (10(-3) and 10(-5), respectively); (4) clonal type I::J::C determined following an established protocol (mec A::Tn 554 ::PFGE); (5) sequence type ST247 (3-3-1-12-4-4-16), obtained by multilocus sequence typing (MLST); and (6) the staphylococcal cassette chromosome mec (SCC) IA, obtained by multiplex PCR method. This new strain had different characteristics from the epidemic clone circulating in the same hospital from 1997 and designed "Rome clone," which was susceptible to erythromycin, clindamycin, and spectinomycin and belonged to the II::NH::C genetic background. A high genetic similarity between this Rome clone and the previously classified Archaic and Iberian clones was found, because they shared the same allelic profile (ST247), probably originating from the same S. aureus ancestor of the Iberian MRSA strains. Therefore, the strains responsible for the outbreak, with vancomycin MICs 2-4 mg/L, are variant clones, showing the genotype of the "Rome clone," the ST247 in association with SCC mec type IA (ST247-MRSA-IA), and are characterized by a uniform susceptibility to fosfomycin.     

Evolution of sporadic isolates of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and their similarities to isolates of community-acquired MRSA

Forty-one methicillin-resistant Staphylococcus aureus (MRSA) hospital isolates that clearly differed from the six major pandemic clones of MRSA in pulsed-field gel electrophoresis type, mecA and Tn554 polymorphism, and epidemic behavior were selected from an international strain collection for more detailed characterization. SpaA typing, multilocus sequence typing, and SCCmec (staphylococcal cassette chromosome mec) typing demonstrated extensive diversity among these sporadic isolates both in genetic background and also in the structure of the associated SCCmec elements. Nevertheless, the isolates could be grouped into restricted clonal complexes by using the BURST (i.e., based upon related sequence types) program algorithm, which predicted that most sporadic MRSA isolates evolved from pandemic MRSA clones. Several of the sporadic MRSA resembled community-acquired MRSA isolates in properties that included a relatively limited multiresistance pattern, faster growth rates, diversity of genetic backgrounds, and a frequent association with SCCmec type IV.     

Methicillin-resistantStaphylococcus aureus disease in a portuguese hospital: Characterization of clonal types by a combination of DNA typing methods

Fifteen pediatric patients as well as the five nursing staff of the Burn Unit of the Hospital D. Estefania in Lisbon, Portugal, were assayed at weekly intervals over a five-month period in order to identify the nature and number of methicillin-resistantStaphylococcus aureus (MRSA) clones associated with colonization and wound infection. Methicillin resistance was confirmed by amec-specific DNA probe. MRSA isolates were classified into chromosomal types (clones) on the basis of a variety of techniques: (i) ribotyping; (ii) restriction digestion by the endonucleaseClaI followed by Southern hybridization with themecA-specific DNA probe and (iii) by hybridization with Tn554; and (iv) pulsed-field electrophoresis (PFE) ofSmaI digests followed by (v) Southern hybridization with themecA DNA probe. A sixth, physiological technique (population analysis) was used to define the mode of phenotypic expression of methicillin resistance in each isolate. All isolates carried a single, common polymorph (ClaI type III) of themecA gene. Hybridization with Tn554 resolved these isolates to two novel patterns (alpha and beta), of which one (Tn554 alpha) was predominant (90 %). This pattern could be further resolved to four closely related PFE types (A through D). In contrast, all isolates with the Tn554 beta pattern belonged to an additional, grossly different PFE type E. The Tn554 beta class was also unique in that these bacteria carried themecA gene in aSmaI fragment smaller (about 170 kb) than that found in the alpha type strains (194 kb). Most isolates (83 %) showed a single heterogeneous (population analysis Class 3) mode of resistance expression. The data demonstrate the full capacity of the globally rare (ClaI type III) MRSA clone for colonization and virulence. The results also document the stability of the complex heterogeneous resistance phenotype as well as the stability of the chromosomal types under conditions of in vivo carriage over a period of several months. In a few isolates the samemecA polymorph was present in several, grossly different genetic backgrounds, suggesting horizontal transfer of themecA gene.     

Update on the major clonal types of methicillin-resistant Staphylococcus aureus in the Czech Republic

The purpose of our study was the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 21 hospitals in the Czech Republic in the period 2000-2002 and comparison with previous results from 1996-1997. Strains were analyzed by pulsed-field gel electrophoresis (PFGE) of SmaI digests and ribotyping of HindIII digests hybridized with a 16S-23S DNA probe. The prevalence of the most clinically important macrolide (ermA, ermB, ermC, and msrA) and aminoglycoside (aph3', ant4', and aac6'-aph2") resistance genes was evaluated as well. Selected isolates representative of each clonal type were analyzed by multilocus sequence typing and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec) carried by the bacteria. Our results document the displacement of the Brazilian clone (ST239, SCCmec type IIIA, PFGE type B, ribotype H1) by a new clone that we named "Czech clone" (ST239, SCCmec type IIIA, PFGE type F, ribotype H6) and the maintenance of the Iberian clone (ST247, SCCmec type IA, PFGE type A, ribotype H2) exclusively in one hospital in the Czech Republic. In addition, we found a correlation between the distribution of aminoglycoside resistance genes and MRSA clonal types.