Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR.

The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems.

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.

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.

Low prevalence of methicillin-resistant strains among Staphylococcus aureus colonizing young and healthy members of the community in Portugal.

Recent reports suggest that methicillin-resistant Staphylococcus aureus (MRSA) may be emerging as a community pathogen. In Portuguese hospitals, the incidence of MRSA among disease causing isolates is extremely high (48-50%). To determine the prevalence of MRSA in the Portuguese community, nasal samples were obtained from 823 draftees, 484 nonmedical university students, and 107 high-school students. In addition, throat samples were obtained from the 823 draftees and S. aureus isolates were also recovered from 283 (13%) nasopharyngeal samples obtained from 2,111 children attending day-care centers. The rate of nasal colonization of S. aureus was 34%, 25%, and 46% for draftees, nonmedical university students, and high-school students, respectively. The rate of pharyngeal colonization of the draftees was 33%. Of the 1,001 S. aureus isolates obtained, seven were MRSA and eight were borderline oxacillin-resistant S. aureus (BORSA). By molecular typing techniques, five of the seven MRSA were identified as belonging to one of three highly epidemic clones, the Brazilian, Iberian, and Pediatric clones of MRSA, which were identified as endemic in Portuguese hospitals. The eight BORSA were all members of clones previously identified in international samples. In spite of the extremely high prevalence of MRSA in Portuguese hospitals, the carriage rate of MRSA in healthy and young individuals remains low.

Genetic diversity and clonal patterns among antibiotic-susceptible and -resistant Streptococcus pneumoniae colonizing children: day care centers as autonomous epidemiological units.

Characterization by antibiotype of the 1,096 Streptococcus pneumoniae recovered from 2,111 nasopharyngeal samples of children attending 16 day care centers (DCCs) in Lisbon, Portugal, and molecular typing of 413 drug-resistant pneumococci (DRPn) and 89 fully drug-susceptible pneumococci (DSPn) has allowed several conclusions. (i) There was an increase in the frequency of DRPn colonizing children in DCCs from 40% in 1996 to 45% in 1997 to 50% in 1998. (ii) Drug resistance spread by cross-transmission of DRPn clones. A few (8 out of 57) DRPn clones were repeatedly isolated from a large number of children in several DCCs and during each period of surveillance, suggesting the epidemic nature of these clones, which included lineages representing internationally spread S. pneumoniae clones. (iii) Dissemination of resistance determinants among pneumococci colonizing the nasopharynx occurred. Association of identical pulsed-field gel electrophoresis patterns with diverse antibiotypes among pneumococci colonizing children suggests that the high prevalence of DRPn involves not only cross-transmission of resistant strains but also dispersal of resistance genes through recombinational mechanisms. (iv) DCCs are autonomous epidemiological units. Among the 413 DRPn, 57 different lineages were detected; these lineages were dispersed among the 16 DCCs to produce unique microbiological profiles for each of the DCCs. Higher genetic diversity and less sharing of clonal types were observed among the DSPn.

Carriage of internationally spread clones of Streptococcus pneumoniae with unusual drug resistance patterns in children attending day care centers in Lisbon, Portugal.

Over half (259/503) of drug-resistant (DR) pneumococci colonizing healthy children attending day care centers in Lisbon were identified by molecular typing methods as representatives of several internationally spread clones. These included the 2 penicillin-resistant pandemic Spanish/USA and French/Spanish clones (21% of all DR pneumococci) and 5 new lineages with unusual antibiotypes (accounting for an additional 30% of all DR pneumococci). The most characteristic feature of the latter group was the high frequency of resistance to macrolides and tetracycline and very low or no resistance to penicillin. These observations provide support for the notion that the nasopharyngeal flora of children in day care centers may be a global reservoir of worldwide prevalent strains of DR pneumococci.

Molecular characterization of Staphylococcus sciuri strains isolated from humans.

We previously characterized over 100 Staphylococcus sciuri isolates, mainly of animal origin, and found that they all carried a genetic element (S. sciuri mecA) closely related to the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) strains. We also found a few isolates that carried a second copy of the gene, identical to MRSA mecA. In this work, we analyzed a collection of 28 S. sciuri strains isolated from both healthy and hospitalized individuals. This was a relatively heterogeneous group, as inferred from the different sources, places, and dates of isolation and as confirmed by pulsed-field gel electrophoresis analysis. All strains carried the S. sciuri mecA copy, sustaining our previous proposal that this element belongs to the genetic background of S. sciuri. Moreover, 46% of the strains also carried the MRSA mecA copy. Only these strains showed significant levels of resistance to beta-lactams. Strikingly, the majority of the strains carrying the additional MRSA mecA copy were obtained from healthy individuals in an antibiotic-free environment. Most of the 28 strains were resistant to penicillin, intermediately resistant to clindamycin, and susceptible to tetracycline, erythromycin, and gentamicin. Resistance to these last three antibiotics was found in some strains only. The findings reported in this work confirmed the role of S. sciuri in the evolution of the mechanism of resistance to methicillin in staphylococci and suggested that this species (like the pathogenic staphylococci) may accumulate resistance markers for several classes of antibiotics.

Carriage of respiratory tract pathogens and molecular epidemiology of Streptococcus pneumoniae colonization in healthy children attending day care centers in Lisbon, Portugal.

In an effort to establish the rate of carriage of antibiotic resistant respiratory pathogens in children attending urban day care centers (DCC) in Portugal, seven DCC in Lisbon were selected for determining the rate of nasopharyngeal colonization of children between the ages of 6 months to 6 years by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Of the 586 children studied between January and March 1996, 47% carried S. pneumoniae, 72% H. influenzae, and 54% M. catarrhalis. Twenty-four percent of the pneumococci had reduced susceptibility to penicillin, and most of these belonged to serogroups 19, 23, 14, and 6. An additional 19% were fully susceptible to penicillin but showed decreased susceptibility to other antimicrobials. These isolates expressed serogroups 6, 11, 14, 18, 19, and 34. The majority (96%) of M. catarrhalis and 20% of H. influenzae were penicillin resistant due to the production of beta-lactamases. Recent antimicrobial use was associated with carriage of penicillin non-susceptible pneumococci and beta-lactamase producing H. influenzae (p < 0.05). Individual DCC differed substantially from one another in their rates of carriage of antibiotic resistant H. influenzae and S. pneumoniae. Characterization of antibiotic resistant S. pneumoniae isolates by molecular fingerprinting techniques showed that each DCC had a unique microbiological profile, suggesting little, if any, exchange of the resistant microbial flora among them. An exception to this was the presence of isolates belonging to two internationally spread epidemic clones: the multiresistant Spanish/USA clone expressing serotype 23F, and the penicillin and sulfamethoxazole-trimethoprim resistant French/Spanish clone (serotype 14) which were detected in four and three DCC, respectively.

Extensive intra-hospital spread of a methicillin-resistant staphylococcal clone.

OBJECTIVE: Infection by antibiotic-resistant bacteria can pose serious complications to the therapy of cancer patients. The authors introduced DNA fingerprinting techniques for tracking methicillin-resistant Staphylococcus aureus (MRSA) clones recovered at a central cancer hospital of Lisbon (Instituto Português de Oncologia) with the purpose of making an inventory of the MRSA clones endemic during 1995, and compared them with the outbreak-related clones of 1993. DESIGN: A small group (6 strains) of epidemiologically related MRSA isolated during a suspected outbreak in 1993 and all consecutive single-patient isolates of MRSA (34 strains) recovered between January and November of 1995 from infected patients and health care personnel were characterized using DNA probes and pulsed-field gel electrophoresis. RESULTS: The six 1993 strains and more than half of all 1995 isolates, including those recovered from the health care personnel, showed DNA fingerprints characteristic of the "Iberian MRSA," a multiresistant clone widespread in Portuguese and Spanish hospitals. Four patients were infected by another MRSA clone previously seen only in hospitals in Brazil. CONCLUSION: The epidemic Iberian clone was among the index cases involved with the MRSA outbreak in 1993, and this was found to be endemic in a follow-up survey conducted in 1995, colonizing health care personnel and spreading to most hospital wards. A few isolates of another epidemic clone, the Brazilian MRSA, also were detected among 1995 isolates. A better understanding of the mechanism(s) of epidemicity of these rapidly spreading clones is urgently needed.

Clonal distribution of methicillin-resistant Staphylococcus aureus in Poland.

We report on a study of 158 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates obtained from 1990 to 1996 in 18 different hospitals in Poland. All isolates were recovered from infection and carriage sites of patients, carriage sites of health care personnel, and hospital environment samples. Fifty-seven MRSA strains described here were studied previously and these were divided into two different clusters according to the degree of heterogeneity of methicillin resistance expression. The aim of this study was to extend the correlation between the two clusters and identify the clonal identities among all isolates by a combination of different methodologies: (i) analysis of mecA polymorphs and Tn554 insertion patterns and (ii) determination of pulsed-field gel electrophoresis patterns of chromosomal SmaI digests. Ninety-seven of 158 strains showed a heterogeneous expression of resistance to methicillin. Among these, 75 (77.3%) were ClaI-mecA type I, ClaI-Tn554 type NH (NH, no homology with transposon Tn554), and pulsed-field gel electrophoresis (PFGE) pattern A (I::NH::A); 10 isolates were III::B::M (10.3%); and the remaining clones included a few or single isolates. The isolates with homogeneous expression of resistance to methicillin (n = 61) were predominantly ClaI-mecA type III (49 of 61 [80.3%]) but had great variability in their ClaI-Tn554 and PFGE patterns. This study confirmed the existence of two main clusters of MRSA in Poland.

Intercontinental spread of a multidrug-resistant methicillin-resistant Staphylococcus aureus clone.

Two hundred ten methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered between 1990 and 1997 from three Portuguese hospitals located in Lisbon and Oporto were analyzed by molecular fingerprinting techniques. The hybridization of ClaI restriction digests with the mecA- and Tn554-specific DNA probes combined with pulsed-field gel electrophoresis documented the abrupt appearance and extensive intrahospital spread of the Brazilian epidemic MRSA clone in the 1995 samples of each one of the three hospitals analyzed-suggesting the intercontinental transfer of this strain from Brazil to Portugal. The appearance of this clone may challenge the dominance of another highly epidemic imported clone-the Iberian MRSA, currently the most widely spread MRSA clone in Portuguese hospitals.

Ribotype delineation and description of Staphylococcus sciuri subspecies and their potential as reservoirs of methicillin resistance and staphylolytic enzyme genes.

Three subspecies of Staphylococcus sciuri, S. sciuri subsp. sciuri Kloos, Schleifer, and Smith 1976, 23AL emend. Kloos et al. 1997 [corrected], S. sciuri subsp. carnaticus subsp. nov., and S. sciuri subsp. rodentium subsp. nov., are described on the basis of their ribotype patterns, DNA-DNA liquid hybridization data, and phenotypic characteristics. Normalized ribotyping subdivided the S. sciuri patterns into three blocks of patterns, each corresponding to a subspecies. Each subspecies formed a separate, well-defined DNA similarity group when DNA-DNA hybridizations were conducted under stringent (70 degrees C) reassociation conditions. S. sciuri subsp. sciuri could be distinguished from the other subspecies on the basis of its ability to produce acid from D-cellobiose, alkaline phosphatase activity, and inability to produce either clumping factor or protein A. S. sciuri subsp. carnaticus could be distinguished by its ability to produce acid aerobically from D-xylose and maltose, inability to produce acid from D-melezitose, and smaller colony size on P agar. S. sciuri subsp. rodentium could be distinguished by its positive reaction in the latex agglutination test for clumping factor and/or protein A and generally higher frequencies and levels of oxacillin and methicillin resistance. All 40 strains of S. sciuri tested (including representatives of all three subspecies) hybridized with the mecA gene probe. All strains of S. sciuri subsp. sciuri, 79% of the strains of S. sciuri subsp. carnaticus and 89% of the strains of S. sciuri subsp. rodentium exhibited extracellular, staphylolytic enzyme activity. This activity was associated with an enzyme(s) that immunoblotted with a lysostaphin-specific monoclonal antibody; however, only three strains hybridized with a lysostaphin (end) gene probe. The type strain of S. sciuri subsp. carnaticus is DD 791 (= ATCC 700058), and the type strain of S. sciuri subsp. rodentium is DD 4761 (= ATCC 700061).

Characterization of methicillin-resistant Staphylococcus aureus isolates from Portuguese hospitals by multiple genotyping methods.

One hundred and eighty-three methicillin-resistant Staphylococcus aureus (MRSA) isolates from eight different Portuguese hospitals were genetically typed by random amplification of polymorphic DNA (RAPD) employing different oligonucleotide primers. Fourteen different RAPD genotypes were identified. A subset of the same strains was also characterized by pulsed-field gel electrophoresis (PFGE) and/or hybridization using mecA and Tn554 probes. In the majority of cases, the different genotyping methods have identified the same MRSA clones. However, PFGE combined with the DNA probes was clearly the method providing higher resolution. Most strains that have already been identified by PFGE and DNA probes as members of the widely spread Iberian clone of MRSA generated a common RAPD genotype. The most prevalent Iberian clone was not detected in a collection of MRSA from Poland that was also examined by RAPD. On the other hand, MRSA strains second most frequent in prevalence in the Portuguese and Polish collection appear to be identical by RAPD, indicating extensive geographic spread of this particular clone. No correlation was apparent between epidemic behavior and the number of protein A gene repeats in this particular collection of MRSA strains.

Tracing the origin of an outbreak of methicillin-resistant Staphylococcus aureus infections in a Portuguese hospital by molecular fingerprinting methods.

Seventy-six methicillin-resistant Staphylococcus aureus (MRSA) isolates were collected from July 1992 to May 1995 at a 400-bed district hospital in the northeast of Portugal. During the second half of the surveillance period, in July of 1994, an outbreak was detected in the orthopedic ward. Thirty-three (out of the 76) MRSA strains were recovered only in this ward during the outbreak period. All strains were characterized by a variety of genomic fingerprints. Hybridization of ClaI and SmaI restriction digests with the mecA- and Tn554-specific DNA probes was used to identify polymorphism and determine chromosomal location of these determinants, and pulsed-field gel electrophoretic analysis of SmaI digests was used to determine chromosomal backgrounds. All strains recovered during the outbreak in the orthopedic ward were found to belong to a single clone that carried the mecA polymorph I, Tn554 type E in a macrorestriction background called H (clone I::E::H1), which was identified in 18 patients, and 5 health care personnel and from a fomite sample, and was traced to a single transfer patient admitted to the hospital at the beginning of the outbreak. The new clone I::E::H1 differed only in the macrorestriction profile from the MRSA clone previously dominant in this hospital, known as Iberian epidemic clone I::E::A, which has already been identified in several Spanish and Portuguese hospitals.

Ubiquitous presence of a mecA homologue in natural isolates of Staphylococcus sciuri.

In an effort to explore the origin and/or reservoirs of the genetic determinant(s) of methicillin resistance in Staphylococcus aureus, we examined over 200 strains representing 13 different species within the genus Staphylococcus for the presence of the mecA gene, using a DNA probe internal to this gene prepared from a methicillin-resistant strain of S. aureus. Occasional mecA- positive isolates were detected among several staphylococcal species. On the other hand, each one of the 134 isolates of Staphylococcus sciuri, a species considered taxonomically the most primitive among staphylococci and found primarily on rodents and primitive mammals, gave positive reaction with the DNA probe when tested under conditions of high stringency. About two thirds (99) of these isolates, all of which belonged to S. sciuri subspecies "sciuri," as well as 9 of the 11 species carnaticum isolates, showed only marginal, if any, resistance to methicillin (minimal inhibitory concentration of 0.75-6.0 micrograms/ml), while most of the remaining isolates that belonged to the subspecies "rodentius" (13 isolates in all) expressed antibiotic resistance with a heterogeneous phenotype similar to those seen in many methicillin-resistance strains of S. aureus In SmaI digests of chromosomal DNA isolated from such "methicillin-resistant S. aureus-like" strains, the mecA probe hybridized with DNA fragments in the range of 145-180 kb, while in subspecies "sciuri" and carnaticum isolates the mecA hybridizing fragment was located in the SmaI fragment with the highest molecular size (> or = 400 kb). A DNA probe comprising an internal sequence to the regulatory gene mecI from Staphylococcus epidermidis identified the presence of sequences with low degree of homology in isolates of the three S. sciuri subspecies. The mecA-reacting sequences in these bacteria differed from mecA of S. aureus in several respects (e.g., by the absence of a ClaI restriction site from mecA of subspecies "sciuri" and carnaticum, and in some isolates of subspecies "rodentius." The uniform presence of mecA in each one of a large number of S. sciuri strains belonging to distinct ribotypes and macrorestriction patterns and recovered over a 20-year period from a wide variety of animal sources and geographic sites suggests that mecA may be a native genetic element with an as yet unidentified physiologic function in this staphylococcal species.

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.

Evidence for the geographic spread of a methicillin-resistant Staphylococcus aureus clone between Portugal and Spain.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates collected during a 7-month period in 1992 and 1993 at Hospital Pulido Valente (340 beds), Lisbon, Portugal, were characterized by a combination of genotypic and phenotypic methods. Clonal identities were determined by probing ClaI digests (i) with a mecA probe and (ii) with a Tn554 probe and (iii) by pulsed-field gel electrophoresis (PFGE) patterns of chromosomal SmaI digests. mecA-ClaI type I was predominant among these isolates (38 of 43). Most of these (37 of 38 [97.4%]) were associated with a single Tn554 pattern, pattern E, and the majority (23 of 38 [61%]) also showed a relatively uniform chromosomal background, as indicated by PFGE (PFGE pattern A). The major clone (mecA-ClaI type I::Tn554 type E and PFGE pattern A) at Hospital Pulido Valente was indistinguishable by these molecular typing criteria from the dominant clone that had been identified in two major current outbreaks of MRSA disease in Spain (Barcelona and Madrid). The Portuguese and Spanish clones also had a common heterogeneous class 3 phenotype and identical multidrug resistance patterns. The data presented in this work support the notion that MRSA clones can spread across considerable geographic distances.

Multidrug-resistant Iberian epidemic clone of methicillin-resistant Staphylococcus aureus endemic in a hospital in northern Portugal.

Forty-two methicillin-resistant Staphylococcus aureus (MRSA) isolates collected during 1992-1995 at a hospital in the north of Portugal were characterized by a variety of genomic fingerprints. Hybridization of ClaI and SmaI restriction digests with the mecA- and Tn554-specific DNA probes was used to identify polymorphs and determine their localization in chromosomal DNA preparations, and pulsed-field gel electrophoretic analysis of SmaI digests was used to determine chromosomal backgrounds. A major clone (and its variants) carrying the mecA polymorph I, Tn554 type E in the PFGE background of pattern A, accounted for 85% of all MRSA tested in 1992-1993 and 66% in 1994-1995. This clone is closely related to the epidemic Iberian clone that was associated with outbreaks in Spain during 1989-1993 and was endemic in 1992-1993 in two hospitals in Lisbon (Portugal).