Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus--MRSA--clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.

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.

No abstract

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.

Pulsed-field gel electrophoresis (PFGE) of

No abstract

Day care centers (DCCs) are unique settings where young children are at increased risk for colonization by pneumococci and Haemophilus influenzae. Although point prevalence studies in DCCs are frequent, only a few longitudinal studies on the dynamics of colonization have been published. We conducted a 1-year longitudinal study with 11 sampling periods on nasopharyngeal carriage of pneumococci and H. influenzae among 47 children who attended a single DCC. All isolates were antibiotyped and genotyped by pulsed-field gel electrophoresis. Pneumococci were also serotyped. Of the 414 samples obtained, 61.4% contained pneumococci, and 87% contained H. influenzae. Only 8.3% of the samples were negative for both species. Twenty-one pneumococcal clones and 47 H. influenzae clones were identified. Introduction of clones occurred during all year. Ninety-eight percent and 96% of all pneumococcal and H. influenzae isolates, respectively, belonged to clones shared by more than one child. Children were sequentially colonized with up to six pneumococcal clones (mean, 3.6) and five serotypes and nine H. influenzae clones (mean, 7.1). Clones with increased capacity for transmission and/or prolonged colonization were identified in both species. These two fitness properties appeared to be independent. In conclusion, among DCC attendees, a high rate of acquisition and turnover of strains was observed, and all children were overwhelmingly colonized by clones shared with others. DCCs are units where permanent introduction of new clones occurs, and attendees, as a whole, provide a pool of hosts where the fittest clones find privileged opportunities to persist and expand.Studies conducted during the last decade have highlighted the important role of day care centers (DCCs) as unique places where young children with immature immune systems and poor hygienic behavior are crowded together, resulting in an increased risk for colonization and transmission of upper respiratory tract pathogens such as Streptococcus pneumoniae and Haemophilus influenzae (1,4,5,10,23,30).While point prevalence studies in DCCs to study colonization by these bacteria have been conducted in several countries (reviewed in reference 7), longitudinal studies are less frequent and have often focused on the individual host and not on a particular epidemiological setting (9,14,16). By looking at the DCC as a unit, one would expect to obtain additional information on the fitness capacities (for transmission and persistence) of individual clones, as they would be exposed to the same pool of hosts (the attendees). To our best knowledge, extended longitudinal studies that have systematically applied genotyping techniques to study pneumococci and H. influenzae in DCCs with such objectives in mind are very scarce. Trottier et al. studied H. influenzae colonization among 38 DCC attendees for 4 months (29), and Yagupsy et al. conducted a 7-month study focusing on the transmission of drug-resistant pneumococci among 48 children from two DCCs (30). A third study by Raymond et al. f...

One hundred thirty-two methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from patients with S. aureus infections between January 1998 and February 1999 in two hospitals, one located in Taipei, Taiwan, and another in Nanjing, People's Republic of China, were examined for antibiotic susceptibility and for clonal type by a combination of three methods: hybridization of ClaI restriction digests with mecA-and Tn554-specific DNA probes and pulsed-field gel electrophoresis of chromosomal SmaI digests. Selected isolates representing each clonal type were also analyzed by spaA typing, multilocus sequence typing, and a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec) carried by the bacteria. The overwhelming majority of isolates (126 of 132 or 95%) belonged to minor variants of a single clonal type resembling the Brazilian and Hungarian epidemic MRSA clones, which showed a common spaA type and which were either sequence type 239 (ST239) or ST241 (a single-locus variant of ST239) in association with SCCmec type III or IIIA.