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.

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Pneumococcal vaccination did not change the frequency of carriage of drug-resistant strains being the initially dominant vaccine serotypes replaced by others expressing nonvaccine serotypes. Reduction in the carriage of DRPn may require a combination of the conjugate vaccine and a decrease in antibiotic pressure.

OBJECTIVE: To determine the nature (clonal type and antibiotic resistance pattern) of methicillin-resistant Staphylococcus aureus (MRSA) strains recovered from the largest teaching hospital in Portugal and to detect temporal trends in clonal types during three consecutive surveillance periods in 1992--93, 1994--95 and 1996. METHODS: MRSA strains were characterized by chromosomal SmaI macrorestriction patterns using pulsed-field gel electrophoresis (PFGE) and by DNA fingerprints---applied to ClaI digests---capable of probing two specific areas of the staphylococcal chromosome: (1) the vicinity of the mecA gene, and (2) the attachment site(s) and copy number of transposon Tn554. The combination of these methods can generate 'clonal types' useful for epidemiological tracking of MRSA strains. RESULTS: During the 1992--93 collection period, 65% of MRSA strains carried the mecA polymorph I, Tn554 pattern E and PFGE pattern A (I::E::A)---a clonal type that was used to define the 'Iberian MRSA', which is widely spread throughout southern Europe. The representation of this clone decreased to 42% in 1994--95 and to 20% in 1996. At the same time, a second multiresistant MRSA strain carrying mecA polymorph XI, Tn554 type B and PFGE pattern B (XI::B::B)---a clonal type characteristic of the so-called 'Brazilian MRSA'---increased from 5% in 1992--93 to 36% in 1994--95 and 29% in 1996. CONCLUSIONS: Throughout the four years of surveillance, the Iberian and Brazilian MRSA types and their single subtype variants together have been responsible for the overwhelming majority (close to 90%) of all MRSA infections in the largest teaching hospital of Portugal. The mechanism of epidemicity of these two multiresistant international MRSA clones remains to be elucidated.

Of the nasopharyngeal cultures recovered from 942 day care center (DCC) attendees in Lisbon, Portugal, 591 (62%) yielded Streptococcus pneumoniae during a surveillance performed in February and March of 1999. Forty percent of the isolates were resistant to one or more antimicrobial agents. In particular, 2% were penicillin resistant and 20% had intermediate penicillin resistance. Multidrug resistance to macrolides, lincosamides, and tetracycline was the most frequent antibiotype (17% of all isolates). Serotyping and molecular typing by pulsed-field gel electrophoresis were performed for 202 out of 237 drug-resistant pneumococci (DRPn). The most frequent serotypes were 6B (26%), 14 (22%), 19F (16%), 23F (10%), and nontypeable (12%). The majority (67%) of the DRPn strains were representatives of nine international clones included in the Pneumococcal Molecular Epidemiology Network; eight of them had been detected in previous studies. Fourteen novel clones were identified, corresponding to 26% of the DRPn strains. The remaining 7% of the strains were local clones detected in our previous studies. Comparison with studies conducted since 1996 in Portuguese DCCs identified several trends: (i) the rate of DRPn frequency has fluctuated between 40 and 50%; (ii) the serotypes most frequently recovered have remained the same; (iii) nontypeable strains appear to be increasing in frequency; and (iv) a clone of serotype 33F emerged in 1999. Together, our observations highlight that the nasopharynxes of children in DCCs are a melting pot of successful DRPn clones that are important to study and monitor if we aim to gain a better understanding on the epidemiology of this pathogen.