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.

The multidrug-resistant "Iberian" clone of methicillin-resistant Staphylococcus aureus (MRSA) was first identified on the basis of its unique DNA fingerprints as the strain responsible for the massive 1989 outbreak of MRSA disease in the hospital Princeps d'Espanya, Barcelona, Spain. Most Iberian MRSA carry a constitutive beta-lactamase. They are resistant to most beta-lactam antibiotics, macrolides, aminoglycosides, tetracycline, rifampin and ciprofloxacin and are susceptible to fosfomycin, fusidic acid, mupirocin, sulfamethoxazole/trimethoprim and vancomycin. The characteristic DNA fingerprints of the clone include the mecA polymorph I, Tn554 pattern E (or its variants), a chromosomal macrorestriction pattern (pulsed-field gel electrophoretic type) A (or its subtype variants), the lack of the mecI regulatory gene and a homogeneous, high level of expression of methicillin resistance. Molecular surveillance studies have documented the extensive spread of this clone to many Portuguese hospitals during the 1990s. In this article, we describe the spread of the Iberian MRSA to hospitals in Rome, Italy, and Scotland.

The primary purpose of the multicenter international study "RESIST" was to obtain an update on the degree of multidrug resistance among methicillin-resistant staphylococci collected from a geographically diverse sample. A total of 3,307 staphylococcal isolates were recovered from single patients and primarily from clinical specimens that were collected at 20 collaborating regional health centers located in several countries in Europe, Asia, and Latin America during a 3- to 4-month period each in 1997 and 1998. All strains were deposited at the Laboratory of Molecular Genetics at ITQB/UNL in Oeiras, Portugal, for quality control and for testing by microbiological and molecular typing techniques; the Laboratory of Microbiology at The Rockefeller University serving as organizational center. The majority of strains, 3,100, were methicillin-resistant, of which 1,749 were coagulase positive (methicillin-resistant Staphylococcus aureus, MRSA), and 1,351 were coagulase negative (methicillin-resistant coagulase negative staphylococci, MRCNS). The overall frequency of drug resistance traits among the 1,749 MRSA strains was high (over 70% and up to and over 90% of the strains) to ciprofloxacin, erythromycin, clindamycin, gentamicin, and tetracycline, and was somewhat less frequent to sulfamethoxazole-trimethoprim (45%), chloramphenicol (30%), and rifampin (38%). None of the 3,307 staphylococcal isolates showed reduced susceptibility to vancomycin except for a single methicillin-resistant coagulase-negative isolate. The great majority of staphylococci were also susceptible to the new antimicrobial Synercid. In contrast, resistance to teicoplanin was significant among methicillin-resistant strains of coagulase-negative staphylococci, particularly among Staphylococcus haemolyticus. MRSA isolates showed marked geographic variation in their patterns of multiresistance, most likely reflecting the properties of unique multiresistant MRSA clones dominant in the hospitals that provided the MRSA isolates from the various geographic areas. The multiresistance patterns of MRSA strains and strains of methicillin-resistant coagulase-negative staphylococci originating at the same country source also showed striking differences, suggesting that resistance to antimicrobial agents emerged under different antibiotic pressures in these bacterial species.

Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysin. It shares 95% amino acidic identity with the endolysin of Staphylococcus aureus phage SAP6, which in turn is distantly related to all known CHAP endolysins of S. aureus phages. Lys170 seems to be a natural chimera assembling catalytic and cell-wall-binding domains of different origin. Both endolysins showed a clear preference to act against E. faecalis and they were able to lyse a high proportion of clinical isolates of this species. Specifically, Lys168 and Lys170 lysed more than 70% and 90% of the tested isolates, respectively, which included a panel of diverse and typed strains representative of highly prevalent clonal complexes. Lys170 was active against all tested E. faecalis VRE strains. The quasi specificity toward E. faecalis is discussed considering the nature of the enzymes' functional domains and the structure of the cell wall peptidoglycan.