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.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has not been recognised previously as a cause of MRSA infections in Spain. Nineteen patients carrying Panton-Valentine leukocidin (PVL)-positive MRSA were identified in a Barcelona hospital, of whom 15 were immigrants, mostly from South America. Twelve developed skin and soft-tissue infections. The associated isolates carried the PVL gene and staphylococcal chromosomal cassette (SCC)mecIV. A dominant clone belonging to sequence type (ST)8 and related to the USA300 clone was identified by pulsed-field gel electrophoresis. This clone is emerging in Spain, primarily among immigrants from South America, but dissemination to the native Spanish population could increase.

A cohort study was carried out on hospitalized adult non-critically ill patients (January 2003-December 2004) to identify factors associated with the acquisition of multidrug-resistant Pseudomonas aeruginosa (MDR-PA). A total of 246 non-critically patients were included, 162 (66%) who revealed MDR-PA in the first isolate and 84 (34%) who had carbapenem-resistant P. aeruginosa (CR-PA) isolates. Multivariate analysis identified nosocomial acquisition (odds ratio [OR] 2.7, 95% confidence interval [CI] 1.1-6.3), urinary catheter (OR 2.1, 95%CI 1.1-4.3), and the prior use of fluoroquinolones (OR 2.6, 95%CI 1.0-6.7) as independent risk factors associated with MDR-PA acquisition. Our results show that antibiotics, most notably, fluoroquinolones, may play a major role in the emergence of MDR-PA.

Thirty-four isolates of pan-resistant Pseudomonas aeruginosa producing VIM-2 metallo-beta-lactamase (MBL) were detected at a university hospital in Spain (July 2004-September 2006). Eleven (32%) patients had clinically significant infections, and three (27%) of these patients died. A single clone of MBL-producing P. aeruginosa was identified by pulsed-field gel electrophoresis. A cluster of isolates associated with the vascular surgery ward involved ten patients and appeared as a series of low-grade, sustained and misdiagnosed endemic infections in the hospital. The identification of MBL-positive P. aeruginosa should be considered mandatory in the surveillance of pan-resistant P. aeruginosa and requires a high index of suspicion in the context of endemic infections with a low attack rate.