METHICILLIN-RESISTANT STAPHYLOCOCCUS-AUREUS DISEASE IN A PORTUGUESE HOSPITAL - CHARACTERIZATION OF CLONAL TYPES BY A COMBINATION OF DNA TYPING METHODS

Fifteen pediatric patients as well as the five nursing staff of the Bu rn Unit of the Hospital D. Estefania in Lisbon, Portugal, were assayed at weekly intervals over a fivemonth period in order to identify the nature and number of methicillin-resistant Staphylococcus aureus (MRSA ) clones associated with colonization and wound infection. Methicillin resistance was confirmed by a mec-specific DNA probe. MRSA isolates w ere classified into chromosomal types (clones) on the basis of a varie ty of techniques: (i) ribotyping; (ii) restriction digestion by the en donuclease ClaI followed by Southern hybridization with the mech-speci fic DNA probe and (iii) by hybridization with Tn554; and (iv) pulsed-f ield electrophoresis (PFE) of SmaI digests followed by (v) Southern hy bridization with the mecA DNA probe. A sixth, physiological technique (population analysis) was used to define the mode of phenotypic expres sion of methicillin resistance in each isolate. All isolates carried a single, common polymorph (ClaI type III) of the mecA gene. Hybridizat ion with Tn554 resolved these isolates to two novel patterns (alpha an d beta), of which one (Tn554 alpha) was predominant (90 %). This patte rn could be further resolved to four closely related PFE types (A thro ugh D). In contrast, all isolates with the Tn554 beta pattern belonged to an additional, grossly different PFE type E. The Tn554 beta class was also unique in that these bacteria carried the mecA gene in a SmaI fragment smaller (about 170 kb) than that found in the alpha type str ains (194 kb). Most isolates (83 %) showed a single heterogeneous (pop ulation analysis Class 3) mode of resistance expression. The data demo nstrate the full capacity of the globally rare (ClaI type III) MRSA cl one for colonization and virulence. The results also document the stab ility of the complex heterogeneous resistance phenotype as well as the stability of the chromosomal types under conditions of in vivo carria ge over a period of several months. In a few isolates the same mecA po lymorph was present in several, grossly different genetic backgrounds, suggesting horizontal transfer of the mecA gene.