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The present study was undertaken for the purpose of defining the epidemiology and genetic relatedness of Staphylococcus aureus strains in a region of Italy by investigating the molecular background for which resistance to methicillin, mediated by the acquisition of another penicillin-binding protein gene, is embedded. Restriction fragment length polymorphism (RFLP) data were used for phylogenetic analyses, since genetic distance values can be used as a general measure of the number of events generating distinct clones. The percentages of methicillin-resistant S. aureus (MRSA) isolates in a six-month period from inpatients and outpatients were, respectively, 12% (22 out of 180) and 0.4% (1 out of 257). On the basis of RFLP obtained after pulsed field gel electrophoresis (PFGE), it was possible to designate isolates as indistinguishable, closely related, possibly related and unrelated. We were able to demonstrate the occurrence of at least five distinct MRSA cross-infection episodes in two hospitals, four involving two patients each and one involving four patients. Phylogenetic analyses overcame the simple pairwise comparison of common bands between strains, and provided a comprehensive epidemiological scenario, identifying three major clusters of MRSA, including different levels of genetic relatedness, while excluding the circulation of a single clone in Italy. Moreover, multidimensional scaling analysis of the obtained genetic distance confirmed that MRSA strains belong to a restricted set of clones, thus demonstrating the relatedness of broad evolutionary lineages within the species S. aureus.