JCM Accepts, published online ahead of print on 4 November 2009

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J. Clin. Microbiol. doi:10.1128/JCM.00542-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Diversity of Staphylococcus spp. strains based on partial kat (catalase) gene sequences and design of a PCR-RFLP assay for identification and differentiation of coagulasepositive species (S. aureus, S. delphini, S. hyicus, S. intermedius, S. pseudintermedius, and S. schleiferi subsp. coagulans)

Giuseppe Blaiotta^*, Vincenzina Fusco, Danilo Ercolini, Olimpia Pepe, and Salvatore Coppola

Department of Food Science, School of Agriculture and School of Biotechnological Sciences, University of Naples Federico II, via Università 100, 80055 Portici, Italy; National Research Council, Institute of Sciences of Food Production, Via Amendola 122/o, 70126, Bari, Italy

^* To whom correspondence should be addressed. Email: blaiotta{at}unina.it.

A set of degenerate PCR primers was designed and used to amplify and sequence about 75 % of the catalase (kat) gene of 49 staphylococcal strains. In some strains of Staphylococcus (S.) xylosus, S. saprophyticus and S. equorum two catalase genes, katA and katB, were found. A phylogenetic tree was generated showing diversities among 66 partial (about 900-bp) staphylococcal kat nucleotide sequences (including 17 sequences found in GenBank) representing 26 different species. The topology of this tree showed a distribution of staphylococcal species similar, but not identical, to those previously reported for 16S rDNA, hsp60, sodA, rpoB, tuf and gap genes. The kat gene sequences were less conserved than those of 16S rDNA, rpoB, hsp60, tuf genes and slightly more conserved than those of the gap gene. Therefore kat gene sequence analysis may represent an additional marker for inferring phylogenetic relationships of staphylococci. Moreover, the discrete nucleotide polymorphism revealed in this gene could be exploited for rapid low-cost identification of staphylococcal species through PCR-Restriction Fragment Length Polymorphism (RFLP) analysis. In this study a PCR-RFLP assay, performed by only Taq I restriction enzyme, was successfully developed for rapid unequivocal identification/differentiation, at species and subspecies level, of coagulase-positive staphylococci (CPS). The assay was validated by testing the DNA from 100 staphylococcal strains, including reference and wild CPS strains, isolated from different environments. This rapid (about 6 h from DNA isolation to results), reliable and low-cost (< 5 euro for each strain) approach allowed unambiguous identification of all the strains assayed, including the newly described S. delphini and S. pseudointermedius CPS species.