Edinburgh Research Explorer Population genetic structure of the Staphylococcus intermedius group: Insights into agr diversification and the emergence of methicillin-resistant strains

The population genetic structure of the animal pathogen Staphylococcus intermedius is poorly understood. We carried out a multilocus sequence phylogenetic analysis of isolates from broad host and geographic origins to investigate inter- and intraspecies diversity. We found that isolates phenotypically identiﬁed as S. intermedius are differentiated into three closely related species, S. intermedius , Staphylococcus pseudintermedius , and Staph- ylococcus delphini . S. pseudintermedius , not S. intermedius , is the common cause of canine pyoderma and occasionally causes zoonotic infections of humans. Over 60 extant STs were identiﬁed among the S. pseudin- termedius isolates examined, including several that were distributed on different continents. As the agr quorum-sensing system of staphylococci is thought to have evolved along lines of speciation within the genus, we examined the allelic variation of agrD , which encodes the autoinducing peptide (AIP). Four AIP variants were encoded by S. pseudintermedius isolates, and identical AIP variants were shared among the three species, suggesting that a common quorum-sensing capacity has been conserved in spite of species differentiation in largely distinct ecological niches. A lack of clonal association of agr alleles suggests that assortive recombination may have contributed to the distribution of agr diversity. Finally, we discovered that the recent emergence of methicillin-resistant strains was due to multiple acquisitions of the mecA gene by different S. pseudintermedius clones found on different continents. Taken together, these data have resolved the population genetic structure of the S. intermedius group, resulting in new insights into its ancient and recent evolution. products) was included. Gene loci selected for DNA sequence analysis. Five loci, the 16S rRNA, cpn60 ( hsp60 ), tuf , pta , and agrD genes, were selected for DNA sequence analysis. 16S rRNA, cpn60 , and tuf have been used previously in single-locus approaches to differentiating staphylococcal species (7, 13, 24, 25), and cpn60 , tuf , and pta have been incorporated into multilocus sequence-typing schemes for differentiating strains of several different bacterial species (2, 10, 43). The accessory gene regulator ( agr ) quorum-sensing system found in the majority of staphylococcal species examined (8) is thought to have diversiﬁed along lines of speciation, giving rise to a number of subspecies groups that have the capacity for intra- and interspecies inhibition of virulence (30, 42). We examined the allelic variation of agrD , which encodes the autoinducing peptide (AIP), to further investigate both inter- and intraspecies differentiation. PCR ampliﬁcation of gene fragments. Oligonucleotide primers (Sigma-Geno-sys Invitrogen) were designed for the 16S rRNA, cpn60 , and agrD genes based on gene sequences for S. intermedius in GenBank (accession AY965912, and AF346723), and pta primers were designed based on the S. intermedius pta nucleotide sequence (unpublished data). Oligonucleotide sequences speciﬁc for the tuf and mecA genes were designed in previous studies (21, 31). The oligonucleotide sequences and predicted PCR product sizes for speciﬁed gene as follows: 16S (cid:4)