Genetic Diversity of Streptococcus Equi Subsp. Zooepidemicus Clinical Isolates from Horses in Italy

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of horses and many other animals. SEZ is frequently isolated from horses with pneumonia, pleuropneumonia or endometritis, and cases of strangles-like diseases have also been reported. SEZ can cause different diseases also in humans and its transmission from horses to humans has been described. SEZ are genetically heterogeneous bacteria and several techniques have been used so far for subtyping them. The aim of this work was to investigate the genetic diversity of SEZ strains isolated from Italian horses with respiratory diseases or from the genital tract before breeding. A total of 100 archived DNA samples of SEZ were subtyped by PCR for the 16S-23S rRNA gene intergenic spacer, as previously reported by other authors. On the basis of the preliminary results, some samples were selected for amplification and sequencing of a partial sequence of the 16S rRNA (about 1,000 bp). Finally, 33 sequences were compared with all SEZ 16S rRNA sequences available in GenBank and were used for phylogenetic analysis. PCRs detected at least one strain of each type of 16S-23S rRNA gene intergenic spacer (A1, A2, B1, B2, C1, C2, D1 and D2) and type A1 was the most frequent (33%), as previously reported in UK. In addition, a new type of product was found and named A1/A2a. Surprisingly, multiple intergenic spacer products (e.g. A1/A2, B1D2, etc.) were obtained from 43% samples, while in bibliography the frequency of such products was much lower. Alignment (regions V2-V6) of all SEZ 16S rRNA sequences available in GenBank showed that many strains differed from each other for single or multiple polymorphisms usually located at specific nucleotide positions. Phylogenetic analysis of our sequences showed that most strains were close to those already included in GenBank, although some polymorphisms were present. Furthermore, groups of strains showed new polymorphisms. No significant correlations among polymorphisms in the 16S rRNA sequences and types of 16S-23S rRNA gene intergenic spacer or source of samples (respiratory/genital samples) were found. Sometimes, SEZ collected at the same time from animals living in the same horse farm had different 16S rRNA sequences and/or different 16S-23S rRNA gene intergenic spacer types, confirming that different strains can co-circulate in the same farm. These data show a high heterogeneity among strains of SEZ collected from the respiratory and genital tracts of horses farmed in Italy and serve as a basis for further genetic typing investigations (e.g. MLST).