Abstract
Background and aim: Streptococci are a heterogeneous group that includes more than 30 different species. Most of the species are usual components of the human flora and some are important human pathogens. To date, molecular tools have not been developed for studying the diversity of the genus Streptococcus in clinical samples. We have developed a PCR-based analytical tool to investigate the presence of new yet uncultured pathogenic streptococci in clinical samples. Methods: A 16S rRNA gene-targeted oligonucleotide (1043F) covering all species of the genus Streptococcus has been designed and used in combination with the universal eubacterial primer, 1492R, to produce approximately 450 bp PCR fragments that enclose three (V7, V8 and V9) hypervariable regions. PCR products are suitable for separation through denaturing gradient gel electrophoresis (DGGE). The inclusivity of the PCR assay was verified on 16 different Streptococcus species representing all current taxonomic groups. The exclusivity of the primers was tested on 29 other bacterial species representing all major phylogenetic lineages. Results: Positive PCR results were obtained for all Streptococcus species and Enterococcus faecalis, whereas PCR was negative for the other specimens. The amplification limit was 170 genome equivalents per reaction (95% probability). The oligonucleotide set was tested on colon biopsies and bronchoalveolar lavage samples. Thus, from the 33 sequences retrieved, 16 (48.5%) shared identity to cultured streptococci, whereas 11 (33.3%) of the sequences belonged to uncultured streptococci and three (9%) to other genera. Three sequences (9.7%) corresponded to new Streptococcus phylotypes (< 95% identified to previously known sequences). Conclusion: The use of this new primer set in combination with DGGE has proven to be a suitable method for studying the specific composition of streptococcal populations, particularly those from infected tissues. Moreover, this method can be of use in the identification of new Streptococcus species that are potentially involved in polymicrobial infections.
Acknowledgements
We thank Dr Xavier Aldeguer and Dr José Maria Sirvent of the Endoscopy Service and ICU of Hospital Josep Trueta, respectively, and Dr Carles Lopez from the Hospital Santa Caterina for kindly providing samples. Drs Marga Martinez and Laia Calvó are acknowledged for critically reading the manuscript.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.