Alternative approaches proposed the utilization of MALDI-TOF for species identification based on the sodA gene (Hinse et al., 2011). Most of these assays were developed using blood-derived clinical cultures of the SBSEC or restricted to a single species. In contrast to blood samples, raw dairy products were shown to contain a large diversity of different lactococci, enterococci, Streptococcus thermophilus, or Streptococcus

agalactiae (Delbès et al., 2007; Younan & Bornstein, 2007; Franciosi et al., 2009; Giannino et al., 2009; Jans, 2011), which increases the requirements regarding specificity of the primers. Even though the genes groESL or sodA provide improved capability to differentiate check details between species and subspecies, the 16S rRNA gene is still regarded as the recommended target for the initial identification of novel bacteria for which the higher degree of conservation of the 16S rRNA gene can be of advantage (Glazunova et al., 2009). This gene is one of the most important genotypic markers for bacterial taxonomy (Yarza

et al., 2008), and a large number of 16S rRNA gene sequences are available for downstream comparison and further analysis (Benson et al., 2009). It therefore represents an ideal target for the analysis of complex and Cobimetinib less-studied ecological niches such as the human microbiota (Grice et al., 2008; Liu et al., 2008) or spontaneous food fermentations, e.g., the African dairy environment. The high-density and complex microbial communities in these niches could result in unexpected genetic modifications through horizontal gene transfer (HGT), which was observed for African S. infantarius subsp. infantarius as well as for S. thermophilus and other LAB (Hols et al., 2005; PTK6 Makarova et al., 2006; Jans, 2011).

HGT is affecting nearly all genes within prokaryotic genomes; some genes including the 16S rRNA gene are, however, hypothesized to be less affected by HGT (Jain et al., 1999). Therefore, the objective was to utilize the high conservation of the 16S rRNA gene to develop an identification assay applicable to all species within the SBSEC allowing clear differentiation from other streptococci, enterococci, and lactococci regularly found in the dairy environment. The availability of large sets of nucleotide sequences from all members of the SBSEC including dairy isolates (Jans, 2011) enabled the design of a subsequent RFLP for the discrimination of SBSEC species groups. Furthermore, the primers were designed to work with Sanger sequencing for downstream sequence analysis. The assay was then evaluated against reference strains and isolated species of dairy microbial communities. Bacterial reference strains listed in Table 1 were obtained from the Culture Collection University of Gothenburg (CCUG, Gothenburg, Sweden), the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and the National Collection of Type Cultures (NCTC, Porton Down, UK).