Molecular Detection of some Staphylococcal species with the newly developed folp gene sequences

Abstract

16SrRNA gene sequencing, universally accepted for bacterial identification could not effectively discriminate some closely related Staphylococcal species. For this reason, for bacterial species identification, other DNA sequencing genes are required. In this study, folp gene, encoding DHPS, was tested as a possible and alternative phylogenetic marker for more closely related Staphylococcal species. In this new method, 813 bp were implemented for classification instead of 1447 of 16S rRNA mostly used. Phylogenetic analysis was performed based on DNA sequences obtained from the GenBank including most important Staphylococcal species. As a result, the 16S rRNA-based tree clearly delineated 7 distinct clusters with high bootstrap values (1000) at distance adjusted to 5. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and Staphylococcus saprophyticus were identified in separate clusters. However, 2 mixed clusters were resolved including Staphylococcus epidermidis and Staphylococcus warneri in one cluster, while Staphylococcus pasteuri and Staphylococcus warneri in the other one. Comparatively, the folp gene-based tree yielded 9 clusters with at distance adjusted to 5 and 1000 bootstrap value. Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus ludunensis, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus pasteuri were resolved in separate clusters, however, Staphylococcus warneri and Staphylococcus epidermidis were identified in one mixed cluster. In addition, DNA-DNA relatedness studies indicated high sequence divergence of folp gene exhibiting 61.58-96.48 % interspecies homology compared to 16S rRNA with sequences similarities of 97.09-99.32 %. At the intraspecies level, the nucleotide substitution rates were ranged between 0-25%, mostly at low level for most Staphylococcal species compared to 98.65-99.93 identified in 16S rRNA gene. Moreover, in blind tests, this method was able for correct identification of 13 bacteria isolates of different Staphylococcal species. As a conclusion, folp gene sequences provide better resolution compared to the 16S rRNA gene sequences for both interspecies and interspecies DNA analysis of Staphylococci.