Identification of genus Campylobacter up to species level using internal features of 16S rRNA gene sequences

Abstract

Introduction: 16S rRNA sequencing of novel isolates is one of the preliminary steps in characterization of bacteria, especially when the isolates are of medical relevance. The genus Campylobacter belongs to Class e-proteobacteria under the Phylum Proteobacteria. It represents economically important species which are gastrointestinal pathogens in humans and livestock animals. Currently, more than 400 16S rDNA sequences belonging to 28 species of this genus are present in the RDP database. But heterogeneity has led to the misplacement of many of these sequences within wrong species. Also, various sequences belonging to Campylobacter have been deposited as orphans. The current study aimed to explore the internal features of 16S rRNA gene sequences in order to develop methods for identification of Campylobacter up to species level. Methods: 428 16S rRNA sequences of 28 species of Campylobacter were analyzed. 392 sequences (>1200 nucleotides, nts) of 16 species were considered for (i) phylogenetic framework analysis and (ii) in silico restriction digestion. 28 uncharacterized sequences present in the database were also investigated in the present study. Results: Phylogenetic framework analysis allowed the identification of genetic variability within Campylobacter species and helped to segregate certain uncharacterized sequences up to species level. 12 out of the 16 species under study showed homogenous behavior, but heterogeneity was observed between C. jejuni and C. coli and C. helveticus and C. upsaliensis respectively. Unique restriction enzymes were identified for six species. Conclusions: The present approach clearly showed that internal features of 16S rRNA is a useful tool for characterization of novel isolates up to the species level. Studies have revealed that niche overlap and consequent increase in the horizontal gene transfer between C. coli and C. jejuni, due to anthropogenic factors, maybe the reason for their heterogeneous nature. This explains the difficulties faced in segregation of the members of these species. 16S rRNA gene proved to be a viable and excellent marker for characterizing the uncharacterized Campylobacter strains leading to a significant diminution in database redundancy. Further, the approaches used in the study might assist in easier identification of the various Campylobacter sequences present in the database.