16S rRNA Variable Region Coverage in Salmonella enterica: Insights for Molecular Surveillance and Diagnostic Accuracy

Background Salmonella enterica is a serious foodborne pathogen that has a high genetic diversity and, as a result, it must be effectively identified using molecular means, which is crucial in preventing outbreaks and surveillance. It has a 16S rRNA gene which is the most popular bacterial taxonomic gene that has hypervariable regions (V1-V9) which vary in their discriminatory ability. Methods In this study, there were four Salmonella strains of the type Enterica (AK01-AK04) that were analyzed. The amplification of a partial, approximately 900 bp, 16S rRNA gene (positions 8-907 of the E. coli template), was done by use of 8F-907R primers, sequenced by the Sanger method, and assembled into consensus sequences. Variable regions V1-V5 were arranged against E. coli reference and evaluated in terms of recovery. Isolated sequences were positioned in comparison between the strains and coverage patterns were viewed on secondary structure maps. Results V1 region was lacking in four strains. V2 had no presence in AK01-AK02, was partially recovered in AK03 (19 bp), and partially recovered in AK04 (24 bp). V3 was completely amplified in AK01-AK03 (70 bp) and only partial in AK04 (27 bp). In contrast, V4 (127 bp) and V5 (57 bp) were always and totally recovered in all the isolates. These findings indicate that downstream regions, in particular V4 and V5 are more efficient in amplification and can provide more signals in phylogenetic information as compared to the upstream regions. Discussion The experimental results obtained demonstrate V4 and V5 as powerful targets of phylogenetic analysis and molecular diagnostics of Salmonella , whereas V1-V2 could not be easily accessed with the existing primer design. Whereas V3 was good in most isolates, it was partially covered in one strain indicating variability in the amplification effectiveness. It is because of its limited discriminatory power that 16S is not a sufficient technique but must be used in combination with multilocus sequence typing (MLST) or whole-genome sequencing (WGS) to achieve a resolution on a strain level. Conclusion V4 and V5 are the strongest hypervariable regions that yield a high-quality sequence data of the strains of the Salmonella enterica. Such areas are good examples of where molecular typing and epidemiological applications are possible although it would be advisable to integrate with more rigorous genomic applications in order to have a full surveillance of the area.