Barcoding and Genetic Diversity in Microbes

Abstract

Nucleic acid amplification tests offer versatility, sensitivity and rapidness in bacterial detection. Micro-diverse clusters of 23S rRNA genes were perused to barcode E.coli (candidate) from its colony morphovars; C. koseri, E. aerogenes and K. pneumoniae (non-candidates) by targeting regions of Single Nucleotide Polymorphism. This study also connotes investigating the prospect of 23S rRNA gene for establishing the clonal nature of candidate microbe. An alternative en-route by coupling broad-range PCR with restriction analysis was ventured over routine molecular methods of bacterial identification. Multiple sequence alignment within and among the ribosomal repeats were evaluated for intra-genic and inter-genic diversity. Distributive pattern of 199 conventional restriction enzymes were mapped across 430 ribosomal sequences to choose the ideal enzyme for identification and diversity studies. Two broad range primer sets; 23S P1880 and 23S P2682 were designed; each amplifying a separate region common to all four strains but differing in restriction profiles. Amplified Ribosomal DNA Restriction Analysis (ARDRA) of 23S P1880 with BfaI discriminated E. coli from other members in the enterobacteriaceae family whereas Hae III digestion of 23S P2682 amplicons assisted in establishing predominant clones of E. coli. Analysis of dendrogram constructs of 682 bp amplicon sequence and whole genomic data of 57 E. coli strains confirmed that polymorphic patterns of the core genome can also be corroborated at pan-genomic level. Blast search across individual bacterial families (n=931) and secondary RNA structure prediction confirmed the primer specificity and conservation of recognition sites. ATCC cultures of clinical, commensal, and environmental origin were used for validation; indigenous Perl programs were developed for enable sequence extraction to virtual restriction profiling. The generic nature, conservation and barcode gap of repeats of 23S RNA gene makes it an ideal choice and substitute for 16S RNA gene.