4.7. Ribotyping

Ribotyping is a method for bacterial identification and characterization that, unlike certain previously described molecular typing methods, employs rRNA based phylogenetic analysis. Given that that rRNA genes (such as 16S rRNA) are highly conserved within a bacterial species, identifying 16S rRNA gene polymorphisms is a reflection of the evolutionary lineage of the bacterial species, and can shed light on bacterial classification, taxonomy, epidemiological investigation, and population biology [202]. Ribotyping typically involves a multi-step process starting with restriction enzymes that target the genomic sequence of interest, followed by southern blot transfer and hybridization with probes, and analysis of ribotype RFLP bands. However, with advances in molecular tools and knowledge of genomic sequences, several modifications to this technique have been published [202]. It is important to note that for the purpose of primer and probe design, ribotyping requires some prior knowledge of the genome sequence under study. In one study, PCR-ribotyping was employed to characterize 99 strains of Clostridium difficile isolated from patients with nosocomial diarrhea. Following DNA extraction and PCR amplification of select regions of the 16S rRNA and 23S rRNA genes, amplified products were fractionated by electrophoresis [203]. The banding pattern revealed 41 different PCR-ribotypes with high reproducibility and discriminatory power. In a modification of this method, PCR-ribotyping was directly employed on stool samples for detection and typing of C. difficile strains [204]. Primer modifications targeting both, the 16S-23S rRNA intergenic spacer region and 16S and 23S genes itself, resulted in increased specificity for direct typing. With these new primers, PCR-ribotype could be detected directly from stool samples in 86 out of 99 cases, with a high degree of concordance with PCR-ribotyping done from isolated colonies.