Classification of Parabacteroides distasonis and other Bacteroidetes using O- antigen virulence gene: RfbA-Typing and hypothesis for pathogenic vs. probiotic strain differentiation

Parabacteroides distasonis (Pdis) is the type species for the new Parabacteroides genus, and a gut commensal of the Bacteroidetes phylum. Emerging reports (primarily based on reference strain/ATCC-8503) concerningly propose that long-known opportunistic pathogen Pdis is a probiotic. We posit there is an urgent need to characterize the pathogenicity of Pdis strain-strain variability. Unfortunately, no methods/insights exist to classify Bacteroidetes for this purpose. Herein, we developed a virulence gene-based classification system for Pdis and Bacteroidetes to facilitate pathogenic-vs-probiotic characterization. We used DNA in silico methods to develop a system based on the virulence (lipopolysaccharide/bacterial wall) 'rfbA O-antigen-synthesis gene'. We then performed phylogenetic analysis of rfbA from fourteen Pdis complete genomes (21 genes), other Parabacteroides, Bacteroidetes, and Enterobacteriaceae; and proposed a PCR-based Restriction-Fragment Length Polymorphism method. Cluster analysis revealed that Pdis can be classified into four lineages (based on gene gaps/insertions) which we designated rfbA-Types I, II, III, and IV. In context, we found 14 additional rfbA-types (I-XVIII) interspersed with numerous Bacteroidetes and pathogenic Enterobacteriaceae forming three major rfbA-superclusters. For laboratory rfbA-Typing implementation, we developed a PCR-primer strategy to amplify Pdis rfbA genes (100%-specificity) to conduct MboII-RFLP and sub-classify Pdis. In-silico primers for other Bacteroidetes are proposed/discussed. Comparative analysis of lipopolysaccharide/lipid-A gene lpxK confirmed rfbA as highly discriminant. In conclusion, rfbA-Typing classifies Bacteroidetes/Pdis into unique clusters/superclusters given rfbA copy/sequence variability. Analysis revealed that most pathogenic Pdis strains are single-copy rfbA-Type I . The relevance of the rfbA strain variability in disease might depend on their hypothetical modulatory interactions with other O-antigens/lipopolysaccharides and TLR4 lipopolysaccharide-receptors in human/animal cells.

Automated summary

In this study, a classification system based on the rfbA gene was developed to characterize the pathogenicity and probiotic properties of Pdis and Bacteroidetes. Analysis of 14 Pdis complete genomes revealed four lineages within Pdis. In addition, a PCR primer strategy for other Bacteroidetes was proposed. Comparative analysis confirmed the high discriminant power of rfbA.