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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.

Although it is generally accepted that dietary fiber is health promoting, the underlying immunological and molecular mechanisms are not well defined, especially with respect to cellulose, the most ubiquitous dietary fiber. Here, the impact of dietary cellulose on intestinal microbiota, immune responses and gene expression in health and disease was examined. Lack of dietary cellulose disrupted the age-related diversification of the intestinal microbiota, which subsequently remained in an immature state. Interestingly, one of the most affected microbial genera was Alistipes which is equipped with enzymes to degrade cellulose. Absence of cellulose changed the microbial metabolome, skewed intestinal immune responses toward inflammation, altered the gene expression of intestinal epithelial cells and mice showed increased sensitivity to colitis induction. In contrast, mice with a defined microbiota including A. finegoldii showed enhanced colonic expression of intestinal IL-22 and Reg3γ restoring intestinal barrier function. This study supports the epidemiological observations and adds a causal explanation for the health promoting effects of the most common biopolymer on earth.

Comensal Bacteroidota ( Bacteroidota ) and Enterobacteriacea are often linked to gut inflammation. However, the causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic dualism in Bacteroidota remain unclear. By using the classical lipopolysaccharide/O-antigen ' rfb operon' in Enterobacteriaceae as a surface antigen model (5- rfb- gene-cluster rfbABCDX ), and a recent rfbA- typing strategy for strain classification, we characterized the integrity and conservancy of the entire rfb operon in Bacteroidota . Through exploratory analysis of complete genomes and metagenomes, we discovered that most Bacteroidota have the rfb operon fragmented into nonrandom patterns of gene-singlets and doublets/triplets, termed ' rfb- gene-clusters', or rfb-'minioperons' if predicted as transcriptional. To reflect global operon integrity, contiguity, duplication, and fragmentation principles, we propose a six-category (infra/supra-numerary) cataloging system and a Global Operon Profiling System for bacteria. Mechanistically, genomic sequence analyses revealed that operon fragmentation is driven by intra-operon insertions of predominantly Bacteroides -DNA ( thetaiotaomicron/fragilis ) and likely natural selection in gut-wall specific micro-niches or micropathologies. Bacteroides -insertions, also detected in other antigenic operons (fimbriae), but not in operons deemed essential (ribosomal), could explain why Bacteroidota have fewer KEGG-pathways despite large genomes. DNA insertions, overrepresenting DNA-exchange-avid ( Bacteroides ) species, impact our interpretation of functional metagenomics data by inflating by inflating gene-based pathway inference and by overestimating 'extra-species' abundance. Of disease relevance, Bacteroidota species isolated from cavitating/cavernous fistulous tract (CavFT) microlesions in Crohn's Disease have supra-numerary fragmented operons, stimulate TNF-alpha from macrophages with low potency, and do not induce hyperacute peritonitis in mice compared to CavFT Enterobacteriaceae . The impact of 'foreign-DNA' insertions on pro-inflammatory operons, metagenomics, and commensalism/opportunism requires further studies to elucidate their potential for novel diagnostics and therapeutics, and to elucidate the role of co-existing pathobionts in Crohn's disease microlesions.

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.

Although it is generally accepted that dietary fiber is health promoting, the underlying immunological and molecular mechanisms are not well defined, especially with respect to cellulose, the most ubiquitous dietary fiber. Here, the impact of dietary cellulose on intestinal microbiota, immune responses and gene expression in health and disease was examined. Lack of dietary cellulose disrupted the age-related diversification of the intestinal microbiota, which subsequently remained in an immature state. Interestingly, one of the most affected microbial genera was Alistipes which is equipped with enzymes to degrade cellulose. Absence of cellulose changed the microbial metabolome, skewed intestinal immune responses toward inflammation, altered the gene expression of intestinal epithelial cells and mice showed increased sensitivity to colitis induction. In contrast, mice with a defined microbiota including A. finegoldii showed enhanced colonic expression of intestinal IL-22 and Reg3γ restoring intestinal barrier function. This study supports the epidemiological observations and adds a causal explanation for the health promoting effects of the most common biopolymer on earth.