Paraphocaeicola brunensis gen. nov., sp. nov., Carrying Two Variants of nimB Resistance Gene from Bacteroides fragilis, and Caecibacteroides pullorum gen. nov., sp. nov., Two Novel Genera Isolated from Chicken Caeca

Three difficult-to-cultivate, strictly anaerobic strains, AN20(T), AN421(T), and AN502, were analyzed within a project studying possible probiotics for newly hatched chickens. Phylogenetic analyses showed that strains AN20(T), AN421(T), and AN502 formed two well-separated phylogenetic lineages in all phylogenetic and phylogenomic trees comprising members of the family Bacteroidaceae. Comparison to reference genomes of type species Bacteroides fragilis NCTC 9343(T), Phocaeicola abscessus CCUG 55929(T), and Capsularis zoogleoformans ATCC 33285(T) showed low relatedness based on the calculated genome-to-genome distance and orthologous average nucleotide identity. Analysis of fatty acid profiles showed iso-C(15:0), anteiso-C(15:0), C(16:0), C(18:1) ω9c, and iso-C(17:0) 3OH as the major fatty acids for all three strains and additionally C(16:0) 3OH for AN421(T) and AN502. A specific combination of respiratory quinones different from related taxa was found in analyzed strains, MK-5 plus MK-11 in strain AN20(T) and MK-5 plus MK-10 in strains AN421(T) and AN502. Strains AN421(T) and AN502 harbor complete CRISPR loci with CRISPR array, type II-C, accompanied by a set of cas genes (cas9, cas1, and cas2) in close proximity. Interestingly, strain AN20(T) was found to harbor two copies of nimB gene with >95% similarity to nimB of B. fragilis, suggesting a horizontal gene transfer between these taxa. In summary, three isolates characterized in this study represent two novel species, which we proposed to be classified in two novel genera of the family Bacteroidaceae, for which the names Paraphocaeicola brunensis sp. nov. (AN20(T) = CCM 9041(T) = DSM 111154(T)) and Caecibacteroides pullorum sp. nov. (AN421(T)= CCM 9040(T) = DSM 111155(T)) are proposed. IMPORTANCE This study represents follow-up research on three difficult-to-cultivate anaerobic isolates originally isolated within a project focused on strains that are able to stably colonize newly hatched chickens, thus representing possible probiotics. This project is exceptional in that it successfully isolates several miscellaneous strains that required modified and richly supplemented anaerobic media, as information on many gut-colonizing bacteria is based predominantly on metagenomic studies. Superior colonization of newly hatched chickens by Bacteroides spp., Phocaeicola spp., or related taxa can be considered of importance for development of future probiotics. Although different experiments can also be performed with provisionally characterized isolates, precise taxonomical definition is necessary for subsequent broad communication. The aim of this study is therefore to thoroughly characterize these isolates that represent novel genera and precisely determine their taxonomic position among related taxa to facilitate further research and communication involving these strains.