Barnesiella viscericola gen. nov., sp. nov., a novel member of the family Porphyromonadaceae isolated from chicken caecum.

Two bacterial strains isolated from chicken caecum, C46T and C47, were characterized using a polyphasic taxonomic approach that included analysis of the phenotypic and biochemical features, cellular fatty acid profiles, menaquinone profiles and phylogenetic position (using 16S rRNA gene sequence analysis). The 16S rRNA gene sequence analysis showed that these strains belonged to the family Porphyromonadaceae. These strains shared 100 % 16S rRNA gene sequence similarity with each other and were related to Parabacteroides distasonis (showing 86 % sequence similarity). The strains were found to be obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. Growth of the strains was inhibited on medium containing 20 % bile. The major menaquinones of the isolates were MK-11 and MK-12. This menaquinone composition was different from those of other genera of the family Porphyromonadaceae, such as Parabacteroides (in which the predominant menaquinones are MK-9 and MK-10), Porphyromonas (MK-9 and MK-10) and Tannerella (MK-10 and MK-11). This is an important chemotaxonomic characteristic of these micro-organisms. The DNA G+C content of strain C46T is 52.0 mol%. On the basis of these data, strains C46T and C47 represent a novel genus and species, for which the name Barnesiella viscericola gen. nov., sp. nov. is proposed. The type strain of Barnesiella viscericola is C46T (=JCM 13660T=DSM 18177T).

Bacteroides barnesiae sp. nov., Bacteroides salanitronis sp. nov. and Bacteroides gallinarum sp. nov., isolated from chicken caecum.

Eight bacterial strains isolated from the caecum of chicken, BL2(T), BL66, EG3, EG6, M27, BL78(T), C35(T) and C43, were characterized by determining their phenotypic characteristics, cellular fatty acid profiles, menaquinone profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these isolates belonged to the genus Bacteroides. One group of five strains (BL2(T), BL66, EG3, EG6 and M27) was related most closely to Bacteroides coprocola JCM 12979(T), with approximately 93 % 16S rRNA gene sequence similarity, and to Bacteroides plebeius JCM 12973(T), with about 92 % similarity, and shared >or=99.6 % similarity with each other. Strain BL78(T) exhibited 90.5 % similarity to B. plebeius JCM 12973(T) and 89.8 % similarity to B. coprocola JCM 12979(T) and differed from the above group of five strains at >or=10 % sequence divergence. Strains C35(T) and C43 were related most closely to Bacteroides eggerthii JCM 12986(T), with 95.1 % sequence similarity, to Bacteroides stercoris JCM 9496(T), with 94.6 % similarity, and to Bacteroides uniformis JCM 5828(T), with 94.4 % similarity, and shared 100 % similarity with each other. From results of phenotypic examination, cellular fatty acid composition analysis, menaquinone composition analysis and DNA G+C contents, the group of five strains as well as strain BL78(T) were shown to differ from the type strains of B. coprocola and B. plebeius. Strain BL78(T) differed from the others based on its menaquinone composition, which included MK-11 and MK-12. Strains C35(T) and C43 could also be differentiated from the type strains of B. eggerthii, B. stercoris and B. uniformis. The group of five strains, strain BL78(T), B. coprocola JCM 12979(T) and B. plebeius JCM 12973(T) showed low levels of DNA-DNA relatedness (<35 %) with each other. High levels of DNA-DNA relatedness were obtained within the group of five strains (>75 %). Strains C35(T) and C43 exhibited a high level of DNA-DNA relatedness (>88 %) with each other, but low levels with B. eggerthii JCM 12986(T) (<40 %), B. stercoris JCM 9496(T) (<37 %) and B. uniformis JCM 5828(T) (<16 %). On the basis of these data, three novel Bacteroides species are proposed: Bacteroides barnesiae sp. nov. (type strain BL2(T)=JCM 13652(T)=DSM 18169(T)), Bacteroides salanitronis sp. nov. (type strain BL78(T)=JCM 13657(T)=DSM 18170(T)) and Bacteroides gallinarum sp. nov. (type strain C35(T)=JCM 13658(T)=DSM 18171(T)).

Effects of two probiotic Lactobacillus strains on jejunal and cecal microbiota of broiler chicken under acute heat stress condition as revealed by molecular analysis of 16S rRNA genes.

We examined the effects of probiotic Lactobacillus strains of Lactobacillus agilis JCM 1048 and Lactobacillus salivarius subsp. salicinius JCM 1230 on jejunal and cecal microbiota of broiler chicken under heat stress condition using terminal restriction fragment length polymorphism (T-RFLP) analysis. The jejunal bacterial community was limited to a few bacterial groups, mostly Lactobacillus spp. A relatively abundant and higher prevalence of Lactobacillus spp. were observed in the jejunal and cecal microbiota of the probiotic chickens compared with those of the control chickens under heat stress condition. In general, the probiotic strains did not significantly affect the abundance of L. agilis and L. salivarius in chicken intestine but clearly contributed to increasing their prevalence in the probiotic chickens. The probiotic Lactobacillus strains enriched the diversity of Lactobacillus flora in chicken jejunum and cecum by increasing the abundance and prevalence of Lactobacillus spp. inhabiting the intestine. The richness of Lactobacillus species tended to be similar among the jejunal and cecal microbiota. The bacterial community of cecum was complex and age-dependent. The major components of the cecal microbiota were clostridia and lactobacilli. The Clostridium subcluster XIVa was the most predominant group in chicken cecum. Probiotic Lactobacillus strains restored the microbial balance and maintained the natural stability of indigenous bacterial microbiota following heat stress-induced changes.

Impact of two probiotic Lactobacillus strains feeding on fecal lactobacilli and weight gains in chicken.

Two probiotic strains, Lactobacillus agilis JCM 1048 and L. salivarius subsp. salicinius JCM 1230 isolated from chicken intestine, exhibited probiotic characteristics that can be applied for chicken production. After 7 days of probiotic feeding (FD7), the count of intestinal lactobacilli in the probiotic group (group P, n=10) was significantly (p<0.05) higher than that in the control group (group C, n=9). After 40 days of probiotic feeding (FD40), the lactobacilli and enterococci counts were stable but the Enterobacteriaceae number was significantly reduced (p<0.05). A total of 163 isolated lactobacilli were identified as the L. acidophilus/gallinarum group (49.7%), L. agilis (30.7%), L. salivarius (9.2%), L. reuteri (9.2%), and Lactobacillus spp. (1.2%). The probiotic lactobacilli positively affected the Lactobacillus biota in chickens at FD7, with a significant increase in the number (p<0.05) of L. agilis and group P. The viable counts of each Lactobacillus species at FD40, however, showed no differences between two groups. An increasing incidence of L. agilis was also noted with probiotic feeding. The probiotic effect of two strains resulted in significantly increased weight gains (10.7%) of group P in comparison with group C at FD40 (p<0.01).

Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries.

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.