A novel bifidobacteria (designated S053-2T) was isolated from the gut of honeybee (Apis mellifera). Strain S053-2T was characterized using a polyphasic taxonomic approach. The result of 16S rRNA gene sequence analysis indicated that strain S053-2T was phylogenetically related to the type strains of Bifidobacterium asteroides , Bifidobacterium indicum , Bifidobacterium actinocoloniiforme , Bifidobacterium xylocopae , Bifidobacterium coryneforme , Bifidobacterium apousia , Bifidobacterium choladohabitans and Bifidobacterium polysaccharolyticum , and had 95.5–99.7 % 16S rRNA gene sequence similarities. Based on the 16S rRNA gene sequence analysis, strain S053-2T was most closely related to the type strain of B. asteroides , having 99.7 % 16S rRNA gene sequence similarity. Strain S053-2T had relatively low (91.6–95.7 %) pheS, atpA, clpC, dnaG, fusA, glnA, glyS, hsp60, argS, pyrG and recA sequence similarities to the type strain of B. asteroides . Strain S053-2T had 94.5–95.3% atpA, clpC, dnaG, dnaK and pyrG sequence similarities to the type strain of B. apousia . The phylogenomic tree indicated that strain S053-2T belonged to the B. asteroides group, and was most closely related to the type strains of B. asteroides , B. apousia , B. choladohabitans and B. polysaccharolyticum , and distantly related to type strains of other phylogenetically related species in the B. asteroides group. Strain S053-2T shared the highest average nucleotide identity (ANI, 93.8 %), digital DNA–DNA hybridization (dDDH, 52.4 %) and average amino acid identity (AAI, 95.6%) values with B. apousia W8102T. Strain S053-2T shared 91.1 % ANI, 41.9 % dDDH and 92.5 % AAI values with B. asteroides DSM 20089T. Acid production from l-arabinose, d-xylose, d-mannose, amygdalin, cellobiose, maltose, melibiose, sucrose, raffinose, gentiobiose and l-fucose, and activity of esterase lipase (C8) and α-fucosidase could differentiate strain S053-2T from B. asteroides DSM 20089T. Acid production from d-mannose, maltose, sucrose, melezitose and gentiobiose, and activity of α-fucosidase could differentiate strain S053-2T from B. apousia W8102T. Based upon the data obtained in the present study, a novel species, Bifidobacterium mizhiense sp. nov., is proposed, and the type strain is S053-2T (=JCM 34710T=CCTCC AB 2021129T).

Two Gram-stain-positive bacterial strains, designated 213-9(3)T and 30-1(2)T, were isolated from traditional Chinese pickle, and were characterized using a polyphasic taxonomic approach. Results of 16S rRNA gene sequence analysis indicated that strain 213-9(3)T was most closely related to Levilactobacillus paucivorans TMW 1.1424T, Levilactobacillus huananensis 151-2BT and Levilactobacillus lindianensis 220-4T, having 99.7–99.9 % 16S rRNA gene sequence similarities; strain 30-1(2)T was most closely related to Lapidilactobacillus achengensis 247-4T, with 99.4 % 16S rRNA gene sequence similarity. Strain 213-9(3)T shared the highest pheS (93.9 %), rpoA (99.3 %) and concatenated pheS and rpoA (97.5 %) sequence similarities to L. paucivorans TMW 1.1424T. Strain 30-1(2)T had the highest pheS (82.4 %), rpoA (95.5 %) and concatenated pheS and rpoA (91.2 %) sequence similarities to L. achengensis 247-4T. The phylogenetic relationships based on concatenated pheS and rpoA sequences and whole genome sequences were identical to those based on 16S rRNA gene sequences. Strain 213-9(3)T exhibited the highest average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values (92.7 and 48.8 %, respectively) to L. paucivorans DSM 22467T. Strain 30-1(2)T had the highest ANI (84.4 %) and dDDH (32.8 %) values with L. achengensis 247-4T. Acid production from d-galactose, d-glucose, d-mannose, N-acetyl-β-glucosaminidase, arbutin, salicin, cellobiose, maltose, gentiobiose, d-tagatose and gluconate, hydrolysis of aesculin, and activity of cystine arylamidase could differentiate strain 213-9(3)T from L. paucivorans DSM 22467T. Acid production from l-arabinose, d-ribose, d-xylose and d-galactose, and activity of alkaline phosphatase, esterase (C4), α-mannosidase and α-fucosidase could differentiate strain 30-1(2)T from L. achengensis 247-4T. Based upon the data obtained in the present study, two novel species, Levilactobacillus humaensis sp. nov. and Lapidilactobacillus luobeiensis sp. nov., are proposed and the type strains are 213-9(3)T (=CCM 9241T=CCTCC AB 2022115T=JCM 35554T) and 30-1(2)T (=CCM 9240T=CCTCC AB 2022114T=JCM 35553T), respectively.

Currently, Lactococcus garvieae contains two subspecies: L. garvieae subsp. bovis and L. garvieae subsp. garvieae . In a study by Varsha and Nampoothiri, high pheS (99.7 %) and rpoA (99.6 %) sequence similarities indicated that L. garvieae subsp. bovis and Lactococcus formosensis probably have a close taxonomic relationship; low pheS (92.2 %) and rpoA (97.8 %) sequence similarities and relatively low DNA–DNA hybridization value (75.8 %) indicated that L. garvieae subsp. bovis and L. garvieae subsp. garvieae probably represent two different species. In the present study, the taxonomic relationships between L. garvieae subsp. bovis , L. garvieae subsp. garvieae and L. formosensis were re-examined based on sequence analyses of 16S rRNA, pheS, recA, rpoA and rpoB genes, average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH) values, average amino acid identity (AAI), fatty acid methyl ester analysis and phenotypic characterization. L. garvieae subsp. bovis LMG 30663T exhibited 97.3 % ANI, 78.3 % dDDH and 96.4 % AAI values to L. formosensis NBRC 109475T, higher than the threshold for species demarcation (95–96, 70 and 95–96 %, respectively), indicating that L. garvieae subsp. bovis LMG 30663T and L. formosensis NBRC 109475T belong to the same species. L. garvieae subsp. bovis LMG 30663T had 91.2 % ANI, 43.3 % dDDH and 92.9–93.0% AAI values with the type strain of L. garvieae subsp. garvieae , indicating that they represent two different species. Because L. formosensis has been proposed and validated before L. garvieae subsp. bovis , L. garvieae subsp. bovis is transferred to L. formosensis as L. formosensis subsp. bovis comb. nov. The type strain of L. formosensis subsp. bovis is BSN307T (=DSM 100577T=MCC 2824T=KCTC 21083T=LMG 30663T). The type strain of L. formosensis subsp. formosensis is 516T (=NBRC 109475T=BCRC 80576T).