Lactobacillus huangpiensis sp. nov. and Lactobacillus laiwuensis sp. nov., isolated from the gut of honeybee (Apis mellifera)

Four Gram-stain-positive bacterial strains were isolated from the gut of honeybee (Apis mellifera) in China. These strains were characterized using a polyphasic taxonomic approach. The data demonstrated that three of the four strains represented two novel species of the genus Lactobacillus, strains F306-1(T) and F551-2(T) were designated as the type strains. Results of 165 rRNA gene sequence analysis indicated that strains F306-1(T), F447 and F551-2(T) were phylogenetically related to the type strains of Lactobacillus kimbladii and Lactobacillus kullabergensis, having 99.1-99.7% 16S rRNA gene sequence (about 1400 bp) similarities. The phylogenetic tree based on concatenated pheS, rpoA, gyrB, hsp60, recA, rpoB and tuf sequences (4114 bp) and the phylogenomic tree based on whole genome sequences indicated that strains F306-1(T) and F447 were most closely related to L. kullabergensis Biut2N(T), and strain F551-2(T) was most closely related to L. kimbladii Hma2N(T). Strains F306-1(T) and F447 shared 99.9% average nucleotide identity (ANI), 99.7% digital DNA-DNA hybridization (dDDH) and 99.9% average amino acid identity (AAI) values, indicating that they belong to the same species. Strain F306-1(T) exhibited the highest ANI (94.4%), dDDH (56.7%) and AAI (94.7%) values to L. kullabergensis Biut2N(T). Strain F551-2(T) had the highest ANI (94.0%). dDDH (54.3%) and AAI (95.8%) values with L. kimbladii Hma2N(T). Acid production from amygdalin, maltose, starch. gentiobiose and turanose, activity of esterase (C4) and alpha-glucosidase, growth with 3% NaCl at 37 degrees C under strict anaerobic condition (on mMRS agar plates), and growth with 1-6% NaCl at 37 degrees C under aerobic condition (on mMRS agar plates supplemented with 0.05% cysteine or with 1% cysteine and 2% fructose) could differentiate strains F306-1(T) and F447 from L. kullabergensis DSM 26262(T). Acid production from D-glucose, arbutin and gentiobiose, growth with 3% NaCl at 37 degrees C under strict anaerobic condition (on mMRS agar plates), and growth at 45 degrees C under strict anaerobic condition (on mMRS agar plates) could differentiate strain F551-2(T) from L. kimbladii DSM 26263(T). Based upon the data obtained in the present study, two novel species. Lactobacillus huangpiensis sp. nov. and Lactobacillus laiwuensis sp. nov., are proposed and the type strains are F306-1(T) (=LMG 32144(T) =JCM 34361(T)=CCTCC AB 2020300(T)) and F551-2(T) (=JCM 34502(T)=CCTCC AB 2021027(T)), respectively.