pheS Gene Sequence Similarities Research Articles

PMA-qPCR method for the selective quantitation of viable lactic acid bacteria in fermented milk.

The number of viable lactic acid bacteria (LAB) is a key indicator of the quality of fermented milk. Currently, the combination of propidium monoazide (PMA) and qPCR has been applied in the quantification of viable bacteria in various matrices. In this research, the PMA-qPCR method was used to detect the number of viable bacteria of each LAB species in fermented milk. By analyzing pheS gene and 16S rRNA gene sequence similarities in five species of LAB, namely Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum, Streptococcus thermophilus, Lactobacillus helveticus, and Lactococcus lactis subsp. lactis, the pheS gene resolved species identities better and was thus selected to design specific primers and probes. The pheS gene was cloned into the pUC19 vector and used to construct a standard curve for absolute quantification. Standard curves for quantification were constructed for each LAB species for serial dilutions between 1011 and 106 CFU/mL, with R2 > 0.99. The number of viable bacteria in the fermented milk detected by PMA-qPCR was significantly lower than that of qPCR (P < 0.05), indicating that PMA inhibited the amplification of DNA from dead cells. This was corroborated by the results from bacterial staining and plate count experiments. The proposed PMA-qPCR method provided rapid qualitative and quantitative determination of the number of viable bacteria for each LAB species in fermented milk within 3 h.

Proposal of Lactobacillus kosoi Chiou et al. 2018 as a later heterotypic synonym of Lactobacillus micheneri McFrederick et al. 2018, elevation of Lactobacillus plantarum subsp. argentoratensis to the species level as Lactobacillus argentoratensis sp. nov., and Lactobacillus zhaodongensis sp. nov., isolated from traditional Chinese pickle and the intestinal tract of a honey bee (Apis mellifera).

Lactobacillus kosoi Chiou et al. 2018 and Lactobacillus micheneri McFrederick et al. 2018 are closely related, and they share 100 % 16S rRNA gene sequence similarity, 99.6 % pheS gene sequence similarity, 100 % rpoA gene sequence similarity, 97.3 % average nucleotide identity (ANI) value and 76.6 % in silico DNA-DNA hybridization (isDDH) value, indicating that they represent the same species. Fatty acid methyl esters (FAME) analysis and phenotypic characterization also indicated that L. kosoi and L. micheneri are very similar. We propose L. kosoi Chiou et al. 2018 as a later heterotypic synonym of L. micheneri McFrederick et al. 2018. The taxonomic position of Lactobacillus plantarum subsp. argentoratensis in the L. plantarum group was re-examined using a polyphasic approach, including sequence analyses of 16S rRNA, pheS, rpoA and recA genes, average nucleotide identity analysis, in silico DNA-DNA hybridization, fatty acid methyl ester analysis and phenotypic characterization. Results of 16S rRNA gene sequence analysis indicated that L. plantarum subsp. argentoratensis was closely related to L. plantarum subsp. plantarum, L. pentosus and L. paraplantarum in the L. plantarum group, sharing 99.6-99.7 % 16S rRNA gene sequence similarities. Results of pheS, rpoA and recA gene sequence analyses indicated that L. plantarum subsp. argentoratensis was most closely related to L. plantarum subsp. plantarum, having 91.8 % pheS gene sequence similarity, 98.9 % rpoA gene sequence similarity and 93.1 % recA gene sequence similarity. L. plantarum subsp. argentoratensis DSM 16365T shared 95.6 % ANI value and 62.9 % isDDH value with L. plantarum subsp. plantarum ATCC 14917T. The low isDDH value confirmed that L. plantarum subsp. argentoratensis and L. plantarum subsp. plantarum represent two different species, rather than two different subspecies in the L. plantarum group. On the basis of the data from polyphasic characterization obtained in the present study and in previous studies, L. plantarum subsp. argentoratensis is elevated to the species level and represents a novel species of the genus Lactobacillus, for which the name Lactobacillus argentoratensis sp. nov. is proposed and the type strain is DKO 22T (=CIP 108320T=DSM 16365T=JCM 16169T). Two novel Gram-stain-positive bacterial strains, designated 1206-1T and F027-1-2, were isolated from traditional pickle in Heilongjiang Province, PR China, and from the intestinal tract of a honey bee (Apis mellifera) in Hubei Province, PR China, respectively. The two bacteria were characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester analysis, average nucleotide identity analysis, in silico DNA-DNA hybridization analysis and an analysis of phenotypic features. The results of 16S rRNA gene sequence analysis indicated that strains 1206-1T and F027-1-2 were distantly related to Lactobacillus sharpeae, Lactobacillus hulanensis, Lactobacillus songhuajiangensis, Lactobacillus pantheris, Lactobacillus thailandensis, Lactobacillus camelliae, Lactobacillus jixianensis, Lactobacillus nasuensis, Lactobacillus baoqingensis, Lactobacillus manihotivorans and Lactobacillus porcinae. Strain 1206-1T exhibited 94.2-96.4 % 16S rRNA gene sequence similarities, 69.5-83.3 % pheS gene sequence similarities and 73.1-90.3 % rpoA gene sequence similarities to type strains of phylogenetically related species. ANI and isDDH values between strain 1206-1T and the type strains of phylogenetically related species were 52.7-73.7 % and 21.1-30.1 %, respectively. On the basis of the data obtained in the present study, a novel species, Lactobacillus zhaodongensis sp. nov. is proposed and the type strain is 1206-1T (=CCM 8981T=CCTCC AB 2019200T=LMG 31620T).

Lactobacillus hegangensis sp. nov., Lactobacillus suibinensis sp. nov., Lactobacillus daqingensis sp. nov., Lactobacillus yichunensis sp. nov., Lactobacillus mulanensis sp. nov., Lactobacillus achengensis sp. nov., Lactobacillus wuchangensis sp. nov., Lactobacillus gannanensis sp. nov., Lactobacillus binensis sp. nov. and Lactobacillus angrenensis sp. nov., isolated from Chinese traditional pickle and yogurt.

Fourteen Gram-stain-positive bacterial strains were isolated from Chinese traditional pickle and yogurt. The strains were characterized using a polyphasic taxonomic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester analysis, determination of DNA G+C content, determination of average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH) and an analysis of phenotypic features. The data demonstrated that the 14 strains represented ten novel species belonging to the genus Lactobacillus, strains 73-4T, 247-3T, 143-4(a)T, 33-1T, 143-6T, 247-4T, 17-4T, 143-1T, 735-2T and M1530-1T were designated as the type strains. Strains 73-4T and 247-3T were phylogenetically related to the type strains of Lactobacillus camelliae and Lactobacillus jixianensis, having 97.0-98.9 % 16S rRNA gene sequence similarities, 83.9-87.2 % pheS gene sequence similarities and 86.8-93.3 % rpoA gene sequence similarities. Strains 143-4(a)T and 33-1T were phylogenetically related to the type strains of Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus casei, having 93.6-96.5 % 16S rRNA gene sequence similarities, 73.9-77.2 % pheS gene sequence similarities and 76.1-77.6 % rpoA gene sequence similarities. Strains 143-6T, 247-4T, 17-4T and 143-1T were phylogenetically related to the type strains of Lactobacillus concavus, Lactobacillus dextrinicus and Lactobacillus bayanensis, exhibiting 95.5-99.9 % 16S rRNA gene sequence similarities, 76.5-83.1 % pheS gene sequence similarities and 83.6-98.3 % rpoA gene sequence similarities. Strain 735-2T was phylogenetically related to the type strains of Lactobacillus zhaoyuanensis, Lactobacillus jiayinensis and Lactobacillus coryniformis, having 98.2-99.1 % 16S rRNA gene sequence similarities, 82.8-84.1 % pheS gene sequence similarities and 93.0-93.9 % rpoA gene sequence similarities. Strain M1530-1T was phylogenetically related to the type strains of Lactobacillus suantsaiihabitans and Lactobacillus brevis, having 99.5 and 99.0 % 16S rRNA gene sequence similarities, 90.3 and 81.7 % pheS gene sequence similarities and 97.7 and 91.1 % rpoA gene sequence similarities. The ANI and isDDH values between strains 73-4T, 247-3T, 143-4(a)T, 33-1T, 143-6T, 247-4T, 17-4T, 143-1T, 735-2T, M1530-1T and type strains of phylogenetically related species were less than 86.8 % and 33.9 % respectively, confirming that they represent ten novel species within the genus Lactobacillus. Based upon the data of polyphasic characterization obtained in the present study, ten novel species, Lactobacillus hegangensis sp. nov., Lactobacillus suibinensis sp. nov., Lactobacillus daqingensis sp. nov., Lactobacillus yichunensis sp. nov., Lactobacillus mulanensis sp. nov., Lactobacillus achengensis sp. nov., Lactobacillus wuchangensis sp. nov., Lactobacillus gannanensis sp. nov., Lactobacillus binensis sp. nov. and Lactobacillus angrenensis sp. nov., are proposed and the type strains are 73-4T (=NCIMB 15177T=CCM 8912T=CCTCC AB 2018407T), 247-3T (=NCIMB 15176T=JCM 33275T), 143-4(a)T (=NCIMB 15173T=CCM 8948T=JCM 33273T=CCTCC AB 2018390T), 33-1T (=NCIMB 15169T=CCM 8947T=JCM 33272T=CCTCC AB 2018405T), 143-6T (=NCIMB 15162T=CCM 8951T=JCM 33274T=CCTCC AB 2018411T), 247-4T (=NCIMB 15155T=CCM 8897T=LMG 31059T=CCTCC AB 2018410T), 17-4T (=NCIMB 15161T=CCM 8946T=JCM 33271T=CCTCC AB 2018406T), 143-1T (=NCIMB 15157T=CCM 8937T=CCTCC AB 2018409T), 735-2T (=NCIMB 15190T=CCM 8925T=LMG 31186T) and M1530-1T (=NCIMB 15150T=CCM 8893T=LMG 31046T=CCTCC AB 2018402T), respectively.

Weissella sagaensis sp. nov., isolated from traditional Chinese yogurt.

Three Gram-stain-positive bacterial strains, designated X0750T, X0278 and X0401, isolated from traditional yogurt in Tibet Autonomous Region, PR China, were characterized by a polyphasic approach, including sequence analyses of the 16S rRNA gene and three housekeeping genes (pheS, rpoA and recA), determination of average nucleotide identity (ANI) and average amino acid identity (AAI), in silico DNA-DNA hybridization (isDDH), fatty acid methyl ester (FAME) analysis and phenotypic characterization. Strain X0750T was phylogenetically related to the type strains of Weissella hellenica, Weissella bombi, Weissella paramesenteroides, Weissella jogaejeotgali, Weissella thailandensis, Weissella oryzae, Weissella cibaria and Weissella confusa, having 94.4-100 % 16S rRNA gene sequence similarities, 76.7-90.0 % pheS gene sequence similarities, 88.9-99.4 % rpoA gene sequence similarities and 77.6-92.8 % recA gene sequence similarities, respectively. ANI, isDDH and AAI values between strain X0750T and type strains of phylogenetically related species were less than 90.4, 40.9 and 92.8 % respectively, confirming that strain X0750T represents a novel species within the genus Weissella. Based upon the data obtained in the present study, a novel species, Weissella sagaensis sp. nov., is proposed and the type strain is X0750T(=NCIMB 15192T=CCM 8924T=LMG 31184T=CCTCC AB 2018403T).

Lactobacillus yilanensis sp. nov., Lactobacillus bayanensis sp. nov., Lactobacillus keshanensis sp. nov., Lactobacillus kedongensis sp. nov., Lactobacillus baiquanensis sp. nov., Lactobacillus jidongensis sp. nov., Lactobacillus hulinensis sp. nov., Lactobacillus mishanensis sp. nov. and Lactobacillus zhongbaensis sp. nov., isolated from Chinese traditional pickle and yogurt.

Fourteen Gram-stain-positive bacterial strains were isolated from Chinese traditional pickle and yogurt. The strains were characterised using a polyphasic taxonomic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, determination of DNA G+C content, determination of average nucleotide identity, fatty acid methyl ester analysis and an analysis of phenotypic features. The data demonstrated that the fourteen strains represented nine novel species belonging to the genus Lactobacillus, strains 54-2T, 54-5T, 33-7T, 116-2T, 184-8T, 204-8T, 8-1(1)T, 256-3T and M1575T were designated as the type strains. Strain 54-2T was phylogenetically related to the type strains of Lactobacillus composti and Lactobacillus floricola, having 96.5 and 91.6 % 16S rRNA gene sequence similarities, less than 74.6 % pheS gene sequence similarities, less than 81.6 % rpoA gene sequence similarities and less than 72.5 % ANI values. Strain 54-5T was phylogenetically related to the type strains of Lactobacillus dextrinicus and Lactobacillus concavus, exhibiting 99.1 and 97.3 % 16S rRNA gene sequence similarities, less than 83.1 % pheS gene sequence similarities, less than 93.1 % rpoA gene sequence similarities and less than 79.9 % ANI values. Strains 33-7T, 116-2T, 184-8T, 204-8T, 8-1(1)T, 256-3T and M1575T were phylogenetically related to the type strains of Lactobacillus tucceti, Lactobacillus nodensis, Lactobacillus insicii, Lactobacillus allii, Lactobacillus metriopterae, Lactobacillus terrae, Lactobacillus versmoldensis and Lactobacillus furfuricola, sharing 95.6-100 % 16S rRNA gene sequence similarities, less than 91.6 % pheS gene sequence similarities, less than 98.2 % rpoA gene sequence similarities and less than 89.4 % ANI values. Based upon the data of polyphasic characterisation obtained in the present study, nine novel species, Lactobacillus yilanensis sp. nov., Lactobacillus bayanensis sp. nov., Lactobacillus keshanensis sp. nov., Lactobacillus kedongensis sp. nov., Lactobacillus baiquanensis sp. nov., Lactobacillus jidongensis sp. nov., Lactobacillus hulinensis sp. nov., Lactobacillus mishanensis sp. nov. and Lactobacillus zhongbaensis sp. nov., are proposed and the type strains are 54-2T (=NCIMB 15154T=CCM 8896T=KCTC 21120T=LMG 31058T), 54-5T (=NCIMB 15151T=CCM 8894T), 33-7T (=NCIMB 15153T=CCM 8936T=KCTC 21118T=LMG 31166T), 116-2T (=NCIMB 15158T=CCM 8899T=KCTC 21124T=LMG 31051T), 184-8T (=NCIMB 15152T=CCM 8895T=KCTC 21131T=LMG 31050T), 204-8T (=NCIMB 15159T=CCM 8900T=KCTC 21133T=LMG 31054T), 8-1(1)T (=NCIMB 15156T=CCM 8898T=KCTC 21115T=LMG 31047T), 256-3T (=NCIMB 15160T=CCM 8901T=LMG 31048T) and M1575T (=NCIMB 15149T=CCM 8892T=LMG 31045T), respectively.

Lactobacillus pingfangensis sp. nov., Lactobacillus daoliensis sp. nov., Lactobacillus nangangensis sp. nov., Lactobacillus daowaiensis sp. nov., Lactobacillus dongliensis sp. nov., Lactobacillus songbeiensis sp. nov. and Lactobacillus kaifaensis sp. nov., isolated from traditional Chinese pickle.

Seven novel lactic acid bacterial strains, isolated from traditional Chinese pickle, were characterized using a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, determination of DNA G+C content, determination of average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH), fatty acid methyl ester (FAME) analysis and an analysis of phenotypic features. Strains 382-1T, 116-1AT, 381-7T, 203-3T, 218-3T and 398-2T were phylogenetically related to the type strains of Lactobacillus plantarumsubsp. plantarum, Lactobacillus plantarumsubsp. argentoratensis, Lactobacillus pentosus, Lactobacillus paraplantarum, Lactobacillus fabifermentans, Lactobacillus herbarum, Lactobacillus mudanjiangensis, Lactobacillus xiangfangensis, Lactobacillus plajomi and Lactobacillus modestisalitolerans, having 97.1-99.9 % 16S rRNA gene sequence similarities, less than 89.9 % pheS gene sequence similarities, less than 98.0 % rpoA gene sequence similarities, less than 91.2 % ANI values and less than 43.3 % isDDH values. Strain 778-3T was phylogenetically related to the type strains of Lactobacillus hokkaidonensis, Lactobacillus wasatchensis, Lactobacillus oligofermentans, Lactobacillus nenjiangensis, Lactobacillus vaccinostercus and Lactobacillus suebicus, exhibiting 97.0-99.4 % 16S rRNA gene sequence similarities, 78.2-82.1 % pheS gene sequence similarities, 80.0-91.5 % rpoA gene sequence similarities, less than 78.6 % ANI values and less than 22.9 % isDDH values. Based upon the data of polyphasic characterization obtained in the present study, seven novel species, Lactobacillus pingfangensis sp. nov., Lactobacillus daoliensis sp. nov., Lactobacillus nangangensis sp. nov., Lactobacillus daowaiensis sp. nov., Lactobacillus dongliensis sp. nov., Lactobacillus songbeiensis sp. nov. and Lactobacillus kaifaensis sp. nov., are proposed and the type strains are 382-1T (=NCIMB 15187T=CCM 8935T=LMG 31176T), 116-1AT (=NCIMB 15181T=CCM 8934T=LMG 31171T), 381-7T (=NCIMB 15186T=CCM 8930T), 203-3T (=NCIMB 15183T=CCM 8933T=LMG 31172T), 218-3T (=NCIMB 15184T=CCM 8932T=LMG 31173T), 398-2T (=NCIMB 15189T=CCM 8931T=LMG 31174T) and 778-3T (=NCIMB 15191T=CCM 8929T=LMG 31177T), respectively.

Lactobacillus huachuanensis sp. nov., isolated from Chinese traditional pickle.

A Gram-stain-positive bacterial strain, 395-6.2T, was isolated from traditional pickle in Heilongjiang Province, PR China. The bacterium was characterised by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester (FAME) analysis, determination of DNA G+C content, average nucleotide identity (ANI) analysis, in silico DNA-DNA hybridisation (isDDH) and an analysis of phenotypic features. Analysis of the 16S rRNA gene sequence showed that strain 395-6.2T was phylogenetically related to Lactobacillus farciminis, Lactobacillus formosensis, Lactobacillus futsaii, Lactobacillus crustorum, Lactobacillus nuruki, Lactobacillus heilongjiangensis, Lactobacillus musae, Lactobacillus nantensis, Lactobacillus mindensis, Lactobacillus kimchiensis, Lactobacillus zhachilii, Lactobacillus alimentarius, Lactobacillus bobalius, Lactobacillus kimchii and Lactobacillus paralimentarius. Strain 395-6.2T exhibited 95.7-99.4 % 16S rRNA gene sequence similarities, 85.0-94.0 % pheS gene sequence similarities, 94.2-98.0 % rpoA gene sequence similarities to type strains of phylogenetically related species. ANI and isDDH values between strain 395-6.2T and type strains of phylogenetically related species were 77.9-87.1 % and 22.5-33.5 %, respectively. Based upon the data obtained in the present study, a novel species, Lactobacillus huachuanensis sp. nov., is proposed and the type strain is 395-6.2T (=CCM 8927T=NCIMB 15188T=LMG 31179T).

Enterococcus florum sp. nov., isolated from a cotton flower (Gossypium hirsutum L.).

A Gram-stain-positive and catalase negative coccus, designated strain Gos25-1T, isolated from a cotton flower (Gossypium hirsutum L.) collected from Khao Wong district, Kalasin province, Thailand. The taxonomic position of this strain was systematically studied based upon polyphasic taxonomic methods. The strain was facultatively anaerobic and produced l-lactic acid from glucose. The predominant cellular fatty acids were the straight-chain fatty acids C18 : 1ω9c and C16 : 0. According to 16S rRNA and phenylalanyl-tRNA synthase alpha subunit (pheS) gene sequence similarity, this strain was closely related to Enterococcus pallens NBRC 100697T, E. hermanniensis CIP 108559T, E. avium NBRC 100477T and E. raffinosus NBRC 100492T with 98.9-99.1 % and 77.0-82.0 % sequence similarities, respectively. Phylogenetic analysis indicated that strain Gos25-1T was clearly distinguished from closely related species of the genus Enterococcus. Draft genome of Gos25-1T had a size of 3.99 Mb which was contained 3788 coding sequences with in silico G+C content of 42.4 mol%. The ANIb and a digital DNA-DNA hybridisation (dDDH) values between strain Gos25-1T and the closest related species, E. pallens NBRC 100697T were 73.65 and 21.10 %, respectively. According to polyphasic characterisation, this strain represents a novel species of the genus Enterococcus, for which the name Enterococcus florum sp. nov. is proposed. The type strain is Gos25-1T (=CIP 110956T=LMG 29007T=NBRC 111461T=TISTR 2382T).

Lactobacillus suantsaicola sp. nov. and Lactobacillus suantsaiihabitans sp. nov., isolated from suan-tsai, a traditional fermented mustard green product of Taiwan.

Four Gram-stain-positive strains, R7T, R11, R19T and R27, were isolated from suan-tsai, a traditional fermented mustard green product of Taiwan. Cells were rod-shaped, non-motile, non-haemolytic, asporogenous, facultatively anaerobic, heterofermentative, and did not exhibit catalase and oxidase activities. Comparative analyses of 16S rRNA, pheS and rpoA gene sequences demonstrated that these novel strains were members of the genus Lactobacillus. 16S rRNA and the concatenated pheS and rpoA gene sequence similarities between strains R7T and R11, and strains R19T and R27 were very high (>99.8 % similarity), respectively. On the basis of 16S rRNA gene sequence similarities, the type strains of Lactobacillus paralimentarius (98.5 %), Lactobacillus kimchii (98.5 %), Lactobacillus alimentarius (98.1 %) and Lactobacillus bobalius (98.1 %) were the closest neighbours to strains R7T and R11, and the type strains of Lactobacillus brevis (98.9 %), Lactobacillus cerevisiae (98.4 %), Lactobacillus hammesii (98.4 %), Lactobacillus koreensis (98.4 %) and Lactobacillus yonginensis (98.0 %) were the closest neighbours to strains R19T and R27, respectively. The average nucleotide identity values of R7T and R19T with the closely related type strains were 78.9-80.1% and 75.7-80.5 %, respectively. The digital DNA-DNA hybridization values were 22.8-23.6% and 21.0-23.1 %, respectively. Phenotypic and genotypic test results demonstrated that these strains represent two novel species of the genus Lactobacillus, for which the name Lactobacillus suantsaicola sp. nov. (R7T=BCRC 81127T=NBRC 113530T) and Lactobacillus suantsaiihabitans sp. nov. (R19T=BCRC 81129T=NBRC 113532T) are proposed.

Lactobacillus jixianensis sp. nov., Lactobacillus baoqingensis sp. nov., Lactobacillus jiayinensis sp. nov., Lactobacillus zhaoyuanensis sp. nov., Lactobacillus lindianensis sp. nov., Lactobacillus huananensis sp. nov., Lactobacillus tangyuanensis sp. nov., Lactobacillus fuyuanensis sp. nov., Lactobacillus tongjiangensis sp. nov., Lactobacillus fujinensis sp. nov. and Lactobacillus mulengensis sp. nov., isolated from Chinese traditional pickle.

Thirty Gram-stain-positive bacterial strains were isolated from Chinese traditional pickle. The strains were characterized using a polyphasic taxonomic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester analysis, determination of DNA G+C content, determination of average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH), determination of average amino acid identity (AAI) and an analysis of phenotypic features. The data demonstrated that the 30 strains represented 11 novel species belonging to the genus Lactobacillus, strains 159-4T, 47-3T, 257-1T, 187-3T, 220-4T, 151-2BT, 137-3T, 244-4T, 218-10T, 218-6T and 112-3T were designated as the type strains. Strains 159-4T and 47-3T were related to the type strains of Lactobacillus porcinae, Lactobacillus manihotivorans, Lactobacillus nasuensis, Lactobacillus camelliae, Lactobacillus pantheris, Lactobacillus thailandensis, Lactobacillus sharpeae and Lactobacillus songhuajiangensis, having 92.0-98.9 % 16S rRNA gene sequence similarities, 58.1-84.7 % pheS gene sequence similarities and 61.4-90.3 % rpoA gene sequence similarities. Strains 257-1T and 187-3T were related to the type strains of Lactobacillus coryniformis, Lactobacillus iwatensis, Lactobacillus backii, Lactobacillus rennini and Lactobacillus bifermentans, having 93.5-99.3 % 16S rRNA gene sequence similarities, 67.7-81.8 % pheS gene sequence similarities and 77.0-96.2 % rpoA gene sequence similarities. Strains 220-4T, 151-2BT, 137-3T, 244-4T, 218-10T, 218-6T and 112-3T were closely related to the type strains of Lactobacillus paucivorans, Lactobacillus brevis, Lactobacillus hammesii, Lactobacillus senmaizukei, Lactobacillus parabrevis, Lactobacillus yonginensis, Lactobacillus koreensis and Lactobacillus cerevisiae, having 95.6-99.9 % 16S rRNA gene sequence similarities, less than 93.9 % pheS gene sequence similarities and 87.0-99.7 % rpoA gene sequence similarities. ANI, isDDH and AAI values between strains 159-4T, 47-3T, 257-1T, 187-3T, 220-4T, 151-2BT, 137-3T, 244-4T, 218-10T, 218-6T, 112-3T and type strains of phylogenetically related species were less than 92.7, 48.4 and 96.6 %, respectively, confirming that they represent 11 novel species within the genus Lactobacillus. Based upon the data of polyphasic characterization obtained in the present study, eleven novel species, Lactobacillusjixianensis sp. nov., Lactobacillusbaoqingensis sp. nov., Lactobacillusjiayinensis sp. nov., Lactobacilluszhaoyuanensis sp. nov., Lactobacilluslindianensis sp. nov., Lactobacillushuananensis sp. nov., Lactobacillustangyuanensis sp. nov., Lactobacillusfuyuanensis sp. nov., Lactobacillustongjiangensis sp. nov., Lactobacillusfujinensis sp. nov. and Lactobacillusmulengensis sp. nov., are proposed and the type strains are 159-4T (=NCIMB 15175T=CCM 8911T), 47-3T (=NCIMB 15165T=CCM 8903T=LMG 31064T), 257-1T (=NCIMB 15166T=CCM 8904T=LMG 31065T), 187-3T (=NCIMB 15172T =CCM 8910T), 220-4T (=NCIMB 15163T =CCM 8902T=KCTC 21136T), 151-2BT (=NCIMB 15164T=CCM 8913T=KCTC 21129T=LMG 31063T), 137-3T (=NCIMB 15170T=CCM 8907T=KCTC 21125T=LMG 31053T), 244-4T (=NCIMB 15168T=CCM 8906T=KCTC 21137T=LMG 31052T), 218-10T (=NCIMB 15167T=CCM 8905T=KCTC 21135T =LMG 31055T), 218-6T (=NCIMB 15171T=CCM 8908T=KCTC 21134T =LMG 31067T) and 112-3T (=NCIMB 15174T=CCM 8909T=KCTC 21123T=LMG 31049T), respectively.

Lactobacillus hulanensis sp. nov., isolated from suancai, a traditional Chinese pickle.

A Gram-stain-positive bacterial strain, ZW163T, isolated from suancai, a traditional pickle of Heilongjiang province in China, was characterized by a polyphasic approach, including phenotypic characterization, sequence analyses of the 16S rRNA gene and two housekeeping genes (pheS and rpoA), whole genome sequencing and cellular fatty acid composition. Strain ZW163T was phylogenetically related to Lactobacillus sharpeae, Lactobacillus songhuajiangensis, Lactobacillus pantheris and Lactobacillus thailandensis, having 93.8-98.7 % 16S rRNA gene sequence similarities, 76.5-87.1 % pheS gene sequence similarities and 74.3-95.7 % rpoA gene sequence similarities to the type strains of L. sharpeae, L. songhuajiangensis, L. pantheris and L. thailandensis, respectively. Average nucleotide identity and in silico DNA-DNA hybridization values between strain ZW163T and the type strains of L. sharpeae, L. songhuajiangensis, L. pantheris and L. thailandensis were less than 77.7 % and 23.6 % respectively. Based upon the data obtained in the present study, a novel species, Lactobacillushulanensis sp. nov., is proposed and the type strain is ZW163T (=NCIMB 15193T=CCM 8926T=CCTCC AB 2019015T).

Lactobacillus homohiochii is a later heterotypic synonym of Lactobacillus fructivorans.

The taxonomic relationship of Lactobacillus fructivorans and Lactobacillus homohiochii was re-evaluated. The type strains of these two species shared 100 % 16S rRNA gene sequence similarity, 100 % pheS gene sequence similarity, 100 % rpoA gene sequence similarity, a 99.8 % ANI (average nucleotide identity) value and a 99.1 % in silico DNA-DNA hybridization value, showing that they have the same taxonomic position. Lactobacillus homohiochii should be reclassified as Lactobacillus fructivorans. Lactobacillus homohiochii is a later heterotypic synonym of Lactobacillus fructivorans.

Lactobacillus terrae is a later heterotypic synonym of Lactobacillus metriopterae.

Lactobacillus metriopterae and Lactobacillus terrae are closely related, and they share 99.5 % 16S rRNA gene sequence similarity, 98.4 % pheS gene sequence similarity, 99.6 % rpoA gene sequence similarity, 97.9 % average nucleotide identity value and 81.3 % in silico DNA-DNA hybridization value, showing that they have the same taxonomic position. L. terrae should be reclassified as L. metriopterae. L. terrae is a later heterotypic synonym of L. metriopterae.

Lactobacillus bambusae sp. nov., isolated from traditional fermented ma bamboo shoots in Taiwan.

A Gram-stain-positive strain, BS-W1T, was isolated from a traditional fermented ma bamboo shoots (Dendrocalamus latiflorus Munro) product of Taiwan. It was rod-shaped, non-motile, non-haemolytic, asporogenous, facultatively anaerobic, heterofermentative and did not exhibit catalase or oxidase activities. Comparative analysis of 16S rRNA, pheS, rpoA and gyrB gene sequences demonstrated that the novel strain BS-W1T was a member of the genus Lactobacillus. On the basis of 16S RNA gene sequence similarity, the type strains of Lactobacillus oryzae (94.4 % similarity), Lactobacillus acidifarinae (93.8 %), Lactobacillus namurensis (93.7 %) and Lactobacillus zymae (93.7 %) were the closest neighbours to strain BS-W1T. The pheS, rpoA and gyrB gene sequence similarities of strain BS-W1T to closely related these species were less than 80.2 %. DNA-DNA reassociation values with these type strains were 21.0-33.8 %. The DNA G+C content was 46.6 mol%. The average nucleotide identity values between BS-W1T and the closest relatives were lower than 70 %. Phenotypic and genotypic features demonstrated that the strain represents a novel species of the genus Lactobacillus, for which the name Lactobacillus bambusae sp. nov. is proposed. The type strain is BS-W1T (=BCRC 80970T=NBRC 112377T).

Lactobacillus rodentium sp. nov., from the digestive tract of wild rodents

Three strains of regular, long, Gram-stain-positive bacterial rods were isolated using TPY, M.R.S. and Rogosa agar under anaerobic conditions from the digestive tract of wild mice (Mus musculus). All 16S rRNA gene sequences of these isolates were most similar to sequences of Lactobacillus gasseri ATCC 33323T and Lactobacillus johnsonii ATCC 33200T (97.3% and 97.2% sequence similarities, respectively). The novel strains shared 99.2-99.6% 16S rRNA gene sequence similarities. Type strains of L. gasseri and L. johnsonii were also most related to the newly isolated strains according to rpoA (83.9-84.0% similarities), pheS (84.6-87.8%), atpA (86.2-87.7%), hsp60 (89.4-90.4%) and tuf (92.7-93.6%) gene sequence similarities. Phylogenetic studies based on 16S rRNA, hsp60, rpoA, atpA and pheS gene sequences, other genotypic and many phenotypic characteristics (results of API 50 CHL, Rapid ID 32A and API ZYM biochemical tests; cellular fatty acid profiles; cellular polar lipid profiles; end products of glucose fermentation) showed that these bacterial strains represent a novel species within the genus Lactobacillus. The name Lactobacillus rodentium sp. nov. is proposed to accommodate this group of new isolates. The type strain is MYMRS/TLU1T (=DSM 24759T=CCM 7945T).

Enterococcus xiangfangensis sp. nov., isolated from Chinese pickle

A Gram-stain-positive bacterial strain, 11097(T), was isolated from traditional pickle in Heilongjiang Province, China. The bacterium was characterized using a polyphasic approach, including 16S rRNA gene sequence analysis, phenylalanyl-tRNA synthase (pheS) gene sequence analysis, RNA polymerase α subunit (rpoA) gene sequence analysis, fatty acid methyl ester (FAME) analysis, determination of DNA G+C content, DNA-DNA hybridization and an analysis of phenotypic features. Strain 11097(T) was phylogenetically related to Enterococcus devriesei, E. pseudoavium, E. viikkiensis, E. avium, E. malodoratus, E. gilvus and E. raffinosus. Strain 11097(T) had 99.1-99.9% 16S rRNA gene sequence similarities, 78.2-83.2% pheS gene sequence similarities and 93.8-96.6% rpoA gene sequence similarities with type strains of phylogenetically related species. Based upon polyphasic characterization data obtained in the present study, a novel species of the genus Enterococcus, Enterococcus xiangfangensis sp. nov., is proposed with the type strain 11097(T) ( = LMG 27495(T) = NCIMB 14834(T)).

Lactobacillus mudanjiangensis sp. nov., Lactobacillus songhuajiangensis sp. nov. and Lactobacillus nenjiangensis sp. nov., isolated from Chinese traditional pickle and sourdough

Three Gram-stain-positive bacterial strains, 11050(T), 7-19(T) and 11102(T), were isolated from traditional pickle and sourdough in Heilongjiang Province, China. These bacteria were characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, dnaK gene sequence analysis, fatty acid methyl ester (FAME) analysis, determination of DNA G+C content, DNA-DNA hybridization and an analysis of phenotypic features. Strain 11050(T) belonged to the Lactobacillus plantarum species group and shared 98.0-98.4 % 16S rRNA gene sequence similarities and 84.7-88.9 % dnaK gene sequence similarities with type strains of Lactobacillus plantarum subsp. plantarum, Lactobacillus plantarum subsp. argentoratensis, Lactobacillus pentosus, Lactobacillus paraplantarum, Lactobacillus fabifermentans and Lactobacillus xiangfangensis and had 75.9-80.7 % pheS gene sequence similarities and 90.7-92.5 % rpoA gene sequence similarities with Lactobacillus plantarum subsp. plantarum LMG 6907(T), Lactobacillus plantarum subsp. argentoratensis LMG 9205, Lactobacillus pentosus LMG 10755(T), Lactobacillus paraplantarum LMG 16673(T), Lactobacillus fabifermentans LMG 24284(T) and Lactobacillus xiangfangensis 3.1.1(T), respectively. Strain 7-19(T) was phylogenetically related to Lactobacillus thailandensis, Lactobacillus pantheris and Lactobacillus sharpeae, having 94.1-96.7 % 16S rRNA gene sequence similarities, 71.5-82.3 % pheS gene sequence similarities and 71.2-83.4 % rpoA gene sequence similarities with type strains of Lactobacillus thailandensis, Lactobacillus pantheris and Lactobacillus sharpeae, respectively. Strain 11102(T) was phylogenetically related to Lactobacillus oligofermentans, Lactobacillus suebicus, Lactobacillus vaccinostercus and Lactobacillus hokkaidonensis. Strain 11102(T) had 99.2 % 16S rRNA gene sequence similarity, 81.3 % pheS gene sequence similarity and 96.1 % rpoA gene sequence similarity with Lactobacillus oligofermentans LMG 22743(T), respectively. Strain 11102(T) shared 96.0-96.8 % 16S rRNA gene sequence similarities, 73.3-81.0 % pheS gene sequence similarities and 74.6-76.9 % rpoA gene sequence similarities with type strains of Lactobacillus suebicus, Lactobacillus vaccinostercus and Lactobacillus hokkaidonensis, respectively. Based upon the data from polyphasic characterization obtained in the present study, three novel species, Lactobacillus mudanjiangensis sp. nov., Lactobacillus songhuajiangensis sp. nov. and Lactobacillus nenjiangensis sp. nov., are proposed and the type strains are 11050(T) ( = LMG 27194(T) = CCUG 62991(T)), 7-19(T) ( = LMG 27191(T) = NCIMB 14832(T) = CCUG 62990(T)) and 11102(T) ( = LMG 27192(T) = NCIMB 14833(T)), respectively.

Leuconostoc mesenteroides subsp. suionicum subsp. nov.

Strains LMG 8159 and LMG 11499 were reclassified by a polyphasic approach, including 16S rRNA gene sequence analysis, 16S-23S rRNA intergenic spacer (IGS) sequence analysis, (GTG)(5)-PCR fingerprinting, RAPD fingerprinting, fatty acid methyl ester analysis and an analysis of phenotypic features using API 50 CH. The two strains were closely related to the type strains of the three defined subspecies of Leuconostoc mesenteroides, showing 99.7-99.9% 16S rRNA gene sequence similarity, 99.2% 16S-23S rRNA gene intergenic spacer sequence similarity, 97.1-97.4% pheS gene sequence similarity and 98.0-98.2% rpoA gene sequence similarity. Low atpA gene sequence similarity (91.4-91.7%), (GTG)(5)-PCR fingerprinting, RAPD fingerprinting, fatty acid compositions and phenotypic features allowed us to differentiate strains LMG 8159 and LMG 11499 from all established subspecies within L. mesenteroides. Based upon the data obtained in the present and previous studies, a novel subspecies is proposed within the species L. mesenteroides, Leuconostoc mesenteroides subsp. suionicum subsp. nov., with the type strain LMG 8159(T) (=ATCC 9135(T) =DSM 20241(T) =NCIMB 6992(T)).

Lactobacillus heilongjiangensis sp. nov., isolated from Chinese pickle

A Gram-stain-positive bacterial strain, S4-3(T), was isolated from traditional pickle in Heilongjiang Province, China. The bacterium was characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, dnaK gene sequence analysis, fatty acid methyl ester (FAME) analysis, determination of DNA G+C content, DNA-DNA hybridization and an analysis of phenotypic features. Strain S4-3(T) showed 97.9-98.7 % 16S rRNA gene sequence similarities, 84.4-94.1 % pheS gene sequence similarities and 94.4-96.9 % rpoA gene sequence similarities to the type strains of Lactobacillus nantensis, Lactobacillus mindensis, Lactobacillus crustorum, Lactobacillus futsaii, Lactobacillus farciminis and Lactobacillus kimchiensis. dnaK gene sequence similarities between S4-3(T) and Lactobacillus nantensis LMG 23510(T), Lactobacillus mindensis LMG 21932(T), Lactobacillus crustorum LMG 23699(T), Lactobacillus futsaii JCM 17355(T) and Lactobacillus farciminis LMG 9200(T) were 95.4, 91.5, 90.4, 91.7 and 93.1 %, respectively. Based upon the data obtained in the present study, a novel species, Lactobacillus heilongjiangensis sp. nov., is proposed and the type strain is S4-3(T) ( = LMG 26166(T) = NCIMB 14701(T)).

Lactobacillus sobrius Konstantinov et al. 2006 is a later synonym of Lactobacillus amylovorus Nakamura 1981

While studying the taxonomy of six lactic acid bacterium isolates from Finnish porcine intestine and faeces, the taxonomic positions of Lactobacillus sobrius type strain DSM 16698T and strain AD5 based on comparative 16S rRNA sequence analysis were found to be controversial, as they showed high similarity to Lactobacillus amylovorus strains. Therefore, the taxonomy of these species was addressed in a polyphasic taxonomy study that included, in addition to re-evaluating the 16S rRNA gene sequence and DNA-DNA reassociation results, multilocus sequence analysis (MLSA) of the housekeeping genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) as well as numerical analysis of HindIII and EcoRI ribotypes. 16S rRNA gene sequence analysis demonstrated a very high similarity between the L. sobrius and L. amylovorus type and reference strains and representative Finnish porcine isolates (99.6-99.9 %). The MLSA data showed the close phylogenetic relationship of these strains; pheS and rpoA gene sequence similarities were 98.5-100 % and 99.6-99.8 %, respectively. Numerical analyses of HindIII/EcoRI ribotypes placed these strains in a single cluster by both enzymes. Finally, the DNA-DNA reassociation experiments revealed high reassociation levels (higher than 79 %) between the strains. These results indicate that DSM 16698T, AD5 and the related porcine lactobacilli strains from Finland constitute a single species, Lactobacillus amylovorus, and that the name Lactobacillus sobrius should be considered as a later synonym of Lactobacillus amylovorus.