rpoA Gene Sequences Research Articles

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 suantsaii sp. nov., isolated from suan-tsai, a traditional Taiwanese fermented mustard green.

A Gram-stain-positive, rod-shaped, non-motile, catalase-negative and facultative anaerobic strain, L88T, was isolated from suan-tsai, a traditional Taiwanese fermented mustard green. Comparative analyses of 16S rRNA, pheS and rpoA gene sequences demonstrated that strain L88Twas a member of the genus Lactobacillus. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus acidifarinae (98.2 % similarity), Lactobacillus namurensis (98.1 %), Lactobacillus zymae (98.1 %) and Lactobacillus spicheri (96.8 %) were the closest neighbours to this novel strain. The average nucleotide identity, digital DNA‒DNA hybridization and average amino acid identity values between L88T and its closest relatives were lower than 80, 30 and 90 %, respectively. Phenotypic and genotypic test results demonstrated that strain L88T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus suantsaii sp. nov., is proposed. The type strain is L88T (=BCRC 12945T=NBRC 113535T).

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 metriopterae sp. nov., a novel lactic acid bacterium isolated from the gut of grasshopper Metrioptera engelhardti.

A strain (Hime 5-1T) of lactic acid bacterium was isolated from the gut of the grasshopper Metrioptera engelhardti from a mountainous area of Nagano Prefecture, Japan. Strain Hime 5-1T had a low 16S rRNA gene sequence similarity to known lactic acid bacteria, with the closest recognized relatives being Lactobacillus tucceti (96.7 %), Lactobacillus furfuricola (96.5 %), Lactobacillus versmoldensis (96.3 %) and Lactobacillus nodensis (96.1 %). Comparative analyses of the rpoA and pheS gene sequences indicated that Hime 5-1T is not closely related to other Lactobacillus species. Strain Hime 5-1T is a Gram-stain-positive, catalase-negative and homofermentative bacterium with yellowish colonies, which contrasts with the whitish colonies of its closest recognized relatives. Based on phenotypic and genotypic properties, we conclude that the isolated bacterium represents a novel species of the genus Lactobacillus, for which the name Lactobacillus metriopterae sp. nov. is proposed. The type strain is Hime 5-1T (=JCM 31635T=DSM 103730T). 16S rRNA gene based high-throughput sequencing revealed that L. metriopterae is the dominant microbiota in the gut of Metrioptera engelhardti.

Enterococcus crotali sp. nov., isolated from faecal material of a timber rattlesnake.

A facultatively anaerobic, Gram-stain-positive bacterium, designated ETRF1T, was found in faecal material of a timber rattlesnake (Crotalus horridus). Based on a comparative 16S rRNA gene sequence analysis, the isolate was assigned to the genus Enterococcus. The 16S rRNA gene sequence of strain ETRF1T showed >97 % similarity to that of the type strains of Enterococcus rotai, E. caccae, E. silesiacus, E haemoperoxidus, E. ureasiticus, E. moraviensis, E. plantarum, E. quebecensis, E. ureilyticus, E. termitis, E. rivorum and E. faecalis. The organism could be distinguished from these 12 phylogenetically related enterococci using conventional biochemical testing, the Rapid ID32 Strep system, comparative pheS and rpoA gene sequence analysis, and comparative whole genome sequence analysis. The estimated in silico DNA-DNA hybridization values were <70 %, and average nucleotide identity values were <96 %, when compared to these 12 species, further validating that ETRF1T represents a unique species within the genus Enterococcus. On the basis of these analyses, strain ETRF1T (=CCUG 65857T=LMG 28312T) is proposed as the type strain of a novel species, Enterococcus crotali sp. nov.

Isolation of Vibrio tapetis from two native fish species (Genypterus chilensis and Paralichthys adspersus) reared in Chile and description of Vibrio tapetis subsp. quintayensis subsp. nov.

A group of seven Chilean isolates presumptively belonging to Vibrio tapetis was isolated from diseased fine flounders (Paralichthys adspersus) and red conger eel (Genypterus chilensis) experimentally reared in Quintay (Chile). All isolates were confirmed as members of V. tapetis on the basis of matrix-assisted laser desorption ionization time-of-flight MS, 16S rRNA gene sequencing, DNA-DNA hybridization values and G+C content. The ERIC-PCR and REP-PCR patterns were homogeneous among those isolates recovered from the same host (red conger or fine flounders), but distinct from the type strains V. tapetis subsp. tapetis CECT 4600T and V. tapetis subsp. britannicus CECT 8161T. On the basis of atpA, rpoA, rpoD, recA and pyrH gene sequence similarities (99.7-100 %) and clustering in the phylogenetic trees, the red conger isolates (Q20, Q047, Q48 and Q50) were confirmed as representing V. tapetis subsp. tapetis. However, they differed from V. tapetis subsp. tapetis CECT 4600T in their lipase, alpha quimiotripsin and non-acid phosphatase production. On the other hand, the fine flounder isolates (QL-9T, QL-35 and QL-41) showed rpoD, recA and pyrH gene sequence similarities ranging from 91.6 to 97.7 % with the type strains of the two V. tapetis subspecies (CECT 4600T and CECT 8161T) and consistently clustered together as an independent phylogenetic line within V. tapetis. Moreover, they could be differentiated phenotypically from strains CECT 4600T and CECT 8161T by nine and three different biochemical tests, respectively. In conclusion, the presence of V. tapetis in diseased red conger eel and fine flounder was demonstrated, extending the known host range and geographical location for this pathogen. Furthermore, this study demonstrates that the three isolates from fine flounder represent a novel subdivision within V. tapetis, for which the name V. tapetis subsp. quintayensis subsp. nov. is proposed and with QL-9T (=CECT 8851T=LMG 28759T) as the type strain. Although QL-9T was isolated from kidney of diseased fine flounder specimens, the challenge assays showed that it was non-pathogenic for this species.

Diversity of Lactic Acid Bacteria Associated with Banana Fruits in Taiwan.

Banana is a popular fruit worldwide. The lactic acid bacteria (LAB) microflora in banana fruits has not been studied in detail. A total of 164 LAB were isolated from banana fruits in Taiwan. These isolates were initially divided into nine groups (r1 to r9) using restriction fragment length polymorphism analysis and 16S ribosomal DNA sequencing. Isolates belonging to Lactobacillus plantarum group were further divided into three additional groups using multiplex PCR assay targeting the recA gene. The most common bacterial genera found in banana fruits were Lactobacillus and Weissella. The distribution of LAB indicated that, in most cases, neighboring regions shared common strains, but there were still some differences between regions. On the basis of phylogenetic analysis of 16S rRNA, rpoA, and pheS gene sequences, two strains included in the genera Lactobacillus were identified as potential novel species or subspecies. In addition, a total 36 isolates were found to have bacteriocin-producing abilities. These results suggest that various LAB are associated with banana fruits in Taiwan. This is the first report describing the distribution and varieties of LAB associated with banana fruits. In addition, one potential novel LAB species was also found in this study.

Enterococcus Xinjiangensis sp. nov., Isolated from Yogurt of Xinjiang, China.

A Gram-strain-positive bacterial strain 48(T) was isolated from traditional yogurt in Xinjiang Province, China. The bacterium was characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, polymerase α subunit (rpoA) gene sequence analysis, determination of DNA G+C content, DNA-DNA hybridization with the type strain of Enterococcus ratti and analysis of phenotypic features. Strain 48(T) accounted for 96.1, 95.8, 95.8, and 95.7% with Enterococcus faecium CGMCC 1.2136(T), Enterococcus hirae ATCC 9790(T), Enterococcus durans CECT 411(T), and E. ratti ATCC 700914(T) in the 16S rRNA gene sequence similarities, respectively. The sequence of rpoA gene showed similarities of 99.0, 96.0, 96.0, and 96% with that of E. faecium ATCC 19434(T), Enterococcus villorum LMG12287, E. hirae ATCC 9790(T), and E. durans ATCC 19432(T), respectively. Based upon of polyphasic characterization data obtained in the study, a novel species, Enterococcus xinjiangensis sp. nov., was proposed and the type strain was 48(T)(=CCTCC AB 2014041(T)=JCM 30200(T)).

Lactobacillus plajomi sp. nov. and Lactobacillus modestisalitolerans sp. nov., isolated from traditional fermented foods.

Three Lactobacillus-like strains, NB53T, NB446T and NB702, were isolated from traditional fermented food in Thailand. Comparative 16S rRNA gene sequence analysis indicated that these strains belong to the Lactobacillus plantarum group. Phylogenetic analysis based on the dnaK, rpoA, pheS and recA gene sequences indicated that these three strains were distantly related to known species present in the L. plantarum group. DNA-DNA hybridization with closely related strains demonstrated that these strains represented two novel species; the novel strains could be differentiated based on chemotaxonomic and phenotypic characteristics. Therefore, two novel species of the genus Lactobacillus, Lactobacillus plajomi sp. nov. (NB53T) and Lactobacillus modestisalitolerans sp. nov. (NB446T and NB702), are proposed with the type strains NB53T ( = NBRC 107333T = BCC 38054T) and NB446T ( = NBRC 107235T = BCC 38191T), respectively.

Lactobacillus mixtipabuli sp. nov. isolated from total mixed ration silage.

Using a polyphasic taxonomic approach, we investigated three bacterial strains - IWT30T, IWT8 and IWT75 - isolated from total mixed ration silage prepared in Hachimantai, Iwate, Japan. The isolates comprised Gram-stain positive, non-motile, non-spore-forming, catalase-negative, rod-shaped bacteria. Good growth occurred at 15-45 °C and at pH 4.0-7.5. Their major cellular fatty acids were C18:1ω9c and C19:1 cyclo 9,10.The G+C content of genomic DNA of strain IWT30T was 44.6 mol%. Comparative 16S rRNA gene sequence analysis showed that these novel strains belonged to the genus Lactobacillus. These strains shared 100 % 16S rRNA gene sequence similarity and were most closely related to the type strains of Lactobacillus silagei, Lactobacillus odoratitofui, Lactobacillus similis, Lactobacillus collinoides, Lactobacillus paracollinoides and Lactobacillus kimchicus, with sequence similarity values of 99.5, 98.8, 98.7, 97.8, 97.8 and 96.8 %, respectively. The level of DNA-DNA relatedness between these strains and their closest phylogenetic neighbours was less than 30 %. On the basis of additional phylogenetic analysis of pheS and rpoA gene sequences and phenotypic and chemotaxonomic characteristics, we conclude that these three strains represent a novel species of the genus Lactobacillus, for which we propose the name Lactobacillus mixtipabuli sp. nov. The type strain is IWT30T ( = JCM 19805T = DSM 28580T).

Vibrio olivae sp. nov., isolated from Spanish-style green-olive fermentations.

Three isolates originating from Spanish-style green-olive fermentations in a manufacturing company in the province of Seville, Spain, were taxonomically characterized by a polyphasic approach. This included a phylogenetic analysis based on 16S rRNA gene sequences and multi-locus sequence analysis (MLSA) based on pyrH, recA, rpoA, gyrB and mreB genes. The isolates shared 98.0 % 16S rRNA gene sequence similarity with Vibrio xiamenensis G21T. Phylogenetic analysis based on 16S rRNA gene sequences using the neighbour-joining and maximum-likelihood methods showed that the isolates fell within the genus Vibrio and formed an independent branch close to V. xiamenensis G21T. The maximum-parsimony method grouped the isolates to V. xiamenensis G21T but forming two clearly separated branches. Phylogenetic trees based on individual pyrH, recA, rpoA, gyrB and mreB gene sequences revealed that strain IGJ1.11T formed a clade alone or with V. xiamenensis G21T. Sequence similarities of the pyrH, recA, rpoA, gyrB and mreB genes between strain IGJ1.11T and V. xiamenensis G21T were 86.7, 85.7, 97.3, 87.6 and 84.8 %, respectively. MLSA of concatenated sequences showed that strain IGJ1.11T and V. xiamenensis G21T are two clearly separated species that form a clade, which we named Clade Xiamenensis, that presented 89.7 % concatenated gene sequence similarity, i.e. less than 92 %. The major cellular fatty acids (>5 %) of strain IGJ1.11T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). Enzymic activity profiles, sugar fermentation patterns and DNA G+C content (52.9 mol%) differentiated the novel strains from the closest related members of the genus Vibrio. The name Vibrio olivae sp. nov. is proposed for the novel species. The type strain is IGJ1.11T ( = CECT 8064T = DSM 25438T).

Photobacterium sanctipaulisp. nov. isolated from bleachedMadracis decactis(Scleractinia) in the St Peter &amp; St Paul Archipelago, Mid-Atlantic Ridge, Brazil

Five novel strains of Photobacterium (A-394T, A-373, A-379, A-397 and A-398) were isolated from bleached coral Madracis decactis (scleractinian) in the remote St Peter & St Archipelago (SPSPA), Mid-Atlantic Ridge, Brazil. Healthy M. decactis specimens were also surveyed, but no strains were related to them. The novel isolates formed a distinct lineage based on the 16S rRNA, recA, and rpoA gene sequences analysis. Their closest phylogenetic neighbours were Photobacterium rosenbergii, P. gaetbulicola, and P. lutimaris, sharing 96.6 to 95.8% 16S rRNA gene sequence similarity. The novel species can be differentiated from the closest neighbours by several phenotypic and chemotaxonomic markers. It grows at pH 11, produces tryptophane deaminase, presents the fatty acid C18:0, but lacks C16:0 iso. The whole cell protein profile, based in MALDI-TOF MS, distinguished the strains of the novel species among each other and from the closest neighbors. In addition, we are releasing the whole genome sequence of the type strain. The name Photobacterium sanctipauli sp. nov. is proposed for this taxon. The G + C content of the type strain A-394T (= LMG27910T = CAIM1892T) is 48.2 mol%.

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.

Pseudocitrobacter gen. nov., a novel genus of the Enterobacteriaceae with two new species Pseudocitrobacter faecalis sp. nov., and Pseudocitrobacter anthropi sp. nov, isolated from fecal samples from hospitalized patients in Pakistan

Four isolates of Gram-negative facultatively anaerobic bacteria, three of them producing NDM-1 carbapenemase, were isolated from hospitalized patients and outpatients attending two military hospitals in Rawalpindi, Pakistan, and studied for their taxonomic position. Initially the strains were phenotypically identified as Citrobacter species. Comparative analysis of 16S rRNA gene sequences then showed that the four strains shared >97%, but in no case >98.3%, 16S rRNA gene sequence similarities to members of the genera Citrobacter, Kluyvera, Pantoea, Enterobacter and Raoultella, but always formed a separate cluster in respective phylogenetic trees. Based on multilocus sequence analysis (MLSA) including partial recN, rpoA, thdF and rpoB gene sequence and respective amino acid sequence analysis it turned out that the strains also here always formed separate clusters. Based on further comparative analyses including DNA–DNA hybridizations, genomic fingerprint analysis using rep- and RAPD-PCRs and physiological tests, it is proposed to classify these four strains into the novel genus Pseudocitrobacter gen. nov. with a new species Pseudocitrobacter faecalis sp. nov. with strain 25 CITT (=CCM 8479T=LMG 27751T) and Pseudocitrobacter anthropi sp. nov. with strain C138T (=CCM 8478T=LMG 27750T), as the type strains, respectively.

Description of Lactobacillus iwatensis sp. nov., isolated from orchardgrass (Dactylis glomerata L.) silage, and Lactobacillus backii sp. nov.

Two bacterial strains, designated IWT246(T) and IWT248, were isolated from orchardgrass (Dactylis glomerata L.) silage from Iwate prefecture, Japan, and examined for a taxonomic study. Both organisms were rod-shaped, Gram-stain-positive, catalase-negative, facultatively anaerobic and homofermentative. The cell wall did not contain meso-diaminopimelic acid and the major fatty acids were C18 : 1ω9c and C19 cyclo 9,10/:1. Comparative analyses of 16S rRNA, pheS and rpoA gene sequences revealed that these strains were novel and belonged to the genus Lactobacillus. Based on 16S rRNA gene sequence similarity, the isolates were most closely related to the type strains of the following members of the genus Lactobacillus: Lactobacillus coryniformis subsp. coryniformis (96.7 % similarity), L. coryniformis subsp. torquens (96.6 %), L. bifermentans (95.5 %) and L. rennini (94.1 %). However, the 16S rRNA gene sequences of both IWT246(T) and IWT248 were 99.7 % similar to that of 'Lactobacillus backi' JCM 18665; this name has not been validly published. Genotypic, phenotypic and chemotaxonomic analyses confirmed that these novel strains occupy a unique taxonomic position. DNA-DNA hybridization experiments demonstrated genotypic separation of the novel isolates from related Lactobacillus species. The name Lactobacillus iwatensis sp. nov. is proposed for the novel isolates, with strain IWT246(T) ( = JCM 18838(T) = DSM 26942(T)) as the type strain. Our results also suggest that 'L. backi' does represent a novel Lactobacillus species. The cells did not contain meso-diaminopimelic acid in their cell-wall peptidoglycan and the major fatty acids were C16 : 0, C19 cyclo 9,10/:1 and summed feature 10 (one or more of C18 : 1ω11c, C18 : 1ω9t, C18 : 1ω6t and unknown ECL 17.834). We therefore propose the corrected name Lactobacillus backii sp. nov., with the type strain JCM 18665(T) ( = LMG 23555(T) = DSM 18080(T) = L1062(T)).

Isolation and characterization of lactic acid bacteria from Yan-tsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan

To isolate, characterize and identify lactic acid bacteria (LAB) in yan-tsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan. A total of 226 LAB were isolated; 111 cultures were isolated from samples collected from seven different markets and 115 from six fresh broccoli samples. These isolates were characterized phenotypically and then initially divided into nine groups (r1 to r9) using restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Some isolates were further divided into four additional groups by other genetic analysis. The most common bacterial genera in yan-tsai-shin and fresh broccoli were Weissella, Lactococcus and Lactobacillus. Regional similarities in the LAB, with differences in diversity, were observed in this study. On the basis of phylogenetic analysis of 16S rRNA, rpoA, rpoB and pheS gene sequences, two strains were included in the genera Enterococcus and Lactococcus, respectively, and identified as potential novel species or subspecies. In addition, the novel enterococcal strain, and 33 L.lactis subsp. lactis and two Weissella cibaria strains were found to have bacteriocin-like inhibitory substance (BLIS) producing abilities. These results suggest that the LAB play important roles in the fermentation of yan-tsai-shin. This is the first report describing the distribution and varieties of LAB existing in yan-tsai-shin and fresh broccoli. In addition, two potential novel LAB species or subspecies and one potential novel BLIS were also found in this study.