RESULTS:

Obligately anaerobic Gram-stain-positive small-chain coccobacilli strains 12EGH17T and 18CBH55 were isolated from faecal samples of healthy Japanese humans. Strain 12EGH17T showed the highest 16S rRNA gene sequence similarity to Sellimonas intestinalis BR72T (95.5 %) Coprococcus comes ATCC 27758T (94.4 %) and Clostridium nexile DSM 1787T (93.7 %). The percentage of conserved proteins values between the genome of strain 12EGH17T and that of the members of the genus Sellimonas were >54 % suggesting that strain 12EGH17T belongs to the genus Sellimonas . The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strains 12EGH17T and 18CBH55 were higher than the cut-off values of species demarcation (90 % dDDH and 99 % ANI) indicating these two strains are the same species. However the dDDH and ANI values of these strains were lower than the cut-off values of species demarcation against other strains (<30 % dDDH and <79 % ANI). These results indicate that the isolates should be considered to represent a new species of the genus Sellimonas . The isolates were differentiated from the type species S. intestinalis by the ability of aesculin hydrolysis. Based on the collected data strains 12EGH17T and 18CBH55 represent a novel species in the genus Sellimonas for which the name Sellimonas catena sp. nov. is proposed. The type strain of S. catena is 12EGH17T (=JCM 35622T=DSM 114916T).

Two obligately anaerobic Gram-stain-positive rod-shaped bacteria were isolated from faecal samples of healthy humans in Japan. 16S rRNA gene sequence analysis indicated that these two strains (8CFCBH1T and 9CBH6) belonged to the genus Adlercreutzia which is known as an equol-producing bacterium. The closest neighbours of strain 8CFCBH1T were Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450T (98.6%) Adlercreutzia equolifaciens subsp. celatus do03T (98.4%) Adlercreutzia muris WCA-131-CoC-2T (96.6%) Parvibacter caecicola NR06T (96.4%) Adlercreutzia caecimuris B7T (95.3%) and Adlercreutzia mucosicola Mt1B8T (95.3%). The closest relatives to strain 9CBH6 were A. equolifaciens subsp. equolifaciens DSM 19450T (99.8%) A. equolifaciens subsp. celatus do03T (99.6%) and A. muris WCA-131-CoC-2T (96.8%). Strain 8CFCBH1T showed 22.3–53.5% digital DNA–DNA hybridization (dDDH) values with its related species. In addition the average nucleotide identity (ANI) values between strain 8CFCBH1T and its related species ranged from 75.4 to 93.3%. On the other hand strain 9CBH6 was considered as A. equolifaciens based on the dDDH and ANI values (>70% dDDH and >95–96% ANI). Strain 9CBH6 showed daidzein-converting activity as expected from the result of genome analysis. The genome of strain 8CFCBH1T lacked four genes involved in equol production. Growing cells of strain 8CFCBH1T were not capable of converting daidzein. Based on the collected data strain 8CFCBH1T represents a novel species in the genus Adlercreutzia for which the name Adlercreutzia hattorii sp. nov. is proposed. The type strain of A. hattorii is 8CFCBH1T (=JCM 34083T=DSM 112284T).

Faecalibacterium prausnitzii is one of the most important butyrate-producing bacteria in the human gut. Previous studies have suggested the presence of several phylogenetic groups with differences at the species level in the species and a taxonomic re-evaluation is thus essential for further understanding of ecology of the important human symbiont. Here we examine the phenotypic physiological chemotaxonomic and phylogenomic characteristics of six F. prausnitzii strains (BCRC 81047T=ATCC 27768T A2-165T=JCM 31915T APC918/95b=JCM 39207 APC942/30−2=JCM 39208 APC924/119=JCM 39209 and APC922/41−1T=JCM 39210T) deposited in public culture collections with two reference strains of Faecalibacterium butyricigenerans JCM 39212T and Faecalibacterium longum JCM 39211T. Faecalibacterium sp. JCM 17207T isolated from caecum of broiler chicken was also included. Three strains of F. prausnitzii (BCRC 81047T JCM 39207 and JCM 39209) shared more than 96.6 % average nucleotide identity (ANI) and 69.6 % digital DNA–DNA hybridization (dDDH) values indicating that the three strains are members of the same species. On the other hand the remaining three strains of F. prausnitzii (JCM 31915T JCM 39208 and JCM 39210T) were clearly separated from the above three strains based on the ANI and dDDH values. Rather JCM 39208 showed ANI and dDDH values over the cut-off values of species discrimination (>70 % dDDH and >95–96 % ANI) with F. longum JCM 39211T whereas JCM 31915T JCM 39210T and JCM 17207T did not share dDDH and ANI values over the currently accepted cut-off values with any of the tested strains including among them. Furthermore the cellular fatty acid patterns of these strains were slightly different from other F. prausnitzii strains. Based on the collected data F. prausnitzii JCM 31915T F. prausnitzii JCM 39210T and Faecalibacterium sp. JCM 17207T represent three novel species of the genus Faecalibacterium for which the names Faecalibacterium duncaniae sp. nov. (type strain JCM 31915T=DSM 17677T=A2-165T) Faecalibacterium hattorii sp. nov. (type strain JCM 39210T=DSM 107841T=APC922/41-1T) and Faecalibacterium gallinarum sp. nov. (type strain JCM 17207T=DSM 23680T=ic1379T) are proposed.

Obligately anaerobic Gram-stain-positive bacilli strains 12BBH14T 9CFEGH4 and 10CPCBH12 were isolated from faecal samples of healthy Japanese people. Strain 12BBH14T showed the highest 16S rRNA gene sequence similarity to Sellimonas monacensis Cla-CZ-80T (97.5 %) and ‘Lachnoclostridium phocaeense’ Marseille-P3177T (97.2 %). Strain 12BBH14T was also closely related to Eubacterium sp. c-25 with 99.7 % 16S rRNA gene sequence similarity. The 16S rRNA gene sequence analysis showed that strains 12BBH14T 9CFEGH4 and 10CPCBH12 formed a monophyletic cluster with Eubacterium sp. c-25. Near this monophyletic cluster S. monacensis Cla-CZ-80T and ‘L. phocaeense’ Marseille-P3177T formed a cluster and did not form a cluster with other Sellimonas species. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strains 12BBH14T 9CFEGH4 10CPCBH12 and Eubacterium sp. c-25 were higher than the cut-off values of species demarcation (>88 % dDDH and >98 % ANI) indicating that these four strains are the same species. On the other hand the dDDH and ANI values of these strains were lower than the cut-off values of species demarcation against other strains (<29 % dDDH and <76 % ANI). Moreover the average amino acid identity values among these strains were higher than the genus boundary. These results indicate that the isolates should be considered to belong to a new genus of the family Lachnospiraceae . Based on the collected data strains 12BBH14T 9CFEGH4 and 10CPCBH12 represent a novel species of a novel genus for which the name Claveliimonas bilis gen. nov. sp. nov. is proposed. The type strain of C. bilis is 12BBH14T (=JCM 35899T=DSM 115701T). Eubacterium sp. c-25 belongs to C. bilis. In addition S. monacensis is transferred to the genus Claveliimonas as Claveliimonas monacensis comb. nov.

A strain of the recently validated species Faecalibacterium hominis shares 99.0 % 16S rRNA gene sequence similarity with the type strain of Faecalibacterium duncaniae . The aim of this study was to evaluate the taxonomic relationship between F. hominis and F. duncaniae. F. duncaniae JCM 31915T showed 73.0 % digital DNA–DNA hybridization (dDDH) value with F. hominis JCM 39347T. The average nucleotide identity (ANI) value between these two strains was 96.7 %. These results indicate that F. duncaniae JCM 31915T and F. hominis JCM 39347T represent members of the same species. Based on these data we propose Faecalibacterium hominis as a later heterotypic synonym of Faecalibacterium duncaniae . An emended description is provided.

A rod-shaped Gram-stain-negative strictly anaerobic catalase-negative and endospore-forming bacterial strain CSC2T was isolated from corn silage preserved in Tochigi Japan. The strain CSC2T grew at 15–40 °C at pH 5.0–7.7 and with up to 0.5 % (w/v) NaCl. The main cellular fatty acids were C14 : 0 C16 : 0 and C16 : 0 dimethyl acetal. The cellular polar lipids detected were diphosphatidylglycerol phosphatidylglycerol phosphatidylethanolamine phosphatidic acid lysophosphatidylethanolamine phosphatidylserine lysophosphatidylcholine and two unidentified polar lipids. Phylogenetic analysis of the 16S rRNA gene showed that strain CSC2T was a member of the genus Clostridium and closely related to Clostridium polyendosporum DSM 57272T (95.6 % gene sequence similarity) and Clostridium fallax ATCC 19400T (95.3 %). The genomic DNA G+C content of strain CSC2T was 31.1 mol% (whole genome analysis). The average nucleotide identity based on blast and digital DNA–DNA hybridization values between strain CSC2T and the type strains of phylogenetically related species were below 71 and 24 % respectively. On the basis of the genotypic phenotypic and chemotaxonomic characteristics it is proposed to designate strain CSC2T as representing Clostridium zeae sp. nov. The type strain is CSC2T (=MAFF212476T=JCM 33766T=DSM 111242T).

A taxonomic study of a Gram-stain-positive rod-shaped non-motile non-spore-forming catalase-negative bacterium strain YK43T isolated from spent mushroom substrates stored in Nagano Japan was performed. Growth was detected at 15–45 °C pH 5.0–8.5 and 0–10 % (w/v) NaCl. The genomic DNA G+C content of strain YK43T was 43.6 mol%. The predominant fatty acids were C16 : 0 C18 : 1 ω9c and summed feature 8. Based on 16S rRNA gene sequence analysis the type strains of Lactobacillus acidipiscis (sequence similarity 97.6 %) and Lactobacillus pobuzihii (97.4 %) were most closely related to YK43T. The average nucleotide identities were 74.1 % between strain YK43T and L. acidipiscis DSM 15836T and 74.0 % between YK43T and L. pobuzihii E100301T. Based on a multilocus sequence analysis comparative genomic analysis and a range of phenotypic and chemotaxonomic characteristics strain YK43T represents a novel species of the genus Lactobacillus for which the name Lactobacillus salitolerans sp. nov. is proposed. The type strain is YK43T (=JCM 31331T = DSM 103433T).

The taxonomic status of the species Clostridium methoxybenzovorans was assessed. The 16S rRNA gene sequence whole-genome sequence and phenotypic characterizations suggested that the type strain deposited in the American Type Culture Collection ( C. methoxybenzovorans ATCC 700855T) is a member of the species Eubacterium callanderi . Hence C. methoxybenzovorans ATCC 700855T cannot be used as a reference for taxonomic study. The type strain deposited in the German Collection of Microorganism and Cell Cultures GmbH (DSM 12182T) is no longer listed in its online catalogue. Also both the 16S rRNA gene and the whole-genome sequences of the original strain SR3T showed high sequence identity with those of Lacrimispora indolis (recently reclassified from Clostridium indolis ) as the most closely related species. Analysis of the two genomes showed average nucleotide identity based on blast and digital DNA–DNA hybridization values of 98.3 and 87.9 % respectively. Based on these results C. methoxybenzovorans SR3T was considered to be a member of L. indolis .

In this study we isolated a novel strain of lactic acid bacteria AF129T from alfalfa silage prepared locally in Morioka Iwate Japan. Polyphasic taxonomy was used to characterize the bacterial strain. The bacterium was rod-shaped Gram-stain-positive non-spore-forming and catalase-negative. The strain grew at various temperatures (15–40°C) and pH levels (4.0–8.0). The optimum growth conditions were a temperature of 30°C and a pH of 6.0. AF129T exhibited growth at salt (NaCl) concentrations of up to 6.5 % (w/v). The G+C content of the strain’s genomic DNA was 41.5 %. The major fatty acids were C16 : 0 C18 : 1ω9c C19 : 0cyclo ω8c and summed feature 8. 16S rRNA gene sequencing revealed that AF129T represents a member of the genus Ligilactobacillus and it has higher sequence similarities with Ligilactobacillus pobuzihii (98.4 %) Ligilactobacillus acidipiscis (97.5 %) and Ligilactobacillus salitolerans (97.4 %). The digital DNA–DNA hybridization values for AF129T and phylogenetically related species of the genus Ligilactobacillus ranged from 19.8% to 24.1%. The average nucleotide identity of the strain with its closely related taxa was lower than the threshold (95 %–96 %) used for species differentiation. In the light of the above-mentioned physiological genotypic chemotaxonomic and phylogenetic evidence we confirm that AF129T represents a member of the genus Ligilactobacillus and constitutes a novel species; we propose the name Ligilactobacillus pabuli sp. nov. for this species. The type strain is AF129T =MAFF 518002T =JCM 34518T=BCRC 81335T.

Two Gram-stain-positive rod-shaped non-motile non-spore-forming catalase-negative bacteria designated strains SG162T and NK01 were isolated from Japanese rice grain silage and total mixed ration silage respectively. They were initially identified as Lactobacillus buchneri based on the 16S rRNA gene sequence similarities. However the two strains were separated into a distinct clade from L. buchneri DSM 20057T (=JCM 1115T) through whole-genome sequence-based characterization forming an infraspecific subgroup together with strains CD034 and S42 whose genomic sequences were available in the public sequence database. Strains within the subgroup shared 99.4–99.7 % average nucleotide identity (ANI) and 97.5–99.0 % digital DNA–DNA hybridization (dDDH) with each other albeit 96.9–97.0 % ANI and 76.0–76.6 % dDDH against DSM 20057T. Strains SG162T and NK01 could utilize more substrates as sole carbon sources than DSM 20057T potentially owing to the abundance of genes involved in carbon metabolism especially the Entner–Doudoroff pathway. The inability of γ-aminobutyric acid (GABA) production was evidenced by the lack of glutamate decarboxylase and glutamate/GABA antiporter genes in the new subgroup strains. Strain SG162T grew at 10–45 °C (optimum 30 °C) pH 3.5–8.0 and 0–8 % (w/v) NaCl. Its genomic DNA G+C content was 44.1 mol%. The predominant fatty acids were C16 : 0 C19 : 0 cyclo ω8c and summed feature 8. On the basis of the polyphasic characterization findings strains SG162T and NK01 represent a novel subspecies of L. buchneri for which the name Lactobacillus buchneri subsp. silagei subsp. nov. is proposed. The type strain is SG162T (=JCM 32599T=DSM 107969T) and strains CD034 and S42 are also transferred to L. buchneri subsp. silagei.