RESULTS:

A Gram-stain-negative endospore-forming rod-shaped indole-producing bacterial strain designated YZC6T was isolated from fermented cabbage. Strain YZC6T grew at 10–37  °C pH 5.5–8.5 and with up to 2  % (w/v) NaCl. The major cellular fatty acids were C16 : 0 and C18 : 1 cis 11 dimethyl acetal. Phylogenetic analysis of the 16S rRNA gene revealed that strain YZC6T belonged to the genus Lacrimispora and was closely related to Lacrimispora aerotolerans DSM 5434T (98.3  % sequence similarity) Lacrimispora saccharolytica WM1T (98.1  %) and Lacrimispora algidixylanolytica SPL73T (98.1  %). The average nucleotide identity based on blast (below 87.8  %) and digital DNA–DNA hybridization (below 36.1 %) values between the novel isolate and its corresponding relatives showed that strain YZC6T could be readily distinguished from its closely related species. Based on genotypic phenotypic and chemotaxonomic data a novel Lacrimispora species Lacrimispora brassicae sp. nov. was proposed with YZC6T as the type strain (=MAFF 212518T=JCM 32810T=DSM 112100T). This study also proposed Clostridium indicum Gundawar et al. 2019 as a later heterotypic synonym of Lacrimispora amygdalina (Parshina et al. 2003) Haas and Blanchard 2020 and Clostridium methoxybenzovorans Mechichi et al. 1999 as a later heterotypic synonym of Lacrimispora indolis (McClung and McCpy 1957) Haas and Blanchard 2020.

Obligately anaerobic Gram-stain-negative wavy rods strains 17YCFAHCo10 18YCFAH0.3Co2 and 19YCFAH0.3Co2 were isolated from faecal samples of healthy Japanese people. The three isolates showed the highest 16S rRNA gene sequence similarity to Waltera intestinalis WCA3-601-WT-6HT (99.2–100 %) and Brotolimicola acetigignens f_CXYT (99.2–99.7 %). The 16S rRNA gene sequence analysis showed that the three isolates formed a cluster with W. intestinalis WCA3-601-WT-6HT. Strain 19YCFAH0.3Co2 formed a subcluster with the type strain of W. intestinalis and did not form a cluster with the other two isolates. B. acetigignens f_CXYT also formed a cluster with W. intestinalis WCA3-601-WT-6HT and three isolates. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain 19YCFAH0.3Co2 and W. intestinalis WCA3-601-WT-6HT were higher (72 % dDDH and 97 % ANI) than the cut-off values for species delimitation indicating that strain 19YCFAH0.3Co2 is W. intestinalis. On the other hand the dDDH and ANI values between strains 17YCFAHCo10 and 18YCFAH0.3Co2 and the type strain of W. intestinalis were lower (<34 % dDDH and <87 % ANI) than the cut-off values for species delimitation indicating that these two isolates are different species from W. intestinalis. The percentage of conserved proteins and the average amino acid identity values support the assignment of the isolates to the genus Waltera. Strains 17YCFAHCo10 and 18YCFAH0.3Co2 could be distinguished from W. intestinalis by their inability to ferment melibiose and ribose and lack of activity for β-glucuronidase. In addition the dDDH and ANI values between two strains (17YCFAHCo10 and 18YCFAH0.3Co2) and B. acetigignens f_CXYT were higher (>78 % dDDH and >97 % ANI) indicating these two strains and B. acetigignens are the same species. As the genus Waltera has priority B. acetigignens is transferred to the genus Waltera as Waltera acetigignens comb. nov. The type strain of W. acetigignens is f_CXYT (=JCM 34988T=DSM 107528T).

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.

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).

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.

The genus Prevotella plays an important role in polysaccharide degradation and fermentation in the rumen. To further understand the function of the phylogenetically diverse genus Prevotella it is necessary to explore the individual characteristics at the species level. In this study Gram-negative anaerobic bacterial strains isolated from the rumen of Holstein cows were identified. Strain R5019T was classified within the genus Prevotella based on 16S rRNA gene sequence-based phylogenetic analysis. The values of 16S rRNA gene sequence similarity average nucleotide identity and digital DNA–DNA hybridization between strain R5019T and its phylogenetically nearest species Prevotella multisaccharivorax PPPA20T were 89.8 82.6 and 29.3 % respectively. The genome size of R5019T was estimated to be ca. 4.19 Mb with a genomic G+C content of 49.5 mol%. The major cellular fatty acids and menaquinones were C15 : 0 anteiso and C17 : 0 anteiso and MK-11 and MK-12 respectively. Succinate lactate malate acetate and formate were produced as the fermentation end products using glucose. Based on phylogenetic physiological biochemical and genomic differences between 11 strains and other phylogenetically related Prevotella species a novel species Prevotella lacticifex sp. nov. is proposed within the genus Prevotella . The type strain is R5019T (=JCM 34664T=DSM 112675T).

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).

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 .

Three strains (YZ01T YZ02 and YZ03) of Gram-stain-positive facultatively anaerobic rods were isolated from the forestomach contents collected from a captive male proboscis monkey (Nasalis larvatus) at Yokohama Zoo in Japan. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belonged to the genus Lactobacillus . Based on the sequence similarity of the 16S rRNA gene Lactobacillus delbrueckii subsp. indicus JCM 15610T was the closest phylogenetic neighbour to YZ01T. Sequence analyses of two partial concatenated housekeeping genes the RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS) also indicated that the novel strains belonged to the genus Lactobacillus . The average nucleotide identity and digital DNA–DNA hybridization (dDDH) between L. delbrueckii subsp. indicus and YZ01T were 85.9 and 31.4 % respectively. The phylogenetic tree based on the whole genomic data of strains YZ01T YZ02 and YZ03 suggested that these three strains formed a single monophyletic cluster in the genus Lactobacillus indicating that it belonged to a new species. The DNA G+C content of strain YZ01T was 51.6 mol%. The major fatty acids were C16 : 0 and C18 : 1 ω9c. Therefore based on phylogenetic phenotypic and physiological evidence strains YZ01T YZ02 and YZ03 represent a novel species of the genus Lactobacillus for which the name Lactobacillus nasalidis sp. nov. is proposed with the type strain YZ01T (=JCM 33769T=DSM 110539T).

Two strains of lactic acid bacteria designated Hs20B0-1T and Hs30E4-3T were isolated from the gut of the damp-wood termite Hodotermopsis sjostedti. These strains were characterized genetically and phenotypically. Strain Hs20B0-1T was related to Lactococcus piscium DSM 6634T showing 96.3 and 84.2 % sequence similarity in 16S rRNA gene and rpoB gene sequences respectively. Strain Hs30E4-3T was related to Lactococcus plantarum DSM 20686T showing 94.8 and 82.2 % sequence similarity in 16S rRNA gene and rpoB gene sequences respectively. The 16S rRNA gene sequence similarity between strains Hs20B0-1T and Hs30E4-3T was 95.7 %. Furthermore genomic comparisons using pairwise average nucleotide identity (ANI) and digital DNA–DNA hybridization (DDH) analyses between strain Hs20B0-1T and L. piscium DSM 6634T resulted in values of 73.5 and 20.1 % respectively. Strain Hs30E4-3T had 72.8 % ANI similarity and 21.3 % DDH similarity to L. plantarum DSM 20686T. Strains Hs20B0-1T and Hs30E4-3T had 75.4 % ANI similarity and 21.1 % DDH similarity to each other. The cell-wall peptidoglycan types of strains Hs20B0-1T and Hs30E4-3T were A4α Lys-Asp and A3α Lys–Thr–Ala respectively. The two strains Hs20B0-1T and Hs30E4-3T are distinguishable from each other and other established Lactococcus species phylogenetically and phenotypically. In conclusion two novel species of the genus Lactococcus are proposed namely Lactococcus insecticola Hs20B0-1T (=JCM 33485T=DSM 110147T) and Lactococcus hodotermopsidis Hs30E4-3T (=JCM 33486T=DSM 110148T) respectively.