The diversity among the species Tetragenococcus halophilus including new isolates from a lupine seed fermentation.

BACKGROUND: Tetragenococcus (T.) halophilus can be isolated from a variety of fermented foods, such as soy sauce, different soy pastes, salted fish sauce and from cheese brine or degraded sugar beet thick juice. This species contributes by the formation of short chain acids to the flavor of the product. Recently, T. halophilus has been identified as a dominant species in a seasoning sauce fermentation based on koji made with lupine seeds. RESULTS: In this study we characterized six strains of T. halophilus isolated from lupine moromi fermentations in terms of their adaptation towards this fermentation environment, salt tolerance and production of biogenic amines. Phylogenic and genomic analysis revealed three distinctive lineages within the species T. halophilus with no relation to their isolation source, besides the lineage of T. halophilus subsp. flandriensis. All isolated strains from lupine moromi belong to one lineage in that any of the type strains are absent. The strains form lupine moromi could not convincingly be assigned to one of the current subspecies. Taken together with strain specific differences in the carbohydrate metabolism (arabinose, mannitol, melibiose, gluconate, galactonate) and amino acid degradation pathways such as arginine deiminase pathway (ADI) and the agmatine deiminase pathway (AgDI) the biodiversity in the species of T. halophilus is greater than expected. Among the new strains, some strains have a favorable combination of traits wanted in a starter culture. CONCLUSIONS: Our study characterized T. halophilus strains that were isolated from lupine fermentation. The lupine moromi environment appears to select strains with specific traits as all of the strains are phylogenetically closely related, which potentially can be used as a starter culture for lupine moromi. We also found that the strains can be clearly distinguished phylogenetically and phenotypically from the type strains of both subspecies T. halophilus subsp. halophilus and T. halophilus subsp. flandriensis.

Vagococcus silagei sp. nov., isolated from brewer's grain used to make silage in Taiwan.

A Gram-stain-positive, coccus- or oval-shaped, non-motile, haemolytic, asporogenous, catalase- and oxidase-negative, and facultatively anaerobic strain, 2B-2T, was isolated from a brewer's grain used to make silage in Taiwan. Comparative analyses of 16S rRNA, hsp60 and pheS gene sequences demonstrated that strain 2B-2T was a member of the genus Vagococcus. On the basis of 16S rRNA gene sequence similarity, the type strains of Vagococcus teuberi (98.4 % similarity), Vagococcus carniphilus (98.4 %), Vagococcus martis (98.2 %), Vagococcus penaei (98.2 %) and Vagococcus fluvialis (98.0 %) were the closest neighbours to this novel strain. The similarity levels of concatenated housekeeping gene sequences (hsp60 and pheS) between strain 2B-2T and these closely related species ranged from 84.5 to 88.0 %. The average nucleotide identity and in silico DNA-DNA hybridization values between strain 2B-2T and its closest relatives were lower than 72.9 and 21.6 %, respectively. The DNA G+C content was 34.7 mol%. Phenotypic and genotypic features demonstrated that strain 2B-2T represents a novel species of the genus Vagococcus, for which the name Vagococcus silagei sp. nov. is proposed. The type strain is 2B-2T (=BCRC 81132T=NBRC 113536T).

Vagococcus xieshaowenii sp. nov., isolated from snow finch (Montifringilla taczanowskii) cloacal content.

A Gram-stain-positive, coccus-shaped, non-motile bacterium, designated CF-49T, was isolated from the cloacal content of a snow finch, which was incidentally captured in a plateau pika burrow on the Qinghai-Tibet Plateau, PR China. Analysis of the 16S rRNA gene sequence showed that strain CF-49T was closely related to Vagococcus elongatus CCUG 51432T (96.5 % similarity), Vagococcus fluvialis NCFB 2497T (96.0 %) and Vagococcus lutrae CCUG 39187T (95.9 %), whereas the similarity to another isolate (CF-210) was 99.9 %. Strains CF-49T and CF-210 grew optimally at 37 °C and pH 7.0 and in the presence of 0.5 % (w/v) NaCl. Acid was produced from N-acetylglucosamine, cellobiose, d-fructose, d-glucose, d-mannose, d-mannitol, maltose, d-ribose and salicin. The cell-wall peptidoglycan type was A4α (l-Lys-d-Asp). The major cellular fatty acids (>10 %) were C16 : 0 (35.6 %), C14 : 0 (17.3 %), C18 : 1 ω9c (16.2 %) and C16 : 1 ω9c (10.6 %). The predominant respiratory quinone was menaquinone MK-7 (68.8 %). The G+C content of the genomic DNA was 35.9 mol%. Digital DNA-DNA hybridization of strain CF-49T with V. fluvialis DSM 5731T, V. elongatus CCUG 51432Tand V. lutrae CCUG 39187T resulted in relatedness values of 21.4, 23.3 and 24.6 %, respectively. Based on results from polyphasic analyses, our two isolates are proposed to represent a novel species in the genus Vagococcus, with the name Vagococcus xieshaowenii. The type strain is CF-49T (=CGMCC 1.6436T=GDMCC 1.1588T=JCM 33477T).

Identification of the putative N-acetylglucosaminidase CseA associated with daughter cell separation in Tetragenococcus halophilus.

The lactic acid bacterium Tetragenococcus halophilus, which is used as a starter to brew soy sauce, comprises both cluster-forming strains and dispersed strains. The cluster-forming strains are industrially useful for obtaining clear soy sauce, because the cell clusters are trapped by filter cloth when the soy sauce mash is pressed. However, the molecular mechanism underlying cell cluster formation is unknown. Whole genome sequence analysis and subsequent target sequence analysis revealed that the cluster-forming strains commonly have functional defects in N-acetylglucosaminidase CseA, a peptidoglycan hydrolase. CseA is a multimodular protein that harbors a GH73 domain and six peptidoglycan-binding LysM domains. Recombinant CseA hydrolyzed peptidoglycan and promoted cell separation. Functional analysis of truncated CseA derivatives revealed that the LysM domains play an important role in efficient peptidoglycan degradation and cell separation. Taken together, the results of this study identify CseA as a factor that greatly affects the cluster formation in T. halophilus.

Vagococcus bubulae sp. nov., isolated from ground beef, and Vagococcus vulneris sp. nov., isolated from a human foot wound.

Two unusual catalase-negative, Gram-stain-positive, Vagococcus-like isolates that were referred to the CDC Streptococcus Laboratory for identification are described. Strain SS1994T was isolated from ground beef and strain SS1995T was isolated from a human foot wound. Comparative 16S rRNA gene sequence analysis of isolates SS1994T and SS1995T against Vagococcus type strain sequences supported their inclusion in the genus Vagococcus. Strain SS1994T showed high sequence similarity (>97.0 %) to the two most recently proposed species, Vagococcus martis (99.2 %) and Vagococcus teuberi (99.0 %) followed by Vagococcus penaei (98.8 %), strain SS1995T (98.6 %), Vagococcus carniphilus (98.0 %), Vagococcus acidifermentans (98.0 %) and Vagococcus fluvialis (97.9 %). The 16S rRNA gene sequence of strain SS1995T was most similar to V. penaei (99.1 %), followed by SS1994T (98.6 %), V. martis (98.4 %), V. teuberi (98.1 %), V. acidifermentans (97.8 %), and both V. carniphilus and V. fluvialis (97.5 %). A polyphasic taxonomic study using conventional biochemical and the rapid ID 32 STREP system, MALDI-TOF MS, cell fatty acid analysis, pairwise sequence comparisons of the 16S rRNA, rpoA, rpoB, pheS and groL genes, and comparative core and whole genome sequence analyses revealed that strains SS1994T and SS1995T were two novel Vagococcus species. The novel taxonomic status of the two isolates was confirmed with core genome phylogeny, average nucleotide identity <84 % and in silico DNA-DNA hybridization <28 % to any other Vagococcus species. The names Vagococcusbubulae SS1994T=(CCUG 70831T=LMG 30164T) and Vagococcusvulneris SS1995T=(CCUG 70832T=LMG 30165T) are proposed.

Identification and typing of Vagococcus salmoninarum using genomic and proteomic techniques.

This study reports on the characterization of Vagococcus salmoninarum using phenotypic, serological, antigenic, genetic and proteomic methods. All strains of V. salmoninarum were resistant to most of the antimicrobials tested, and only 10% of strains were sensitive to florfenicol. Serological analysis demonstrated a high antigenic homogeneity within the species. No cross-reaction was detected with other fish pathogenic species causing streptococcosis (Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, Streptococcus agalactiae, Carnobacterium maltaromaticum) using serum against V. salmoninarum CECT 5810. Electrophoretic analysis of cell surface proteins and immunoblot supported the antigenic homogeneity within V. salmoninarum strains. Moreover, limited diversity was detected using genomic (RAPD, ERIC-PCR and REP-PCR) and MALDI-TOF-MS analyses. The phenotypic, genomic and proteomic methods tested allowed the rapid differentiation of V. salmoninarum from the other species causing streptococcosis. However, MALDI-TOF-MS is the most promising method for typing and characterization of V. salmoninarum.

Burying beetles regulate the microbiome of carcasses and use it to transmit a core microbiota to their offspring.

Necrophagous beetles utilize carrion, a highly nutritious resource that is susceptible to intense microbial competition, by treating it with antimicrobial anal and oral secretions. However, how this regulates the carcass microbiota remains unclear. Here, we show that carcasses prepared by the burying beetle Nicrophorus vespilloides undergo significant changes in their microbial communities subsequent to their burial and "preparation." Prepared carcasses hosted a microbial community that was more similar to that of beetles' anal and oral secretions than to the native carcass community or the surrounding soil, indicating that the beetles regulated the carcass microbiota. A core microbial community (Xanthomonadaceae, Enterococcaceae, Enterobacteriaceae and Yarrowia yeasts) was transmitted by the beetles to the larvae via the anal and oral secretions and the carcass surface. These core taxa proliferated on the carcass, indicating a growth conducive environment for these microbes when associated with beetles. However, total bacterial loads were higher on decomposing carcasses without beetles than on beetle-prepared carcasses, indicating that the beetles and/or their associated symbionts suppress the growth of competing microbes. Thus, apart from being a nutritional resource, the carcass provides a medium for vertical transmission of a tightly regulated symbiotic microbiota, whose activity on the carcass and in the larval gut may involve carcass preservation as well as digestion.

Culturable symbionts associated with the reproductive and digestive tissues of the Neotropical brown stinkbug Euschistus heros.

Symbionts are widely distributed in eukaryotes, and potentially affect the physiology, ecology and evolution of their host. Most insects harbour free-living bacteria in their haemocoel and gut lumen, intracellular-living bacteria in a range of tissues or bacteria in host-derived specialized cells. Stinkbugs, as do many arthropods, harbour extracellular bacteria in the gut that may affect the fitness of their host. This study identified the culturable symbionts associated with the ovaries, spermatheca, seminal vesicle and posterior midgut region (V4) of males and females of Euschistus heros (F.) (Hemiptera: Pentatomidae). Several culture media were used to isolate the bacteria associated with these structures. The selected colonies (morphotypes) were cultured in liquid medium, subjected to genomic DNA extraction, 16S rRNA gene amplification, and restriction fragment length polymorphism (RFLP) analyses. Morphotypes with distinct RFLP patterns were purified and sequenced, and the sequences obtained were used for putative identification and phylogenetic analysis. Comparison of the sequences with those available in the EzTaxon-e database and the use of a matrix of paired distances grouped the isolates in phylotypes belonging to the Phylum Proteobacteria. Proteobacteria was represented by γ-Proteobacteria phylotypes belonging to Enterobacteriaceae, while Firmicutes had Bacilli phylotypes distributed in Enterococcaceae and Staphylococcaceae. Some of the phylotypes identified were associated exclusively with single structures, such as ovaries, spermatheca and the V4 midgut region of males and females. All culturable bacteria associated with the seminal vesicle were also associated with other tissues.

Vagococcus humatus sp. nov., isolated from soil beneath a decomposing pig carcass.

A Gram-stain-positive, non-motile, coccus-shaped bacterium, designated strain C25T, was isolated from the soil beneath a decomposing pig carcass in Korea and was characterized using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that strain C25T belongs to the genus Vagococcus in the family Enterococcaceae of the Lactobacillales. 16S rRNA gene sequence analysis showed that strain C25T was closely related to Vagococcus lutrae CCUG 39187T (96.5 % similarity) and Enterococcus termitis LMG 8895T (95.8 %). The chemotaxonomic properties of strain C25T were consistent with those of the genus Vagococcus; the major cellular fatty acids consisted of C16 : 0, C16 : 1ω9c and C18 : 1ω9c, and the cell-wall peptidoglycan type was based on meso-diaminopimelic acid. The G+C content of the genomic DNA was 44 mol%. On the basis of phylogenetic inference, fatty acid profile, and chemotaxonomic and other phenotypic properties, strain C25T is clearly differentiated from closely related type strains of the genus Vagococcus and represents a novel species in this genus, for which the name Vagococcus humatus sp. nov. is proposed. The type strain is C25T (=KEMB 562-002T=JCM 31581T).

Vagococcus martis sp. nov., isolated from the small intestine of a marten, Martes flavigula.

A novel coccus-shaped, Gram-stain-positive, non-motile and facultative aerobic bacterium, designated strain D7T301T, was isolated from the small intestine of a marten, Martes flavigula, which was killed on the road in Pocheon-si, Gyeonggi-do, Republic of Korea. Grown on a tryptic soy yeast agar plate, colonies had a creamy colour and irregular form. The new isolate formed a monophyletic clade with Vagococcus penaei CD276T on a phylogenetic consensus tree based on the 16S rRNA gene sequence. The isolate grew optimally at 37 °C and pH 7 in the presence of 0.5 % (w/v) NaCl. The isolate was catalase- and oxidase-negative. The cell-wall peptidoglycan was type A4α l-Lys-d-Asp. The major cellular fatty acids were C16 : 0, C14 : 0, and C16 : 1ω9c. The predominant respiratory quinone was menaquinone MK-7 (85.1 %). The DNA G+C content based on genome sequencing was 33.8 mol%. The average nucleotide identity value obtained from comparative genomic analysis between strain D7T301T and V. penaei CIP 109914T was 72.6 %. On the basis of the phenotypic, phylogenetic, biochemical, chemotaxonomic, and genotypic analyses, Vagococcusmartis is proposed as a novel species of the genus Vagococcus. The type strain is D7T301T (=KCTC 21069T=JCM 31178T).

Analysis of the gull fecal microbial community reveals the dominance of Catellicoccus marimammalium in relation to culturable Enterococci.

Gulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB. Enterococcaceae and Enterobacteriaceae were the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealed Catellicoccus marimammalium as the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcus indoxyl-ß-D-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified as Enterococcus spp., 1.2% were identified as Streptococcus spp., and none were identified as C. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions of C. marimammalium 16S rRNA gene sequences (>50-fold) relative to typical mEI culturable Enterococcus spp. C. marimammalium therefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment.

Vagococcus entomophilus sp. nov., from the digestive tract of a wasp (Vespula vulgaris).

Three unknown Gram-stain-positive, catalase-negative, facultatively anaerobic and coccus-shaped strains of bacteria were isolated from the digestive tracts of wasps (Vespula vulgaris). Analysis of 16S rRNA gene sequences revealed that these strains had identical sequences and showed that Vagococcus salmoninarum, with 96.2% sequence similarity, was the closest phylogenetic neighbour. Further analyses based on hsp60 and pheS gene sequences of representatives of the family Enteroccocaceae and genotypic and phenotypic characterization using (GTG)5-PCR fingerprintings, EcoRI ribotyping, DNA G+C content, whole-cell protein profiling, cellular fatty acid profiles analysis and extensive biotyping confirmed that the investigated strains were representatives of a novel bacterial species within the genus Vagoccocus for which the name Vagoccocus entomophilus sp. nov. is proposed. The type strain is VOSTP2(T) ( = DSM 24756(T) = CCM 7946(T)).

Isolation and characterization of halophilic lactic acid bacteria acting as a starter culture for sauce fermentation of the red alga Nori (Porphyra yezoensis).

AIMS: A screening test was conducted for environmental samples to isolate halophilic lactic acid bacteria (HLAB) that can act as a starter in a Nori (Porphyra yezoensis)-sauce culture. METHOD AND RESULTS: After 9 months of incubation of enrichment cultures added with 25 kinds of environmental samples, growth of HLAB-like microorganisms was observed in six cultures salted at a 15% w/w level, including culture samples originally from mesopelagic water taken from 321 m-depth and from mountain snow taken at 2450 m-height. Ten strains were isolated and characterized as Tetragenococcus halophilus based on sequence analysis of the 16S rRNA gene. The isolates were inoculated into a newly prepared Nori-sauce culture and were confirmed to be able to act as a starter culture while three reference strains of T. halophilus obtained from a culture collection could not grow in the same culture. CONCLUSIONS: Halophilic lactic acid bacteria strains that can make growth in a highly salted Nori-sauce culture were isolated from environmental samples for the first time. All the isolates were identified as T. halophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated strains are expected to be utilized as a starter culture for manufacturing fermented seaweed-sauce, which will be the first fermented food products obtained from algae.

Short communication: effect of milk and milk containing Lactobacillus casei on the intestinal microbiota of mice.

BALB/c mice were fed milk or Lactobacillus casei BL23 in milk for 14d and fecal samples were collected at d 0, 4, and 7 as well as 1 and 8d after the last administration. According to high-throughput DNA sequencing of the 16S rRNA genes extracted from the fecal microbiota, the bacterial diversity in the fecal samples of all mice increased over time. After 14d of administration, the consumption of milk and milk containing L. casei BL23 resulted in distinct effects on the microbial composition in the intestine. Specifically, the proportions of bacteria in the Lactobacillaceae, Porphyromonadaceae, and Comamonadaceae were significantly higher in mice fed the L. casei BL23-milk culture compared with one or more of the other groups of mice. The relative amounts of Lachnospiraceae were higher and Streptococcaceae were lower in mice fed milk alone. The changes were not found at d 4 and 7 during milk and L. casei feeding and were no longer detected 8d after administration was stopped. This study shows that consumption of milk or probiotic L. casei-containing milk results in non-overlapping, taxa-specific effects on the bacteria in the distal murine intestine.

Characterization of Tetragenococcus strains from sugar thick juice reveals a novel species, Tetragenococcus osmophilus sp. nov., and divides Tetragenococcus halophilus into two subspecies, T. halophilus subsp. halophilus subsp. nov. and T. halophilus subsp. flandriensis subsp. nov.

Most bacteria recovered so far from sugar thick juice during storage represent strains of the species Tetragenococcus halophilus. Recently, several Gram-positive, non-motile, non-spore-forming cocci with other physiological and genetic traits were isolated from sugar thick juice samples from different origins. In this study, representative isolates were investigated using a polyphasic taxonomic approach. The 16S rRNA gene sequence similarity between these isolates and their closest relative, Tetragenococcus muriaticus, was 97.4%. The level of DNA-DNA relatedness between isolate T1(T), representing the newly found Tetragenococcus isolates, and T. muriaticus was 57%. Isolate T1(T) had a DNA G+C content of 36.7 mol%. Phylogenetic data and genomic and phenotypic features demonstrated that the isolates represent a novel species, for which the name Tetragenococcus osmophilus sp. nov. is proposed with T1(T) as the type strain (=LMG 26041(T) =DSM 23765(T)). Additionally, T. halophilus isolates from high-salt and high-sugar environments showed clear differences in several physiological and genetic characteristics like RAPD fingerprints and 16S rRNA gene sequences. DNA-DNA hybridizations, however, showed 79 to 80% relatedness between osmophilic and halophilic T. halophilus isolates, demonstrating that the different strains belong to the same species. Based on the phenotypic and genotypic differences observed, as well as the different origins of the strains and the industrial relevance of thick juice degradation, two subspecies of T. halophilus are described in this manuscript: T. halophilus subsp. halophilus subsp. nov. for the strains isolated from salt media and T. halophilus subsp. flandriensis subsp. nov. for the strains isolated from sugar-rich environments, which were first isolated in Flanders, Belgium. The type strains for the subspecies are IAM 1676(T) (=LMG 11490(T) =DSM 20339(T)) and T5(T) (=LMG 26042(T) =DSM 23766(T)), respectively.

Rapid lactic acid bacteria identification in dairy products by high-resolution melt analysis of DGGE bands.

AIM: To investigate the application of high-resolution melt (HRM) analysis for rapid species-level identification of lactic acid bacteria (LAB) communities in dairy products, as well as for bacterial community profiling and monitoring. METHODS AND RESULTS: First, comparisons of HRM profiles of known reference strains of LAB and their denaturing gradient gel electrophoresis (DGGE) bands showed very good agreement, allowing species recognition and identification from DGGE bands by HRM. Second, samples of cheese, kefir grains and kefir were characterized by PCR-DGGE, and melting profiles of DGGE bands were compared with known reference strains. Of the 13 DGGE bands, ten were identified by HRM by comparison with the reference strains and only three required sequencing for identification. Use of HRM profiling for comparison and monitoring of total LAB communities from dairy products or starter cultures was also evaluated, and good agreement was found when comparing clustering of DGGE band profiles with clustering of HRM melting profiles. CONCLUSION: Identification of DGGE bands is possible by comparison of HRM melting profiles with known reference strains. SIGNIFICANCE AND IMPACT OF THE STUDY: HRM profiling is suggested as an additional approach for identification of DGGE bands.

Intestinal gluconeogenesis shapes gut microbiota, fecal and urine metabolome in mice with gastric bypass surgery.

Intestinal gluconeogenesis (IGN), gastric bypass (GBP) and gut microbiota positively regulate glucose homeostasis and diet-induced dysmetabolism. GBP modulates gut microbiota, whether IGN could shape it has not been investigated. We studied gut microbiota and microbiome in wild type and IGN-deficient mice, undergoing GBP or not, and fed on either a normal chow (NC) or a high-fat/high-sucrose (HFHS) diet. We also studied fecal and urine metabolome in NC-fed mice. IGN and GBP had a different effect on the gut microbiota of mice fed with NC and HFHS diet. IGN inactivation increased abundance of Deltaproteobacteria on NC and of Proteobacteria such as Helicobacter on HFHS diet. GBP increased abundance of Firmicutes and Proteobacteria on NC-fed WT mice and of Firmicutes, Bacteroidetes and Proteobacteria on HFHS-fed WT mice. The combined effect of IGN inactivation and GBP increased abundance of Actinobacteria on NC and the abundance of Enterococcaceae and Enterobacteriaceae on HFHS diet. A reduction was observed in the amounf of short-chain fatty acids in fecal (by GBP) and in both fecal and urine (by IGN inactivation) metabolome. IGN and GBP, separately or combined, shape gut microbiota and microbiome on NC- and HFHS-fed mice, and modify fecal and urine metabolome.

Vagococcus acidifermentans sp. nov., isolated from an acidogenic fermentation bioreactor.

A Gram-staining-positive, coccus-shaped, non-spore-forming, facultatively anaerobic bacterium, designated AC-1(T), was isolated from an acidogenic fermentation bioreactor treating food wastewater. On the basis of 16S rRNA gene sequence analysis, strain AC-1(T) was shown to belong to the genus Vagococcus. The closest phylogenetic relatives were Vagococcus elongatus PPC9(T) (97.4 % 16S rRNA gene sequence similarity), Vagococcus penaei CD276(T) (96.7 %) and Vagococcus carniphilus ATCC BAA-640(T) (96.6 %). The major fatty acids were C(18 : 1)ω9c (24.8 %) and C(16 : 0) (19.5 %) and the G+C content of genomic DNA was 44.2 mol%, which supported the affiliation of strain AC-1(T) to the genus Vagococcus. Strain AC-1(T) and V. elongatus DSM 21480(T) exhibited 11 % DNA-DNA relatedness. Physiological and biochemical tests differentiated strain AC-1(T) from the type strains of recognized species of the genus Vagococcus. Therefore, strain AC-1(T) is considered to represent a novel species, for which the name Vagococcus acidifermentans sp. nov. is proposed. The type strain is AC-1(T) ( = KCTC 13418(T)  = LMG 24798(T)).

Description of Vagococcus coleopterorum sp. nov., isolated from the intestine of the diving beetle, Cybister lewisianus, and Vagococcus hydrophili sp. nov., isolated from the intestine of the dark diving beetle, Hydrophilus acuminatus, and emended description of the genus Vagococcus.

A polyphasic taxonomic approach was used to characterize two novel bacterial strains, HDW17AT and HDW17BT, isolated from the intestine of the diving beetle Cybister lewisianus, and the dark diving beetle Hydrophilus acuminatus, respectively. Both strains were Gram-positive and facultative anaerobic cocci forming cream-colored colonies. The isolates grew optimally at 25°C, pH 7, in the presence of 0.3% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences and genome sequences showed that the isolates were members of the genus Vagococcus, and strain HDW17AT was closely related to Vagococcus fessus CCUG 41755T (98.9% of 16S rRNA gene sequence similarity and 74.3% of average nucleotide identity [ANI]), whereas strain HDW17BT was closely related to Vagococcus fluvialis NCFB 2497T (98.9% of 16S rRNA gene sequence similarity and 76.6% of ANI). Both strains contained C16:0, and C18:1ω9c as the major cellular fatty acids, but C16:1ω9c was also observed only in strain HDW17BT as the major cellular fatty acid. The respiratory quinone of the isolates was MK-7. The major polar lipid components were phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. The genomic DNA G + C content of strains HDW17AT and HDW17BT were 36.6 and 34.4%, respectively. Both strains had cell wall peptidoglycan composed of the amino acids L-alanine, glycine, D-glutamic acid, L-tryptophan, L-lysine, and L-aspartic acid, and the sugars ribose, glucose, and galactose. Based on phylogenetic, phenotypic, chemotaxonomic, and genotypic analyses, strains HDW17AT and HDW17BT represent two novel species in the genus Vagococcus. We propose the name Vagococcus coleopterorum sp. nov. for strain HDW17AT (= KACC 21348T = KCTC 49324T = JCM 33674T) and the name Vagococcus hydrophili sp. nov. for strain HDW17BT (= KACC 21349T = KCTC 49325T = JCM 33675T).

Vagococcus zengguangii sp. nov., isolated from yak faeces.

Two unknown Gram-stain-positive, catalase- and oxidasenegative, non-motile, and coccus-shaped bacteria, designated MN-17T and MN-09, were isolated from yaks faeces (Bos grunniens) in the Qinghai-Tibet Plateau of China. 16S rRNA gene sequence-based comparative analyses revealed that the two strains were grouped within the genus Vagococcus, displaying the highest similarity with Vagococcus xieshaowenii CGMCC 1.16436T (98.6%) and Vagococcus elongatus CCUG 51432T (96.4%). Both strains grew optimally at 37°C and pH 7.0 in the presence of 0.5% (w/v) NaCl. The complete genome of MN-17T comprises 2,085 putative genes with a total of 2,190,262 bp and an average G + C content of 36.7 mol%. The major fatty acids were C16:0 (31.2%), C14:0 (28.5%), and C18:1ω9c (13.0%); the predominant respiratory quinone was MK-7 (68.8%); the peptidoglycan type was A4α(L-Lys-D-Asp); and the major polar lipid was diphosphatidylglycerol. Together, these supported the affiliation of strain MN-17T to the genus Vagococcus. In silico DNA-DNA hybridization and the average nucleotide identity values between MN-17T and all recognized species in the genus were 21.6-26.1% and 70.7-83.0%, respectively. MN-17T produced acid from D-cellobiose, D-fructose, glycerol, D-glucose, N-acetyl-glucosamine, gentiobiose, D-mannose, D-maltose, D-ribose, D-saccharose, salicin, D-trehalose, and D-xylose. These results distinguished MN-17T and MN-09 from closely related species in Vagococcus. Thus, we propose that strains MN-17T and MN-09 represent a novel species in the genus Vagococcus, with the name Vagococcus zengguangii sp. The type strain is MN-17T (= CGMCC 1.16726T = GDMCC 1.1589T = JCM 33478T).