Lactobacillus equigenerosi sp. nov., a coccoid species isolated from faeces of thoroughbred racehorses

Two strains of lactic acid bacteria were isolated from faeces of two actively racing thoroughbred horses. The isolates formed a subcluster in the Lactobacillus reuteri phylogenetic group, closely related to Lactobacillus fermentum, L. gastricus, L. ingluviei and L. mucosae, by phylogenetic analysis based on 16S rRNA gene sequences. Levels of DNA–DNA relatedness revealed that the isolates belonged to the same taxon and were genetically separated from their phylogenetic relatives. Biochemical and physiological characteristics also distinguished the isolates from their phylogenetic relatives. The isolates produced spherical or oval cells, and tetrad-like cells were rarely seen. To the best of our knowledge, this is the first report of this morphological characteristic within the genus Lactobacillus. Thus, the isolates represent an atypical novel species of the genus Lactobacillus, for which the name Lactobacillus equigenerosi sp. nov. is proposed. The type strain is NRIC 0697^T (=JCM 14505^T =DSM 18793^T).

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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains NRIC 0696 and NRIC 0697^T are respectively AB288049 and AB288050.

16S rRNA gene sequence-based maximum-likelihood and maximum-parsimony trees are available as supplementary material with the online version of this paper.

The gastrointestinal tracts of animals harbour complex microbiota, and lactic acid bacteria are common members of the microbiota of many animals. As lactic acid bacteria have been reported to have several health-promoting effects on host animals (Ouwehand et al., 2002), the microbiota of the organisms in faeces and/or gastrointestinal contents have been actively studied for many animals (Brashears et al., 2003; Casey et al., 2004; Walter et al., 2001).

During a study of lactic acid bacteria inhabiting the gastrointestinal tract of thoroughbred racehorses, 35 strains of lactic acid bacteria were isolated from faeces of six thoroughbreds. Thirty of the isolates were identified as Lactobacillus equi, L. johnsonii, L. kitasatonis, L. mucosae, L. salivarius, Streptococcus bovis, S. equinus or Weissella confusa, and the other five strains could not be identified (unpublished data). Two of the five strains were phylogenetic relatives of Lactobacillus gastricus, one was a phylogenetic relative of Lactobacillus salivarius and the other two belonged to the Streptococcus phylogenetic cluster. The phylogenetic relative of L. salivarius was recently classified within a novel Lactobacillus species, Lactobacillus hayakitensis (Morita et al., 2007). This paper deals with the taxonomic study of the two phylogenetic relatives of L. gastricus.

Faecal samples were collected from six actively racing thoroughbred horses at the Miho or Ritto Training Center in Ibaraki or Shiga prefecture of Japan in 2003, and the faeces were immediately brought to our laboratory under anaerobic conditions by using anaerobic jars (GasPak System; BBL). Strains NRIC 0696 and NRIC 0697^T were isolated from faeces of different thoroughbreds collected at the Ritto Training Center by using LBS agar (BBL) under anaerobic conditions in anaerobic jars. After isolation, they were maintained on MRS agar (Oxoid) containing 5.0 g calcium carbonate l^–1. L. gastricus DSM 16045^T was obtained from the DSMZ and the strain was used as a reference strain in the present study. The reference strain was also maintained on MRS agar.

16S rRNA gene sequences of the two isolates were determined by methods described previously (Endo & Okada, 2005). The closest recognized relatives of the isolates were determined by performing DataBase searches, and sequences of closely related species were retrieved from DDBJ. Multiple alignments of the sequences were carried out with the program CLUSTAL_X, version 1.18 (Thompson et al., 1997). Distance matrices for the aligned sequences were calculated by using the two-parameter method of Kimura (1980). The neighbour-joining method was used to construct a phylogenetic tree (Saitou & Nei, 1987). The robustness of individual branches was estimated by bootstrapping with 1000 replicates (Felsenstein, 1985). Phylogenetic trees were also constructed by using the maximum-likelihood (Cavalli-Sforza & Edwards, 1967) and maximum-parsimony (Kluge & Farris, 1969) methods with PHYLIP version 3.65 as described previously (Endo & Okada, 2006). The determined 16S rRNA gene sequences of the isolates were compared with each other, and the sequence of NRIC 0697^T was used to search for sequence similarity with DataBase. Approximately 1450 bp of the 16S rRNA gene sequences of the isolates and related species were used for constructing phylogenetic trees. The sequence similarity between NRIC 0696 and NRIC 0697^T was 99.9 %. The highest sequence similarity of NRIC 0697^T to the type strains of known species of lactic acid bacteria was found with L. gastricus, L. mucosae, L. ingluviei and L. fermentum (97.9, 95.8, 95.2 and 93.4 %, respectively). The DataBase search also showed that the partial sequence of NRIC 0697^T was identical to the sequence of the predominant DNA (GenBank accession no. AB250240) derived from faeces of thoroughbreds by using Lactobacillus-group-specific denaturing gradient gel electrophoresis analysis in our other study (Endo et al., 2007). This probably shows that the isolates represent one of the predominant Lactobacillus species in faeces of thoroughbreds. The isolates formed a subcluster with Lactobacillus fermentum, L. gastricus, Lactobacillus ingluviei and L. mucosae in the Lactobacillus reuteri phylogenetic group (Schleifer & Ludwig, 1995; Hammes & Hertel, 2006) on the basis of neighbour-joining analysis (Fig. 1). Identical tree topologies were obtained by using the maximum-parsimony and maximum-likelihood analysis (see Supplementary Figs S1 and S2 in IJSEM Online). L. gastricus, L. ingluviei and L. mucosae were originally isolated from the gastrointestinal contents of animals (Roos et al., 2000, 2005; Baele et al., 2003) and L. fermentum has been isolated from various environmental samples including animal faeces (Dellaglio et al., 2004). This suggests that the major habitat of members of this subcluster is the animal gastrointestinal tract.

(45K): Fig. 1. Phylogenetic relationships of strains NRIC 0696 and NRIC 0697T (L. equigenerosi sp. nov.) to related species based on 16S rRNA gene sequences. The tree was constructed by the neighbour-joining method. L. delbrueckii ATCC 9649T was used as an outgroup. Bootstrap percentages above 70 % are given at branching points. Bar, evolutionary distance (Knuc) of 0.01.

Levels of DNA–DNA relatedness between the isolates and L. gastricus DSM 16045^T and the G+C contents of the isolates were determined by methods described previously (Endo & Okada, 2006). Extraction and isolation of bacterial DNAs were performed by the method of Marmur (1961) as modified by Ezaki et al. (1983). Strains of L. fermentum, L. ingluviei and L. mucosae were not used for the determination of DNA–DNA relatedness because the sequence similarities between the isolates and the type strains of these three species were lower than the recommended value for species differentiation described by Stackebrandt & Goebel (1994). Recently, a revised recommended value for species differentiation was suggested, at 98.7–99 % (Stackebrandt & Ebers, 2006). The isolates showed a high level of DNA–DNA relatedness (93 %). Therefore, we concluded that the isolates belonged to the same taxon. In contrast, the isolates showed low levels of DNA–DNA relatedness to L. gastricus DSM 16045^T (18–23 %). The G+C contents of the isolates were 42 mol%.

Biochemical and physiological characteristics of the isolates were determined by methods described previously (Endo & Okada, 2005). The detailed characteristics of the isolates are given in the species description, and the characteristics were compared with those of phylogenetic relatives L. fermentum, L. gastricus, L. ingluviei and L. mucosae (Table 1). The isolates were obligately heterofermentative lactic acid bacteria, and produced DL-lactic acid, carbon dioxide and ethanol or acetic acid from D-glucose. The isolates were distinguished from L. gastricus by their acid production patterns from carbohydrates and growth at 45 °C (Table 1). Furthermore, values of DNA G+C content of the isolates were very different from those of L. fermentum, L. ingluviei and L. mucosae (Table 1).

Table 1. Differential characteristics between L. equigenerosi sp. nov. (strains NRIC 0696 and NRIC 0697T) and closely related species Species: 1, L. equigenerosi sp. nov.; 2, L. gastricus (unless indicated, data from Roos et al., 2005); 3, L. fermentum (Dellaglio et al., 2004); 4, L. ingluviei (Baele et al., 2003); 5, L. mucosae (Roos et al., 2000). +, Positive; –, negative; V, variable; W, weakly positive.

Morphological characteristics of NRIC 0697^T and L. gastricus DSM 16045^T were determined by using a scanning electron microscope (SEM). The type strains were cultured for 1 to 2 days in MRS broth at 37 °C under anaerobic conditions. After culturing, cells were washed with 0.2 M phosphate buffer (pH 7.0), fixed with 2 % glutaraldehyde for 2 h at 4 °C, post-fixed with 1 % OsO₄ for 3 h at 4 °C, dehydrated with a series of increasing ethanol concentrations (70, 80, 90 and 95 % and twice at 100 %, for 15 min each) and soaked in 3-methyl butyl acetate for 1 day. The prepared cells were subsequently critical-point-dried in a critical-point drying unit (model HCP-2; Hitachi), sputtered with Pt/Pd (model E-102; Hitachi) and observed with an SEM (model S-4000; Hitachi). Scanning electron micrographs of NRIC 0697^T and L. gastricus DSM 16045^T are shown in Fig. 2. The micrographs showed that the cells of both strains were spherical or oval cocci and occurred singly or in pairs. Tetrad-like coccoid cells were rarely seen for either strain. L. gastricus has been reported to produce rod-shaped cells (Roos et al., 2005), but we were unable to observe rod-shaped cells for either type strain in various growth phases and/or in various media (data not shown). We then determined the morphological characteristics of the original type strain of L. gastricus, Kx156A7^T, preserved at the Swedish University of Agricultural Sciences. In our analysis, this strain formed coccoid cells that occurred in pairs or in tetrads, but not rod-shaped cells. Therefore, we concluded that L. gastricus is a coccus. The species of the genus Lactobacillus are well known as producing rod-shaped cells (Rogosa, 1974; Hammes & Vogel, 1996), but the phylogenetic cluster of the genus Lactobacillus comprises bacilli with cocci, which are the tetrad-forming cocci in the genus Pediococcus (Collins et al., 1991; Schleifer & Ludwig, 1995). Almost all members of the genus Pediococcus formed a subcluster within the Lactobacillus cluster (Liu et al., 2006) but, exceptionally, Pediococcus dextrinicus did not belong to the Pediococcus subcluster and formed a subcluster with Lactobacillus species (Liu et al., 2006; Tong & Dong, 2005). As P. dextrinicus was a tetrad-forming coccus (Garvie, 1986), the species was classified as a member of the genus Pediococcus (Simpson & Taguchi, 1996; Garvie, 1986). However, more recently, classification of lactic acid bacteria at the genus level has been based on phylogenetic position using 16S rRNA gene sequences (Schleifer & Ludwig, 1995) rather than morphological characteristics. The genus Weissella, one of the genera of lactic acid bacteria, also comprises bacilli and cocci (Collins et al., 1993). As mentioned above, as the new isolates and L. gastricus formed a subcluster with Lactobacillus species by phylogenetic analysis (Fig. 1), the isolates and L. gastricus should be regarded as coccoid species in the genus Lactobacillus based on the current classification method of lactic acid bacteria (Schleifer & Ludwig, 1995). With application of this classification method, P. dextrinicus should also be a member of the genus Lactobacillus rather than the genus Pediococcus, although this is not discussed further in the present study.

(172K): Fig. 2. Scanning electron micrographs of cells of L. equigenerosi NRIC 0697T (a, b) and L. gastricus DSM 16045T (c, d). Bars, 1 µm.

Based on the data provided, the isolates can be assigned as members of the genus Lactobacillus by their phylogenetic position, but physiological and biochemical characteristics and levels of DNA–DNA relatedness distinguished the isolates from their phylogenetic relatives. Moreover, morphological characteristics demonstrated that the isolates represented an atypical species in the genus Lactobacillus. Thus, the isolates represent a novel species, for which the name Lactobacillus equigenerosi sp. nov. is proposed. In addition, as the morphological characteristics of L. gastricus do not conform to the original description of the species (Roos et al., 2005), an emended description of Lactobacillus gastricus is presented below.

Description of Lactobacillus equigenerosi sp. nov.
Lactobacillus equigenerosi (e'qui.ge.ne.ro'si. L. n. equus a horse; L. adj. generosus of noble birth, well-bred; N.L. gen. n. equigenerosi of a thoroughbred horse).

Cells are Gram-positive, non-motile and spherical or oval cocci measuring 0.5–0.8x0.8–1.5 µm. Cells usually occur singly or in pairs, and tetrad-like cells are uncommon. Facultatively anaerobic and catalase-negative. Growth in broth is enhanced under anaerobic conditions. Colonies are not formed under aerobic conditions but are formed under anaerobic conditions. Colonies on MRS agar under anaerobic conditions are beige, smooth and approximately 1–2 mm in diameter after incubation for 2 days at 37 °C. Obligately heterofermentative and produces DL-lactic acid, carbon dioxide and ethanol or acetic acid from D-glucose. Nitrate is not reduced. Acid is produced from D-glucose, D-xylose, maltose, melibiose and sucrose and is produced weakly from D-ribose and D-fructose, but acid is not produced from L-rhamnose, cellobiose, D-salicin, trehalose, melezitose, D-mannitol, D-sorbitol or starch. Acid production from L-arabinose, D-galactose, lactose and raffinose is strain-dependent. Cells grow at 30–45 °C and grow slowly at 25 °C, but not at 20 or 50 °C. Both known strains grow at pH 4.0 but not at pH 3.5. Growth is observed in MRS broth containing 2.5 % (w/v) NaCl but not 5.0 % (w/v) NaCl. The DNA G+C content of both known strains is 42 mol%.

The type strain is NRIC 0697^T (=JCM 14505^T =DSM 18793^T). Strains NRIC 0697^T and NRIC 0696 were isolated from faeces of thoroughbred racehorses collected at the Ritto Training Center in Shiga prefecture, Japan, in 2003.

Emended description of Lactobacillus gastricus Roos et al. 2005
Lactobacillus gastricus (gas'tri.cus. N.L. masc. adj. gastricus from Gr. adj. gastrikos of the stomach).

The description of L. gastricus is as given in detail by Roos et al. (2005) with the following change. Cells are spherical or oval cocci measuring 0.6–0.8x1.0–2.0 µm. Cells usually occur singly or in pairs, and tetrad-like cells are uncommon. The type strain is Kx156A7^T (=LMG 22113^T =DSM 16045^T =CCUG 48454^T).