Clostridium lavalense sp. nov., a glycopeptide-resistant species isolated from human faeces

Accurate analysis of the microbial ecosystem in the human digestive tract is always a challenging topic for microbiologists because of the interactions between complex bacterial communities and host cells and also because of the implications for human health (Backhed et al., 2005; Hooper et al., 2002; Zoetendal et al., 2006). The microbiota of the human gut has been investigated extensively, using both traditional culture methods and molecular techniques, and has been found to contain a large number of unknown species belonging to the 19 phylogenetic clusters of the genus Clostridium (Collins et al., 1994; Eckburg et al., 2005; Suau et al., 1999). The bacteria most frequently detected in human faeces can be classified as belonging to three major groups. One group comprises the low-G+C, Gram-positive organisms (46–58 %), as follows: the Clostridium coccoides–Eubacterium rectale group (also known as cluster XIVa) (22.7–28 %), the Clostridium leptum group (also known as cluster IV) (21.1–25.2 %), the Lactobacillus–Enterococcus group (<0.1–1.8 %), the Eubacterium cylindroides group (1.1–1.4 %), the Veillonella group (<0.1–1.3 %) and the Phascolarctobacterium group (0.6 %). The next category consists of Gram-negative organisms (10–30 %): the Bacteroides–Prevotella group (8.5–28 %), the family Enterobacteriaceae (0.1–0.2 %) and the genus Akkermansia (1.3 %). Finally, there are the high-G+C, Gram-positive organisms (8–17 %) represented by the genera Atopobium (3.1–11.9 %) and Bifidobacterium (4.4–4.8 %) (Zoetendal et al., 2006).

Two strains, CCRI-9929 (=NML 03-A-014=MLG245) and CCRI-9842^T (=NML 03-A-015^T) were isolated during screening of human faecal specimens to detect carriers of vancomycin-resistant enterococci (Ballard et al., 2005; Domingo et al., 2005; Stinear et al., 2001). In this study, we characterized the phenotypic profiles and phylogenetic relationships of these two strains. The data from this polyphasic taxonomic analysis, presented here, revealed that these strains represent a novel species of the genus Clostridium.

During a screening programme for the detection of vancomycin-resistant enterococci in clinical settings in Québec (Canada) and Melbourne (Australia), two anaerobic bacterial strains, CCRI-9929 and CCRI-9842^T, were isolated, respectively, from faecal specimens of patients tested for colonization by vancomycin-resistant enterococci (Ballard et al., 2005; Domingo et al., 2005; Stinear et al., 2001). No further information was collected from these patients. The isolates were stored at −80 °C in brain heart infusion (BHI; Difco) +20 % glycerol media until the identification tests could be performed. These strains were subcultured twice on BHI blood agar containing haemin (0.005 mg ml⁻¹) and vitamin K (0.001 mg ml⁻¹). Phenotypic identification tests were performed as described in the Wadsworth and VPI anaerobic manuals (Holdeman et al., 1977; Jousimies-Somer et al., 2002) at the Centre de Recherche en Infectiologie, Université Laval, Québec, Canada, and at the National Microbiology Laboratory, Winnipeg, Canada. Morphology was observed using light microscopy (Leitz) and transmission electron microscopy (1200EX; JEOL). Growth was observed in peptone-yeast broth enriched with glucose, with or without bile, serum, Tween 80 or formate-fumarate (Jousimies-Somer et al., 2002). Special-potency discs (Oxoid) containing colistin (10 μg), kanamycin (1000 μg), vancomycin (5 μg) and metronidazole (5 μg) were used to group the anaerobic bacteria, as recommended in the Wadsworth manual (Jousimies-Somer et al., 2002). Minimum inhibitory concentrations (MICs) for vancomycin and metronidazole were determined using the agar dilution method for anaerobes according to the Clinical and Laboratory Standard Institute (NCCLS, 2004). The MIC for teicoplanin was determined using the Etest method (AB Biodisk). Fermentation tests were performed for various sugars using prereduced, anaerobically sterilized peptone-yeast sugar broth tubes (Med-Ox Diagnostics). Other tests, such as those for catalase, aesculin, indole, lecithinase, lipase, nitrate and oxidase, were performed as described previously (Jousimies-Somer et al., 2002). The novel isolates were examined (according to the manufacturers' instructions) using enzyme test kits including the Rapid ID 32A and API ZYM systems (bioMérieux) and the Rapid ANA II system (Remel). GLC analysis of the metabolic end products of fermentation was performed as described previously (Bernard et al., 2002) except that anaerobe system broth was acquired from Med-Ox Diagnostics. Cellular fatty acid composition and library generation analyses were performed using the MIDI Sherlock system and LGS software (MIDI), as described previously (Bernard et al., 2002).

Strains CCRI-9842^T and CCRI-9929 comprised strictly anaerobic, Gram-positive, rod-shaped cells (0.8–1.5 μm wide by 2.5–6.0 μm long) with tapered ends. Subterminal spores were observed (Fig. 1⇓). The isolates grew well anaerobically, but no growth occurred following subculture under 5 % O₂. Growth was enhanced in the presence of glucose and formate-fumarate for both strains, and in the presence of bile and serum in the case of strain CCRI-9929 (Table 1⇓). Neither strain was inhibited by Tween 80. After 48 h incubation on blood agar at 37 °C under anaerobic conditions, colonies of both strains were 2–3 mm in diameter, greyish white in colour and had irregular edges. The centres of the colonies were slightly raised and no zones of haemolysis were observed on blood agar. Colonies of strain CCRI-9842^T were strongly adherent on blood agar whereas those of strain CCRI-9929 were non-adherent on the same medium. This adherence of strain CCRI-9842^T declined progressively after several subcultures on agar medium. Cells of both novel isolates were motile. The isolates were indole-positive and catalase-negative. Nitrate was reduced by strain CCRI-9842^T but not by strain CCRI-9929. No liquefaction of gelatin or peptonization of milk occurred in either strain. Neither strain hydrolysed urea or starch. Analysis using special potency discs showed that both strains were susceptible to 1000 μg kanamycin and 5 μg metronidazole and that both were resistant to 10 μg colistin and 5 μg vancomycin. MIC determinations showed that both strains were resistant to vancomycin (MIC>256 μg ml⁻¹) but were susceptible to teicoplanin (MIC<1.5 μg ml⁻¹) and metronidazole (MIC<0.064 μg ml⁻¹). The enzyme profile obtained using Rapid ID 32A and API ZYM tests is summarized in Table 1⇓. Both strains belong to the genus Clostridium, being obligatory anaerobic, endospore-forming, Gram-positive bacilli. On the basis of their cultural and biochemical characteristics, as well their glycopeptide susceptibility profile, it was evident that these isolates closely resembled each other and probably belonged to the same species.

Total DNA from strains CCRI-9842^T and CCRI-9929 was purified using the GNOME DNA kit (Qbiogene). Purified genomic DNA was used in PCRs to amplify a 1466 bp region of the 16S rRNA genes as described previously (Paradis et al., 2005). Sequencing of the specific amplification products was also performed as described previously (Domingo et al., 2005). To identify the taxonomic neighbours of strains CCRI-9842^T and CCRI-9929, 16S rRNA gene sequences were used for an initial blast search against the GenBank database. Subsequently, bacterial species closely related to both strains were used for a phylogenetic analysis. Multiple sequence alignments were performed using clustal w from the GCG package (Wisconsin Package, version 10.3; Accelrys). The phylogenetic analysis was carried out using the neighbour-joining (Saitou and Nei, 1987) and maximum-parsimony (Fitch, 1971) methods with mega, version 4 (Tamura et al., 2007). Evolutionary distance matrices were generated according to the Kimura two-parameter model (Kimura, 1980). Bootstrap values (%) were calculated from 1000 resamplings. The final phylogenetic tree based on 16S rRNA gene sequences was rooted with type strains of Fusobacterium nucleatum and Propionigenium modestum (cluster XIX) as the outgroup. The comparative 16S rRNA gene sequence analysis showed that isolates CCRI-9842^T and CCRI-9929 were genetically very closely related to each other, displaying 99.2 % sequence similarity. Searches in the sequence database revealed that the novel isolates were closely related to members of cluster XIVa of the genus Clostridium (Collins et al., 1994). On the basis of 16S rRNA gene sequence similarity, the closest relatives of the unidentified isolates were type strains of Clostridium asparagiforme (97 %), Clostridium bolteae (96 %) and Clostridium clostridioforme (95 %). A phylogenetic tree generated using a larger number of 16S rRNA gene sequences and constructed using the neighbour-joining and maximum-parsimony methods showed that the unknown anaerobic isolates fitted into the Clostridium coccoides rRNA complex (cluster XIVa of the genus Clostridium) (Collins et al., 1994) (Fig. 2⇓ and Supplementary Figs S1 and S2, available with IJSEM Online). Cluster XIVa contains various genera, including Clostridium, Ruminococcus, Eubacterium, Acetitomaculum, Roseburia and Coprococcus (Collins et al., 1994). To elucidate the phylogenetic relationships between the novel isolates and the Clostridium species belonging to cluster XIVa, phylogenetic trees based on 16S rRNA gene sequences were constructed by using three different tree-making algorithms. The neighbour-joining (Fig. 2⇓), maximum-parsimony (see Supplementary Fig. S3, available with IJSEM Online) and maximum-likelihood (Supplementary Fig. S4 in IJSEM Online) trees clearly demonstrated that the two novel isolates represent a hitherto unknown subline within cluster XIVa of the genus Clostridium. The isolates clustered within a distinct subgroup that included Clostridium asparagiforme, Clostridium bolteae, Clostridium clostridioforme, Clostridium citroniae and Clostridium aldenense. It is generally accepted that organisms displaying 16S rRNA gene sequence dissimilarities close to 3 % or more do not belong to the same species (Stackebrandt & Goebel, 1994; Stackebrandt & Ebers, 2006). Several characteristics, including morphology, biochemical profiles and the 16S rRNA gene sequence tree topology, were used to distinguish strains CCRI-9842^T and CCRI-9929 from the closely related species Clostridium asparagiforme. Strains CCRI-9842^T and CCRI-9929 were resistant to vancomycin and harboured the vanB gene cluster. The cellular morphology of these strains was different from the asparagus-shaped morphology of cells of Clostridium asparagiforme (Fig. 1⇑). These novel isolates also differed from Clostridium asparagiforme in that they produced acid from arabinose, mannose and xylose (Table 1⇑). Enzymic activities for the novel isolates differed from those of Clostridium asparagiforme DSM 15981^T: the novel isolates showed negative reactions for α-glucosidase, α-fucosidase, α-galactosidase and β-glucuronidase. For both novel isolates, acetic acid was the major end product, with minor yields of lactate and succinate from glucose metabolism; in contrast, acetic acid, lactate and ethanol were the major end products for Clostridium asparagiforme DSM 15981^T. Long-chain cellular fatty acid profiles showed that strains CCRI-9842^T and CCRI-9929 differed from Clostridium asparagiforme DSM 15981^T in several respects (Table 2⇓). Moreover, the branching pattern in the tree based on 16S rRNA gene sequences clearly shows that the novel bacterial isolates represent a separate species within cluster XIVa of the genus Clostridium. Despite the close relationships demonstrated to exist between the novel isolates and Clostridium asparagiforme, the data from the polyphasic taxonomic analysis presented here strongly support the classification of strains CCRI-9842^T and CCRI-9929 within a novel species of the genus Clostridium, for which the name Clostridium lavalense sp. nov. is proposed.