Veillonella magna sp. nov., isolated from the jejunal mucosa of a healthy pig, and emended description of Veillonella ratti

Footnotes

The GenBank/EMBL/DDBJ accession numbers for the dnaK and 16S rRNA gene sequences of strain lac18^T are EU096494 and EU096495, respectively.

Intra- and interspecific similarity percentages for the dnaK sequences of strain lac18^T and strains in the genus Veillonella are presented in a supplementary table available with the online version of this paper.

The type strains of all recognized Veillonella species have been isolated from their main natural habitats, namely the oral cavity and the gastrointestinal tract of homeothermic vertebrates (Smith, 1965; Rogosa, 1984; Kolenbrander, 2006). The species V. parvula, V. atypica, V. dispar, V. montpellierensis, V. denticariosi and V. rogosae have been isolated from man and V. criceti, V. ratti, V. rodentium and V. caviae have been isolated from rodents. In food animals, Veillonella strains are also detected regularly as indigenous inhabitants of all sections of the gastrointestinal tract (Johns, 1951; Alexander & Davies, 1963; Smith & Jones, 1963; Tannock & Smith, 1970; McGillivery & Cranwell, 1992; Leser et al., 2002; Tsukahara & Ushida, 2002; Murphy et al., 2005; Gong et al., 2007; Wise & Siragusa, 2007).

The veillonellae possess a truncated glycolytic system (Rogosa et al., 1965) but utilize the metabolic end products of co-existing carbohydrate-fermenting bacteria, i.e. three- and four-carbon organic acids, as energy and carbon sources via the methylmalonyl-CoA pathway (Delwiche et al., 1985; Denger & Schink, 1992; Seeliger et al., 2002). In the gastrointestinal tract, strains of the genus Veillonella thereby constitute an essential link with indigenous lactic acid bacteria in a natural microbial food-chain (Marounek & Bartos, 1987; Pacheco Delahaye et al., 1994; Kolenbrander, 2006). Mixed cultures of veillonellae and, inter alia, lactic acid bacteria, isolated from the caeca of chickens, are inhibitory to enteropathogenic bacteria (Hinton et al., 1991; Hinton et al., 1992; Corrier et al., 1995). One such mixed culture is commercially available as a health-promoting competitive exclusion culture for poultry production (Nisbet, 2002).

Strain lac18^T was isolated in 2007 in the course of a cultural study of lactic acid bacteria from porcine mucosal jejunum. A cross-bred fattening pig was reared at our Institute and euthanized at 62 days by means of an overdose of sodium pentobarbital, in accordance with the legal requirements of the relevant local authority for animal welfare. Fresh, washed, pea-size mucosal samples were inoculated into 10 ml of a medium selective for lactic acid bacteria and containing porcine gastric mucin (Type III; Sigma) as the main carbon and nitrogen source. This medium, henceforth referred to as mucin medium, contained the following (l^–1): 10.0 g mucin, 0.01 g peptone, 0.01 g yeast extract, 0.01 g glucose, 0.3 g NaCl, 0.1 g CaCl₂, 6.0 g KH₂PO₄, 5 ml Rogosa's salt solution (Rogosa et al., 1951), 1 ml modified (lacking elements already included in Rogosa's salt solution) Pfennig's SL8 trace element solution (Bast, 2001), 0.2 ml vitamin solution (Stams et al., 1993) and 0.5x10^–3 g resazurin. All of the components were autoclaved, except the vitamins, which were filter-sterilized. The pH of the medium was adjusted to 5.0 using 37–38 % HCl prior to autoclaving. Incubations were performed in loosely screw-capped 15 ml tubes at 37 °C under anaerobic, CO₂-enriched (18 % by vol.) conditions in an anaerobic jar using Anaerocult A gas packs (Merck). The incubation time between initial transfers was always 7–14 days. Single colonies were obtained on mucin medium containing 0.4–0.75 % agar. Strain lac18^T was eventually recovered from a co-culture with an Enterococcus isolate (lac32) on MRS agar (Roth). It was presumptively identified to the genus level using the VITEK Anaerobe Identification system (bioMérieux). Strain lac18^T appeared to be distinct from a total of 32 isolates that were recovered from the gastric and jejunal mucosae of two pigs (data not shown). We decided, therefore, to classify and characterize strain lac18^T by means of a polyphasic approach. On the basis of the data from this study, we describe the first species of the genus Veillonella with a type strain of non-human or non-rodent origin.

Definitive identification of strain lac18^T, including its taxonomic assignment to the genus Veillonella, was obtained using 16S rRNA gene sequence analysis. Genomic DNA was extracted from cells with a NucleoSpin Tissue kit (Macherey-Nagel). The 16S rRNA gene was amplified using a HotStarTaq Master Mix kit (Qiagen) and primers 27f and 1492r (Lane, 1991). Amplified PCR products were purified using a High Pure PCR Product Purification kit (Roche) and sequenced commercially (MWG Biotech). A 16S rRNA gene sequence of 1449 bp was obtained and subsequently analysed with the Ribosomal Database Project (RDP) II classification algorithm (; Maidak et al., 2001; Wang et al., 2007) and the BLAST algorithm (; Altschul et al., 1990; McGinnis & Madden, 2004). Phylogenetic analysis with MEGA software (version 4.0) (Tamura et al., 2007) was conducted to infer an evolutionary tree using the neighbour-joining method (Saitou & Nei, 1987) based on the Kimura two-parameter model (Kimura, 1980). The taxonomic position of strain lac18^T in the genus Veillonella was further resolved by means of partial sequence (703 bp) analysis of the 70 kDa heat-shock-protein gene (dnaK) as proposed by Marchandin et al. (2003). Phylogenetic analysis in BLAST was carried out as described for the 16S rRNA gene. As there is currently no published threshold for dnaK gene sequence-based species delineation for the genus Veillonella (Marchandin et al., 2005), a pairwise BLAST 2 SEQUENCES analysis of all known dnaK sequences in the genus was also performed (; Tatusova & Madden, 1999). DNA–DNA hybridization with V. ratti DSM 20736^T, V. criceti DSM 20734^T and V. parvula DSM 2008^T was determined using a membrane filter technique (Johnson, 1994) with digoxigenin-based products from Roche (DIG DNA labelling kit, DIG Easy Hyb granules, Anti-DIG-AP, CDP-Star) according to the manufacturer's protocols. The amount of genomic DNA immobilized on the nylon membranes was 4000 ng. The actual hybridization was performed at 42 °C for 12 h. Signal intensities were visualized by means of long exposure to a charge-coupled device camera (SensiCam Qe, CamWare software version 2.20; PCO) and the similarities (%) were determined using the PCBAS software program (version 2.09e; Raytest).

The 16S rRNA gene sequence analysis indicated that strain lac18^T was most closely related to the V. ratti–V. criceti cluster (Johnson & Harich, 1983) of the genus Veillonella, with V. ratti ATCC 17746^T (96.6 %), V. criceti ATCC 17747^T (96.6 %), V. denticariosi RBV106^T (95.1 %) and V. atypica ATCC 17744^T (95.1 %) as the closest relatives with validly published names. The sequence similarity with respect to V. parvula ATCC 10790^T was 94.0 %. High similarity (99.4 %) was obtained with the 1309 bp sequence of one unpublished isolate, Veillonella sp. MY-P9 (GenBank accession no. DQ979378 deposited by M. Y. Jung & Y.-H. Chang, 2006). The genetic distinctness of strain lac18^T with respect to the V. ratti–V. criceti cluster was strongly supported by a high bootstrap value (100 %) in the neighbour-joining analysis (Fig. 1). The dnaK gene sequence analysis indicated that the taxonomic position of the novel strain was closer to V. ratti ATCC 17746^T (84.5 %) than to V. criceti ATCC 17747^T (83.4 %). The sequence similarity of the novel strain was highest with the Veillonella sp. isolate MY-P9 (99.7 %). The BLAST 2 SEQUENCES analysis showed that the interspecific similarity of the dnaK gene sequences of recognized strains in the genus Veillonella (except Veillonella sp. ADV 4313.2, Veillonella sp. ADV 360.1 and Veillonella sp. MY-P9) varied between 77.9 % (lac18^T versus V. dispar ATCC 17748^T) and 95.7 % (V. denticariosi RBV81 versus V. rodentium ATCC 17743^T). The intraspecific similarities of the dnaK gene sequences of strains of recognized species varied between 98.4 % and 99.8 % (V. montpellierensis). This comparison produced a 96–98 % threshold for dnaK gene-based species delineation in the genus Veillonella (see Supplementary Table S1, available in IJSEM Online). DNA–DNA hybridization results confirmed the genotypic distinctness of strain lac18^T within the genus Veillonella: the DNA–DNA relatedness values with V. ratti DSM 20736^T, V. criceti DSM 20734^T and V. parvula DSM 2008^T were 40.0, 39.0 and 27.0 %, respectively.

Cell morphology was observed under a Zeiss light microscope at x1000 magnification and under a digital scanning electron microscope (DSM 950; Zeiss) at x5000–20 000 magnification (Taras, 2001). Gram-staining was performed using a Gram-colour staining set (Merck) according to the manufacturer's instructions. Spore formation and motility were analysed using conventional methods (Bast, 2001). Commercial tests (Fluka) were applied for the detection of cytochrome oxidase and nitrate reduction. The benzidine reaction with porphyrin and catalase activity were assayed according to Deibel & Evans (1960). The production of H₂O₂ was studied as described by Juárez Tomás et al. (2004) with the following exceptions: prolonged incubation was performed under both anaerobic and microaerobic (8–10 % CO₂, 5–7 % O₂, by vol.; Anaerocult C gas packs, Merck) conditions for up to 4 days and exposure to air was for 24 h. The H₂O₂ assays were evaluated as described by Otero & Nader-Macías (2006). H₂S production was analysed in a defined modified Veillonella medium with 0.6x10^–3 M L-cysteine (Rogosa & Bishop, 1964a). For biochemical profiling, the Rapid ID 32A and API ZYM systems (bioMérieux) were used according to the manufacturer's instructions. Blood agar base No. 2 (LabM) plus 5 % defibrinated horse blood was chosen as the second culture medium with Rapid ID 32A. Putrescine auxotrophy was analysed in the defined modified Veillonella medium, supplemented with putrescine at 0.003 and 0.005 g l^–1 (Rogosa & Bishop, 1964b; Ritchey & Delwiche, 1975). Fermentation of fructose was studied likewise using supplementation with 0.02 M D-fructose. Short-chain fatty acids produced as metabolic end products from the fermentation of lactate were determined as described previously (Schäfer, 1995) and gas production was assayed by measuring increases in pressure in air-tight, screw-capped roll tubes. Growth characteristics under various pH and temperature values were studied using standard anaerobic methods (Hungate, 1969). The response to oxygen was assessed using aerobic and microaerobic (Anaerocult C gas packs) incubation with or without the reducing agent sodium thioglycolate (0.75 g l^–1).

The results of the phenotypic characterization, including differential features, are given in the species description. Phenotypic characteristics that serve to differentiate strain lac18^T from V. ratti DSM 20736^T and V. criceti DSM 20734^T are given in Table 1. On the basis of the results of 16S rRNA and dnaK gene sequencing and the differential phenotypic data, strain lac18^T represents a novel species of the genus Veillonella, for which the name Veillonella magna is proposed.

Table 1. Results of differential phenotypic characterization of strain lac18T, V. ratti DSM 20736T and V. criceti DSM 20734T Taxa: 1, Strain lac18T; 2, V. ratti DSM 20736T; 3, V. criceti DSM 20734T. +, Positive; –, negative; W, weak. In the API ZYM, all strains were negative for the following: lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase. Values for the API ZYM are means of the activity mark±SD, n=10. The activity mark is a measure of the quantity of hydrolysed substrate (nmol): 0=<5; 1=5–<10; 2=10–<20; 3=20–<30; 4=30–<40; 5=≥40.

Cells are spherical to coccoid, 0.65–0.85 µm in diameter (mean, approx. 0.75 µm) and are arranged singly or in pairs (rarely in short chains or small masses). Adjacent sides of cell pairs are flattened and the cell surface is convoluted (Fig. 2). Cells stain Gram-negative, with an enhanced tendency to resist decolorization after 48 h incubation. They are non-spore-forming and non-motile. After anaerobic incubation on modified Veillonella agar at 37 °C for 24 h, colonies are circular, up to 1.2 mm in diameter, have entire margins and smooth, shiny surfaces and are raised, opaque, creamy grey, soft and moist. After 48 h, colonies look similar but are up to 2.0 mm in diameter and greyish beige. Cells are non-haemolytic on blood agar. Cytochrome oxidase is not present. Nitrate is reduced. The benzidine test for porphyrin is positive. Catalase activity is negative or delayed (approx. 30–45 s) and weak. The type of catalase is not determined in the benzidine test. Benzidine reacts non-specifically with the porphyrin compounds of the haem group in respiratory systems including (besides catalase) cytochromes and nitrite reductase (Bascomb & Manafi, 1998). H₂O₂ accumulation is weak under anaerobic conditions and moderate under microaerobic conditions. H₂S is produced from L-cysteine. Biochemical profiling with Rapid ID 32A yields positive results for arginine dihydrolase, reduction of nitrates, alkaline phosphatase and histidine arylamidase (identification profile 2000500001). The leucine arylamidase reaction is ambiguous using modified Veillonella medium [identification profile 200050(0/2)001]. API ZYM results are indicated in Table 1. Putrescine is required for normal growth. Negative for fermentation of fructose. Lactate is metabolized anaerobically to more propionate and less acetate. Gas is also produced, often abundantly. The temperature range for growth is approximately 21–45 °C. Growth is very weak at approximately 21 °C, absent at 46 °C, good at 30 °C and very good at 37 and 40 °C. The pH range for growth is 5.5–9.5 (optimum, pH 6.5–7.5). Alkalinization of modified Veillonella medium occurs at pH 5.5–6.0 (maximally to pH 6.5) and acidification occurs at pH 6.5–9.5 (maximally to pH 6.2). Cells are anaerobic and microaerotolerant (less than approx. 5 % O₂, by vol.), showing no growth on agar plates but producing vigorous growth and long-lasting viability (up to at least 2 weeks) in broth medium suspended in air-tight, screw-capped roll tubes under aerobic, unreduced conditions at 37 °C.

Emended description of Veillonella ratti (Rogosa 1965) Mays et al. 1982
The description remains as given by Rogosa (1984), except that the strain V. ratti DSM 20736^T is capable of fructose fermentation.