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1 Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas, PO Box 73, Burjassot, 46100 Valencia, Spain
2 Departament de Microbiología i Ecología, Facultat de Biología, Universitat de València, Campus de Burjassot, 46100 Valencia, Spain
Correspondence
Rosa Aznar
rosa.aznar{at}uv.es
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Published online ahead of print on 21 October 2005 as DOI 10.1099/ijs.0.64021-0.
A table detailing the reference strains used to test L. rennini-specific PCR primers and a dendrogram derived from 16S-ARDRA analyses are available as supplementary material in IJSEM Online.
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), includes 97 recognized species and novel taxa are continuously arising within this group. Species of the genus Lactobacillus are widespread in nature and can be found on plants or material of plant origin, in manure and man-made habitats such as sewage, in fermenting or spoiling food and also in association with the intestinal tracts and mucous membranes of man and many animals. The identification of novel isolates as separate species is not always possible by classical methods of physiological testing as they may share many phenotypic traits. At present, it is well established that, for an unequivocal identification, DNA and RNA analysis (hybridization or gene sequencing) form the basis of systematic taxonomy (Hammes & Vogel, 1995).
Chenoll et al. (2003) obtained rDNA-RFLP profiles for 91 lactic acid bacteria (LAB) reference strains, including 45 species of Lactobacillus (of which 40 were type strains). The rDNA-RFLP profiles were derived from DdeI and HaeIII digests of the internal spacer region (ISR; ISR-DdeI, ISR-HaeIII) and amplified rDNA restriction analysis (ARDRA) of the 16S rRNA gene digested with the same restriction enzymes (16S-ARDRA-DdeI and 16S-ARDRA-HaeIII). Analysis of the rDNA-RFLP profiles revealed that some reference Lactobacillus strains, deposited as Lactobacillus sp., had unique rDNA-RFLP profiles (Chenoll et al., 2003). Based on 16S rRNA gene sequence analysis, it was suggested that these strains might represent novel species since the similarity values were clearly below 97 %. Two of these strains, CECT 5922 (=DSM 20253) and CECT 5923 (=DSM 20254) isolated from rennin and causing spoilage in cheese, were deposited at the DSMZ by J. Stadhouders. In 1967, Stadhouders and Veringa reported the presence of salt-tolerant lactobacilli multiplying in rennet that caused defects in Dutch cheese (Stadhouders & Veringa, 1967). The spoiled cheeses showed many eyes and holes and, at a later stage of ripening, also showed small and large cracks and often developed a phenolic flavour. Lactobacilli were isolated from inefficiently filtered rennet and appeared to be related to species Lactobacillus casei and Lactobacillus plantarum, but differed from them, amongst other things, in their higher resistance to salt. The lactobacillus strains were able to persist in cheese for a long time during ripening and remained active by their breakdown of amino acids, which led to the production of off-flavours, acetic acid, ammonia and gas (CO2). By using this contaminated rennet, it was demonstrated that cheese could be produced which showed the defects in question (Stadhouders & Veringa, 1967).
The two strains, CECT 5922T and CECT 5923, share 99·9 % 16S rRNA gene sequence similarity and have less than 94·3 % similarity with any other species of the genus; their nearest phylogenetic neighbour is Lactobacillus coryniformis (Chenoll et al., 2003). In this work, phenotypic and genetic characterization of the two strains has confirmed that they belong to a novel species in the genus Lactobacillus.
Strains CECT 5922T and CECT 5923 were routinely grown in MRS (Oxoid) at 30 °C under microaerophilic conditions and stored in liquid growth medium containing 20 % (v/v) glycerol at –80 °C. Biochemical characterization of these strains, together with L. coryniformis subsp. torquens, was performed following standardized methods. All strains are Gram-positive and catalase-negative. Colonies of strains CECT 5922T and CECT 5923 grown on MRS agar plates for 3 days are very small (up to 1 mm in diameter), grey, smooth, circular and with entire edges. Cells are non-motile and non-spore-forming, rod-shaped, approximately 2–4x1 µm in size, though occasionally longer cells are observed, and occur mainly singly or in pairs. Strains CECT 5922T and CECT 5923 grow at 13, 25 and 37 °C, but not at 4 or 45 °C, and at pH 3·7, 4·5 and pH 8·0. Both strains grow in medium with 5 and 10 % (w/v) NaCl. Strains CECT 5922T and CECT 5923 do not produce gas from glucose or gluconate as examined with Durham tubes, revealing their homofermentative metabolism. Production of ammonia from arginine, determined as described by Kato et al. (2000), is negative. The lactic acid isomer, determined enzymically using the DL-lactate test kit (Boehringer Mannheim), is L(D).
Carbohydrate fermentation patterns and aesculin hydrolysis were evaluated using API 50 CHL galleries (bioMérieux) according to the manufacturer's instructions. Strains were incubated at 30 °C for 48 h. Strains CECT 5922T and CECT 5923 show similar fermentation profiles, but strain CECT 5922T also ferments D-mannitol, maltose, lactose and sucrose and is able to hydrolyse aesculin. Differential phenotypic traits are shown in Table 1. L. coryniformis subsp. torquens CECT 4129T differed from the novel strains in its ability to ferment 2-ketogluconate and in its inability to ferment L-arabinose, D-ribose or D-xylose and its inability to hydrolyse aesculin (Table 1).
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The 16S rRNA gene sequences of about 1500 bp of strains CECT 5922T (GenBank accession number AJ576007) and CECT 5923 (GenBank accession number AJ576008) were obtained in a previous study (Chenoll et al., 2003). Comparative analysis was performed with the full dataset of more than 22 000 reference sequences from the ARB database (http://www.arb-home.de) using the ARB program package (Ludwig et al., 2004). The ARB tools were used for automated sequence alignment and tree reconstruction. The alignment was checked visually and corrected manually using the sequence editor ARB_EDIT. Sequence similarity values were calculated by comparing nucleotides at corresponding positions and the high similarity between strains CECT 5922T and CECT 5923 (99·9 %) was confirmed. A closely related sequence, submitted to EMBL by D. Ouwerkerk & A. V. Klieve in 2000 (GenBank accession number AF316586), has 99·8 % similarity with both novel strains. This sequence is documented as belonging to a feedlot manure isolate, YE07, and identified as Lactobacillus sp. However, strains CECT 5922T and CECT 5923 had less than 94·3 % sequence similarity to any species with validly published names. The phylogenetic tree constructed using the neighbour-joining method shows that L. coryniformis subsp. torquens and L. coryniformis subsp. coryniformis are the closest phylogenetic neighbours to the novel strains (Fig. 1). Following the recommendations of Ludwig et al. (1998), alternative treeing methods (maximum-parsimony and maximum-likelihood) were used which confirmed the position of CECT 5922T and CECT 5923 in the vicinity of L. coryniformis and Lactobacillus bifermentans (data not shown).
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) which was similar to those of Lactobacillus parabuchneri and L. coryniformis subsp. torquens (see Supplementary Fig. S1 in IJSEM Online).
DNA–DNA hybridization experiments were performed as described by Ziemke et al. (1998) with DNA of strains CECT 5922T, CECT 5923 and L. coryniformis subsp. torquens CECT 4129T. The reference DNA was that of strain CECT 5922T and the results are expressed as mean percentage values based on three independent hybridization experiments. The relative binding ratio values of strain CECT 5922T to strain CECT 5923 were always above 80 % (mean 86·5, max. 93, min. 80, SD 9·2, n=3). The hybridization values between strain CECT 5922T and L. coryniformis subsp. torquens CECT 4129T were always below 60 % (mean 55·5, max. 60, min. 46·3, SD 6·4, n=3). All these results confirm that strains CECT 5922T and CECT 5923 are members of the same species, with separate status within the genus Lactobacillus.
Species-specific primers targeted to the 16S rRNA gene for the novel strains were designed using the appropriate ARB software package tool (Ludwig et al., 2004). Following the nomenclature of Alm et al. (1996), they are S-S-Lren-0060-a-S (5'-GTCGCACGCACAACTGTT-3') as the sense primer and S-S-Lren-0175-a-A (5'-GTCGCGTATTTCTATGCG-3') as the antisense primer. The primers were proved to be specific by testing in silico using the BLAST tool. In addition, specificity was tested in vitro by PCR amplification of 52 LAB reference strains (seven Carnobacterium species, 36 Lactobacillus species and nine Leuconostoc species). The strains used are listed in Supplementary Table S1 (see IJSEM Online). PCR was carried out at an annealing temperature of 56 °C, as described by Macián et al. (2004). The specific fragment of 133 bp was only amplified from strains CECT 5922T and CECT 5923.
Based on the data reported here, strains CECT 5922T and CECT 5923 are members of a novel species within the genus Lactobacillus. They show phenotypic characters that distinguish them from other homofermentative lactobacillus species and L. coryniformis, their closest phylogenetic relative (Table 1
), as well as genotypic traits that enable them to be differentiated by any of the following rDNA-RFLP profiles: 16S-ARDRA-DdeI, 16S-ARDRA-HaeIII, ISR-DdeI or ISR-HaeIII (see Supplementary Fig. S1). We propose the name Lactobacillus rennini sp. nov. for the two novel strains isolated from rennin by J. Stadhouders. The type strain is CECT 5922T (=DSM 20253T) and CECT 5923 (=DSM 20254) is a reference strain.
Description of Lactobacillus rennini sp. nov.
Lactobacillus rennini (ren.ni'ni. N.L. n. renninum rennin, an enzyme that coagulates milk and is used in making cheese; N.L. gen. n. rennini of rennin).
Cells are rod-shaped, 1 µm wide by 2–4 µm long, Gram-positive, catalase-negative, non-spore-forming and non-motile. They are found singly and in pairs. Grows better in MRS broth than on agar plates and under anaerobic conditions. After 3 days of incubation in MRS agar at 30 °C, colonies are small (up to 1 mm in diameter), circular, translucent and grey. Grows at 13 °C, but not at 4 or 45 °C. Homofermentative. No gas is produced from glucose or gluconate. Arginine is not hydrolysed. Both D- and L-lactic acid are produced. Strains ferment L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose and N-acetylglucosamine. Does not ferment glycerol, erythritol, D-arabinose, L-xylose, adonitol, methyl 
-xyloside, L-sorbose, L-rhamnose, dulcitol, inositol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, amygdalin, arbutin, salicin, D-cellobiose, melibiose, D-trehalose, inulin, melezitose, D-raffinose, starch, glycogen, xylitol, gentibiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. The type strain also ferments D-mannitol, maltose, lactose and sucrose and hydrolyses aesculin. Peptidoglycan type is L-lys–D-Asp.
The type strain, CECT 5922T (=DSM 20253T), was isolated from rennin by J. Stadhouders. Strain CECT 5923 (=DSM 20254), also isolated from rennin by J. Stadhouders, is a reference strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Stadhouders, J. & Veringa, H. A. (1967). Texture and flavour defects in cheese caused by bacteria from contaminated rennet. Neth Milk & Dairy J 21, 192–207.
Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48, 179–186.
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