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1 Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada le Grazie, 15, I-37134 Verona, Italy
2 Dipartimento di Scienze Biomediche, Sezione di Microbiologia Sperimentale e Clinica, Viale San Pietro 43b, I-07100 Sassari, Italy
3 BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
4 Laboratory of Microbiology, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
Correspondence
F. Dellaglio
franco.dellaglio{at}univr.it
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was described as follows on the basis of the characteristics of seven strains isolated from pigeon crops and intestines. Cells are Gram-positive (rapidly decolourizing in the Gram-stain procedure), non-motile, very short, plump rods that mostly occur singly or in pairs. Better growth is obtained at 42 °C than at 37 or 30 °C; no growth occurs at 25 °C. Acid is produced from L-arabinose, D-fructose, methyl 
-xyloside, ribose, sucrose and D-xylose; fermentation of D-glucose, maltose, D-mannose, D-raffinose, gluconate and aesculin is variable. The DNA G+C content is 49 mol%. This species, based on 16S rRNA gene sequence analysis, belongs to the Lactobacillus reuteri phylogenetic group and highest sequences similarities were obtained to the sequences of Lactobacillus fermentum and Lactobacillus mucosae.
Lactobacillus thermotolerans Niamsup et al. 2003 was described on the basis of morphological, physiological and biochemical characteristics of five thermotolerant strains isolated from chicken faeces in Thailand. Cells are Gram-positive, non-motile, non-spore-forming, catalase-negative rods, 1x2–3 µm in size, which occur singly, in pairs or as short chains. Obligately heterofermentative, they produce both D- and L-lactic acid isomers. Growth is observed up to 50 °C, but not at 15 °C (the optimum temperature is 42 °C). meso-Diaminopimelic acid is not present in the cell wall and the DNA G+C content of the type strain is 50·5 mol%.
A comparative sequence analysis based on 16S rRNA-encoding genes obtained from GenBank (accession numbers AF333975 for L. ingluviei KR3T and AF317702 for L. thermotolerans G 35T) revealed very high similarity; only two nucleotides were found to be different (99·87 % similarity over a total of 1504 bp). Moreover, the physiological traits reported by the authors, the habitats from which these bacteria have been isolated and the observation that the two species were described in the same year (and no reciprocal comparison was therefore made) suggest the necessity of clarifying their taxonomic relationship with additional genetic approaches.
L. ingluviei LMG 20380T and L. thermotolerans DSM 14792T (=LMG 22056T) were grown in MRS broth at 37 °C under anaerobiosis and bacterial cultures were checked for purity.
In order to look at the difference between the two species, two protein-encoding genes, hsp60 and recA, were sequenced and compared. It has already been reported that sequences of protein-encoding genes have higher discriminatory power than 16S rRNA gene sequences (Felis et al., 2001; Rosselló-Mora & Amann, 2001). Sequence comparison of these genes has proven to be helpful to describe the relationships among closely related taxa (Felis et al., 2001; Torriani et al., 2001; Dellaglio et al., 2004, 2005; Bringel et al., 2005), although no threshold of sequence similarity for these protein-encoding genes has yet been proposed.
DNA of type strains LMG 20380T and DSM 14792T was extracted following the procedure of Marmur (1961). Partial hsp60 gene sequences were amplified using two degenerate primers, cpn-f and cpn-r, as described by Dellaglio et al. (2005) and amplification products of about 1000 bp were obtained. Sequencing reactions were performed at the Biomolecular Research Centre (BMR), University of Padua, Italy, with the primers used during the PCR. Sequences of 954 bp, representing about the 57 % of the complete gene (considering the complete hsp60 gene sequences NT01LP0651 and NT01LJ0492 of the genome sequences of Lactobacillus plantarum WCSF1 and Lactobacillus johnsonii NCC 533, respectively), were obtained for the two type strains and compared. A total of 11 nucleotides were found to be different.
Partial recA gene sequences were obtained using the degenerate primers recEXT-f and recEXT1-r as described by Dellaglio et al. (2005). Thermal conditions were optimized to improve amplification efficiency as follows: an initial denaturation of 5 min at 94 °C, 35 cycles of 45 s at 94 °C, 120 s at 50 °C and 105 s at 72 °C and a final extension at 72 °C for 7 min. PCR products of 750 bp, about half of the complete gene sequence considering the complete recA gene of L. plantarum WCSF1 and L. johnsonii NCC 533, were obtained for the L. ingluviei and L. thermotolerans type strains. Sequence comparison revealed seven differences from a total of 452 bp.
In order to clarify the taxonomic status of the two species further, a DNA–DNA hybridization test was performed between the two type strains L. ingluviei LMG 20380T and L. thermotolerans LMG 22056T and their G+C contents were redetermined. For strain LMG 20380T, DNA was extracted from 0·75–1·25 g wet weight of cells using the protocol described by Pitcher et al. (1989) with the following modifications: the washed cell pellet was resuspended and lysed in a buffer (10 mM Tris/HCl, 100 mM EDTA, pH 8·0) containing RNase (200 µg ml–1; Sigma), mutanolysin (100 U ml–1; Sigma) and lysozyme (25 mg ml–1; SERVA) for 1 h at 37 °C. Before addition of GES reagent (5 M guanidine thiocyanate, 100 nM EDTA, 0·5 % Sarkosyl in water), proteinase K (200 µg ml–1; Merck) was added to the mixture for 15 min. For strain LMG 22056T, DNA was extracted from 0·75–1·25 g wet weight of cells by using the protocol described by Gevers et al. (2001), using a combination of glass beads and enzymes, but with the following modifications. Volumes were increased tenfold for application on a large scale. SDS-treated cells were vortexed with beads for 30 s. After addition and gentle shaking with 16·5 ml buffer (10 mM Tris/HCl, 100 mM EDTA, pH 8·0) and 5 ml 5 M NaCl, the suspension was incubated at 65 °C for 10 min. Subsequent chloroform/isoamyl alcohol extraction, precipitation, spooling of DNA on a glass rod, washing with ethanol and RNase treatment was performed as described by Marmur (1961). For DNA–DNA hybridizations, the microplate method was used as described by Ezaki et al. (1989) and Goris et al. (1998) using an HTS7000 Bio Assay Reader (Perkin Elmer) for fluorescence measurements. Biotinylated DNA was hybridized with unlabelled single-stranded DNA, which was bound non-covalently to microplate wells. Hybridizations were performed at 41 °C in hybridization mixture (2x SSC, 5x Denhardt's solution, 2·5 % dextran sulphate, 50 % formamide, 100 µg denatured salmon sperm DNA ml–1, 1·25 µg biotinylated probe DNA ml–1). The percentage DNA relatedness presented is a mean value, based on four independent hybridization experiments. Reciprocal reactions (i.e. AxB and BxA) were performed and are also considered as independent hybridization experiments.
For determination of the DNA G+C content, DNA was enzymically degraded into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture was then separated by HPLC using a Waters SymmetryShield C8 column maintained at a temperature of 37 °C. The solvent was 0·02 M (NH4)H2PO4 (pH 4·0) with 1·5 % acetonitrile. Non-methylated 
phage DNA (Sigma) was used as the calibration reference.
Results showed that the type strains of L. ingluviei and L. thermotolerans had a binding level of 79±3 %; consequently, as currently delineated (Rosselló-Mora & Amann, 2001; Stackebrandt & Goebel, 1994), the two species constitute a single taxonomic unit. The G+C contents of the type strains were 50 mol% for L. ingluviei LMG 20380T and 49 mol% for L. thermotolerans DSM 14792T.
Considering the high value of total DNA hybridization obtained, the possibility of describing two subspecies was evaluated to group strains of the two former species. Therefore, a few fermentation tests were performed: data reported in the original species descriptions were compared and it was found that melibiose was fermented by L. thermotolerans but not by L. ingluviei. Both species are able to metabolize D-fructose and not lactose. These experiments were repeated by growing L. ingluviei LMG 20380T and L. thermotolerans DSM 14792T at 30 °C for 24–48 h in phenol red medium broth with lactose, melibiose or fructose (1 % final concentration) added as the sole carbon source. Lactose and fructose were used as negative and positive controls, respectively. The two type strains yield identical metabolic profiles, as both metabolized fructose, but neither fermented melibiose or lactose, in contrast to the description of the species L. thermotolerans (Niamsup et al., 2003).
Although the two species were isolated from slightly different environments (pigeons and chickens) and from very distant places (Europe and Asia) and display sequences of two protein-encoding genes that are not identical, the delineation of intraspecific clusters was not considered in this study. Therefore it is proposed that the two species Lactobacillus ingluviei and Lactobacillus thermotolerans should be united under the same name. According to the rules of priority (Rules 38 and 42 of the Bacteriological Code; Lapage et al., 1992), the name Lactobacillus ingluviei should be retained with its type strain KR3T (=CCUG 45722T=DSM 15946T=JCM 12531T=LMG 20380T). Consequently, strains of L. thermotolerans should be reclassified as belonging to L. ingluviei. Due to the very similar traits reported in the descriptions of the two species, an emended description of L. ingluviei is not considered necessary. The species L. ingluviei belongs to the L. reuteri phylogenetic group within the genus Lactobacillus as delineated by Hammes & Hertel (2003).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bringel, F., Castioni, A., Olukoya, D. K., Felis, G. E., Torriani, S. & Dellaglio, F. (2005). Lactobacillus plantarum subsp. argentoratensis subsp. nov., isolated from vegetable matrices. Int J Syst Evol Microbiol 55, 1629–1634.
Dellaglio, F., Torriani, S. & Felis, G. E. (2004). Reclassification of Lactobacillus cellobiosus Rogosa et al. 1953 as a later synonym of Lactobacillus fermentum Beijerinck 1901. Int J Syst Evol Microbiol 54, 809–812.
Dellaglio, F., Felis, G. E., Castioni, A., Torriani, S. & Germond, J.-E. (2005). Lactobacillus delbrueckii subsp. indicus subsp. nov., isolated from Indian dairy products. Int J Syst Evol Microbiol 55, 401–404.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Felis, G. E., Dellaglio, F., Mizzi, L. & Torriani, S. (2001). Comparative sequence analysis of a recA gene fragment brings new evidence for a change in the taxonomy of the Lactobacillus casei group. Int J Syst Evol Microbiol 51, 2113–2117.[Abstract]
Gevers, D., Huys, G. & Swings, J. (2001). Applicability of rep-PCR fingerprinting for differentiation of Lactobacillus species. FEMS Microbiol Lett 205, 31–36.[CrossRef][Medline]
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA-DNA hybridization method compared with the initial renaturation method. Can J Microbiol 44, 1148–1153.[CrossRef]
Hammes, W. P. & Hertel, C. (2003). The genera Lactobacillus and Carnobacterium. In The Prokaryotes. An Evolving Electronic Resource for the Microbiological Community, release 3.15. Edited by M. Dworkin and others. http://141.150.157.117:8080/prokPUB/index.htm
Lapage, S. P., Sneath, P. H. A., Lessel, E. F., Skerman, V. B. D., Seelinger, H. P. R. & Clark, W. A. (editors) (1992). International Code of Nomenclature of Bacteria (1990 Revision). Bacteriological Code. Washington, DC: American Society for Microbiology.
Marmur, J. (1961). A procedure for the isolation of DNA from microorganisms. J Mol Biol 3, 208–218.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Niamsup, P., Sujaya, I. N., Tanaka, M. & 8 other authors (2003). Lactobacillus thermotolerans sp. nov., a novel thermotolerant species isolated from chicken faeces. Int J Syst Evol Microbiol 53, 263–268.
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156.
Rosselló-Mora, R. & Amann, R. (2001). The species concept for prokaryotes. FEMS Microbiol Rev 25, 39–67.[Medline]
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Torriani, S., Felis, G. E. & Dellaglio, F. (2001). Differentiation of Lactobacillus plantarum, L. pentosus and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Appl Environ Microbiol 67, 3450–3454.
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