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1 Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden
2 Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden
Correspondence
Stefan Roos
stefan.roos{at}mikrob.slu.se
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Lactobacillus gastricus Kx156A7T, Lactobacillus antri Kx146A4T, Lactobacillus kalixensis Kx127A2T and Lactobacillus ultunensis Kx146C1T are AY253658, AY253659, AY253657 and AY253660, respectively.
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The strains were isolated from human gastric mucosa obtained from healthy individuals from Kalix, a town in northern Sweden. Primary isolates were produced by spreading homogenates of biopsies from both the antrum and the corpus region of the stomach on Rogosa agar (Merck). The plates were incubated for 3 days in anaerobic jars under a CO2+N2 atmosphere (GasPak System, BBL) at 37 °C. All further cultivation was performed at 37 °C in anaerobic jars on MRS agar or in MRS broth (Oxoid) if not otherwise stated. Strains Kx127A2T (=LMG 22115T=DSM 16043T=CCUG 48459T), Kx146A4T (=LMG 22111T=DSM 16041T=CCUG 48456T), Kx146C1T (=LMG 22117T=DSM 16047T=CCUG 48460T), Kx146C2 (=LMG 22489=DSM 16044=CCUG 48458), Kx156A7T (=LMG 22113T=DSM 16045T=CCUG 48454T), Kx293C1 (=LMG 22490=DSM 16048=CCUG 48461), Kx293C4 (=LMG 22488=DSM 16046=CCUG 48455) and Kx329A2 (=LMG 22112=DSM 16042=CCUG 48457) have been deposited with the BCCM, DSMZ and CCUG.
Bacterial DNA was isolated using the DNeasy Tissue kit (Qiagen). The almost complete 16S rRNA gene sequences for the strains were amplified by PCR with domain Bacteria-specific primers (Weizenegger et al., 1992
). The resulting PCR products were purified by using the Qiagen PCR purification kit. The first part (about 500 bp) of the purified fragments was sequenced according to standard methods. For some strains, the whole fragment was sequenced. The primers used for amplification, together with additional internal primers, were used for sequencing of the PCR products. The sequences obtained from the isolates were used for searches in the GenBank database and levels of DNA similarity were analysed with the SIMILARITY MATRIX tool at Ribosomal Database Project II (http://rdp.cme.msu.edu/html). Sequences representing the closest matches were retrieved and then aligned using the CLUSTAL W program (Thompson et al., 1994). For all sequences, approximately 1450 nt were used. A distance matrix was calculated using the DNADIST program of the PHYLIP package (Felsenstein, 1993) with the F84 parameter model, and a phylogenetic tree was constructed with the NEIGHBOR program. Statistical significance of groupings was estimated by bootstrapping (1000 replicates) using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE, all of which are from the PHYLIP package. The phylogenetic tree was displayed by using the TREEVIEW program (Page, 1996).
Cell morphologies of the bacteria were observed by phase-contrast microscopy. Determination of Gram reactions was performed using the KOH method of Gregersen (1978). Sugar-fermentation patterns and aesculin hydrolysis were assessed using the API 50 CHL system (bioMérieux) in duplicate at 37 °C. Lactic acid configuration was determined using a test kit from Boehringer Mannheim. Catalase activity was determined by transferring fresh colonies from MRS agar to a glass slide and adding 5 % H2O2. The production of gas from glucose was assayed by growing the bacteria in MRS tubes containing Durham tubes. Electrophoretic analysis of whole-cell proteins was performed by the BCCM. The preparation of whole-cell protein extracts and SDS-PAGE analysis were performed as described by Pot et al. (1994). The normalized and digitized patterns were numerically analysed and clustered with reference profiles in the SDS-PAGE protein database of the BCCM. Cell-wall analysis was performed at the DSMZ. Preparation of cell walls and determination of peptidoglycan structure were carried out by the methods described by Schleifer (1985) and Schleifer & Kandler (1972) with the modification that TLC on cellulose sheets was used instead of paper chromatography. The G+C content of the DNA and DNA–DNA relatedness values were determined at the DSMZ. DNA was isolated by chromatography on hydroxyapatite by the procedure of Cashion et al. (1977). The G+C content was thereafter determined by HPLC as described by Mesbah et al. (1989) and DNA–DNA hybridizations were carried out as described by De Ley et al. (1970), with the modifications described by Huß et al. (1983), using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-controlled 6x6 multicell changer and a temperature controller with in-situ temperature probe (Varian).
Analysis of the 16S rRNA gene sequences of the 15 isolates differing from previously described lactobacilli revealed that they could be divided into four groups. Group G1 contained six isolates from three individuals, G2 contained two isolates from two individuals, G3 contained five isolates from two individuals and G4 contained two isolates from two individuals. Two representative isolates from each group were chosen for further analysis: G1, Kx156A7T and Kx293C4; G2, Kx146A4T and Kx329A2; G3, Kx127A2T and Kx146C2; G4, Kx146C1T and Kx293C1.
Group G1
The complete 16S rRNA gene sequence of Kx156A7T showed the highest levels of similarity to Lactobacillus mucosae, Lactobacillus thermotolerans and Lactobacillus ingluviei, all members of the Lactobacillus reuteri subgroup of lactobacilli, with 96·4, 95·4 and 95·3 % similarity, respectively. A 425 bp sequence from the 16S rRNA gene of Kx293C4 revealed 100 % similarity to that of Kx156A7T. The low level of 16S rRNA gene sequence similarity with recognized lactobacilli strongly suggests that the isolates in G1 represent a previously undescribed species. Comparison of DNA G+C content, peptidoglycan type and other characteristics with the members of the L. reuteri subgroup (Table 1) confirmed this suggestion. Furthermore, electrophoretic analysis of whole-cell proteins showed that strains Kx156A7T and Kx293C4 are distinct from the other species in this subgroup (Fig. 1). We propose that the isolates be classified in the genus Lactobacillus as Lactobacillus gastricus sp. nov. Additional characteristics of strains Kx156A7T and Kx293C4 are given under the species description.
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), indicating that Kx146A4T represents a novel species. Comparison of DNA G+C content, peptidoglycan type and other characteristics with members of the L. reuteri subgroup (Table 1
) confirmed this suggestion. Electrophoretic analysis of whole-cell proteins also showed that strains Kx146A4T and Kx329A2 are distinct from the other species in this subgroup (Fig. 1). We propose that the isolates be classified in the genus Lactobacillus as Lactobacillus antri sp. nov. Additional characteristics of strains Kx146A4T and Kx329A2 are given under the species description.
Group G3
The complete 16S rRNA gene sequence of Kx127A2T showed the highest levels of similarity to Lactobacillus intestinalis, Lactobacillus amylolyticus and Lactobacillus crispatus, all members of the Lactobacillus delbrueckii subgroup of lactobacilli, with 96·4, 95·7 and 95·6 % similarity, respectively. A 518 bp sequence from the 16S rRNA gene of Kx146C2 showed 100 % similarity to that of Kx127A2T. The low levels of 16S rRNA gene sequence similarity with recognized lactobacilli strongly suggests that the isolates in G3 represent a previously undescribed species. Comparison of DNA G+C content, peptidoglycan type and other characteristics with the closest related members of the L. delbrueckii subgroup (Table 2) confirmed this suggestion. Furthermore, electrophoretic analysis of whole-cell proteins showed that strains Kx127A2T and Kx146C2 are distinct from the other species in this subgroup (Fig. 2). We propose that the isolates be classified in the genus Lactobacillus as Lactobacillus kalixensis sp. nov. Additional characteristics of strains Kx127A2T and Kx146C2 are given under the species description.
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), indicating that Kx146C1T represents a previously undescribed species. Comparison of DNA G+C content, peptidoglycan type and other characteristics with the closest related members of the L. delbrueckii subgroup (Table 2) confirmed this suggestion. In addition, electrophoretic analysis of whole-cell proteins showed that strains Kx146C1T and Kx293C1 are distinct from the other species in this subgroup (Fig. 2). We propose that the isolates be classified in the genus Lactobacillus as Lactobacillus ultunensis sp. nov. Additional characteristics of strains Kx146C1T and Kx293C1 are given under the species description.
The 16S rRNA gene sequences of the four novel species were aligned with those of all members of the L. reuteri subgroup and the most relevant members of the L. delbrueckii subgroup. This alignment was then used for a tree analysis. The analysis placed the novel species in positions of the phylogenetic tree that correspond to what was found in the similarity rank analyses (Fig. 3).
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Cells are Gram-positive, non-motile, non-spore-forming, catalase-negative rods, 0·9x1·2 µm in size and occurring as single cells or in pairs. After anaerobic growth at 37 °C for 48 h, colonies on MRS agar are 2 mm in diameter; they are white, smooth and convex. Growth on MRS agar under aerobic conditions is very weak. In MRS broth, growth occurs at 25 and 42 °C, but not at 20 or 45 °C. Both D- and L-lactate are produced. Gas is produced from glucose. Acid is produced from L-arabinose (one of two strains), ribose (one of two strains), galactose, D-glucose, D-fructose, D-mannose, methyl 
-D-glucopyranoside, N-acetylglucosamine (one of two strains), amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, D-raffinose, 
-gentiobiose, D-turanose and gluconate. Acid is not produced from glycerol, erythritol, D-arabinose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, L-sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl -D-mannoside, inulin, melezitose, starch, glycogen, xylitol, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconate or 5-ketogluconate. Aesculin is not hydrolysed. The DNA G+C content of strain Kx156A7T is 41·3 mol% and the peptidoglycan type is A4 L-orn–D-Asp. Phylogenetic analysis of the 16S rRNA gene sequence places the species in the L. reuteri subgroup of lactobacilli.
The type strain is Kx156A7T (=LMG 22113T=DSM 16045T=CCUG 48454T), isolated from a biopsy of the healthy human gastric mucosa.
Description of Lactobacillus antri sp. nov.
Lactobacillus antri [an'tri. L. gen. n. antri of a cave (the antrum region of the stomach)].
Cells are Gram-positive, non-motile, non-spore-forming, catalase-negative rods, 1x1·2–2 µm in size and occurring as single cells or in pairs. After anaerobic growth at 37 °C for 48 h, colonies on MRS agar are 2–3·5 mm in diameter; they are white, smooth and slightly convex. Growth on MRS agar under aerobic conditions is very weak. In MRS broth, growth occurs at 25 and 45 °C, but not 20 °C. Both D- and L-lactate are produced. Gas is produced from glucose. Acid is produced from L-arabinose, ribose, D-xylose (one of two strains), galactose, D-glucose, D-fructose, mannitol (weak reaction), methyl -D-glucopyranoside (weak reaction), maltose, melibiose, sucrose, D-raffinose, D-turanose (weak reaction), D-arabitol and gluconate (weak reaction). Acid is not produced from glycerol, erythritol, D-arabinose, L-xylose, adonitol, methyl -D-xyloside, D-mannose, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl -D-mannoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, lactose, trehalose, inulin, melezitose, starch, glycogen, xylitol, -gentiobiose, D-lyxose, D-tagatose, D-fucose, L-fucose, L-arabitol, 2-ketogluconate or 5-ketogluconate. Aesculin is hydrolysed (weak reaction). The DNA G+C content of strain Kx146A4T is 44·9 mol% and the peptidoglycan type is A4 L-lys–D-Asp. Phylogenetic analysis of the 16S rRNA gene sequence places the species in the L. reuteri subgroup of lactobacilli.
The type strain is Kx146A4T (=LMG 22111T=DSM 16041T=CCUG 48456T), isolated from a biopsy of the healthy human gastric mucosa.
Description of Lactobacillus kalixensis sp. nov.
Lactobacillus kalixensis (ka.lix.en'sis. N.L. masc. adj. kalixensis pertaining to Kalix, a town in northern Sweden, where the gastric biopsies were sampled).
Cells are Gram-positive, non-motile, non-spore-forming, catalase-negative rods, 1x1·5–10 µm in size and occurring as single cells, pairs or chains. After anaerobic growth at 37 °C for 48 h, colonies on MRS agar are 2 mm in diameter; they are white, smooth and convex. Growth on MRS agar under aerobic conditions is weak. In MRS broth, growth occurs at 25 and 45 °C, but not at 20 °C. Both D- and L-lactate are produced. Gas is not produced from glucose. Acid is produced from galactose, D-glucose, D-fructose, D-mannose, mannitol (one of two strains), N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, D-raffinose, starch and -gentiobiose. Acid is not produced from glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl -D-mannoside, methyl -D-glucopyranoside, inulin, melezitose, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Aesculin is hydrolysed. The DNA G+C content of strain Kx127A2T is 35·5 mol% and the peptidoglycan type is A4 L-lys–D-Asp. Phylogenetic analysis of the 16S rRNA gene sequence places the species in the L. delbrueckii subgroup of lactobacilli.
The type strain is Kx127A2T (=LMG 22115T=DSM 16043T=CCUG 48459T), isolated from a biopsy of the healthy human gastric mucosa.
Description of Lactobacillus ultunensis sp. nov.
Lactobacillus ultunensis (ul.tun.en'sis. N.L. masc. adj. ultunensis pertaining to Ultuna, the site of Swedish University of Agricultural Sciences in Uppsala, Sweden).
Cells are Gram-positive, non-motile, non-spore-forming, catalase-negative rods, 1x2–30 µm in size and occurring as single cells, pairs or filaments. After anaerobic growth at 37 °C for 48 h, colonies on MRS agar are 2–3 mm in diameter; they are white, irregular and have a dry appearance. Growth on MRS agar under aerobic conditions is very weak. In MRS broth, growth occurs at 25 and 42 °C, but not at 20 or 45 °C. Both D- and L-lactate are produced. Gas is not produced from glucose. Acid is produced from galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, arbutin, salicin, cellobiose, maltose, lactose, melibiose (one of two strains, weak reaction), sucrose, trehalose, D-raffinose (one of two strains), starch (one of two strains) and -gentiobiose. Acid is not produced from glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, L-sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl -D-mannoside, methyl -D-glucopyranoside, amygdalin, inulin, melezitose, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Aesculin is hydrolysed. The DNA G+C content of strain Kx146C1T is 35·7 mol% and the peptidoglycan type is A4 L-lys–D-Asp. Phylogenetic analysis of the 16S rRNA gene sequence places the species in the L. delbrueckii subgroup of lactobacilli.
The type strain is Kx146C1T (=LMG 22117T=DSM 16047T=CCUG 48460T), isolated from a biopsy of the healthy human gastric mucosa.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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90 % strains positive; –, 
