International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1526–1533
DOI 10.1099/ijs.0.054924-0
Lactobacillus rodentium sp. nov., from the digestive tract of wild rodents J. Killer,1,2 J. Havlı´k,2 E. Vlkova´,2 V. Rada,2 R. Pechar,2 O. Benada,3,4 J. Kopecˇny´,1 O. Kofronˇova´3 and H. Sechovcova´1 Correspondence J. Killer [email protected] or killer@ iapg.cas.cz
1
Institute of Animal Physiology and Genetics v.v.i., Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, Prague 4 – Krcˇ 142 20, Czech Republic
2
Czech University of Life Sciences, Faculty of Agrobiology, Food and Natural Resources, Department of Microbiology, Nutrition and Dietetics, Kamy´cka´ 129, Prague 6 – Suchdol 165 21, Czech Republic
3
Laboratory of Molecular Structure Characterization, Institute of Microbiology, Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, Prague 4 – Krcˇ 142 20, Czech Republic
4
Department of Biology, Faculty of Science, J. E. Purkyneˇ University in Ustı´ nad Labem, Za Va´lcovnou 1000/8, 400 96 U´stı´ nad Labem, Czech Republic
Three strains of regular, long, Gram-stain-positive bacterial rods were isolated using TPY, M.R.S. and Rogosa agar under anaerobic conditions from the digestive tract of wild mice (Mus musculus). All 16S rRNA gene sequences of these isolates were most similar to sequences of Lactobacillus gasseri ATCC 33323T and Lactobacillus johnsonii ATCC 33200T (97.3 % and 97.2 % sequence similarities, respectively). The novel strains shared 99.2–99.6 % 16S rRNA gene sequence similarities. Type strains of L. gasseri and L. johnsonii were also most related to the newly isolated strains according to rpoA (83.9–84.0 % similarities), pheS (84.6–87.8 %), atpA (86.2–87.7 %), hsp60 (89.4–90.4 %) and tuf (92.7–93.6 %) gene sequence similarities. Phylogenetic studies based on 16S rRNA, hsp60, rpoA, atpA and pheS gene sequences, other genotypic and many phenotypic characteristics (results of API 50 CHL, Rapid ID 32A and API ZYM biochemical tests; cellular fatty acid profiles; cellular polar lipid profiles; end products of glucose fermentation) showed that these bacterial strains represent a novel species within the genus Lactobacillus. The name Lactobacillus rodentium sp. nov. is proposed to accommodate this group of new isolates. The type strain is MYMRS/TLU1T (5DSM 24759T5CCM 7945T).
Representatives of the genus Lactobacillus are phylogenetically classified into the bacterial phylum Firmicutes, class Bacilli, order Lactobacillales and family Lactobacillaceae. Abbreviations: ITS, intergenic transcribed spacer; MLST, multilocus sequence typing. The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA gene sequences of strains MYMRS/TLU1T, MYTPY/TEN3 and MYMRS/TEN2 are HQ851022, HQ851020 and HQ851021, respectively. The accession numbers for the partial hsp60, pheS, rpoA and atpA gene sequences of strains MYMRS/TLU1T, MYTPY/TEN3 and MYMRS/TEN2 are JQ363678, JQ363679 and KF841362; JQ363687, JQ363688 and KF841360; JQ363696, KF841355 and KF841356; and JQ363703, KF841367 and KF841368, respectively. The accession numbers for the partial Tuf gene sequences of strains MYMRS/TLU1T and MYTPY/TEN3 are JQ363713 and JQ363714, respectively. The accession number for the partial ITS of strain MYMRS/ TLU1T is JQ363710. Six supplementary figures and one supplementary table are available with the online version of this paper.
1526
Various species of lactobacilli have been isolated from different environments rich in complex organic substrates. They are found on plant surfaces, and in plant-origin fermented products, milk, dairy and meat products, fish and marinated fish, oral cavities of mammals, urogenital tracts of mammals and gastrointestinal tract of vertebrates and insects (Hammes & Hertel, 2009; Forsgren et al., 2010). More than 90 different species of the genus Lactobacillus have been described (Hammes & Hertel, 2009). However, the List of Prokaryotic names with Standing in Nomenclature (available at http://www.bacterio.net/lactobacillus.html) includes one hundred and seventy species and subspecies of the genus Lactobacillus at the time of writing. Many bacterial species that were assigned to the genus Lactobacillus have recently been reclassified into new genera within the family Lactobacillaceae (Mora et al., 2003; Endo & Okada, 2008; Salvetti et al., 2011). Lactobacilli are generally considered as safe, non-pathogenic bacteria. In addition, many species of lactobacilli are used as probiotics in human and veterinary 054924 G 2014 IUMS Printed in Great Britain
Lactobacillus rodentium sp. nov.
medicine (Ohashi & Ushida, 2009). Nevertheless, some species of lactobacilli, e.g. Lactobacillus rhamnosus, may act as pathogens and opportunistic pathogens (Gouriet et al., 2012). Although most species of lactobacilli inhabiting the gastrointestinal tract of humans and animals are found in various animal species, some species of lactobacilli appear to be host-specific. Typical examples are some of the species inhabiting the digestive tract of horses, poultry and rodents (Mitsuoka & Fujisawa, 1987; Mukai et al., 2003; Morita et al., 2009). Species such as Lactobacillus hamsteri, Lactobacillus intestinalis, Lactobacillus johnsonii and Lactobacillus murinus are common bacterial symbionts of rodents (Mitsuoka & Fujisawa, 1987; Fujisawa et al., 1990; Clavel et al., 2010; Tannock et al., 2012). New bacterial strains isolated from the digestive tract of wild mice are characterized in this study. They represent a novel species of the genus Lactobacillus related to L. johnsonii and Lactobacillus gasseri, thus bacterial species that are often used in human medicine as probiotics (Morita et al., 2006; Yamano et al., 2006). Two individuals of wild mice (Mus musculus) were captured in cage traps in Central Bohemia (locality Jesˇetice) in 2010. Live animals were transported to the laboratory and euthanized with carbon dioxide. Fresh samples from the rectum and small intestine were aseptically transferred into tubes containing anaerobic TPY broth (Scardovi, 1986) and serially diluted in the same medium. One millilitre of each serially diluted sample was placed on TPY, M.R.S. or Rogosa agar (Oxoid) and incubated under anaerobic conditions (Anaerobic jars, Oxoid) at 37 uC for 72 h. Bacterial colonies were then immediately transferred into tubes containing anaerobic TPY or M.R.S. broth and cultivated at 37 uC for 24 h. Nearly complete 16S rRNA gene fragments were amplified in bacterial isolates using the fD1 and rP2 primers (Weisburg et al., 1991). Purified 16S rRNA gene fragments were subsequently sequenced by using an automatic genetic analyser, ABI PRISM 3130xl (Applied Biosystems). Resulting sequences were compared with sequences deposited in GenBank using the BLAST tool. Similarity of 16S rRNA gene sequences of bacterial strains from wild rodents to those of the most closely related bacterial species were calculated by the jPHYDIT program based on results of BLAST searching. The 16S rRNA gene sequences of type strains of the most closely related species were selected for similarity calculations. Pure bacterial isolates morphologically most similar to lactobacilli (regular long rods occurring singly or in short chains) were marked as MYMRS/TLU1T, MYTPY/TEN3 and MYMRS/TEN2. Strains were isolated from the colon (MYMRS/TLU1T) and small intestine (MYTPY/TEN3 and MYMRS/TEN2) of mice. Highest observed similarity of their 16S rRNA gene sequences was with L. gasseri ATCC 33323T (97.3 %; GenBank accession number AF519171) and L. johnsonii ATCC 33200T (97.2 %; AJ002515). Strains from the digestive tract of mice showed ¢99.2 % sequence similarity to each other. At the present time, it should be http://ijs.sgmjournals.org
mandatory to test the genomic uniqueness of a novel bacterial species if 16S rRNA gene sequence similarity to other species is in the range 98.7–99 % (Stackebrandt & Ebers, 2006). However, the MLST (multilocus sequence typing) technique was chosen to confirm the status of the novel bacterial species in this study. This technique has been used for all three strains. Partial sequences of hsp60 (heatshock protein 60 kDa; Dobson et al., 2002), pheS (phenylalanyl t-RNA synthase alpha subunit; Naser et al., 2005b), rpoA (RNA polymerase alpha subunit; Naser et al., 2005b), atpA (ATP synthase alpha subunit; Naser et al., 2005a) and Tuf (putative elongation factor Tu; Ventura et al., 2003) genes were amplified, sequenced and compared with those of the most closely related bacterial strains as described above. The ITS (intergenic transcribed spacer) region was also sequenced in the type strain MYMRS/TLU1T (Dobson et al., 2002). The highest hsp60, pheS, rpoA, atpA and Tuf gene sequence similarities of the strains from wild mice were found to those of L. gasseri CIP 102991T (89.4–89.7 %; GenBank accession number HE573894), L. gasseri LMG 18177 (85.8–87.7 %; AM284240), L. gasseri CIP 102991T (84.0 %; HE573912), L. gasseri LMG 9203T (86.6–86.7 %; AM087875) and L. gasseri ATCC 33323T (92.7–93.6 %; AY372047), respectively. The highest ITS region sequence similarity obtained for strain MYMRS/TLU1T was observed with L. gasseri JCM 1025 (81.5 %; GenBank accession number AF182721). These relatively low levels of sequence similarity confirm the conclusions that new isolates represent a novel species within the genus Lactobacillus. Notably, we found high 16S rRNA, hsp60, rpoA, Tuf and atpA sequence similarities between the type strains of L. gasseri and L. johnsonii with values of 99.2 % (GenBank accession numbers AF519171 and AJ002515), 96.5 % (HE573894 and HE573895), 97.8 % (HE573912 and HE573911), 97.4 % (AY372047 and AY372036) and 93.6 % (AM087875 and AM087879), respectively. In addition, a high value of 99.1 % ITS (GenBank accession numbers AF182721 and EU547283) sequence similarity was determined between these two species of lactobacilli. Based on these results, further sophisticated analyses will be needed to decide whether these bacterial strains really represent two different bacterial (sub)species. It was necessary to confirm the results based on sequence similarities of phylogenetic markers by phylogenetic analysis and determination of phenotypic characteristics. Thus, 16S rRNA genes of type strains of species belonging to the genus Lactobacillus and representatives of some related bacterial genera were obtained from the GenBank database. Sequences were then aligned using the CLUSTAL W algorithm in the MEGA 5.05 program (Tamura et al., 2011). The alignments were improved by removing hypervariable positions using the program Gblocks under the default conditions (Castresana, 2000). The same procedure was also used in the case of phylogenetic analyses based on hsp60, rpoA, atpA and pheS gene sequences. The maximum-likelihood algorithm within the MEGA 5.05 program was used for phylogenetic tree reconstructions. Resulting phylogenetic trees based on 16S 1527
J. Killer and others
1528 T 94 Lactobacillus gallinarum ATCC 33199 (AJ417737) Lactobacillus helveticus DSM 20075T (AM113779) Lactobacillus ultunensis DSM 16047T (AY253660)
0.02
Lactobacillus crispatus ATCC 33820T (AF257097) 96 71
Lactobacillus kitasatonis JCM 1039T (AB107638) Lactobacillus hamsteri CCUG 30726T (AJ306299) Lactobacillus kalixensis Kx127A2T (AY253657)
98
Lactobacillus intestinalis DSM 6629T (AJ306298) Lactobacillus equicursoris DI70T (AB290830) 90
Lactobacillus gasseri ATCC 33323T (AF519171) Lactobacillus johnsonii ATCC 33200T (AJ002515) Lactobacillus iners CCUG 28746T (Y16329)
99 80
Lactobacillus acidophilus DSM 20079T (AY773947) Lactobacillus amylovorus DSM 20531T (AY944408)
100
Lactobacillus rodentium MYMRS/TEN2T (HQ851021) Lactobacillus rodentium MYMRS/TLU1T (HQ851022)
International Journal of Systematic and Evolutionary Microbiology 64
Lactobacillus rodentium MYTPY/TEN3T (HQ851020) Lactobacillus jensenii ATCC 25258T (AF243176) Lactobacillus psittaci DSM 15354T (AJ272391)
90
Lactobacillus amylophilus DSM 20533T (M58806) Lactobacillus vaginalis ATCC 49540T (AF243177) 100
Lactobacillus kimchii DSM 13961T (AB107638) Lactobacillus mindensis DSM 14500T (AJ313530)
Lactobacillus concavus AS 1.5017T (AY683322) Atopostipes suicloacalis DSM 15692T (AF445248)
77
Carnobacterium inhibens CCUG 31728T (Z73313)
100 79
Streptococcus caballi DSM 19004T (EF364098) 83
Enterococcus caccae CCUG 51564T (AY943820) Enterococcus faecium LMG 11423T (AJ301830)
Fig. 1. Unrooted phylogenetic tree of members of the genus Lactobacillus and family Enterococcaceae showing the position of strains representing Lactobacillus rodentium sp. nov. Reconstructed by the maximum-likelihood method based on 16S rRNA gene sequences (length of 1397 nt) using MEGA version 5.05 software and the Jukes–Cantor model. Bootstrap values, expressed as percentages of 1000 datasets, are given at nodes. Numbers in parentheses correspond to GenBank accession numbers. Bar, 0.02 substitutions per nucleotide position.
Lactobacillus rodentium sp. nov.
rRNA (Fig. 1), hsp60, rpoA, atpA and pheS (Figs S1–4, available in the online Supplementary Material) gene sequences revealed that the new bacterial strains MYMRST/ LU1T, MYTPY/TEN3 and MYMRS/TEN2 are situated on a separate phylogenetic branch between the species L. gasseri, L. johnsonii and Lactobacillus iners, respectively. Greater difference in sequence similarity among the strains studied was detected only in the case of the pheS gene. This fact is shown in the phylogenetic tree based on pheS gene sequences (Fig. S4). The concatenation of genes has been shown to be extremely useful in order to infer bacterial phylogeny (Teichmann & Mitchison, 1999). Phylogenetic trees reconstructed on the basis of the concatenated amino acid
sequences of the hsp60, pheS and rpoA genes demonstrated that the new bacterial strains are closely related to each other, but phylogenetically distinct from L. gasseri and L. johnsonii (data not shown). The DNA G+C contents of all new strains, L. gasseri DSM 20243T and L. johnsonii DSM 10533T were determined using the enzymic degradation method with some modifications as described previously (Killer et al., 2011). The DNA G+C content of strain MYMRS/TLU1T was 43.7 mol% (mean of three experiments, SD50.2). The DNA G+C contents for strains MYTPY/TEN3 and MYMRS/TEN2 were 43.4 and 44.1 mol%, respectively (Table 1). A range of values from 32
Table 1. Differences in DNA G+C contents, growth characteristics and results of biochemical tests in strains representing a novel species within the genus Lactobacillus and the most closely related species based on phylogenetic analyses Strains: 1, Lactobacillus rodentium sp. nov. MYMRS/TLU1T; 2, L. rodentium sp. nov. MYMRS/TEN2; 3, L. rodentium sp. nov. MYTPY/TEN3; 4, L. gasseri DSM 20243T; 5, L. johnsonii DSM 10533T. All data from this study. All strains utilized D-glucose, D-fructose, maltose and sucrose. None of the strains produced acids from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, inulin, melezitose, starch, glycogen, xylitol, turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. All strains were positive for a-glucosidase, b-glucosidase, arginine arylamidase, tyrosine arylamidase, phenylalanine arylamidase, leucine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase and serine arylamidase. All strains were negative for urease, arginine dihydrolase, 6-phospho-b-galactosidase, a-arabinosidase, b-glucuronidase, a-fucosidase, nitrate reduction, indole production, alkaline phosphatase, glutamyl glutamic acid arylamidase, esterase lipase (C8), lipase (C14), trypsin, a-chymotrypsin, a-mannosidase, gelatin hydrolysis, catalase and oxidase. +, Positive reaction; 2, negative reaction; W, weakly positive reaction. Characteristic DNA G+C content (mol%) Temperature range for growth (uC) pH range for growth API 50 CHL D-Galactose D-Mannose Amygdalin Aesculin Salicin Arbutin Gentiobiose Cellobiose Lactose Melibiose Raffinose N-Acetylglucosamine D-Tagatose RAPID ID 32A, API ZYM a-Galactosidase b-Galactosidase Glutamic acid decarboxylase Glycine arylamidase Leucyl glycine arylamidase N-Acetyl-b-glucosaminidase Proline arylamidase Pyroglutamic acid arylamidase Esterase (C4) Naphthol-AS-BI-phosphohydrolase
http://ijs.sgmjournals.org
1
2
3
4
5
43.7 15–46 5.0–9.5
44.1 15–46 5.0–9.5
43.4 15–46 5.0–9.5
34.7 20–45 4.5–9.0
34.3 20–45 4.5–9.0
+ 2
2 + 2 2 2 2
+ +
2 + + + + + + + + 2 2 + +
+ + + + + + + + + 2 2 + 2
+ 2 + + 2 + + + 2 2
2 + + 2 + + 2 2 +
W W W
2 + + + + + W
2 + + + + + 2 2 2
W W W
2
W
W
2
W
+ + + +
2 2
2
W
+
2 + + W
2 2 2 2
W
+ + 2 2 2 2 2 2
W
W
W
W
+
+
W
1529
J. Killer and others
to 55 mol% DNA G+C was found among species in the genus Lactobacillus (Hammes & Hertel, 2009). Values observed in isolates from the digestive tract of wild rodents were higher in comparison with those of the type strains of L. gasseri (34.7 mol%) and L. johnsonii (34.3 mol%), the most closely related species within the genus Lactobacillus according to MLST. DNA–DNA hybridization was subsequently used to confirm the status of the novel bacterial species because the 16S rRNA gene similarity to the closest related species was greater than 97 % (Tindall et al., 2010). DNA–DNA hybridizations were performed among strains MYMRS/ TLU1T, L. gasseri DSM 20243T and L. johnsonii DSM 10533T. The new isolates from wild rodents were also tested to confirm whether they really belong to the same bacterial species. DNA was extracted from 0.75–1.25 g (wet wt) by using the protocol described by Gevers et al. (2001). 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 singlestranded DNA, which was bound non-covalently to microplate wells. Hybridizations were performed at 37 uC in hybridization mixture. Relative binding of DNA from strain MYMRS/TLU1T with the type strain of L. gasseri and L. johnsonii gave values of 12.8 (mean of three experiments, SD50.7) and 14.2 % (SD50.3), respectively. Reciprocal values of DNA–DNA relatedness of L. gasseri and L. johnsonii strains with respect to strain MYMRS/TLU1T were 13.4 (SD50.1) and 14.4 % (SD50.3), respectively. The values for DNA–DNA binding among the three new strains were in the range 80.6–90.8 %. The results confirmed that the new strains from wild mice represent a novel taxon within the genus Lactobacillus (Stackebrandt et al., 2002). The substrate utilization and enzyme activity patterns of the three new bacterial strains, L. gasseri DSM 20243T and L. johnsonii DSM 10533T were determined using API 50 CHL, Rapid ID 32A and API ZYM test strips (bioMe´rieux). All strains were also tested for oxidase activity (Lui & Jurtshuk, 1986) and hydrolysis of gelatin by the API 20E system (bioMe´rieux). Effect of temperature and pH on the growth of the above-mentioned bacterial strains was assessed as described previously (Killer et al., 2013). Table 1 shows the main phenotypic differences among the new bacterial strains and the most closely related species according to the results of the 16S rRNA gene sequence similarities. They differ from each other in fermentation of thirteen carbohydrates and production of ten enzymes. Capillary isotachophoresis was used for determination of the end products of hexose catabolism in strain MYMRST/ LU1T according to Killer et al. (2011). The strain was also tested for production of D- and L-lactic acid by the D-/ L-lactic acid kit (Megazyme). Gas production from glucose was assayed using a Durham tube in MRS broth. Lactic acid at a concentration of 124.3 mmol l21 (70 % of all short-chain fatty acids produced) was the main end 1530
product of glucose fermentation. Acetic and propionic acids were also quantified in the strain at concentrations of 44.4 mmol l21 (25 %) and 9.4 mmol l21 (5 %), respectively. These results, along with the inability to utilize pentoses and gluconates, and produce gas from glucose, suggest the new strain belongs to the group of obligately homofermentative lactobacilli (type A of glucose fermentation according to Hammes & Hertel, 2009). Cells of strain MYMRS/TLU1T produced only L-lactic acid from D-glucose. Scanning electron microscopy (Killer et al., 2009) was used to determine detailed cell morphology in strain MYMRS/ TLU1T. Cells of this strain were regular, long rods occurring mostly singly and occasionally in pairs (Fig. S5). Susceptibility to 33 different antibiotics and two chemotherapeutics by the disc diffusion method (Vlkova´ et al., 2006) was assessed as an additional phenotypic characteristic of strain MYMRS/TLU1T. The tested strain was susceptible to cephalosporines (five out of eight tested), macrolides and penicillin-derived antibiotics and resistant to cephalosporines (three out of eight tested), aminoglycosides, fluoroquinolones and sulfonamides. Similar results have recently been found in different species of lactobacilli (Karapetkov et al., 2011). All results of susceptibility testing are shown in Table S1. Cellular fatty acid profiling was performed in all new strains from the digestive tract of wild mice, L. gasseri DSM 20243T and L. johnsonii DSM 10533T by the method
Table 2. Cellular fatty acid profiles of strains representing Lactobacillus rodentium sp. nov. and the most closely related species based on phylogenetic analyses Strains: 1, L. gasseri DSM 20243T; 2, L. johnsonii DSM 10533T; 3, L. rodentium sp. nov. MYMRS/TLU1T; 4, L. rodentium sp. nov. MYMRS/TEN2; 5, L. rodentium sp. nov. MYTPY/TEN3. Relative proportions (%; w/v) of fatty acids were calculated. Other fatty acids were not characterized using 37 comp. FAME Mix standard (Supelco). Fatty acid C8 : 0 C10 : 0 C11 : 0 C12 : 0 C14 : 0 C16 : 1 C17 : 1 C18 : 1v9t C18 : 1v9c C18 : 2v6s C20 : 0 C23 : 0 C22 : 2 C24 : 0
1 1.3 5.2 1.9 2.2 ,1.0 ,1.0 2.8 1.8 1.3 6.4 1.4 40.5 23.4 1.9
2 1.3 5.8 1.6 1.5 ,1.0 1.9 1.8 ,1.0 2.1 5.9 ,1.0 40.8 24.7 ,1.0
3 3.3 5.8 3.2 ,1.0 1.8 5.2 23.9 ,1.0 ,1.0 1.3 3.2 22.4 24.7 13.0
4 2.1 7.4 3.3 ,1.0 2.2 9.9 29.0 ,1.0 ,1.0 1.3 5.5 19.0 20.8 10.4
5 4.0 6.5 3.0 ,1.0 2.8 5.8 19.5 ,1.0 ,1.0 1.5 3.9 20.6 19.4 8.5
International Journal of Systematic and Evolutionary Microbiology 64
Lactobacillus rodentium sp. nov.
described in our previous study (Killer et al., 2009). For the purposes of this analysis, all strains were grown under identical conditions in MRS broth at 37 uC for 48 h. Higher relative proportions of C17 : 1, C24 : 0 and C16 : 1 fatty acids (Table 2) were determined in strains MYMRS/ TLU1T, MYMRS/TEN2 and MYTPY/TEN3 compared with strains L. gasseri DSM 20243T and L. johnsonii DSM 10533T, identified as the most closely related species on the basis of the results of MLST. The latter two species of the genus Lactobacillus had very similar profiles of cellular fatty acids. This fact, together with relatively high levels of sequence similarity of phylogenetic markers, suggests a close relationship of these species. On the other hand, the new strains had lower relative proportions of C23 : 0 fatty acid in comparison to L. gasseri DSM 20243T and L. johnsonii DSM 10533T. Notably, relatively high proportions of C22 : 2 fatty acid were found in all strains tested. The peptidoglycan structure type A4a L-Lys-D-Asp (A11.31 according to the DSMZ catalogue of strains; DSMZ, 2001) was determined in strain MYMRS/TLU1T by the Identification Service Department of DSMZ (Braunschweig, Germany) by methods described previously (Killer et al., 2010). The total hydrolysate of the peptidoglycan contained the amino acids alanine, glycine, threonine, serine, asparagine, glutamic acid and lysine in an approximate ratio of 0.9 : 0.4 : 0.4 : 0.4 : 1.0 : 0.9. The above-mentioned type of peptidoglycan was determined in the majority of known species of lactobacilli (Hammes & Hertel, 2009). The profile of cellular polar lipids was determined in strain MYMRS/TLU1T as an additional chemotaxonomic characteristic. Analysis of cellular polar lipids was provided by the Identification Service Department of DSMZ according to the methods of Bligh & Dyer (1959) and Tindall et al. (2007). Bacterial strain MYMRS/TLU1T was cultivated under anaerobic conditions in MRS medium for 24 h at 37 uC. Bacterial cells were then centrifuged and lyophilized. Diphosphatidylglycerol, phosphatidylglycerol, two phospholipids and several different glycolipids were determined in cells of strain MYMRS/TLU1T (Fig. S6). Phosphatidylethanolamine, phosphatidylglycerol and phospholipids were previously detected in lactobacilli (Arbogast & Henderson, 1975; Kim et al., 2011; Liang et al., 2011). All results of genotypic and phenotypic characteristics suggest that the new bacterial isolates from the gastrointestinal tract of wild rodents may be classified in a novel species for which the name Lactobacillus rodentium sp. nov. is proposed. Description of Lactobacillus rodentium sp. nov. Lactobacillus rodentium (ro.den9ti.um. L. gen. n. rodentium of gnawers, referring to the fact that the organism was isolated from wild rodents). Cells are Gram-positive, catalase- and oxidase-negative. Cell morphology is typical of that for most species of lactobacilli: regular, long rods (0.8–1.2 mm wide and http://ijs.sgmjournals.org
2.1–8.6 mm long) occurring mostly singly and occasionally in pairs. Grows best in anaerobic TPY broth and on M.R.S. agar. However, also grows under microaerophilic conditions (CampyGen, Oxoid). Colonies on M.R.S. agar under anaerobic conditions after 72 h are cream in colour, regular disc-shaped and rigid. The edges of the colonies are sharply defined, regular and blunt in profile. Colony size is in the range from 0.46 to 0.68 mm in diameter. Grows at 15–46 uC and pH 5–9.5. Utilizes D-glucose, Dfructose, maltose, melibiose and sucrose. Does not produce acids from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-Dxylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, inulin, trehalose, arbutin, melezitose, starch, glycogen, xylitol, D-tagatose, turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Variable in utilization of D-galactose, D-mannose, amygdalin, aesculin, salicin, gentiobiose, cellobiose, lactose, raffinose and Nacetylglucosamine. Positive for a-glucosidase, b-glucosidase, b-galactosidase, arginine arylamidase, tyrosine arylamidase, phenylalanine arylamidase, leucine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, esterase (C4) (weakly positive reaction) and serine arylamidase. Negative for urease, arginine dihydrolase, 6-phospho-b-galactosidase, a-arabinosidase, b-glucuronidase, a-fucosidase, pyroglutamic acid arylamidase, nitrate reduction, indole production, alkaline phosphatase, glutamyl glutamic acid arylamidase, proline arylamidase, N-acetyl-b-glucosaminidase, esterase lipase (C8), lipase (C14), trypsin, a-chymotrypsin, a-mannosidase, gelatin hydrolysis, catalase and oxidase. Variable in production of a-galactosidase, glutamic acid decarboxylase, glycine arylamidase, leucyl glycine arylamidase and naphthol-AS-BIphosphohydrolase. The peptidoglycan structure type is A4a L-Lys-D-Asp. Diphosphatidylglycerol, phosphatidylglycerol, two phospholipids and several different glycolipids are present. Susceptible to cephalosporines, macrolides, penicillin-derived antibiotics and chemotherapeutic mupirocine. Resistant to aminoglycosides, fluoroquinolones, sulfonamides and chemotherapeutic metronidazole (Table S1). Major fatty acids in cells are C17 : 1, C22 : 2, C23 : 0 and C24 : 0 (Table 2). DNA G+C content is in the range 43.4 to 44.1 mol%. The type strain, MYMRS/TLU1T (5DSM 24759T5CCM 7945T) was isolated from the colon of wild mice (Mus musculus) captured in cage traps in Central Bohemia (locality Jesˇetice) in 2010. The DNA G+C content of the type strain is 43.7 mol%. Additional strains of the species are MYMRS/TEN2 and MYTPY/TEN3.
Acknowledgements This study was supported by the Grant Agency of the Czech Republic (projects GA CR 304/11/1252 and GA13 - 08803S), by the 1531
J. Killer and others Institutional Research Project of the Institute of Animal Physiology and Genetics, Acad. Sci. CR (RVO: 67985904) and by the Institutional Research Concept RVO 61388971.
sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees. Syst Appl Microbiol 33, 359–366. Killer, J., Kopecˇny´, J., Mra´zek, J., Koppova´, I., Havlı´k, J., Benada, O. & Kott, T. (2011). Bifidobacterium actinocoloniiforme sp. nov. and
References
Bifidobacterium bohemicum sp. nov., from the bumblebee digestive tract. Int J Syst Evol Microbiol 61, 1315–1321.
Arbogast, L. Y. & Henderson, T. O. (1975). Effect of inhibition of
Killer, J., Mra´zek, J., Bunesˇova´, V., Havlı´k, J., Koppova´, I., Benada, O., Rada, V., Kopecˇny´, J. & Vlkova´, E. (2013). Pseudoscardovia suis gen.
protein synthesis on lipid metabolism in Lactobacillus plantarum. J Bacteriol 123, 962–971. Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid
extraction and purification. Can J Biochem Physiol 37, 911–917. Castresana, J. (2000). Selection of conserved blocks from multiple
alignments for their use in phylogenetic analysis. Mol Biol Evol 17, 540–552. Clavel, T., Saalfrank, A., Charrier, C. & Haller, D. (2010). Isolation
of bacteria from mouse caecal samples and description of Bacteroides sartorii sp. nov. Arch Microbiol 192, 427–435. Dobson, C. M., Deneer, H., Lee, S., Hemmingsen, S., Glaze, S. & Ziola, B. (2002). Phylogenetic analysis of the genus Pediococcus,
including Pediococcus claussenii sp. nov., a novel lactic acid bacterium isolated from beer. Int J Syst Evol Microbiol 52, 2003–2010.
nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of wild pigs (Sus scrofa). Syst Appl Microbiol 36, 11–16. Kim, H. J., Eom, S. J., Park, S. J., Cha, C. J. & Kim, G. B. (2011).
Lactobacillus alvi sp. nov., isolated from the intestinal tract of chicken. FEMS Microbiol Lett 323, 83–87. Liang, Z. Q., Srinivasan, S., Kim, Y. J., Kim, H. B., Wang, H. T. & Yang, D. C. (2011). Lactobacillus kimchicus sp. nov., a b-glucosidase-
producing bacterium isolated from kimchi. Int J Syst Evol Microbiol 61, 894–897. Lui, J.-K. & Jurtshuk, P. (1986). N,N,N’-N’-tetramethyl-p-phenylenediamine-dependent cytochrome oxidase analyses of Bacillus species. Int J Syst Bacteriol 36, 38–46.
DSMZ.
Mitsuoka, T. & Fujisawa, T. (1987). Lactobacillus hamsteri, a new species from the intestine of hamsters. Proc Jpn Acad, Ser B, Phys Biol Sci 63, 269–272.
Endo, A. & Okada, S. (2008). Reclassification of the genus Leuco-
Mora, D., Scarpellini, M., Franzetti, L., Colombo, S. & Galli, A. (2003).
DSMZ (2001). Catalogue of Strains, 7th edn, p. 617. Braunschweig:
nostoc and proposals of Fructobacillus fructosus gen. nov., comb. nov., Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb. nov. and Fructobacillus pseudoficulneus comb. nov. Int J Syst Evol Microbiol 58, 2195–2205. 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. Forsgren, E., Olofsson, T., Va´squez, A. & Fries, I. (2010). Novel lactic
acid bacteria inhibiting Paenibacillus larvae in honey bee larvae. Apidologie (Celle) 41, 99–108. Fujisawa, T., Itoh, K., Benno, Y. & Mitsuoka, T. (1990). Lactobacillus
intestinalis (ex Hemme 1974) sp. nov., nom. rev., isolated from the intestines of mice and rats. Int J Syst Bacteriol 40, 302–304.
Reclassification of Lactobacillus maltaromicus (Miller et al. 1974) DSM 20342T and DSM 20344 and Carnobacterium piscicola (Collins et al. 1987) DSM 20730T and DSM 20722 as Carnobacterium maltaromaticum comb. nov. Int J Syst Evol Microbiol 53, 675–678. Morita, H., He, F., Kawase, M., Kubota, A., Hiramatsu, M., Kurisaki, J. & Salminen, S. (2006). Preliminary human study for possible alteration
of serum immunoglobulin E production in perennial allergic rhinitis with fermented milk prepared with Lactobacillus gasseri TMC0356. Microbiol Immunol 50, 701–706. Morita, H., Nakano, A., Shimazu, M., Toh, H., Nakajima, F., Nagayama, M., Hisamatsu, S., Kato, Y., Takagi, M. & other authors (2009). Lactobacillus hayakitensis, L. equigenerosi and L. equi, pre-
dominant lactobacilli in the intestinal flora of healthy thoroughbreds. Anim Sci J 80, 339–346.
Gevers, D., Huys, G. & Swings, J. (2001). Applicability of rep-
Mukai, T., Arihara, K., Ikeda, A., Nomura, K., Suzuki, F. & Ohori, H. (2003). Lactobacillus kitasatonis sp. nov., from chicken intestine. Int J
PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 205, 31–36.
Syst Evol Microbiol 53, 2055–2059.
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. Gouriet, F., Million, M., Henri, M., Fournier, P. E. & Raoult, D. (2012).
Lactobacillus rhamnosus bacteremia: an emerging clinical entity. Eur J Clin Microbiol Infect Dis 31, 2469–2480. Hammes, W. P. & Hertel, C. (2009). Genus Lactobacillus. In Bergey’s
Manual of Systematic Bacteriology, 2nd edn, vol 3, pp. 465–511. Edited by P. De Vos, G. M. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey, K.-H. Schleifer & W. B. Whitman. New York: Springer. Karapetkov, N., Georgieva, R., Rumyan, N. & Karaivanova, E. (2011).
Antibiotic susceptibility of different lactic acid bacteria strains. Benef Microbes 2, 335–339. Killer, J., Kopecny´, J., Mra´zek, J., Rada, V., Benada, O., Koppova´, I., Havlı´k, J. & Straka, J. (2009). Bifidobacterium bombi sp. nov., from the
bumblebee digestive tract. Int J Syst Evol Microbiol 59, 2020–2024.
Naser, S., Thompson, F. L., Hoste, B., Gevers, D., Vandemeulebroecke, K., Cleenwerck, I., Thompson, C. C., Vancanneyt, M. & Swings, J. (2005a). Phylogeny and identification of Enterococci by atpA gene
sequence analysis. J Clin Microbiol 43, 2224–2230. Naser, S. M., Thompson, F. L., Hoste, B., Gevers, D., Dawyndt, P., Vancanneyt, M. & Swings, J. (2005b). Application of multilocus
sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151, 2141–2150. Ohashi, Y. & Ushida, K. (2009). Health-beneficial effects of probiotics:
its mode of action. Anim Sci J 80, 361–371. Salvetti, E., Felis, G. E., Dellaglio, F., Castioni, A., Torriani, S. & Lawson, P. A. (2011). Reclassification of Lactobacillus catenaformis
(Eggerth 1935) Moore and Holdeman 1970 and Lactobacillus vitulinus Sharpe et al. 1973 as Eggerthia catenaformis gen. nov., comb. nov. and Kandleria vitulina gen. nov., comb. nov., respectively. Int J Syst Evol Microbiol 61, 2520–2524. Scardovi, V. (1986). Genus Bifidobacterium Orla-Jensen 1924, 472AL.
Killer, J., Kopecˇny´, J., Mra´zek, J., Havlı´k, J., Koppova´, I., Benada, O., Rada, V. & Kofronˇova´, O. (2010). Bombiscardovia coagulans gen. nov.,
In Bergey’s Manual of Systematic Bacteriology, vol. 2, pp. 1418–1434. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharp & J. G. Holt. Baltimore: Williams and Wilkins.
1532
International Journal of Systematic and Evolutionary Microbiology 64
Lactobacillus rodentium sp. nov. Stackebrandt, E. & Ebers, J. (2006). Taxonomic parameters revisited:
tarnished gold standards. Microbiology Today 33, 152–155. Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A., Ka¨mpfer, P., Maiden, M. C., Nesme, X., Rossello´-Mora, R., Swings, J. & other authors (2002). Report of the ad hoc committee for the re-
evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739. Tannock, G. W., Wilson, C. M., Loach, D., Cook, G. M., Eason, J., O’Toole, P. W., Holtrop, G. & Lawley, B. (2012). Resource partitioning
in relation to cohabitation of Lactobacillus species in the mouse forestomach. ISME J 6, 927–938. Teichmann, S. A. & Mitchison, G. (1999). Is there a phylogenetic
signal in prokaryote proteins? J Mol Evol 49, 98–107. Tindall, B. J., Sikorski, J., Smibert, R. M. & Kreig, N. R. (2007).
Phenotypic characterization and the principles of comparative systematics. In Methods for General and Molecular Microbiology, 3rd edn, pp. 330–393. Edited by C. A. Reddy, T. J. Beveridge, J. A. Breznak,
http://ijs.sgmjournals.org
G. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC, USA: American Society for Microbiology. Tindall, B. J., Rossello´-Mo´ra, R., Busse, H.-J., Ludwig, W. & Ka¨mpfer, P. (2010). Notes on the characterization of prokaryote
strains for taxonomic purposes. Int J Syst Evol Microbiol 60, 249– 266. Ventura, M., Canchaya, C., Meylan, V., Klaenhammer, T. R. & Zink, R. (2003). Analysis, characterization, and loci of the tuf
genes in Lactobacillus and Bifidobacterium species and their direct application for species identification. Appl Environ Microbiol 69, 6908–6922. Vlkova´, E., Rada, V., Popela´rˇova´, P., Trojanova´, I. & Killer, J. (2006).
Antimicrobial susceptibility of bifidobacteria isolated from gastrointestinal tract of calves. Livest Sci 105, 253–259. Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S
ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703. Yamano, T., Iino, H., Takada, M., Blum, S., Rochat, F. & Fukushima, Y. (2006). Improvement of the human intestinal flora by ingestion
of the probiotic strain Lactobacillus johnsonii La1. Br J Nutr 95, 303– 312.
1533
DOI 10.1099/ijs.0.054924-0
Lactobacillus rodentium sp. nov., from the digestive tract of wild rodents J. Killer,1,2 J. Havlı´k,2 E. Vlkova´,2 V. Rada,2 R. Pechar,2 O. Benada,3,4 J. Kopecˇny´,1 O. Kofronˇova´3 and H. Sechovcova´1 Correspondence J. Killer [email protected] or killer@ iapg.cas.cz
1
Institute of Animal Physiology and Genetics v.v.i., Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, Prague 4 – Krcˇ 142 20, Czech Republic
2
Czech University of Life Sciences, Faculty of Agrobiology, Food and Natural Resources, Department of Microbiology, Nutrition and Dietetics, Kamy´cka´ 129, Prague 6 – Suchdol 165 21, Czech Republic
3
Laboratory of Molecular Structure Characterization, Institute of Microbiology, Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, Prague 4 – Krcˇ 142 20, Czech Republic
4
Department of Biology, Faculty of Science, J. E. Purkyneˇ University in Ustı´ nad Labem, Za Va´lcovnou 1000/8, 400 96 U´stı´ nad Labem, Czech Republic
Three strains of regular, long, Gram-stain-positive bacterial rods were isolated using TPY, M.R.S. and Rogosa agar under anaerobic conditions from the digestive tract of wild mice (Mus musculus). All 16S rRNA gene sequences of these isolates were most similar to sequences of Lactobacillus gasseri ATCC 33323T and Lactobacillus johnsonii ATCC 33200T (97.3 % and 97.2 % sequence similarities, respectively). The novel strains shared 99.2–99.6 % 16S rRNA gene sequence similarities. Type strains of L. gasseri and L. johnsonii were also most related to the newly isolated strains according to rpoA (83.9–84.0 % similarities), pheS (84.6–87.8 %), atpA (86.2–87.7 %), hsp60 (89.4–90.4 %) and tuf (92.7–93.6 %) gene sequence similarities. Phylogenetic studies based on 16S rRNA, hsp60, rpoA, atpA and pheS gene sequences, other genotypic and many phenotypic characteristics (results of API 50 CHL, Rapid ID 32A and API ZYM biochemical tests; cellular fatty acid profiles; cellular polar lipid profiles; end products of glucose fermentation) showed that these bacterial strains represent a novel species within the genus Lactobacillus. The name Lactobacillus rodentium sp. nov. is proposed to accommodate this group of new isolates. The type strain is MYMRS/TLU1T (5DSM 24759T5CCM 7945T).
Representatives of the genus Lactobacillus are phylogenetically classified into the bacterial phylum Firmicutes, class Bacilli, order Lactobacillales and family Lactobacillaceae. Abbreviations: ITS, intergenic transcribed spacer; MLST, multilocus sequence typing. The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA gene sequences of strains MYMRS/TLU1T, MYTPY/TEN3 and MYMRS/TEN2 are HQ851022, HQ851020 and HQ851021, respectively. The accession numbers for the partial hsp60, pheS, rpoA and atpA gene sequences of strains MYMRS/TLU1T, MYTPY/TEN3 and MYMRS/TEN2 are JQ363678, JQ363679 and KF841362; JQ363687, JQ363688 and KF841360; JQ363696, KF841355 and KF841356; and JQ363703, KF841367 and KF841368, respectively. The accession numbers for the partial Tuf gene sequences of strains MYMRS/TLU1T and MYTPY/TEN3 are JQ363713 and JQ363714, respectively. The accession number for the partial ITS of strain MYMRS/ TLU1T is JQ363710. Six supplementary figures and one supplementary table are available with the online version of this paper.
1526
Various species of lactobacilli have been isolated from different environments rich in complex organic substrates. They are found on plant surfaces, and in plant-origin fermented products, milk, dairy and meat products, fish and marinated fish, oral cavities of mammals, urogenital tracts of mammals and gastrointestinal tract of vertebrates and insects (Hammes & Hertel, 2009; Forsgren et al., 2010). More than 90 different species of the genus Lactobacillus have been described (Hammes & Hertel, 2009). However, the List of Prokaryotic names with Standing in Nomenclature (available at http://www.bacterio.net/lactobacillus.html) includes one hundred and seventy species and subspecies of the genus Lactobacillus at the time of writing. Many bacterial species that were assigned to the genus Lactobacillus have recently been reclassified into new genera within the family Lactobacillaceae (Mora et al., 2003; Endo & Okada, 2008; Salvetti et al., 2011). Lactobacilli are generally considered as safe, non-pathogenic bacteria. In addition, many species of lactobacilli are used as probiotics in human and veterinary 054924 G 2014 IUMS Printed in Great Britain
Lactobacillus rodentium sp. nov.
medicine (Ohashi & Ushida, 2009). Nevertheless, some species of lactobacilli, e.g. Lactobacillus rhamnosus, may act as pathogens and opportunistic pathogens (Gouriet et al., 2012). Although most species of lactobacilli inhabiting the gastrointestinal tract of humans and animals are found in various animal species, some species of lactobacilli appear to be host-specific. Typical examples are some of the species inhabiting the digestive tract of horses, poultry and rodents (Mitsuoka & Fujisawa, 1987; Mukai et al., 2003; Morita et al., 2009). Species such as Lactobacillus hamsteri, Lactobacillus intestinalis, Lactobacillus johnsonii and Lactobacillus murinus are common bacterial symbionts of rodents (Mitsuoka & Fujisawa, 1987; Fujisawa et al., 1990; Clavel et al., 2010; Tannock et al., 2012). New bacterial strains isolated from the digestive tract of wild mice are characterized in this study. They represent a novel species of the genus Lactobacillus related to L. johnsonii and Lactobacillus gasseri, thus bacterial species that are often used in human medicine as probiotics (Morita et al., 2006; Yamano et al., 2006). Two individuals of wild mice (Mus musculus) were captured in cage traps in Central Bohemia (locality Jesˇetice) in 2010. Live animals were transported to the laboratory and euthanized with carbon dioxide. Fresh samples from the rectum and small intestine were aseptically transferred into tubes containing anaerobic TPY broth (Scardovi, 1986) and serially diluted in the same medium. One millilitre of each serially diluted sample was placed on TPY, M.R.S. or Rogosa agar (Oxoid) and incubated under anaerobic conditions (Anaerobic jars, Oxoid) at 37 uC for 72 h. Bacterial colonies were then immediately transferred into tubes containing anaerobic TPY or M.R.S. broth and cultivated at 37 uC for 24 h. Nearly complete 16S rRNA gene fragments were amplified in bacterial isolates using the fD1 and rP2 primers (Weisburg et al., 1991). Purified 16S rRNA gene fragments were subsequently sequenced by using an automatic genetic analyser, ABI PRISM 3130xl (Applied Biosystems). Resulting sequences were compared with sequences deposited in GenBank using the BLAST tool. Similarity of 16S rRNA gene sequences of bacterial strains from wild rodents to those of the most closely related bacterial species were calculated by the jPHYDIT program based on results of BLAST searching. The 16S rRNA gene sequences of type strains of the most closely related species were selected for similarity calculations. Pure bacterial isolates morphologically most similar to lactobacilli (regular long rods occurring singly or in short chains) were marked as MYMRS/TLU1T, MYTPY/TEN3 and MYMRS/TEN2. Strains were isolated from the colon (MYMRS/TLU1T) and small intestine (MYTPY/TEN3 and MYMRS/TEN2) of mice. Highest observed similarity of their 16S rRNA gene sequences was with L. gasseri ATCC 33323T (97.3 %; GenBank accession number AF519171) and L. johnsonii ATCC 33200T (97.2 %; AJ002515). Strains from the digestive tract of mice showed ¢99.2 % sequence similarity to each other. At the present time, it should be http://ijs.sgmjournals.org
mandatory to test the genomic uniqueness of a novel bacterial species if 16S rRNA gene sequence similarity to other species is in the range 98.7–99 % (Stackebrandt & Ebers, 2006). However, the MLST (multilocus sequence typing) technique was chosen to confirm the status of the novel bacterial species in this study. This technique has been used for all three strains. Partial sequences of hsp60 (heatshock protein 60 kDa; Dobson et al., 2002), pheS (phenylalanyl t-RNA synthase alpha subunit; Naser et al., 2005b), rpoA (RNA polymerase alpha subunit; Naser et al., 2005b), atpA (ATP synthase alpha subunit; Naser et al., 2005a) and Tuf (putative elongation factor Tu; Ventura et al., 2003) genes were amplified, sequenced and compared with those of the most closely related bacterial strains as described above. The ITS (intergenic transcribed spacer) region was also sequenced in the type strain MYMRS/TLU1T (Dobson et al., 2002). The highest hsp60, pheS, rpoA, atpA and Tuf gene sequence similarities of the strains from wild mice were found to those of L. gasseri CIP 102991T (89.4–89.7 %; GenBank accession number HE573894), L. gasseri LMG 18177 (85.8–87.7 %; AM284240), L. gasseri CIP 102991T (84.0 %; HE573912), L. gasseri LMG 9203T (86.6–86.7 %; AM087875) and L. gasseri ATCC 33323T (92.7–93.6 %; AY372047), respectively. The highest ITS region sequence similarity obtained for strain MYMRS/TLU1T was observed with L. gasseri JCM 1025 (81.5 %; GenBank accession number AF182721). These relatively low levels of sequence similarity confirm the conclusions that new isolates represent a novel species within the genus Lactobacillus. Notably, we found high 16S rRNA, hsp60, rpoA, Tuf and atpA sequence similarities between the type strains of L. gasseri and L. johnsonii with values of 99.2 % (GenBank accession numbers AF519171 and AJ002515), 96.5 % (HE573894 and HE573895), 97.8 % (HE573912 and HE573911), 97.4 % (AY372047 and AY372036) and 93.6 % (AM087875 and AM087879), respectively. In addition, a high value of 99.1 % ITS (GenBank accession numbers AF182721 and EU547283) sequence similarity was determined between these two species of lactobacilli. Based on these results, further sophisticated analyses will be needed to decide whether these bacterial strains really represent two different bacterial (sub)species. It was necessary to confirm the results based on sequence similarities of phylogenetic markers by phylogenetic analysis and determination of phenotypic characteristics. Thus, 16S rRNA genes of type strains of species belonging to the genus Lactobacillus and representatives of some related bacterial genera were obtained from the GenBank database. Sequences were then aligned using the CLUSTAL W algorithm in the MEGA 5.05 program (Tamura et al., 2011). The alignments were improved by removing hypervariable positions using the program Gblocks under the default conditions (Castresana, 2000). The same procedure was also used in the case of phylogenetic analyses based on hsp60, rpoA, atpA and pheS gene sequences. The maximum-likelihood algorithm within the MEGA 5.05 program was used for phylogenetic tree reconstructions. Resulting phylogenetic trees based on 16S 1527
J. Killer and others
1528 T 94 Lactobacillus gallinarum ATCC 33199 (AJ417737) Lactobacillus helveticus DSM 20075T (AM113779) Lactobacillus ultunensis DSM 16047T (AY253660)
0.02
Lactobacillus crispatus ATCC 33820T (AF257097) 96 71
Lactobacillus kitasatonis JCM 1039T (AB107638) Lactobacillus hamsteri CCUG 30726T (AJ306299) Lactobacillus kalixensis Kx127A2T (AY253657)
98
Lactobacillus intestinalis DSM 6629T (AJ306298) Lactobacillus equicursoris DI70T (AB290830) 90
Lactobacillus gasseri ATCC 33323T (AF519171) Lactobacillus johnsonii ATCC 33200T (AJ002515) Lactobacillus iners CCUG 28746T (Y16329)
99 80
Lactobacillus acidophilus DSM 20079T (AY773947) Lactobacillus amylovorus DSM 20531T (AY944408)
100
Lactobacillus rodentium MYMRS/TEN2T (HQ851021) Lactobacillus rodentium MYMRS/TLU1T (HQ851022)
International Journal of Systematic and Evolutionary Microbiology 64
Lactobacillus rodentium MYTPY/TEN3T (HQ851020) Lactobacillus jensenii ATCC 25258T (AF243176) Lactobacillus psittaci DSM 15354T (AJ272391)
90
Lactobacillus amylophilus DSM 20533T (M58806) Lactobacillus vaginalis ATCC 49540T (AF243177) 100
Lactobacillus kimchii DSM 13961T (AB107638) Lactobacillus mindensis DSM 14500T (AJ313530)
Lactobacillus concavus AS 1.5017T (AY683322) Atopostipes suicloacalis DSM 15692T (AF445248)
77
Carnobacterium inhibens CCUG 31728T (Z73313)
100 79
Streptococcus caballi DSM 19004T (EF364098) 83
Enterococcus caccae CCUG 51564T (AY943820) Enterococcus faecium LMG 11423T (AJ301830)
Fig. 1. Unrooted phylogenetic tree of members of the genus Lactobacillus and family Enterococcaceae showing the position of strains representing Lactobacillus rodentium sp. nov. Reconstructed by the maximum-likelihood method based on 16S rRNA gene sequences (length of 1397 nt) using MEGA version 5.05 software and the Jukes–Cantor model. Bootstrap values, expressed as percentages of 1000 datasets, are given at nodes. Numbers in parentheses correspond to GenBank accession numbers. Bar, 0.02 substitutions per nucleotide position.
Lactobacillus rodentium sp. nov.
rRNA (Fig. 1), hsp60, rpoA, atpA and pheS (Figs S1–4, available in the online Supplementary Material) gene sequences revealed that the new bacterial strains MYMRST/ LU1T, MYTPY/TEN3 and MYMRS/TEN2 are situated on a separate phylogenetic branch between the species L. gasseri, L. johnsonii and Lactobacillus iners, respectively. Greater difference in sequence similarity among the strains studied was detected only in the case of the pheS gene. This fact is shown in the phylogenetic tree based on pheS gene sequences (Fig. S4). The concatenation of genes has been shown to be extremely useful in order to infer bacterial phylogeny (Teichmann & Mitchison, 1999). Phylogenetic trees reconstructed on the basis of the concatenated amino acid
sequences of the hsp60, pheS and rpoA genes demonstrated that the new bacterial strains are closely related to each other, but phylogenetically distinct from L. gasseri and L. johnsonii (data not shown). The DNA G+C contents of all new strains, L. gasseri DSM 20243T and L. johnsonii DSM 10533T were determined using the enzymic degradation method with some modifications as described previously (Killer et al., 2011). The DNA G+C content of strain MYMRS/TLU1T was 43.7 mol% (mean of three experiments, SD50.2). The DNA G+C contents for strains MYTPY/TEN3 and MYMRS/TEN2 were 43.4 and 44.1 mol%, respectively (Table 1). A range of values from 32
Table 1. Differences in DNA G+C contents, growth characteristics and results of biochemical tests in strains representing a novel species within the genus Lactobacillus and the most closely related species based on phylogenetic analyses Strains: 1, Lactobacillus rodentium sp. nov. MYMRS/TLU1T; 2, L. rodentium sp. nov. MYMRS/TEN2; 3, L. rodentium sp. nov. MYTPY/TEN3; 4, L. gasseri DSM 20243T; 5, L. johnsonii DSM 10533T. All data from this study. All strains utilized D-glucose, D-fructose, maltose and sucrose. None of the strains produced acids from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, inulin, melezitose, starch, glycogen, xylitol, turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. All strains were positive for a-glucosidase, b-glucosidase, arginine arylamidase, tyrosine arylamidase, phenylalanine arylamidase, leucine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase and serine arylamidase. All strains were negative for urease, arginine dihydrolase, 6-phospho-b-galactosidase, a-arabinosidase, b-glucuronidase, a-fucosidase, nitrate reduction, indole production, alkaline phosphatase, glutamyl glutamic acid arylamidase, esterase lipase (C8), lipase (C14), trypsin, a-chymotrypsin, a-mannosidase, gelatin hydrolysis, catalase and oxidase. +, Positive reaction; 2, negative reaction; W, weakly positive reaction. Characteristic DNA G+C content (mol%) Temperature range for growth (uC) pH range for growth API 50 CHL D-Galactose D-Mannose Amygdalin Aesculin Salicin Arbutin Gentiobiose Cellobiose Lactose Melibiose Raffinose N-Acetylglucosamine D-Tagatose RAPID ID 32A, API ZYM a-Galactosidase b-Galactosidase Glutamic acid decarboxylase Glycine arylamidase Leucyl glycine arylamidase N-Acetyl-b-glucosaminidase Proline arylamidase Pyroglutamic acid arylamidase Esterase (C4) Naphthol-AS-BI-phosphohydrolase
http://ijs.sgmjournals.org
1
2
3
4
5
43.7 15–46 5.0–9.5
44.1 15–46 5.0–9.5
43.4 15–46 5.0–9.5
34.7 20–45 4.5–9.0
34.3 20–45 4.5–9.0
+ 2
2 + 2 2 2 2
+ +
2 + + + + + + + + 2 2 + +
+ + + + + + + + + 2 2 + 2
+ 2 + + 2 + + + 2 2
2 + + 2 + + 2 2 +
W W W
2 + + + + + W
2 + + + + + 2 2 2
W W W
2
W
W
2
W
+ + + +
2 2
2
W
+
2 + + W
2 2 2 2
W
+ + 2 2 2 2 2 2
W
W
W
W
+
+
W
1529
J. Killer and others
to 55 mol% DNA G+C was found among species in the genus Lactobacillus (Hammes & Hertel, 2009). Values observed in isolates from the digestive tract of wild rodents were higher in comparison with those of the type strains of L. gasseri (34.7 mol%) and L. johnsonii (34.3 mol%), the most closely related species within the genus Lactobacillus according to MLST. DNA–DNA hybridization was subsequently used to confirm the status of the novel bacterial species because the 16S rRNA gene similarity to the closest related species was greater than 97 % (Tindall et al., 2010). DNA–DNA hybridizations were performed among strains MYMRS/ TLU1T, L. gasseri DSM 20243T and L. johnsonii DSM 10533T. The new isolates from wild rodents were also tested to confirm whether they really belong to the same bacterial species. DNA was extracted from 0.75–1.25 g (wet wt) by using the protocol described by Gevers et al. (2001). 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 singlestranded DNA, which was bound non-covalently to microplate wells. Hybridizations were performed at 37 uC in hybridization mixture. Relative binding of DNA from strain MYMRS/TLU1T with the type strain of L. gasseri and L. johnsonii gave values of 12.8 (mean of three experiments, SD50.7) and 14.2 % (SD50.3), respectively. Reciprocal values of DNA–DNA relatedness of L. gasseri and L. johnsonii strains with respect to strain MYMRS/TLU1T were 13.4 (SD50.1) and 14.4 % (SD50.3), respectively. The values for DNA–DNA binding among the three new strains were in the range 80.6–90.8 %. The results confirmed that the new strains from wild mice represent a novel taxon within the genus Lactobacillus (Stackebrandt et al., 2002). The substrate utilization and enzyme activity patterns of the three new bacterial strains, L. gasseri DSM 20243T and L. johnsonii DSM 10533T were determined using API 50 CHL, Rapid ID 32A and API ZYM test strips (bioMe´rieux). All strains were also tested for oxidase activity (Lui & Jurtshuk, 1986) and hydrolysis of gelatin by the API 20E system (bioMe´rieux). Effect of temperature and pH on the growth of the above-mentioned bacterial strains was assessed as described previously (Killer et al., 2013). Table 1 shows the main phenotypic differences among the new bacterial strains and the most closely related species according to the results of the 16S rRNA gene sequence similarities. They differ from each other in fermentation of thirteen carbohydrates and production of ten enzymes. Capillary isotachophoresis was used for determination of the end products of hexose catabolism in strain MYMRST/ LU1T according to Killer et al. (2011). The strain was also tested for production of D- and L-lactic acid by the D-/ L-lactic acid kit (Megazyme). Gas production from glucose was assayed using a Durham tube in MRS broth. Lactic acid at a concentration of 124.3 mmol l21 (70 % of all short-chain fatty acids produced) was the main end 1530
product of glucose fermentation. Acetic and propionic acids were also quantified in the strain at concentrations of 44.4 mmol l21 (25 %) and 9.4 mmol l21 (5 %), respectively. These results, along with the inability to utilize pentoses and gluconates, and produce gas from glucose, suggest the new strain belongs to the group of obligately homofermentative lactobacilli (type A of glucose fermentation according to Hammes & Hertel, 2009). Cells of strain MYMRS/TLU1T produced only L-lactic acid from D-glucose. Scanning electron microscopy (Killer et al., 2009) was used to determine detailed cell morphology in strain MYMRS/ TLU1T. Cells of this strain were regular, long rods occurring mostly singly and occasionally in pairs (Fig. S5). Susceptibility to 33 different antibiotics and two chemotherapeutics by the disc diffusion method (Vlkova´ et al., 2006) was assessed as an additional phenotypic characteristic of strain MYMRS/TLU1T. The tested strain was susceptible to cephalosporines (five out of eight tested), macrolides and penicillin-derived antibiotics and resistant to cephalosporines (three out of eight tested), aminoglycosides, fluoroquinolones and sulfonamides. Similar results have recently been found in different species of lactobacilli (Karapetkov et al., 2011). All results of susceptibility testing are shown in Table S1. Cellular fatty acid profiling was performed in all new strains from the digestive tract of wild mice, L. gasseri DSM 20243T and L. johnsonii DSM 10533T by the method
Table 2. Cellular fatty acid profiles of strains representing Lactobacillus rodentium sp. nov. and the most closely related species based on phylogenetic analyses Strains: 1, L. gasseri DSM 20243T; 2, L. johnsonii DSM 10533T; 3, L. rodentium sp. nov. MYMRS/TLU1T; 4, L. rodentium sp. nov. MYMRS/TEN2; 5, L. rodentium sp. nov. MYTPY/TEN3. Relative proportions (%; w/v) of fatty acids were calculated. Other fatty acids were not characterized using 37 comp. FAME Mix standard (Supelco). Fatty acid C8 : 0 C10 : 0 C11 : 0 C12 : 0 C14 : 0 C16 : 1 C17 : 1 C18 : 1v9t C18 : 1v9c C18 : 2v6s C20 : 0 C23 : 0 C22 : 2 C24 : 0
1 1.3 5.2 1.9 2.2 ,1.0 ,1.0 2.8 1.8 1.3 6.4 1.4 40.5 23.4 1.9
2 1.3 5.8 1.6 1.5 ,1.0 1.9 1.8 ,1.0 2.1 5.9 ,1.0 40.8 24.7 ,1.0
3 3.3 5.8 3.2 ,1.0 1.8 5.2 23.9 ,1.0 ,1.0 1.3 3.2 22.4 24.7 13.0
4 2.1 7.4 3.3 ,1.0 2.2 9.9 29.0 ,1.0 ,1.0 1.3 5.5 19.0 20.8 10.4
5 4.0 6.5 3.0 ,1.0 2.8 5.8 19.5 ,1.0 ,1.0 1.5 3.9 20.6 19.4 8.5
International Journal of Systematic and Evolutionary Microbiology 64
Lactobacillus rodentium sp. nov.
described in our previous study (Killer et al., 2009). For the purposes of this analysis, all strains were grown under identical conditions in MRS broth at 37 uC for 48 h. Higher relative proportions of C17 : 1, C24 : 0 and C16 : 1 fatty acids (Table 2) were determined in strains MYMRS/ TLU1T, MYMRS/TEN2 and MYTPY/TEN3 compared with strains L. gasseri DSM 20243T and L. johnsonii DSM 10533T, identified as the most closely related species on the basis of the results of MLST. The latter two species of the genus Lactobacillus had very similar profiles of cellular fatty acids. This fact, together with relatively high levels of sequence similarity of phylogenetic markers, suggests a close relationship of these species. On the other hand, the new strains had lower relative proportions of C23 : 0 fatty acid in comparison to L. gasseri DSM 20243T and L. johnsonii DSM 10533T. Notably, relatively high proportions of C22 : 2 fatty acid were found in all strains tested. The peptidoglycan structure type A4a L-Lys-D-Asp (A11.31 according to the DSMZ catalogue of strains; DSMZ, 2001) was determined in strain MYMRS/TLU1T by the Identification Service Department of DSMZ (Braunschweig, Germany) by methods described previously (Killer et al., 2010). The total hydrolysate of the peptidoglycan contained the amino acids alanine, glycine, threonine, serine, asparagine, glutamic acid and lysine in an approximate ratio of 0.9 : 0.4 : 0.4 : 0.4 : 1.0 : 0.9. The above-mentioned type of peptidoglycan was determined in the majority of known species of lactobacilli (Hammes & Hertel, 2009). The profile of cellular polar lipids was determined in strain MYMRS/TLU1T as an additional chemotaxonomic characteristic. Analysis of cellular polar lipids was provided by the Identification Service Department of DSMZ according to the methods of Bligh & Dyer (1959) and Tindall et al. (2007). Bacterial strain MYMRS/TLU1T was cultivated under anaerobic conditions in MRS medium for 24 h at 37 uC. Bacterial cells were then centrifuged and lyophilized. Diphosphatidylglycerol, phosphatidylglycerol, two phospholipids and several different glycolipids were determined in cells of strain MYMRS/TLU1T (Fig. S6). Phosphatidylethanolamine, phosphatidylglycerol and phospholipids were previously detected in lactobacilli (Arbogast & Henderson, 1975; Kim et al., 2011; Liang et al., 2011). All results of genotypic and phenotypic characteristics suggest that the new bacterial isolates from the gastrointestinal tract of wild rodents may be classified in a novel species for which the name Lactobacillus rodentium sp. nov. is proposed. Description of Lactobacillus rodentium sp. nov. Lactobacillus rodentium (ro.den9ti.um. L. gen. n. rodentium of gnawers, referring to the fact that the organism was isolated from wild rodents). Cells are Gram-positive, catalase- and oxidase-negative. Cell morphology is typical of that for most species of lactobacilli: regular, long rods (0.8–1.2 mm wide and http://ijs.sgmjournals.org
2.1–8.6 mm long) occurring mostly singly and occasionally in pairs. Grows best in anaerobic TPY broth and on M.R.S. agar. However, also grows under microaerophilic conditions (CampyGen, Oxoid). Colonies on M.R.S. agar under anaerobic conditions after 72 h are cream in colour, regular disc-shaped and rigid. The edges of the colonies are sharply defined, regular and blunt in profile. Colony size is in the range from 0.46 to 0.68 mm in diameter. Grows at 15–46 uC and pH 5–9.5. Utilizes D-glucose, Dfructose, maltose, melibiose and sucrose. Does not produce acids from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-Dxylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, inulin, trehalose, arbutin, melezitose, starch, glycogen, xylitol, D-tagatose, turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Variable in utilization of D-galactose, D-mannose, amygdalin, aesculin, salicin, gentiobiose, cellobiose, lactose, raffinose and Nacetylglucosamine. Positive for a-glucosidase, b-glucosidase, b-galactosidase, arginine arylamidase, tyrosine arylamidase, phenylalanine arylamidase, leucine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, esterase (C4) (weakly positive reaction) and serine arylamidase. Negative for urease, arginine dihydrolase, 6-phospho-b-galactosidase, a-arabinosidase, b-glucuronidase, a-fucosidase, pyroglutamic acid arylamidase, nitrate reduction, indole production, alkaline phosphatase, glutamyl glutamic acid arylamidase, proline arylamidase, N-acetyl-b-glucosaminidase, esterase lipase (C8), lipase (C14), trypsin, a-chymotrypsin, a-mannosidase, gelatin hydrolysis, catalase and oxidase. Variable in production of a-galactosidase, glutamic acid decarboxylase, glycine arylamidase, leucyl glycine arylamidase and naphthol-AS-BIphosphohydrolase. The peptidoglycan structure type is A4a L-Lys-D-Asp. Diphosphatidylglycerol, phosphatidylglycerol, two phospholipids and several different glycolipids are present. Susceptible to cephalosporines, macrolides, penicillin-derived antibiotics and chemotherapeutic mupirocine. Resistant to aminoglycosides, fluoroquinolones, sulfonamides and chemotherapeutic metronidazole (Table S1). Major fatty acids in cells are C17 : 1, C22 : 2, C23 : 0 and C24 : 0 (Table 2). DNA G+C content is in the range 43.4 to 44.1 mol%. The type strain, MYMRS/TLU1T (5DSM 24759T5CCM 7945T) was isolated from the colon of wild mice (Mus musculus) captured in cage traps in Central Bohemia (locality Jesˇetice) in 2010. The DNA G+C content of the type strain is 43.7 mol%. Additional strains of the species are MYMRS/TEN2 and MYTPY/TEN3.
Acknowledgements This study was supported by the Grant Agency of the Czech Republic (projects GA CR 304/11/1252 and GA13 - 08803S), by the 1531
J. Killer and others Institutional Research Project of the Institute of Animal Physiology and Genetics, Acad. Sci. CR (RVO: 67985904) and by the Institutional Research Concept RVO 61388971.
sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees. Syst Appl Microbiol 33, 359–366. Killer, J., Kopecˇny´, J., Mra´zek, J., Koppova´, I., Havlı´k, J., Benada, O. & Kott, T. (2011). Bifidobacterium actinocoloniiforme sp. nov. and
References
Bifidobacterium bohemicum sp. nov., from the bumblebee digestive tract. Int J Syst Evol Microbiol 61, 1315–1321.
Arbogast, L. Y. & Henderson, T. O. (1975). Effect of inhibition of
Killer, J., Mra´zek, J., Bunesˇova´, V., Havlı´k, J., Koppova´, I., Benada, O., Rada, V., Kopecˇny´, J. & Vlkova´, E. (2013). Pseudoscardovia suis gen.
protein synthesis on lipid metabolism in Lactobacillus plantarum. J Bacteriol 123, 962–971. Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid
extraction and purification. Can J Biochem Physiol 37, 911–917. Castresana, J. (2000). Selection of conserved blocks from multiple
alignments for their use in phylogenetic analysis. Mol Biol Evol 17, 540–552. Clavel, T., Saalfrank, A., Charrier, C. & Haller, D. (2010). Isolation
of bacteria from mouse caecal samples and description of Bacteroides sartorii sp. nov. Arch Microbiol 192, 427–435. Dobson, C. M., Deneer, H., Lee, S., Hemmingsen, S., Glaze, S. & Ziola, B. (2002). Phylogenetic analysis of the genus Pediococcus,
including Pediococcus claussenii sp. nov., a novel lactic acid bacterium isolated from beer. Int J Syst Evol Microbiol 52, 2003–2010.
nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of wild pigs (Sus scrofa). Syst Appl Microbiol 36, 11–16. Kim, H. J., Eom, S. J., Park, S. J., Cha, C. J. & Kim, G. B. (2011).
Lactobacillus alvi sp. nov., isolated from the intestinal tract of chicken. FEMS Microbiol Lett 323, 83–87. Liang, Z. Q., Srinivasan, S., Kim, Y. J., Kim, H. B., Wang, H. T. & Yang, D. C. (2011). Lactobacillus kimchicus sp. nov., a b-glucosidase-
producing bacterium isolated from kimchi. Int J Syst Evol Microbiol 61, 894–897. Lui, J.-K. & Jurtshuk, P. (1986). N,N,N’-N’-tetramethyl-p-phenylenediamine-dependent cytochrome oxidase analyses of Bacillus species. Int J Syst Bacteriol 36, 38–46.
DSMZ.
Mitsuoka, T. & Fujisawa, T. (1987). Lactobacillus hamsteri, a new species from the intestine of hamsters. Proc Jpn Acad, Ser B, Phys Biol Sci 63, 269–272.
Endo, A. & Okada, S. (2008). Reclassification of the genus Leuco-
Mora, D., Scarpellini, M., Franzetti, L., Colombo, S. & Galli, A. (2003).
DSMZ (2001). Catalogue of Strains, 7th edn, p. 617. Braunschweig:
nostoc and proposals of Fructobacillus fructosus gen. nov., comb. nov., Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb. nov. and Fructobacillus pseudoficulneus comb. nov. Int J Syst Evol Microbiol 58, 2195–2205. 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. Forsgren, E., Olofsson, T., Va´squez, A. & Fries, I. (2010). Novel lactic
acid bacteria inhibiting Paenibacillus larvae in honey bee larvae. Apidologie (Celle) 41, 99–108. Fujisawa, T., Itoh, K., Benno, Y. & Mitsuoka, T. (1990). Lactobacillus
intestinalis (ex Hemme 1974) sp. nov., nom. rev., isolated from the intestines of mice and rats. Int J Syst Bacteriol 40, 302–304.
Reclassification of Lactobacillus maltaromicus (Miller et al. 1974) DSM 20342T and DSM 20344 and Carnobacterium piscicola (Collins et al. 1987) DSM 20730T and DSM 20722 as Carnobacterium maltaromaticum comb. nov. Int J Syst Evol Microbiol 53, 675–678. Morita, H., He, F., Kawase, M., Kubota, A., Hiramatsu, M., Kurisaki, J. & Salminen, S. (2006). Preliminary human study for possible alteration
of serum immunoglobulin E production in perennial allergic rhinitis with fermented milk prepared with Lactobacillus gasseri TMC0356. Microbiol Immunol 50, 701–706. Morita, H., Nakano, A., Shimazu, M., Toh, H., Nakajima, F., Nagayama, M., Hisamatsu, S., Kato, Y., Takagi, M. & other authors (2009). Lactobacillus hayakitensis, L. equigenerosi and L. equi, pre-
dominant lactobacilli in the intestinal flora of healthy thoroughbreds. Anim Sci J 80, 339–346.
Gevers, D., Huys, G. & Swings, J. (2001). Applicability of rep-
Mukai, T., Arihara, K., Ikeda, A., Nomura, K., Suzuki, F. & Ohori, H. (2003). Lactobacillus kitasatonis sp. nov., from chicken intestine. Int J
PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 205, 31–36.
Syst Evol Microbiol 53, 2055–2059.
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. Gouriet, F., Million, M., Henri, M., Fournier, P. E. & Raoult, D. (2012).
Lactobacillus rhamnosus bacteremia: an emerging clinical entity. Eur J Clin Microbiol Infect Dis 31, 2469–2480. Hammes, W. P. & Hertel, C. (2009). Genus Lactobacillus. In Bergey’s
Manual of Systematic Bacteriology, 2nd edn, vol 3, pp. 465–511. Edited by P. De Vos, G. M. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey, K.-H. Schleifer & W. B. Whitman. New York: Springer. Karapetkov, N., Georgieva, R., Rumyan, N. & Karaivanova, E. (2011).
Antibiotic susceptibility of different lactic acid bacteria strains. Benef Microbes 2, 335–339. Killer, J., Kopecny´, J., Mra´zek, J., Rada, V., Benada, O., Koppova´, I., Havlı´k, J. & Straka, J. (2009). Bifidobacterium bombi sp. nov., from the
bumblebee digestive tract. Int J Syst Evol Microbiol 59, 2020–2024.
Naser, S., Thompson, F. L., Hoste, B., Gevers, D., Vandemeulebroecke, K., Cleenwerck, I., Thompson, C. C., Vancanneyt, M. & Swings, J. (2005a). Phylogeny and identification of Enterococci by atpA gene
sequence analysis. J Clin Microbiol 43, 2224–2230. Naser, S. M., Thompson, F. L., Hoste, B., Gevers, D., Dawyndt, P., Vancanneyt, M. & Swings, J. (2005b). Application of multilocus
sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151, 2141–2150. Ohashi, Y. & Ushida, K. (2009). Health-beneficial effects of probiotics:
its mode of action. Anim Sci J 80, 361–371. Salvetti, E., Felis, G. E., Dellaglio, F., Castioni, A., Torriani, S. & Lawson, P. A. (2011). Reclassification of Lactobacillus catenaformis
(Eggerth 1935) Moore and Holdeman 1970 and Lactobacillus vitulinus Sharpe et al. 1973 as Eggerthia catenaformis gen. nov., comb. nov. and Kandleria vitulina gen. nov., comb. nov., respectively. Int J Syst Evol Microbiol 61, 2520–2524. Scardovi, V. (1986). Genus Bifidobacterium Orla-Jensen 1924, 472AL.
Killer, J., Kopecˇny´, J., Mra´zek, J., Havlı´k, J., Koppova´, I., Benada, O., Rada, V. & Kofronˇova´, O. (2010). Bombiscardovia coagulans gen. nov.,
In Bergey’s Manual of Systematic Bacteriology, vol. 2, pp. 1418–1434. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharp & J. G. Holt. Baltimore: Williams and Wilkins.
1532
International Journal of Systematic and Evolutionary Microbiology 64
Lactobacillus rodentium sp. nov. Stackebrandt, E. & Ebers, J. (2006). Taxonomic parameters revisited:
tarnished gold standards. Microbiology Today 33, 152–155. Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A., Ka¨mpfer, P., Maiden, M. C., Nesme, X., Rossello´-Mora, R., Swings, J. & other authors (2002). Report of the ad hoc committee for the re-
evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739. Tannock, G. W., Wilson, C. M., Loach, D., Cook, G. M., Eason, J., O’Toole, P. W., Holtrop, G. & Lawley, B. (2012). Resource partitioning
in relation to cohabitation of Lactobacillus species in the mouse forestomach. ISME J 6, 927–938. Teichmann, S. A. & Mitchison, G. (1999). Is there a phylogenetic
signal in prokaryote proteins? J Mol Evol 49, 98–107. Tindall, B. J., Sikorski, J., Smibert, R. M. & Kreig, N. R. (2007).
Phenotypic characterization and the principles of comparative systematics. In Methods for General and Molecular Microbiology, 3rd edn, pp. 330–393. Edited by C. A. Reddy, T. J. Beveridge, J. A. Breznak,
http://ijs.sgmjournals.org
G. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC, USA: American Society for Microbiology. Tindall, B. J., Rossello´-Mo´ra, R., Busse, H.-J., Ludwig, W. & Ka¨mpfer, P. (2010). Notes on the characterization of prokaryote
strains for taxonomic purposes. Int J Syst Evol Microbiol 60, 249– 266. Ventura, M., Canchaya, C., Meylan, V., Klaenhammer, T. R. & Zink, R. (2003). Analysis, characterization, and loci of the tuf
genes in Lactobacillus and Bifidobacterium species and their direct application for species identification. Appl Environ Microbiol 69, 6908–6922. Vlkova´, E., Rada, V., Popela´rˇova´, P., Trojanova´, I. & Killer, J. (2006).
Antimicrobial susceptibility of bifidobacteria isolated from gastrointestinal tract of calves. Livest Sci 105, 253–259. Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S
ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703. Yamano, T., Iino, H., Takada, M., Blum, S., Rochat, F. & Fukushima, Y. (2006). Improvement of the human intestinal flora by ingestion
of the probiotic strain Lactobacillus johnsonii La1. Br J Nutr 95, 303– 312.
1533
