Vol. 40, No. 3
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1990, p. 302-304 0020-7713/90/030302-03$02.oo/o Copyright 0 1990, International Union of Microbiological Societies
NOTES Lactobacillus intestinalis (ex Hemme 1974) sp. nov., norn. rev., Isolated from the Intestines of Mice and Rats TOMOHIKO FUJISAWA,’ KIKUJI ITOH,l YOSHIMI BENNO,* A N D TOMOTARI MITSUOKA173* Laboratory for Intestinal Flora, Frontier Research Program,’ and Japan Collection of Microorganisms,2 The Institute of Physical and Chemical Research, Wako, Saitama 351-01, and Department of Biomedical Science, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113,3Japan The genetic and phenotypic properties of 10 strains identified as Lactobacillus intestinalis sp. nov. were examined. These strains constitute a distinct species which can be differentiated from all of the previously described homofermentativespecies in the genus Lactobacillus by their carbohydrate fermentation pattern. The guanine-plus-cytosinecontents of their DNAs are 33 to 35 mol % DNAs from 10 other Lactobacillus species did not exhibit significant levels of relatedness to representative strains of the new species. The name Lactobacillus intestinalis (ex Hemme) sp. nov., nom. rev. is proposed for these isolates, and strain Th4 (= ATCC 49335 = JCM 7548) is the type strain.
.
During studies on the intestinal flora of mice and rats (3), strains of “Lactobacillus intestinalis” were isolated. This species name was originally proposed by Hemme (D. Hemme, Ph.D. thesis, University of Paris, Paris, France, 1974), but it has never been validly published, was not included on the Approved Lists of Bacterial Names (9), and has no current nomenclatural standing. In a study which demonstrated the heterogeneity of the “Lactobacillus acidophilus group,” Lauer et al. (6) demonstrated that strain CNRZ 219 (corresponding to “L. intestinalis’ ’) represented a separate DNA homology group (group Ie) which did not exhibit high levels of homology with any strain of L . acidophilus or the strains of any other homology group. Johnson et al. (4) investigated four strains that were isolated by Shimohashi and Mutai (8) from rat intestinal tracts in a taxonomic study of the L . acidophilus group and classified them as members of homology group B-2, which is equivalent to group IIb of Lauer et al. (6). The objectives of this study were to determine the phenotypic and genetic characteristics of the strains which were isolated from the intestines of mice and rats and were identified as “ L . intestinalis” and to obtain a better understanding of the taxonomic position of these organisms. Twenty strains were used in this study. Nine strains were isolated from intestinal contents of mice or rats; strains F215, F218, F219, RTD1-1, RTD1-3, and RTD4-5 were isolated from rats, and strains LS-44, LS-47, and LS-49 were isolated from mice. “L. intestinalis” Th4T (T = type strain) was obtained from Hemme. L . acidophilus ATCC 4356T and ATCC 33200, Lactobacillus salivarius ATCC 11741T,Lactobacillus helveticus ATCC 15009T, Lactobacillus jensenii ATCC 2525gT, Lactobacillus crispatus JCM llUT,Lactobacillus gasseri JCM 1131T,Lactobacillus amylovorus JMC 1126T, Lactobacillus animalis JCM 5670T, Lactobacillus agilis JCM 1187T, Lactobacillus acetotolerans JCM 3825T,
and Lactobacillus hamsteri Ha5FlT were obtained from our collection. The strains were characterized by using the methods and procedures described previously (1, 2, 7). A comparison of the morphological, physiological, and biochemical characteristics, DNA base compositions, and TABLE 1. Levels of DNA homology between strains of L. intestinalis and some other Lactobacillus species having low G+C contents % Homology with
Unlabeled DNA from:
3H-labeled DNA from strain:
Th4T
L. intestinalis strains Th4T F215 F218 F219 RTD1-1 RTD1-3 RTD4-5 LS-44 LS-47 LS-49 L. acidophilus ATCC 4356T L. acidophilus ATCC 33200 L. crispatus JCM 1185T L. gasseri JCM 1131T L. amylovorus JCM 1126T L. animalis JCM 5670T L. salivarius ATCC 11741T L. agilis JCM 1187T L. hamsteri Ha5FlT L. helveticus ATCC 15009T L. jensenii ATCC 25258T L. acetotolerans JCM 3825T -, Not tested. Data from reference 5 .
* Corresponding author. 302
100 81 75 85 97 78 84 72 76 76 12 6 7 1 11 10 9 6
G+C content (mol%)
F215
35 32 32 33 32 33 32 34 34 32 32 33 35 33 40b 42b 35b 44b 33 39b 35b 35
VOL.40, 1990
NOTES
303
TABLE 3. Phenotypic characteristics that differentiate L . intestinalis from other homofermentative Lactobacillus species that have low G+C contents and do not grow at 15°C“ Aerobic ~somefls) Species or group
growth
L . intestinalis L . acidophilus group L . helveticus L . salivarius L . jensenii L . animalis-L. murinus L. acetotolerans L . kefiranofaciens L . vitulinus L . ruminis L. aviarius
Of
lactic
Acid produced from:
Su- Escu- Sali- Amygcrose lin cin dalin
DL DL DL L D L DL D , DL D L L, DL
+
-
-
+
+
v
v
-
+
v
v
v
-
-
+
-
+
-
+
-
+ + + + + + + + -+ +- -+ -+ + + + + + + +
a The Characteristics were determined by testing the strains used in this study, including the type strains. + ,Positive reaction; -, negative reaction; v, variable reaction. D, D-Lactic acid makes up 90% or more of the total lactic acid; L, L-lactic acid makes up 90% or more of the total lactic acid; DL, 25 to 75% of the total lactic acid is L-lactic acid.
FIG. 1. Strain Th4T of L . intestinalis sp. nov., nom. rev. (a) Gram-stained cells from a glucose-blood-liver agar culture incubated anaerobically at 37°C for 48 h. Bar = 10 pm. (b) Surface colonies on a glucose-blood-liver agar plate incubated anaerobically at 37°C for 48 h. Bar = 10 mm.
DNA-DNA homologies of the 10 “L. intestinalis” strains studied with the properties of the previously described species of the genus Lactobacillus indicated that these strains represent a distinct species. The name “Lactobacillus intestinalis” has not been validly published previously and was not included on the Approved Lists (9). Therefore, we propose Lactobacillus intestinalis sp. nov., nom. rev. as a valid species of the genus Lactobacillus. Table 1 shows the guanine-plus-cytosine (G +C) contents and levels of DNA homology of the 10 strains of “L. intestinalis” examined as determined by the S1 nuclease method. The G+C contents of the DNAs from the “L. intestinalis” strains were 32 to 35 mol% as determined by the thermal denaturation method. All of the strains had high levels of DNA-DNA homology with each other. The DNAs of 12 other Lactobacillus species exhibited little or no homology with the DNAs of strains Th4T and F215. On the basis of the distinctions described above, we propose the name Lactobacillus intestinalis. A description is given below. Lactobacillus intestinalis (ex Hemme 1970) sp. nov., nom.
TABLE 2. Variable biochemical characteristics of L . intestinalis and type strain Th4
rev. Lactobacillus intestinalis (in.test.in.al’is. N. L. adj. intestinalis, pertaining to the intestine). Gram-positive, nonmotile, nonsporeforming, facultatively anaerobic rods, generally 0.6 to 0.8 by 2.0 to 6.0 pm, that occur singly, in pairs, or occasionally in short chains (Fig. la). All strains grow on glucose-blood-liver agar (2), on MRS agar, in Briggs liver broth, and in MRS broth. Surface colonies on glucoseblood-liver agar after 2 days of anaerobic incubation at 37°C are 0.7 to 2.5 mm in diameter, round, flat, and light brown with rough surfaces and erose edges (Fig. lb). Produces DL-lactic acid from glucose homofermentatively. The final pH of glucose broth is 3.7. No growth occurs at 15°C in Briggs liver broth. Catalase is not produced. Esculin and starch are not hydrolyzed. Gas is not produced from glucose. Acid is produced from glucose, mannose, fructose, galactose, sucrose, maltose, dextrin, and mannitol but not from arabinose, xylose, rhamnose, trehalose, melezitose, starch, sorbitol, esculin, salicin, and amygdalin. The final pH in glucose broth is 3.5 to 3.8. Variable characteristics are shown in Table 2. The G+C content of the DNA is 32.5 to 35.4 mol%. The habitat of the species is the intestines of mice and rats. The type strain is strain Th4 (= ATCC 49335 = JCM 7548). The phenotypic characteristics that differentiate L. intestinalis from other homofermentative Lactobacillus species that have low G+C contents and that do not grow at 15°C are shown in Table 3. LITERATURE CITED
L. intestinalis reactiona
Acid produced from:
10 strains
Ribose Cellobiose Lactose Melibiose Raffinose Dextrin
d d d d d d
8
Type strain Th4
+, Positive reaction; w, weak reaction; s, delayed reaction; d, 11to 89% of the strains are positive.
1. Fujisawa, T., S. Adachi, T. Toba, K. Arihara, and T. Mitsuoka. 1988. Lactobacillus kefiranofaciens sp. nov. isolated from kefir grains. Int. J. Syst. Bacteriol. 38:12-14. 2. Fujisawa, T., S. Shirasaka, J. Watabe, and T. Mitsuoka. 1984. Lactobacillus aviarius sp. nov.: a new species isolated from the intestine of chickens. Syst. Appl. Microbiol. 5:414420. 3. Itoh, K., T. Mitsuoka, K. Sudo, and K. Suzuki. 1983. Comparison of fecal lactobacilli in mice of different strains under different housing conditions. 2. Versuchstierkd. 25:193-200. 4. Johnson, J. L., C. F. Phelps, C. S. Cummins, J. London, and F. Gasser. 1980. Taxonomy of the Lactobacillus acidophilus group.
304
NOTES
Int. J. Syst. Bacteriol. 3053-68. 5. Kandler, O., and N. Weiss. 1986. Genus Lactobacillus Beijerinck 1901, 212*=, p. 1209-1234. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 6. Lauer, E., C. Helming, and 0. Kandler. 1980. Heterogeneity of the species Lactobacillus acidophilus (Moro) Hansen and Moquot as revealed by biochemical characteristics and DNA-DNA hybridisation. Zentralbl. Bakteriol. Parasitenkd. Infektionskr.
INT. J. SYST. BACTERIOL. Hyg. Abt. 1 Orig. Reihe C 1:15&168. 7. Mitsuoka, T. 1969. Vergleichende Untersuchungen uber die Laktobazillen aus den Faeces von Menschen, Schweinen, und Huhnern, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 210:32-51. 8. Shimohashi, H., and M. Mutai. 1977. Specific antigens of Lactobacillus acidophilus. J. Gen. Microbiol. 103:337-344. 9. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath (ed.). 1980. Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30: 225420.
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1990, p. 302-304 0020-7713/90/030302-03$02.oo/o Copyright 0 1990, International Union of Microbiological Societies
NOTES Lactobacillus intestinalis (ex Hemme 1974) sp. nov., norn. rev., Isolated from the Intestines of Mice and Rats TOMOHIKO FUJISAWA,’ KIKUJI ITOH,l YOSHIMI BENNO,* A N D TOMOTARI MITSUOKA173* Laboratory for Intestinal Flora, Frontier Research Program,’ and Japan Collection of Microorganisms,2 The Institute of Physical and Chemical Research, Wako, Saitama 351-01, and Department of Biomedical Science, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113,3Japan The genetic and phenotypic properties of 10 strains identified as Lactobacillus intestinalis sp. nov. were examined. These strains constitute a distinct species which can be differentiated from all of the previously described homofermentativespecies in the genus Lactobacillus by their carbohydrate fermentation pattern. The guanine-plus-cytosinecontents of their DNAs are 33 to 35 mol % DNAs from 10 other Lactobacillus species did not exhibit significant levels of relatedness to representative strains of the new species. The name Lactobacillus intestinalis (ex Hemme) sp. nov., nom. rev. is proposed for these isolates, and strain Th4 (= ATCC 49335 = JCM 7548) is the type strain.
.
During studies on the intestinal flora of mice and rats (3), strains of “Lactobacillus intestinalis” were isolated. This species name was originally proposed by Hemme (D. Hemme, Ph.D. thesis, University of Paris, Paris, France, 1974), but it has never been validly published, was not included on the Approved Lists of Bacterial Names (9), and has no current nomenclatural standing. In a study which demonstrated the heterogeneity of the “Lactobacillus acidophilus group,” Lauer et al. (6) demonstrated that strain CNRZ 219 (corresponding to “L. intestinalis’ ’) represented a separate DNA homology group (group Ie) which did not exhibit high levels of homology with any strain of L . acidophilus or the strains of any other homology group. Johnson et al. (4) investigated four strains that were isolated by Shimohashi and Mutai (8) from rat intestinal tracts in a taxonomic study of the L . acidophilus group and classified them as members of homology group B-2, which is equivalent to group IIb of Lauer et al. (6). The objectives of this study were to determine the phenotypic and genetic characteristics of the strains which were isolated from the intestines of mice and rats and were identified as “ L . intestinalis” and to obtain a better understanding of the taxonomic position of these organisms. Twenty strains were used in this study. Nine strains were isolated from intestinal contents of mice or rats; strains F215, F218, F219, RTD1-1, RTD1-3, and RTD4-5 were isolated from rats, and strains LS-44, LS-47, and LS-49 were isolated from mice. “L. intestinalis” Th4T (T = type strain) was obtained from Hemme. L . acidophilus ATCC 4356T and ATCC 33200, Lactobacillus salivarius ATCC 11741T,Lactobacillus helveticus ATCC 15009T, Lactobacillus jensenii ATCC 2525gT, Lactobacillus crispatus JCM llUT,Lactobacillus gasseri JCM 1131T,Lactobacillus amylovorus JMC 1126T, Lactobacillus animalis JCM 5670T, Lactobacillus agilis JCM 1187T, Lactobacillus acetotolerans JCM 3825T,
and Lactobacillus hamsteri Ha5FlT were obtained from our collection. The strains were characterized by using the methods and procedures described previously (1, 2, 7). A comparison of the morphological, physiological, and biochemical characteristics, DNA base compositions, and TABLE 1. Levels of DNA homology between strains of L. intestinalis and some other Lactobacillus species having low G+C contents % Homology with
Unlabeled DNA from:
3H-labeled DNA from strain:
Th4T
L. intestinalis strains Th4T F215 F218 F219 RTD1-1 RTD1-3 RTD4-5 LS-44 LS-47 LS-49 L. acidophilus ATCC 4356T L. acidophilus ATCC 33200 L. crispatus JCM 1185T L. gasseri JCM 1131T L. amylovorus JCM 1126T L. animalis JCM 5670T L. salivarius ATCC 11741T L. agilis JCM 1187T L. hamsteri Ha5FlT L. helveticus ATCC 15009T L. jensenii ATCC 25258T L. acetotolerans JCM 3825T -, Not tested. Data from reference 5 .
* Corresponding author. 302
100 81 75 85 97 78 84 72 76 76 12 6 7 1 11 10 9 6
G+C content (mol%)
F215
35 32 32 33 32 33 32 34 34 32 32 33 35 33 40b 42b 35b 44b 33 39b 35b 35
VOL.40, 1990
NOTES
303
TABLE 3. Phenotypic characteristics that differentiate L . intestinalis from other homofermentative Lactobacillus species that have low G+C contents and do not grow at 15°C“ Aerobic ~somefls) Species or group
growth
L . intestinalis L . acidophilus group L . helveticus L . salivarius L . jensenii L . animalis-L. murinus L. acetotolerans L . kefiranofaciens L . vitulinus L . ruminis L. aviarius
Of
lactic
Acid produced from:
Su- Escu- Sali- Amygcrose lin cin dalin
DL DL DL L D L DL D , DL D L L, DL
+
-
-
+
+
v
v
-
+
v
v
v
-
-
+
-
+
-
+
-
+ + + + + + + + -+ +- -+ -+ + + + + + + +
a The Characteristics were determined by testing the strains used in this study, including the type strains. + ,Positive reaction; -, negative reaction; v, variable reaction. D, D-Lactic acid makes up 90% or more of the total lactic acid; L, L-lactic acid makes up 90% or more of the total lactic acid; DL, 25 to 75% of the total lactic acid is L-lactic acid.
FIG. 1. Strain Th4T of L . intestinalis sp. nov., nom. rev. (a) Gram-stained cells from a glucose-blood-liver agar culture incubated anaerobically at 37°C for 48 h. Bar = 10 pm. (b) Surface colonies on a glucose-blood-liver agar plate incubated anaerobically at 37°C for 48 h. Bar = 10 mm.
DNA-DNA homologies of the 10 “L. intestinalis” strains studied with the properties of the previously described species of the genus Lactobacillus indicated that these strains represent a distinct species. The name “Lactobacillus intestinalis” has not been validly published previously and was not included on the Approved Lists (9). Therefore, we propose Lactobacillus intestinalis sp. nov., nom. rev. as a valid species of the genus Lactobacillus. Table 1 shows the guanine-plus-cytosine (G +C) contents and levels of DNA homology of the 10 strains of “L. intestinalis” examined as determined by the S1 nuclease method. The G+C contents of the DNAs from the “L. intestinalis” strains were 32 to 35 mol% as determined by the thermal denaturation method. All of the strains had high levels of DNA-DNA homology with each other. The DNAs of 12 other Lactobacillus species exhibited little or no homology with the DNAs of strains Th4T and F215. On the basis of the distinctions described above, we propose the name Lactobacillus intestinalis. A description is given below. Lactobacillus intestinalis (ex Hemme 1970) sp. nov., nom.
TABLE 2. Variable biochemical characteristics of L . intestinalis and type strain Th4
rev. Lactobacillus intestinalis (in.test.in.al’is. N. L. adj. intestinalis, pertaining to the intestine). Gram-positive, nonmotile, nonsporeforming, facultatively anaerobic rods, generally 0.6 to 0.8 by 2.0 to 6.0 pm, that occur singly, in pairs, or occasionally in short chains (Fig. la). All strains grow on glucose-blood-liver agar (2), on MRS agar, in Briggs liver broth, and in MRS broth. Surface colonies on glucoseblood-liver agar after 2 days of anaerobic incubation at 37°C are 0.7 to 2.5 mm in diameter, round, flat, and light brown with rough surfaces and erose edges (Fig. lb). Produces DL-lactic acid from glucose homofermentatively. The final pH of glucose broth is 3.7. No growth occurs at 15°C in Briggs liver broth. Catalase is not produced. Esculin and starch are not hydrolyzed. Gas is not produced from glucose. Acid is produced from glucose, mannose, fructose, galactose, sucrose, maltose, dextrin, and mannitol but not from arabinose, xylose, rhamnose, trehalose, melezitose, starch, sorbitol, esculin, salicin, and amygdalin. The final pH in glucose broth is 3.5 to 3.8. Variable characteristics are shown in Table 2. The G+C content of the DNA is 32.5 to 35.4 mol%. The habitat of the species is the intestines of mice and rats. The type strain is strain Th4 (= ATCC 49335 = JCM 7548). The phenotypic characteristics that differentiate L. intestinalis from other homofermentative Lactobacillus species that have low G+C contents and that do not grow at 15°C are shown in Table 3. LITERATURE CITED
L. intestinalis reactiona
Acid produced from:
10 strains
Ribose Cellobiose Lactose Melibiose Raffinose Dextrin
d d d d d d
8
Type strain Th4
+, Positive reaction; w, weak reaction; s, delayed reaction; d, 11to 89% of the strains are positive.
1. Fujisawa, T., S. Adachi, T. Toba, K. Arihara, and T. Mitsuoka. 1988. Lactobacillus kefiranofaciens sp. nov. isolated from kefir grains. Int. J. Syst. Bacteriol. 38:12-14. 2. Fujisawa, T., S. Shirasaka, J. Watabe, and T. Mitsuoka. 1984. Lactobacillus aviarius sp. nov.: a new species isolated from the intestine of chickens. Syst. Appl. Microbiol. 5:414420. 3. Itoh, K., T. Mitsuoka, K. Sudo, and K. Suzuki. 1983. Comparison of fecal lactobacilli in mice of different strains under different housing conditions. 2. Versuchstierkd. 25:193-200. 4. Johnson, J. L., C. F. Phelps, C. S. Cummins, J. London, and F. Gasser. 1980. Taxonomy of the Lactobacillus acidophilus group.
304
NOTES
Int. J. Syst. Bacteriol. 3053-68. 5. Kandler, O., and N. Weiss. 1986. Genus Lactobacillus Beijerinck 1901, 212*=, p. 1209-1234. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 6. Lauer, E., C. Helming, and 0. Kandler. 1980. Heterogeneity of the species Lactobacillus acidophilus (Moro) Hansen and Moquot as revealed by biochemical characteristics and DNA-DNA hybridisation. Zentralbl. Bakteriol. Parasitenkd. Infektionskr.
INT. J. SYST. BACTERIOL. Hyg. Abt. 1 Orig. Reihe C 1:15&168. 7. Mitsuoka, T. 1969. Vergleichende Untersuchungen uber die Laktobazillen aus den Faeces von Menschen, Schweinen, und Huhnern, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 210:32-51. 8. Shimohashi, H., and M. Mutai. 1977. Specific antigens of Lactobacillus acidophilus. J. Gen. Microbiol. 103:337-344. 9. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath (ed.). 1980. Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30: 225420.
