INTERNATIONAL JOURNAL O F SYSTEMATIC BACTERIOLOGY, OCt. 1991, p. 548-557 0020-7713/91/040548-10$02.00/0 Copyright 0 1991, International Union of Microbiological Societies

Vol. 41, No. 4

Phenotypic Differentiation of Bifidobacteria of Human and Animal Origins FRANCOISE GAVINI,l* ANNE-MARIE POURCHER,2 CHRISTEL NEUT,3 DANIEL MONGET,4 CHARLES ROMOND13CATHERINE OGER,’ A N D DANIEL IZARD135 Institut National de la Recherche Agronomique, Lahoratoire d’Ecologie et de Physiologie du SystPme Digestif, Domaine du C.E.R. T.I.A., 59650 Villeneuve d’Ascq Cedex,’ Service Eaux et Environnement, Institut Pasteur, 59019 Lille Cedex,2 Service de Bactkriologie, FacinltP de Pharmacie, 59045 Lille cede^,^ BioMerieinx-Unite de Bactkriologie, La Balme Les Grottes, 38390 Montalieu VercieuJ4and Service de BactPriologie, FacultP de MPdecine, 59045 Lille cede^,^ France The phenotypes of 153 strains belonging or related to the genus Bijidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of BiJidobacterium pseudocatenulatum and B . catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B . dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIal, and the type strains of B . infantis and B . longum fell into subgroup IIIbl. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group I1 consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B . suis (subgroup IIb), B . thermophilum (subgroup IIf), B . choerinum, and B . boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B . animalis (subgroup Va), B . magnum (subgroup Vb), B . pseudolongum, and B . globosum (subgroup Vc). The strains of human origin (groups I, 111, and VII) were well separated from the animal strains (groups 11, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bijidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:ll-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey’s Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus. -

-

~~

differentiated into four species, B. adolescentis (24), B. dentium, B. catenulatum, and B . angulatum (19), whereas “B. liberorum” and “B. lactensis” have been identified as B . infantis, “B. parvulorum” has been identified as B. breve, and “B. ruminale” has been identified as B. thermophilum (24). Since 1974, 13 species, including 9 species isolated from animals, have been described by Biavati et al. (4) and Scardovi et al. (18, 19, 21, 22, 25). B. magnum (25) and B. cuniculi (22) were found in rabbit feces. B . pullorum (28) and B. gallinarum (29) were isolated from chicken feces. B. boum and B. choerinum, which were first isolated from piglet feces by Zani et al. (30) and were also found in cattle rumina and pig feces, were described in 1979 by Scardovi et al. (22). Two species, B . ruminantium and B. merycicum, which were isolated from bovine rumina, were described recently by Biavati and Mattarelli (3). The human species that have been described most recently are B . pseudocatenulatum (22) and B . gallicum (9). B. minimum and B. subtile (4,21) have been isolated only from sewage. At the present time, the genus Bifidobacterium includes 28 species (10 species of human origin, 13 species of warm-blooded animal origin, 3 species isolated from honey bees, and 2 species isolated from sewage). Acidification tests performed with carbohydrates are not sufficient to identify bifidobacterial species. The results of other techniques that are based on electrophoretic patterns of soluble proteins (4)or transaldolase and 6-phosphogluconate dehydrogenase activities (18) do not correlate well with phenotypic or genomic descriptions of the species.

Since the first description of Bacillus bifidus by Tissier (27) in 1900, bifidobacteria have been studied with increasing interest because of their importance in the physiology of the gastrointestinal tract, particularly the gastrointestinal tract of neonates (2, 5, 14), and in food microbiology. Furthermore, because of their high density in human and animal feces, it has been proposed that these bacteria are potential fecal indicators of water contamination (7, 11, 15). In 1969, the 16 described species of the genus Bijidobacterium were differentiated on the basis of their carbohydrate acidification and serological characteristics (13, 16, 20, 23). The following eight species of human origin had been described by Reuter in 1963 (16):Bifidobacterium infantis, “B. liberorum,” “B. lactensis,” B. longum, B. breve, “B. parvulorum,” B. adolescentis, and B. bijidum. In 1969, Mitsuoka (13) isolated B. thermophilum and B . pseudolongum from bovine rumina and animal feces and described them, as well as B. longum subsp. animalis, which was elevated in 1974 to species level as B. animalis (21). In the same year Scardovi et al. (20, 23) described two species that were isolated from warm-blooded animals, B . glohosum and “B. ruminale,” and three species that were found in honeybees, which they named B. asteroi‘des, B. indicum, and B. coryneforme. B. suis, which was isolated from feces of swine, was described by Matteuzzi et al. in 1971 (12). On the basis of DNA-DNA hybridization results, the strains described by Reuter (16) as B. adolescentis have been

* Corresponding author. 548

VOL. 41. 1991

PHENOTYPIC DIFFERENTIATION OF BIFIDOBACTERIA

549

idase, L-isoleucine arylamidase, L-proline arylamidase, L-threonine arylamidase, and N-CBZ-glycyl-glycyl-arginine arylamidase. The group IV peptidases were p-alanine arylamidase, L-alanyk-phenylalanyl-L-proline arylamidase, L-arginylL-arginine arylamidase, a-L-aspartyl-L-arginine arylamidase, a-L-glutamyl-L-histidine arylamidase, L-alanyl-L-arginine arylarylamiamidase, L-alanyl-L-phenylalanyl-L-prolyl-L-alanine dase, a-L-aspartyl-L-alanine arylamidase, a-L-glutamyl-a-Lglutamic acid arylamidase, and glycyl-L-alanine arylamidase. The group V peptidases were glycyl-L-arginine arylamidase, L-histidyl-L-leucyl-L-histidine arylamidase, L-leucyl-L-alanine MATERIALS AND METHODS arylamidase, L-lysyl-L-alanine arylamidase, L-phenylalanyl-Larginine arylamidase, glycyl-L-tryptophan arylamidase, L-hisBacterial strains. The strains which we used and their tidyl-L-serine arylamidase, L-leucyl-L-leucyl-L-valyl-L-tyrosylsources are listed in Table 1. A total of 153 strains were L-serine arylamidase, L-lysyl-L-lysine arylamidase, and included in the analysis, including 38 type or reference L-phenylalanyl-L-proline arylamidase. The group VI peptistrains that were received from diverse culture collections dases were L-phenylalanyk-prolyl-L-alanine arylamidase, and 115 wild strains. Among the latter, 41 strains were L-seryl-L-methionine arylamidase, N-benzoyl-~-alanine-4isolated from human adults (5 strains from bronchi, 2 strains methoxy-arylamidase, N-CBZ-glycyl-glycyl-L-arginine arylfrom buccal cavities, 2 strains from vaginas, 8 strains from amidase, L-histidyl-L-phenylalanine arylamidase, L-prolyl-Lfeces) and infants (24 strains from intestinal contents or arginine arylamidase, L-valyl-L-tyrosyl-L-serine arylamidase, feces). A total of 56 strains were isolated from feces of various animals (36 strains from pigs, 2 strains from rabbits, N-CBZ-arginyl-4-methoxyarylamidase,N-acetyl-glycyl-L-ly1 strain from a dog, 2 strains from goats, 2 strains from arylamidase. sine arylamidase, and ~-lysyl-~-serine-4-methoxy sheep, 2 strains from hens, 3 strains from horses, 2 strains Esterases tested were C4, C6, C9, C12, C16, C5, C8, C10, C14, from cows, 6 strains from calves). Ten strains were isolated and C18. Several osidases, including a-galactosidase, phosphofrom surface water, and eight strains were isolated from p-galactosidase, a-glucosidase, p-galacturonohydrolase, aurban raw sewage. Isolation was facilitated by the use of maltosidase, N-acetyl-a-glucosaminidase, a-fucosidase, p-LBeerens medium (1). fucosidase, a-mannosidase, a-xylosidase, p-galactosidase, Phenotypic characterization. Strains were considered to be a-arabinosidase, p-glucosidase, P-glucuronidase, p-maltosimembers of the genus Bifdobacteriurn if fructose-6-phosdase, N-acetyl-P-glucosaminidase, P-D-fucosidase, p-lactosiphate phosphoketolase was detected when we used the dase, P-mannosidase, and p-xylosidase, were also tested. technique described by Scardovi (17) and if they had the Enzymatic tests kits were incubated at 39°C for 5 h under following characteristics: anaerobic, gram-positive cells; aerobic conditions. The tests were scored as positive by using catalase negative; no nitrate reduction; indole was not the recommendations of BioMerieux. The 48 carbohydrate produced; and glucose was acidified without gas producacidification tests and the test for hydrolysis of esculin were tion. performed by using API 5OCHL medium supplemented with Enzymatic tests and tests for acidification of carbohydrates 0.05% cysteine hydrochloride. The acidification tests were were performed by using experimental enzymatic strips (90 read after 24 and 48 h of incubation in an anerobic station at tests) and API 50CH kits (49 tests) (BioMerieux, La Balme les 39°C (10% CO,, 10% H,, 80% N,). Tests for growth at 45 and Grottes, Montalieu Vercieu, France). Fermentation tests for 46°C and growth in the presence of different concentrations of the following carbohydrates were used in this analysis: glycsodium chloride (0, 0.8, 3, and 5%) were performed in Tryptierol, L-arabinose, D-xylose, ribose, glucose, mannose, fructose, galactose, saccharose, maltose, cellobiose, lactose, case Phytone yeast broth (17). Reactions were recorded after 2 trehalose, melibiose, raffinose, melezitose, starch, glycogen, and 8 days. Growth at 45°C was tested separately, and the inulin, mannitol, sorbitol, inositol, esculin, salicin, amygdalin, results of this test were not included in the numerical analysis. erythritol, D-arabinose, L-xylose, adonitol, P-methyl-D-xyloNumerical analysis. The results for 26 tests which were side, sorbose, rhamnose, dulcitol, a-methyl-D-mannoside, either positive or negative for all of the strains were not a-methyl-D-glucoside, N-acetylglucosamine, arbutin, xylitol, included in the numerical analysis. The tests that were gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-funegative for all of the strains were the tests for glycerol, cose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate, and inositol, erythritol, D-arabinose, L-xylose, adonitol, sorbose, 5-ketogluconate. The enzymatic activities tested included 59 rhamnose, dulcitol, a-methyl-D-mannoside, D-lyxose, D-fUpeptidase, 10 esterase, and 20 glycosidase activities. The group cose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluI peptidases were as follows: L-tyrosine arylamidase, L-pyrroliconate, 5-ketogluconate, a-glutamyltransferase, N-CBZ-ardone arylamidase, L-lysine arylamidase, L-histidine arylamiginine-4-met hoxy-arylamidase, N-acetyl-glycyl-L-lysine aryldase ,L-arginine arylamidase, glycine arylamidase, L-phenylalamidase, esterase C16, esterase C14, phospho-p-galactosianine arylamidase, L-hydroxyproline arylamidase, L-aspartate dase, and p-galacturonohydrolase; the esterase C18 test was arylamidase, and L-alanine arylamidase. The group I1 peptipositive for all strains. The results of 2 of the 114 tests that dases were a-glutamyltransferase, S-benzyl-cysteine arylamiwere encoded (growth at 46°C and growth in the presence of dase, glycyl-glycine arylamidase, glycyl-proline arylamisodium chloride) were subdivided. These results were coded dase, L-seryl-tyrosine arylamidase, N-benzoyl-leucine arylto give a single quantitative multistate test. The level of amidase, L-methionine arylamidase, glycyl-phenylalanine similarity between bacterial strains was calculated by usarylamidase, and leucyl-glycine arylamidase. The group 111 peptidases were N-CBZ-arginine-4-methoxy-arylamidase, ing the Dice index (8). Grouping of the strains was based on the unweighted pair group average linkage method (6, a-L-glutamate arylamidase, L-ornithine arylamidase, L-serine 26). arylamidase, L-tryptophan arylamidase, L-glutamine arylamTherefore, many questions concerning the ecology and identification of bifidobacteria remain to be answered. The purpose of this study was to assess the distribution of bifidobacteria isolated from animals and humans by performing a numerical analysis that was based on a large number of tests. Most of the tests have not been used previously to study the genus Bijidobacterium, including tests involving new substrates to detect enzymatic activities.

550

INT.J. SYST.BACTERIOL.

GAVINI ET AL. TABLE 1. Strains used in this study

Group

I

Subgroup

Ia Ib

Ic

Id Ungrouped

I1

IIa IIb

IIC

IId IIe IIf

Culture collection or other reference no.a

CUETM 89-158 ATCC 27539* ATCC 15705 CUETM 89-168 DSM 20438T CUETM 89-196 CUETM 89-270 ATCC 27534T CUETM 89-147 CUETM 89-143 CUETM 89-275 CUETM 89-219 CUETM 89-144 CUETM 89-162 ATCC 27678 CUETM 89-141 ATCC 27679 CUETM 89-111 ATCC 15703T CUETM 89-277 CUETM 90-65 CUETM 90-129 ATCC 27670 ATCC 27535T CUETM 90-145 CUETM 90-78 CUETM 90-80 DSM 20211T CUETM 90-152 CUETM 90-155 CUETM 90-141 CUETM 90-153 CUETM 90-44 CUETM 90-158 CUETM 90-138 CUETM 90-85 CUETM 90-40 CUETM 90-105 CUETM 90-52 CUETM 90-140 CUETM 90-77 CUETM 90-86 CUETM 90-82 CUETM 90-41 CUETM 90-146 CUETM 90-90 CUETM 90-139 CUETM 90-45 CUETM 90-150 CUETM 90-111 CUETM 90-110 CUETM 90-79 CUETM 90-119 CUETM 90-51 CUETM 90-49 CUETM 90-50 ATCC 15706 CUETM 90-84 CUETM 89-283 CUETM 90-81 CUETM 90-46 CUETM 90-83 CUETM 90-76 CUETM 90-113 CUETM 90-43 CUETM 90-124 DSM 20210T

Name as received

?

B . catenulatum B . adolescentis

? B . pseudocatenulatum

B . dentium

? ?

? ? 9

B . dentium B . dentium

? ? ?

?

?

B . adolescentis 9

? ?

B . angulatum B . angulatum

B . suis

? ? ?

? ? ? ? ? ? ? ? ? ? 3 9

? 9 9

? ? 9

? ? 9

? ? ?

B . adolescentis ? ? 9 9

?

B . thermophilum

Isolated from:

Adult intestine Adult intestine Adult intestine Adult feces Child feces Child feces Adult feces Dental caries Bronchus Bronchus Buccal cavity Bronchus Bronchus Adult feces Human feces Buccal cavity Human vagina Adult feces Adult intestine Child feces Sewage Dog feces Sewage Human feces Pig feces Pig feces Pig feces Pig feces Pig feces Sewage Pig feces River Pig feces River Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Cow feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Pig feces Adult intestine Pig feces Pig feces Pig feces Pig feces Horse feces Horse feces Horse feces Pig feces Chicken feces Pig feces Continued on following page

VOL.41, 1991

PHENOTYPIC DIFFERENTIATION OF BIFIDOBACTERIA

551

TABLE 1-Continued Group

Subgroup ~

IIIal

IIIa2

IIIa3

IIIbl

IV

V

Ungrouped

Va

Vb vc

Ungrouped VI VII

Name as received

Isolated from:

~~~

Ungrouped

111

Culture collection or other reference no."

VIIa

VIIb

Ungrouped

ATCC 25866 ATCC 27686* CUETM 90-48 CUETM 90-154 ATCC 27917T CUETM 89-184 CUETM 89-175 NCFB 2257T CUETM 89-248 CUETM 89-181 CUETM 89-160 CUETM 89-148 CUETM 90-159 CUETM 89-293 CUETM 90-118 CUETM 90-114 CUETM 89-173 CUETM 89-241 CUETM 89-237 CUETM 89-178 CUETM 90-71 CUETM 89-157 CUETM 90-116 CUETM 90-69 ATCC 15697T CUETM 89-276 CUETM 90-70 CUETM 89-216 ATCC 15707T CUETM 89-177 CUETM 90-66 CUETM 90-67 ATCC 25912T DSM 20089T CUETM 90-47 ATCC 27536 CUETM 90-68 ATCC 25527T CUETM 90-89 CUETM 90-109 DSM 20222T ATCC 27682 ATCC 25864 CUETM 90-104 ATCC 25526T CUETM 90-147 CUETM 90-54 CUETM 90-91 ATCC 25865T DSM 20095 DSM 20094 CUETM 90-55 CUETM 90-148 CUETM 90-149 CUETM 90-156 CUETM 90-64 ATCC 27683 ATCC 27537T CUETM 89-244 CUETM 89-218 CUETM 89-129 CUETM 89-271 CUETM 89-250 CUETM 89-280 CUETM 89-262 CUETM 89-269 CUETM 89-128

B . thermophilum B . choerinum 3

B . boum

? 7 9

B . breve

? 3

? ? 7

? ? 9

B . infantis

? ? ? ? ? 7 '

B . longum

? 9

? ?

B . indicum B . asteroides ?

B . animalis

?

B . animalis

? ?

B . magnum B . magnum B . globosurn ?

B . pseudolongum ? ? ?

B . globosurn B . pseudolongum B . pseudolongum ? 3

? ? 9

B . subtile B . subtile

? ? ? ? ? ? 9

? ?

Bovine rumen Pig feces Pig feces Pig feces Bovine rumen Infant intestine Human vagina Infant intestine Child feces Child feces Adult intestine Child feces River Child feces River River Child feces Child feces Child feces Human vagina Sewage Adult feces River River Infant intestine Child feces River Adult intestine Adult intestine Infant intestine Sewage River Hindgut of honeybee Hindgut of honeybee Chicken feces Chicken feces River Rat feces Sewage Sewage Rabbit feces Rabbit feces Bovine rumen Rabbit feces Pig feces Calf feces Calf feces Sheep feces Bovine rumen Chicken feces Chicken feces Cow feces Goat feces Goat feces Calf feces Calf feces Sewage Sewage Child feces Child feces Child feces (diahrrea) Child feces Child feces Child feces Child feces Child feces Child feces Continued on foiiowing page

552

GAVINI ET AL.

INT. J . SYST.BACTERIOL. TABLE 1-Continued

Group

Culture collection or other reference no."

Subgroup

CUETM 89-166 ATCC 27538T CUETM 90-155 CUETM 90-157 CUETM 89-282 DSM 20093T CUETM 90-151 DSM 20082 CUETM 90-112 CUETM 90-144 CUETM 90-42 CUETM 90-92 ATCC 25911T ATCC 27916T DSM 20433T ATCC 33777* CUETM 89-261 CUETM 89-209 CUETM 90-53

Ungrouped

Name as received

Isolated from:

?

Child feces Sewage Sewage Sewage Child feces Adult intestine Rabbit feces Adult intestine Calf feces Sheep feces Pig feces Pig feces Hindgut of honeybee Rabbit feces Chicken feces Chicken cecum Child feces Human bronchus Calf feces

B . minimum ? ? ?

B . gallicum ? B . biJidum ? ? ? ? B . coryneforme B . cuniculi B . pullorum B . gallinarum ? ? ?

ATCC, American Type Culture Collection, Rockville, Md. ; CUETM, Collection Unit6 Ecotoxicologie Microbienne, Villeneuve d' Ascq, France; DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Germany; NCFB, National Collection of Food Bacteria, Shinfield, Reading, Berks, England.

TABLE 2. Characteristics that differentiate subgroups containing human strains % of positive strains

Characteristic

Enzymatic tests L-Aspartic acid arylamidase L-Alanine arylamidase L-Methionine arylamidase a-L-Glutamyl-L-histidine arylamidase L-Histidine-L-leucyl-Lhistidine arylamidase L-Lysyl-L-alanine arylamidase L-Pro1y 1-L-arginine arylamidase Esterase C4 Esterase C6 Esterase C10 a-Galactosidase P-Galactosidase a-Arabinosidase P-Glucosidase P-Lactosidase Acidification of carbohydrates L-Arabinose D-Xylose Mannose Mannitol Sorbitol a-Methylglucoside Am ygdalin Arbutin Esculin hydrolysis Salicin Trehalose Melezitose Starch GI ycogen

Subgroup Ia ( n = 2)"

Subgroup Ib (n = 4)

Subgroup Ic (n = 12)

Subgroup Id ( n = 2)

Subgroup IIIal (n = 6)

Subgroup IIIa2 ( n = 4)

Subgroup IIIIa3 (n = 5 )

Subgroup IIIbl ( n = 10)

Subgroup VIIa (n = 4)

Subgroup VIIb (n = 4)

25 0 0 0

0 0 0 0

0 0 0 0

100 83 83 0

100 100 0 0

100 80 80 0

100 100 70 60

100 0 0 0

100 75 0

75

0

0

83

75

100

90

25

100

0

0

25

100

33

100

80

50

0

0

0

50

0

0

0

25

100

80

0

25

100 100 0 100 100 50 100 100

100 100

67 67 17 100 100 100 92 83

0 0 0 100 100 50 100 100

83 100 50 100 83 17 83 50

100 100 25 100 100 100 100 100

100 100 0 100 100 100 100 80

100 100 30 90 100 100 10 10

0 0 0 0 0 0 0 0

0 25 0 25 50 0 0 0

100 100 83 83 17 75 100 100 100 100 75 58 100 100

0 0 0 0 100 0 100 100 50 50 0 0 50 50

0 0

25 25 0 25 100 0 100 75 50 50 0 0 50 50

80 100 0 0 100 0 100 100 80 80 60 0 80 100

90 80 10 0 0 0 0 0 0 0 0 40

100 0 0 0 0 0 0 0 25 0 0 100

100 50 50 0 0 0 0 0 50 0 0 100

0 0 0 0

0 0 50 50 100 0 50 0 100 0 0 0 0 0

0

50 75 75 100 25 100 75 0 0

100 0 100 100 100 100 0 0 100 100

67 83 83 0 100 17 100 0 0 0 67 100

0 0

0

0

50

0 0

n is the number of strains in the subgroup. The type strains of the following species belong to the subgroups: subgroup Ia, B . cutenulaturn; subgroup Ib, B . pseudocatenulutum; subgroup Ic, B . dentiurn; subgroup Id, B . adolescentis; subgroup IIIal, B . breve; and subgroup IIIbl, B . infantis and B . longum.

.o

100

% SD

,

groups and

origin

subgroups

of

strains

I

u 11 a r

i

I

Ia

Ib

HUlX3.n

Ic

I

Id

Da

Ilb

IIC

d

d

I1

Animal

IId

Ile

rrf IIIal

ma2

L

ma3

Human

IIIb 1

I

-

c I

I

IV

Animal

V

Animal

Va

Vb

1I ,

vc

I I

I --Ir

-1

Sewage

1

r

f

Vd I VI

I

I

I

VIIa Vllb

VII Human

FIG. 1. Phenotypic dendrogram based on unweighted pair group average linkage. S,, Dice similarity index. 553

554

INT. J . SYST.BACTERIOL.

GAVINI ET AL. TABLE 3. Characteristics that differentiate the seven main groups % of positive strains

Characteristic

Enzymatic tests L-Aspartic acid arylamidase S-Benzoyl-cysteine arylamidase L-Methionine arylamidase L-Seryl-tyrosine arylamidase L-Threonine arylamidase P-Alanine arylamidase a-L- Aspart y I-L-arginine arylamidase L-Histidyl-L-leucyl-L-histidine arylamidase L-Leucyl-L-alanine ary lamidase L-Pheny lalan y 1-L-arginine ary lamidase L-Seryl-L-methionine arylamidase Esterase C5 a-Galactosidase a-Glucosidase P-Glucosidase Acidification of carbohydrates L-Arabinose Ribose D-xylose Mannose Sorbitol Amygdalin Salicin Cellobiose Melezitose Growth at 46°C within 48 h

Group I ( n = 24)"

Group I1 ( n = 48)

Group 111 ( n = 26)

Group IV ( n = 4)

Group V ( n = 21)

Group VI (n = 2 )

Group VII ( n = 9)

13

4

90 85

100 69

50 100

100 100

100 100

100 11

0 0 4 0 0

58 27 65 0 2

65 8 54 0 0

100 75 75 0 0

100 90 81 0 64

100 0 100 100 0

0 0 11 0 0

21

88

85

100

100

0

67

0

38

38

100

100

0

11

4

52

31

25

100

0

0

0

31

8

50

100

50

0

71 88 88 92

98 100 98 100

96 96 85 61

75 100 100 100

100 100 100 86

100 100 100 0

0 11 0 0

75 83 67 44 46 83 75 46 29 0

2 2 4 0 0 85 8 0 13 81

58 69 58 19 58 58 23 11 15 0

25 50 0 25 0 100 75 50 0 25

43 29 24 0 0 5 0 0 0 9

0 100 0 0 100 0 0 0 0 50

100 44 100 22 0 0 0 0 89 0

'' n is the number of strains in the group. The type strains of the following species belong to the groups: group I, B . catenulatitm, B . pseudocutenulaturn, B. dentiurn, B. adolescentis, and B . anguladum; group 11, B. suis, B. thermophilum, B. chnerinum, and B. h u m ; group 111, B. breve, B. infuntis, and B . longum; group IV, B. indic-urn and B. crsteroides; group V, B. animalis, B . magnum, B. globosum, and B. pseudolotigum; and group VI, B. subtile.

RESULTS AND DISCUSSION

Our analysis revealed seven main groups, which were subdivided into 20 subgroups (Fig. 1);18 strains did not fall into any group. A clear separation between groups containing strains of human origin and groups containing strains of animal origin was observed. Groups containing strains of human origin. Group I (Table l), which contained 24 strains, was subdivided into four subgroups. Subgroups Ia and Id, which included only two strains each, contained the type strains of B. catenulatum and B. adolescentis, respectively. Subgroup Ib (four strains) contained the type strain of B. pseudocutenulaturn and B. adolescentis ATCC 15705. Most of the wild strains in subgroups Ia, Ib, and Id were isolated from the intestines or feces of adults. Subgroup Ic (12 strains) was numerically the most important subgroup one in group I; it included collection strains ( B . dentium ATCC 27534T, ATCC 27678, and ATCC 27679) and wild strains that were isolated from dental caries, a vagina, bronchi, and adult feces. Four group I strains (including B. unguluturn ATCC 27535T and ATCC 27670) did not fall into any subgroup. Graup I11 (Table l), which contained 26 strains, was subdivided into the following four subgroups: subgroup IIIal ( B . breve NCFB 2257T and 5 wild strains), subgroup IIIa2 (4

wild strains), subgroup IIIa3 ( 5 wild strains), and subgroup IIIbl (10 strains, including B. infuntis ATCC 15697T and B . longurn ATCC 15707T). A total of 46% of the collection and wild strains were isolated from feces of infants, and the remainder were isolated from adult feces or vaginas (23%) and from river water (31%). Strains isolated from calves have been referred to previously as intermediates between B . longum and B . infantis and were more than 80% related to the reference strains of both species as determined by DNA-DNA hybridization (18); in contrast, Lauer and Kandler (10) found levels of DNA relatedness between B. longum and B. infuntis of 62 to 68%. In our study, no bifidobacteria isolated from calf feces aggregated with these two species. Group I and subgroup IIIa3 contained wild strains that were isolated mainly from adults. Mara and Oragui (11) developed a medium which separated human strains (sorbito1 positive) from animal strains (sorbitol negative). In our analysis we observed no sorbitol acidification by human strains, especially strains belonging to subgroups Ic (B. dentiurn) and IIIbl (B. infuntis and B . longurn). Group VII was composed of 10 wild strains. Subgroups VIIa and VIIb included only strains that were isolated from infant feces and could not be assigned to any species.

PHENOTYPIC DIFFERENTIATION OF BIFlDOBACTERIA

VOL.41, 1991

555

TABLE 4. Characteristics that differentiate groups and subgroups containing animal and sewage strains 5% of positive strains

Characteristic

Enzymatic tests L-Tyrosine arylamidase L- Alanine arylamidase Glycyl-phen ylalanine ary lamidase Leucyl-glycine arylamidase L-SeryI-t yrosine ary lamidase L-Serine arylamidase L-Tryptophan arylamidase N-CBZ-glycyl-glycyl arginine arylamidase P-Alanine arylamidase L-Alan y l-L-phen ylalan y l-Lproline arylamidase L-Histidyl-L-leucyl-Lhistidine arylamidase L-Histidyl-L-serine arylamidase L-Leucyl-1.-alanine ar y lamidase L-Phenylalan yl-1--arginine arylamidase r-Valyl-L-tyrosyl-ia-serine ary lamidase

Subgroup Subgroup Subgroup Subgroup Subgroup Subgroup Group Subgroup Subgroup Subgroup Subgroup Group IIa Ilb IIC IId IIe IIf IV Va Vb vc Vd VI ( n = 3)" ( n = 4) (17 = 25) ( n = 3 ) ( n = 2) ( n = 7) ( n = 4) ( n 5 ) ( n = 2) ( n = 9) ( n = 3) ( n = 2)

100 100 100

100 100 25

36 100 28

0 100 0

0 100 0

0 14 0

75 100 50

100 100 80

100 100 100

100 100 78

100 100 33

100 0 0

100 100

100 100

100 16

100 0

100 0

14 0

100 75

100 100

0 50

89 100

0 67

0 0

100 67 0

100 100 100

100 52 4

100 67 0

100 0 0

0 0 0

100 75 0

100 100 20

100 50 0

100 89 22

100 33 0

100 0 0

0 100

0 100

0 92

0 100

0 50

0 100

0 100

0 100

0 0

0 78

0 100

100 50

100

100

100

100

0

43

100

100

100

100

100

0

0

50

8

33

0

0

0

loo

0

67

100

0

100

100

36

33

0

0

100

100

100

100

100

0

100

100

52

100

50

0

25

100

100

100

100

0

0

0

29

0

58

0

0

20

100

89

0

0

0 ary lamidase ~-Lysyl-~-serine-4-methoxy 67 arylamidase P-Glucosidase 100 Acidification of carbohydrates L-Arabinose 0 0 Ribose D - x ylose 33 67 Galactose 0 Sorbitol 0 Am ygdalin 0 Arbutin 76 Lactose Melibiose . 100 100 Starch Glycogen 100 Growth at 46°C within 48 h 100

100

4

0

0

0

0

0

0

11

0

0

50

0

0

0

0

0

100

0

11

0

0

100

100

100

100

200

100

100

50

89

100

0

25 25 25 100 0 0 0 100 100 100 100 75

0 0 0 80 0 100 12 0 100 100 96 88

0 0 0 100 0 100 0 71 100 0 0 0

0 0 0 0 0 100 100 100 100 50 100 0

0 0 0 86 0 100 0 100 100 100 100 100

25 50 0 75 0 100 75 100 50 25 25 25

20 60 0 80 0 20 0 60 80 0 20 20

100 0 100 100 0 0 0 100 0 0 0 0

67 11 33 67 0 0 0 78 100 89 78 11

0 33 0 0 0 0 0 0 100 0 0 0

0 100 0 100 100 0 0 50 100 100 100 50

N-CBZ-arginyl-4-methox y

'' n is the number of strains in the group or subgroup. The type strains of the following species belong t o the groups o r subgroups: subgroup Ilb, B. suiS; subgroup IIf, R . therrnopphilurn: group IV, R. indicum and B . asteroidrs; subgroup Va. B . anirnalis; subgroup Vb, B . magnum; subgroup Vc. B . pseudolongurn and B . glohosum; and group VI. B. subrile.

According to Biavati et al. (2), the species found most frequently in infant feces were B . infantis, B . longum, and B . breve. However, strains belonging to group VII, despite acidification profiles similar to those of B . infnntis and B . longum, were very different because of the absence of several enzymatic activities on the substrates tested (Table 2). The DNA-DNA relationships between group VII and the two other human groups must be clarified. Phenotypic characteristics of the three human groups are shown in Tables 2 and 3. Tests for L-aspartic acid arylamidase, L-alanine arylamidase, and L-methionine arylamidase activities and acidification of L-arabinose, mannose, mannitol, and amygdalin were most helpful in differentiating among subclusters of the human strains. Groups containing strains of animal or sewage origin.

TABLE 5. Differentiation of human and animal strains by growth at 45°C Strains

Wild Collection

Origin

No. of strain s tested

96 of strains that

Human Animal Human Animal

41 56 14 16

0 96.5 14.3" 75b

grew at 45°C within 48 h

'I The collection strains which grew at 45°C were B. cafenulaturn ATCC 27539T and B. pserrdocatenulatum DSM 20438.". The collection strains which did not grow at 45°C were B. rnugnurn DSM 20122' and ATCC 27682 and B. glohosum ATCC 25864 and ATCC 25865T.

556

GAVINI ET AL.

Group I1 (Table 1) included primarily strains that were isolated from pig feces; only 24% of these strains fell into subgroups that contained collection strains. Group I1 was the most important animal group (48 strains) and was subdivided into six subgroups, three of which did not include any type or collection strains (subgroups IIa, IIc, and IIe). Subgroups IIa, IIb, IIc, and IIe, which contained 3, 4, 25, and 2 strains, respectively, contained primarily organisms that were isolated from pig feces or water. Only three strains aggregated with B. suis DSM 20211T (subgroup IIb), and five strains aggregated with B. thermophilum DSM 20210T and ATCC 25866 (subgroup IIf) (previously named “B. ruminale” by Scardovi et al. [23]). Subgroup IIf comprised wild strains that were isolated from various animals (horse, pig, and chicken feces). Human strain B. adolescentis ATCC 15706 was included in subgroup IId along with wild strains isolated from pig feces. A total of 22 strains isolated from pig feces fell into subgroup IIc, and two strains fell into subgroup IIe. In an ecological study, Zani et al. (30) isolated B. suis from 77% of pig feces samples. In our analysis, subgroup IIb contained only three strains of B. suis (including the type strain) isolated from pig feces. The 25 subgroup IIc strains had a homogeneous acidification pattern, and the identification of these strains as B . suis must be studied by DNA-DNA hybridization. Three subgroups (subgroups IIa, IId, and IIe) which did not contain any type or collection strains were distinguished by their acidification and enzymatic profiles. Subgroup IId and IIe strains which grew at 45°C were differentiated by their inability to grow at 46°C. Group I1 strains B. boum ATCC 27917T and B . choerinum ATCC 27686T did not fall into any subgroup. No wild strains belonging to these species were found in the animal fecal samples. Group V contained 9 type and collection strains and 12 wild strains that were isolated from chicken, rat, rabbit, pig, calf, sheep, cow, and goat feces, sewage, and river water and was divided into four subgroups (subgroups Va through Vd). Subgroup Va (five strains) contained three wild strains isolated from surface water or sewage and two collection strains of B. animalis (strains ATCC 25627T and ATCC 27536), which were 99% related to each other as determined by DNA-DNA hybridization (21). Subgroup Vb, which contained strains that were isolated from rabbits, phenotypically resembled subgroup Vc. This subgroup contained B. magnum DSM 20222T and ATCC 27682, whose DNAs were 75% related to each other (25). Subgroup Vc contained nine strains that were isolated from bovine rumina and rabbit, pig, calf, sheep, and chicken feces. B. globosum ATCC 25865T and ATCC 25864 and B. pseudolongum ATCC 25526T, DSM 20095, and DSM 20094 fell into this subgroup. The wild strains in this subgroup were isolated only from calf and sheep feces; none was isolated from pig feces. These results differed from those of Zani et al. (30), who isolated B. globosum and B. pseudolongum from 41% of their pig feces samples. In our analysis, with the exception of two strains which were ungrouped, all of the wild strains isolated from pig feces fell into subgroups IIa through IIf. Subgroup Vd included two strains that were isolated from goat feces and one strain that was isolated from cow feces. Two strains that were isolated from calf feces did not fall into any subgroup. Group IV contained two collection strains that were isolated from honeybees (B. indicum ATCC 25912T and B. asteroides DSM 20089T) and two wild strains that were isolated from river water and chicken feces. Group VI consisted of two strains, B . subtile ATCC 27537T and ATTC 27683. The clear separation of this group

INT.J. SYST.BACTERIOL.

confirmed the validity of the species B . subtile. No wild strains have been reported since the first description by Scardovi et al. (21), who isolated these organisms from sewage. A few type or collection strains did not fall into any group: B. minimum ATCC 27538T (from sewage), two strains of human origin (B. gallicum DSM 20093T and B. bifdum DSM 20082), and four strains of animal origin (B. pullorum DSM 20433T, B . gallinarum ATCC 33777T, B . cuniculi ATCC 27916T, and B. coryneforme ATCC 25911T). The results of phenotypic tests that are useful for differentiating groups and subgroups of animal strains are shown in Tables 3 and 4. Growth at 45°C seems to discriminate between animal and human strains very well (Table 5 ) since most of the strains that were isolated from animals grew at this temperature, whereas the majority of the human strains did not. In conclusion, strains of human origin (groups I, 111, and VII) were well separated from the animal strains (groups 11, IV, and V). It was not surprising that wild strains that were isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. We also found that acidification patterns are not adequate to differentiate Bifdobacterium species, as discussed previously (14, 17). However, enzymatic tests, such as tests for L-aspartic acid arylamidase, L-alanine arylamidase, and L-methionine arylamidase activities, furnish new taxonomic criteria for the genus. REFERENCES 1. Beerens, H. 1990. An elective and selective isolation medium for Bifidobacteriurn spp. Lett. Appl. Microbiol. 11:155-157. 2. Biavati, B., P. Castagnoli, F. Crociani, and L. D. Trovatelli. 1984. Species of Bijidubacterium in the feces of infants. Microbiologica (Bologna) 7:341-345. 3. Biavati, B., and P. Mattarelli. 1991. Bijidubacrerium rurninantium sp. nov. and Bijidobacterium rnerycicurn sp. nov. from the rumens of cattle. Int. J. Syst. Bacteriol. 41:163-168. 4. Biavati, B., V. Scardovi, and W. E. C. Moore. 1982. Electrophoretic patterns of proteins in the genus BiJidobacteriurn and proposal of four new species. Int. J . Syst. Bacteriol. 32:368--373. 5. Dehnert, J. 1957. Untersuchungen iiber die gram-positive Stuhlflora des Brustmilchkindes. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 169:66-79. 6. Delabre, M., A. Bianchi, and M. Veron. 1973. Etude critique des methodes de taxonomie numCrique. Application a une classification des bacteries aquicoles. Ann. Inst. Pasteur Microbiol. 124Az489-506. 7. Evison, L. M., and A. James. 1975. BiJidobacterium as an indicator of faecal pollution in water. Prog. Water Technol. 757-66. 8. Gavini, F., B. Lefebvre, and H. Leclerc. 1976. Positions taxonomiques d’enterobactkries H,S - par rapport au genre Citrobacter. Ann. Inst. Pasteur Microbiol. 127A:275-295. 9. Lauer, E. 1990. BiJidobacteriurngallicum sp. nov. isolated from human feces. Int. J. Syst. Bacteriol. 40:10&102. 10. Lauer, E., and 0. Kandler. 1983. DNA-DNA homology, murein types, and enzyme patterns in the type strains of the genus Bifidobacteriurn. Syst. Appl. Microbiol. 4:42-64. 11. Mara, D. D., and J. D. Oragui. 1983. Sorbitol-fermenting bifidobacteria as specific indicators of human faecal pollution. J. Appl. Bacteriol. 55349-357. 12. Matteuzzi, D., F. Crociani, G. Zani, and L. D. Trovatelli. 1971. Bifidobacteriurn suis n. sp.: a new species of the genus Bijidobacteriurn isolated from pig feces. Z. Allg. Mikrobiol. 11:387-395. 13. Mitsuoka, T. 1969. Vergleichende Untersuchungen iiber die Bifidobakterien aus dem Verdauungstrakt von Menchen und

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