Bifidobacteria and Probiotic Action
X. IDENTIFICATION BY PHENOTYPE INVESTIGATION
When the bifidobacteria were discovered by Tissier at the beginning of the twentieth century, taxonomy was based entirely on morphological observations. This lack of differ-entiation criteria explains the numerous debates which preceded the creation of the genus Bifidobacterium. Taxonomy subsequently was based on increasingly numerous phenotype characteristics and today can make use of progress in genotyping.
A. Identification of the GenusBifidobacterium
Until the 1960s, the only identification criteria were phenotype characteristics.
Table 5 Distribution of Bifidobacterium Species in Human Colon
Population Predominating species Minor species
Breast-fed infants B. longum B. infantis B. breve
Bottle-fed infants B. adolescentis B. bifidum biovar. b
Children B. infantis
B. breve
B. bifidum biovar. b B. longum
Adults B. adolescentis biovars. a and b B. bifidum biovar. a
B. longum
Older adults B. adolescentis biovars. b B. longum
Hoang-Dung TRAN and friends 1. Morphology
Since a branched appearance is seen in other bacterial genera (Arthrobacter, Propionibacterium, Corynebacterium, Actinomyces), it cannot be considered a specific characteristic but only an indicative criterion.[32]
2. Culture Conditions
Bifidobacteria develop under anaerobic conditions at 378C in species of human origin or 428C and higher for species of animal origin and require an incubation time of 48 hours.[21]
3. Metabolites
The determination by gas chromatography of organic acids produced at the end of fermen-tation and notably an acetic acid/lactic acid ratio of about 3/2 provides excellent identi-fication criteria for the genus Bifidobacterium. In addition, it is important to note that bifidobacteria produce theLþisomer of lactic acid.
4. Enzyme Tests
The association of a branched shape with the presence of fructose-6-phosphate phospho-ketolase (F6PPK) in a strain indicates that it belongs to the genus Bifidobacterium. The detection of F6PPK can be completed by a test fora-galactosidase. The API ZYM system indicatesa-galactosidase activity in bifidobacteria[155]but not in lactobacilli.[156]This test can therefore be used as an identification indicator.[42]
5. Study of Electrophoretic Patterns
All soluble cell protein electrophoretic patterns show a band that migrates over the same distance, with the exception of B. boum, for which this band is located at a slightly greater distance from the anode.[157]The presence of this band therefore appears to provide an appreciable criterion for the identification of the genus.
6. Lipids and Constituents of the Cell Wall Membrane
Bifidobacteria have the following fatty acids: C14:0(myristic acid), C16:0(palmitic acid), C16:1(palmitoleic acid), C18:0(stearic acid), and C18:1(oleic acid). In addition, though the genera Bifidobacterium and Lactobacillus both contain diphosphatidylglycerol and phos-phatidylglycerol, only Bifidobacterium possesses polyglycerolphospholipids and their lyso derivatives, alanylphosphatidylglycerol, and the lyso derivatives of diphosphatidyl-glycerol.[158] Analysis of the cell composition in terms of lipids and phospholipids therefore provides a good criterion for distinguishing between the genus Bifidobac-terium and the Lactobacillaceae. It should be noted that the growth temperature and the composition of the culture medium have a marked influence on the distribution of lipids and phospholipids, although the peptoglycan structure of Bifidobacterium is closer to that of the Lactobacillaceae than the Actinomycetaceae.[159,102]
7. Other Identification Criteria
Other tests can be used to identify the genus Bifidobacterium, notably:[15,160,161]
Rapid and complete coagulation of milk without the formation of gas
Fermentation of glucose, lactose, levulose, fructose, and galactose, accompanied by marked acidification
No acid production from rhamnose, sorbose, adonitol, dulcitol, erythritol, or glycerol
Hoang-Dung TRAN and friends The development of these microorganisms in peptone water
Negative catalase No reduction of nitrates No indole formation No liquefaction of gelatin No fermentation of glycerol No attack of coagulated proteins
B. Species Identification
It is obvious from the multiple taxonomic revisions that have taken place in a few decades how difficult species identification is within the genus Bifidobacterium.
1. Sugar Fermentation
This criterion has been used most frequently to identify and define new species. Until 1957, most researchers classed all bifidobacteria together as a single species: Bifido-bacterium difidum. In 1957, Dehnert[162] was the first to demonstrate the presence of several Bifidobacterium biotypes and used 24 sugar fermentation processes to classify the various species into five groups. A few years later, Reuter[163,164]associated serologi-cal properties to sugar fermentation to identify new human-derived species isolated from the stools of adults and children and their various biotypes.
Using fermentation profiles and the ability to grow at 46.58C enabled Mitsuoka[165]
to separate human strains from animal strains (pig, chicken, calf, sheep, rat, mouse, guinea pig, bee). He proposed two new species, B. thermophilum var. a, b, c, and d, B. pseusolon-gum var. a, b, c, and d, and a new variant, B. lonpseusolon-gum subsp. animalis a and b. B. ruminale (synonym of B. thermophilum) and B. globosum and then B. asteroides, B. indicum, and B.
coryneforme were isolated in the same year.[166,167]
The ability of a strain to ferment certain sugars is the test first used to identify species. Numerous sugars have been tested, and the results obtained have been compared with the identification tables produced by Mitsuoka[5,153]and Scardovi[20](Table 6).This method presents no major operating problems but does have several drawbacks: it is lengthy and tedious because a panel of 30 sugars must be studied for 10 days. In addition, the interpretation of the results using identification tables remains controversial and can at best give an indication of an identification based on the fundamental characteristics not open to doubt, for example:
B. longum ferments melezitose, whereas B. animalis is unable to ferment this sugar.
B. pseudolongum ferments pentoses and starch, whereas B. thermophilum does not ferment pentoses but does ferment starch.
B. breve ferments ribose, mannitol, esculine, and amygdaline but does not ferment arabinose or xylose.
B. infantis does not ferment arabinose, whereas B. longum ferments arabinose and melezitose.
Roy et al.[155]developed a rapid method for identifying bifidobacteria species based on the fermentation of seven sugars: arabinose, cellobiose, lactose, mannose, melezitose, ribose, and salicin. A mixture of these seven sugars is monitored by gas chromatography and should make it possible to identify six to eight typical strains of Bifidobacterium in less than 24 hours.
Table 6 Sugar Fermentation by Bifidobacterium sp.
D-Ribose L-Arabinose Lactate Cellobiose Melezitose Raffinose Sorbitol Starch Gluconate Xylose Mannose Fructose Galactose Sucrose Maltose Trehalose Melibiose Mannitol Inulin Salicin
B. bifidum 2 2 þ 2 2 2 2 2 2 2 2 þ þ v 2 2 v 2 2 2
B. longum þ þ þ 2 þ þ 2 2 2 v v þ þ þ þ 2 þ 2 2 2
B. infantis þ 2 þ 2 2 þ 2 2 2 v v þ þ þ þ 2 þ 2 v 2
B. breve þ 2 þ v v þ v 2 2 2 þ þ þ þ þ v þ v v þ
B. adolescentis þ þ þ þ þ þ v þ þ þ v þ þ þ þ v þ v v þ
B. angulatum þ þ þ 2 2 þ v þ 2 þ þ þ þ þ þ 2 þ 2 þ þ
B. catenulatum þ þ þ þ 2 þ þ 2 v þ 2 þ þ þ þ v þ v v þ
B. pseudocatenulatum þ þ þ v 2 þ v þ v þ þ þ þ þ þ v þ 2 2 þ
B. dentium þ þ þ þ þ þ 2 þ þ þ þ þ þ þ þ þ þ þ 2 þ
B. globosum þ v þ 2 2 þ 2 þ 2 v 2 þ þ þ þ 2 þ 2 2 2
B. pseudolongum þ þ v v v þ 2 þ 2 þ þ þ þ þ þ 2 þ 2 2 2
B. cuniculi 2 þ 2 2 2 2 2 þ 2 2 2 þ þ þ 2 þ 2 2 2
B. choerinum 2 2 þ 2 2 þ 2 þ 2 2 2 2 þ þ þ 2 þ 2 2 2
B. animalis þ þ þ v v þ 2 þ 2 þ v þ þ þ þ v þ 2 2 þ
B. thermophilum 2 2 v v v þ 2 þ 2 2 2 þ þ þ þ v þ 2 v v
B. boum 2 2 v 2 2 þ 2 þ 2 þ 2 2 2 þ þ 2 þ 2 þ 2
B. magnum þ þ þ 2 2 þ 2 2 2 þ 2 þ þ þ þ þ 2 2 2
B. pullorum þ þ 2 2 2 þ 2 2 2 þ þ þ þ þ þ þ þ 2 þ þ
B. suis 2 þ þ 2 2 þ 2 2 2 þ v v þ þ þ 2 þ 2 2 2
B. minimum 2 2 2 2 2 2 2 þ 2 2 2 þ 2 þ þ 2 2 2 2 2
B. subtile þ 2 2 2 þ þ þ þ þ 2 2 þ þ þ þ v þ 2 v v
B. coryneforme þ þ 2 þ 2 þ 2 2 þ þ 2 þ þ þ 2 þ 2 þ
B. asteroides þ þ 2 þ 2 þ 2 2 v þ 2 þ v þ v 2 þ 2 2 þ
B. indicum þ 2 2 þ 2 þ 2 2 þ 2 v þ v þ v 2 þ 2 2 þ
þ ¼positive; 2 ¼ negative; v ¼ variable.
90Ballongue
© 2004 by Marcel Dekker, Inc. All Rights Reserved.
Hoang-Dung TRAN and friends 2. Study of F6PPK Isoenzymes
A test using a colorimetric reaction following starch gel electrophoresis for three isoen-zymes of F6PPK can give an indication of species identity.[35]These isoenzymes catalyze the same reaction but are distinguished by differing electrophoretic patterns. The migration distances are linked to the ecological origin of the species: human (15 cm), mammalian (10 cm), or bee[20](Table 7). In addition, purified preparations of F6PPK from B. globosum (mammalian origin) and B. dentium (human origin) demonstrate activi-ties which vary with regard to optimum pH, the identity of the metal inducing maximum activity, heat inactivation, molecular weight, and affinity toward the substrate.[104]
3. Study of Protein Profiles
A bacterial strain cultured under standard conditions always gives the same protein profiles. The sequence of amino acids, the molecular weight, and the net electrical charge of each protein are determined by the sequence of nucleotides in the DNA. The protein profile of each strain is therefore a fingerprint of the genome. The cell proteins are dissolved using detergents such as SDS, but many studies have been carried out using only the soluble fraction of disintegrated cells.[16]Two types of study can be envisaged for the comparison of Bifidobacterium species with each other.
Electrophoresis in a starch gel (zymogram) of the 14 enzymes of transaldolase and the 19 isoenzyme of 6-phosphogluconate dehydrogenase (6PGD) can be used to compare the electrophoretic mobility of these enzymes in the original strain (Table 8).
A colorimetric method applied to 3-phosphoglyceraldehyde dehydrogenase is able to identify other strains.[168] The electrophoretic migration distances for F6PPK appear to be linked to the ecological origin of the species, but the same is not true for the other glucose metabolism enzymes of Bifidobacterium—transaldolase, transketolase, 6-phosphogluconate dehydrogenase, and aldolase.[169]
Electrophoresis in a polyacrylamide gel of the lysate of a strain provides electro-phoretic profiles of the soluble cell proteins. The distribution of the protein bands is
Table 7 Migration of F6PPK in Bifidobacterium sp.
Species Migration (cm) Species Migration (cm)
B. bifidum 15 B. choerinum n.d.
B. longum 15 B. animalis 10
B. infantis 15 B. thermophilum 10
B. breve 15 B. boum n.d.
B. adolescentis 15 B. magnum 10
B. angulatum 15 B. pullorum 10
B. catenulatum 15 B. suis 10
B. pseudocatenulatum n.d. B. minimum 10
B. dentium 15 B. subtile 10 – 15
B. globosum 10 B. coryneforme 16
B. pseudolongum 10 B. asteroides 16
B. cuniculi n.d. B. indicum 16
Note: n.d., not determined.
Source: Scardovi, 1986.
Hoang-Dung TRAN and friends
then compared with those for a reference strain.[35,157,170]This method is doubtless the most discriminating and is both reliable and sensitive, since it is able to distinguish between strains with DNA-DNA homology levels of up to 80%,[157]but it is an onerous method, requiring reference strains, and is difficult to interpret.
Use of these two types of electrophoresis has given the following results:
1. The homology between B. dentium (the only species thought to be pathogenic) and B. eriksonii (formerly Actinomyces eriksonii) was established by comparing their electrophoretic patterns (zymograms).[169] This identity complies with high DNA-DNA homology (80 – 100%).
2. Electrophoresis in polyacrylamide gel enabled Biavati et al.[157]to recognize four new species: B. minimum, B. subtile, B. cornyeforme, and B. globosum, which is now distinguished from B. pseudolongum.
3. B. adolescentis and B. dentium, which have identical phenotype profiles, can also be differentiated by their zymograms (starch gel electrophoresis), which differ.[169]Polyacrylamide gel electrophoresis also confirms these findings.[157]
Table 8 Migration of Transaldolase and 6PGD in Bifidobacterium sp.
Species
Electrophoretic pattern
Transaldolase 6PGD
B. bifidum 7 7-(8)
B. longum (5)-6-8a 5-(6a)
B. infantis 5-(6)-(8a) (3)-4a-(5)
B. breve 6 (5)-6-7
B. adolescentis 8 5
B. angulantum 5 5
B. catenulatum 5 6a-8
B. pseudocatenulatum 4a-(5) 1a-3
B. dentium 4 (2)
B. globosum 2 (3)-(4)-(5)-6-(7)
B. pseudolongum 2 7
B. cuniculi 1 4
B. choerinum 3 4
B. animalis 5 8-9a
B. thermophilum (7)-8a 7-8-9a
B. boum 6 8a-9
B. magnum 5 7
B. pullorum 2 Absent
B. suis 6 5-8
B. minimum 10 6
B. subtile 3 2
B. coryneforme 6 6
B. asteroides (6)-(7)-8-(9) (9)-10a-(11)-(12)-(13)
B. indicum (6)-7-8-9a 6-(7)-8-(9a)
aNumber of isoenzymes for type strains.
( )Number of isoenzymes in less than 10% of strains.
Source: Scardovi, 1986.
Hoang-Dung TRAN and friends 4. The comparison of zymograms and protein electrophoretic patterns in
poly-acrylamide gel of B. infantis and B. longum is interesting. These two species have the same isoenzymes of transaldolases, that is, three different isoenzymes, which migrate to a distance of 5, 6, or 8 units. The only difference is the inci-dence within the strain: the isoenzyme migrating to a distance of 5 occurs more frequently in B. infantis, and that which goes furthest, to 8, is found most frequently in B. longum.[169] The bands obtained on the electrophoretic diagrams of these two species show an identical distribution. Only the concen-trations of the proteins differ.[35,157]
We are therefore faced by a very unusual phenomenon: these strains, although they belong to different species, present similar profiles, thus defining a “continuum.”Table 8 shows that the transaldolases and 6PGD of B. adolescentis with electrophoretic mobilities of 8 and 5, respectively, are found in 50% of B. longum and in many strains of B. infantis, highlighting what is doubtless a very close degree of relatedness between these species.
In contrast, the electrophoresis patterns of the cell proteins for these three strains differ, confirming that they are indeed three separate species.[36,157,171]
The zymogram of B. bifidum is in contrast highly specific. This species is the only one to show a transaldolase and F6PPK migration distance of 7 units. Only a few strains of B. thermophilum of bovine origin are similar. In this case, the differentiation is based on the mobility of the 3-phosphoglyceraldehyde dehydrogenase.
Study of the protein patterns provides valuable information about a given strain, and the numerical analysis of the patterns of a large number of strains makes it possible to achieve:
Rapid grouping of the strains.
Archiving of a large number of models in a reference data bank.
The attribution of unknown bacteria to their group and their possible identification.
A quick method of determining whether two colony types in a culture are due to variation or contamination.
Determining the homogeneity or heterogeneity of the taxa.
When the preparation conditions for the extracts and their electrophoresis are standardized, a high degree of reproducibility (in excess of 96%) can be obtained.[172]
4. Transaldolase Serology
An immunological approach investigating the serology of the transaldolases can also be used to differentiate between species within the genus Bifidobacterium.Figure 6shows the results of the studies performed.[107,173,174]
This method consists of preparing immunosera against the highly purified transaldo-lases of eight species of Bifidobacterium, B. infantis, B. angulatum, B. globosum, B. ther-mophilum, B. suis, B. cuniculi, B. minimum, and B. asteroides and testing them against 21 bacterial species of the genus in order to determine their immunological distances. These results, expressed as taxonomic distance, are shown in the dendogram (Fig. 6). This dia-gram illustrates the interrelationships existing within the genus and shows that the seven groups defined by Sgorbati and London[107] (A, B, C, D, E, F, and G) detected by this model can be split into four distinct groups closely linked at the ecological origin of the species. These four antigenic groups (I, II, III, and IV) coincide with the arrangement
Hoang-Dung TRAN and friends
of the species of Bifidobacterium based on electrophoretic mobility[36,174,175]and are also confirmed by DNA-DNA hybridization studies.
In addition, this dendogram is able to distinguish two subgroups, A and B, among the strains of human origin (I): (1) B. angulatum, B. catenulatum, B. pseudocatenula-tum, and B. dentium; and (2) B. adolescentis, B. breve, B. longum, B. infantis, and B. bifidum.[174] The species associated with mammalian animal habitats (II) belong to groups C, D, E, and G. The two species found only in the bee (group H) are antigenetically very distant from the other members of the genus (III). The most widely separated species (IV) (group F) were isolated from waste water.
5. Enzymes
B. breve is one of the few species to produceb-glucuronidase activity.[41]It is suspected that this enzyme may convert procarcinogens into carcinogens.[176]In addition, B. longum is the only species to have neitherb-glucosidase nor N-acetylglucosaminidase activity.
6. Composition of the Wall
The sugar composition of the wall varies with strain, particularly with regard to the percentage of rhamnose and glucose[15](Table 9).The sequence of amino acids in the pep-tidoglycan may vary from one species to another, making it possible to separate species from which are relatively close to each other, such as B. boum from B. thermophilum or B. minimum from B. subtile.[102]In addition, Bezirtzoglou[110]noted that only the species B. bifidum has a poly-(1,2)-glycerophosphate skeleton in the lipoteichoic acids, which is substituted in the end position by a polysaccharide.
Figure 6 Immunological relationships among transaldolases in Bifidobacterium. (See text for details.)
Hoang-Dung TRAN and friends
Op Den Camp et al.[177]prepared antibodies to the lipoteichoic acids of B. bifidum by coupling with an immunogenic protein. They were specific towards the polyglycerol phosphate core (essentially poly 1,2) and to a small extent to the polysaccharide portion.
Crossed reaction tests with phenolic extracts of lipoteichoic acids of Bifidobacterium and Lactobacillus have shown that only the former react, making it poss-ible to envisage a serogroup with lipoteichoic acids as group antigens.
7. Processing of the Results
All these identification criteria give responses that must be classified and interpreted. Most of the problems involved in processing data tables resulting from the examination of the physiological and biochemical properties of the bacteria are solved by the use of com-puter-assisted numerical taxonomy. The usefulness of numerical taxonomy depends on several factors:[167]
1. Strain selection
2. Number of characteristics examined (greater than 50) 3. Rigorous standardization of the methods of analysis 4. Weight attributed to each characteristic in the evaluation
5. Classification of the reactions as positive, negative, or noncomparable 6. Type of computer software used
XI. IDENTIFICATION BY STUDY OF THE GENOME