Lactobacillus and Bifidobacterium 43Curr. Issues Intest. Microbiol. (2001) 2(2): 43-53.

© 2001 Horizon Scientific Press

The Lactobacillus and Bifidobacterium Microflora ofthe Human Intestine: Composition and Succession

*For correspondence. Email x; Tel. 030-83852796; Fax. 030-83852792.

Gerhard Reuter

Department of Food Hygiene, Veterinary Faculty,Free University of Berlin, Bruemmerstr. 10, D - 14195 Berlin(Dahlem), Germany

Abstract

Lactobacillus and bifidobacterial cultures areincreasingly used as probiotics in pharmaceuticals andin foods. The selection of strains is performed oftenfor technological rather than for microecologicalreasons. Detailed reports about species and strainscomposition of these microorganisms in the intestinalmicroflora of man are rare. Our investigations wereperformed with samples originating from infants andadults, taken from faeces and from upper sections ofthe intestinal tract including mouth and stomach, andfrom caecum and colon. Post mortem cases as wellas test subjects under physiological conditions wereanalyzed using an automatic capsule system samplingat defined times in different parts of the intestinal tract.The fate of selected strains after oral intake wasstudied, too. Furthermore, influences of the microfloraoriginating from food were considered.

The identification of autochthonous (indigenous)and allochthonous (transient) species could beachieved with descriptions of new species in thegenera Lactobacillus and Bifidobacterium. L. gasseriand L. reuteri proved to be predominantautochthonous Lactobacillus species in infants as wellas in adults. Both species were occasionally presenteven in the stomach. This was also the case with ananaerobic lactic acid bacterium, previously namedCatenabacterium catenaforme, later classified as L.ruminis, a non-motile variant of this species.

The bifidobacterial microflora differed incomposition between infants and adults and indifferent stages of the host’s life. Up to 5 species orspecial strains of bifidobacteria could be present indifferent, individually fixed, combinations. Speciestypical for infants were B. bifidum, B. infantis, B. breve,and B. parvulorum. Typical for adults were 4 differentvariants of B. adolescentis. B. bifidum and B. longumcould often be found in both groups, but in lowernumbers. B. longum showed some oxygen tolerancewhereas B. bifidum and B. adolescentis required strictanaerobic and fastidious conditions for cultivation.

The autochthonous Lactobacillus andBifidobacterium microflora in man will remain stable

life-long. With lactobacilli, however, some successionsmay be caused by transient species derived from foodor from the oral cavity, thus giving the impression ofan altered microflora. Nevertheless L. gasseri, L.reuteri, L. ruminis, and to some degree, L. salivarius,may be present as autochthonous species all of thetime. With bifidobacteria, a decreasing tendency incounts and in multiple composition in elderly peopleexists. Furthermore, this microflora is also influencedby consumption habits, which are probably caused bygeographical circumstances.

Introduction

The gastrointestinal microflora is a very complexcommunity. Within the gastrointestinal tract, differenthabitats have to be recognized, e.g. mouth, stomach, smallintestine (especially lower jejunum and ileum) largeintestine (caecum, colon) and rectum. Normally, nearstability exists in these habitats. The balance is influencedprimarily by the host’s individuality. This means thatinterpersonal (individual) variations exist. Each person willhave an individually fixed microflora as far as qualitativestructure and the quantities of lactobacilli and bifidobacteriaare concerned (Reuter,1963 1965a, b). This fact is of greatinterest, as more than 400 species within the intestinalmicroflora can be identified and may attain population levelsnearly as high as 1012/g in the colon (Mitsuoka, 1982, 1992;Tannock, 1999a).

An indigenous microflora can be recognized, consistingof autochthonous species (e.g. species, which are able tocolonize the mucosal surface of the gastrointestinal tractdue to special adhesion factors including compatibility withthe immunological system of the host). Thesemicroorganisms have to be distinguished fromallochthonous species, which may be present in theintestine, too, but which may only have a transientcharacter. The presence of these strains in the intestinaltract will last for a limited time, probably only a few days(Tannock, 1999a). Especially with lactobacilli, a strictdistinction between these two groups must be considered,due to the fact that large amounts of lactobacilli are enrichedin fermented foods which will be transferred day by daythrough the stomach and small intestine into the largebowel. This supplementation depends on individualconsumption habits (Reuter, 1965a). Some species will notsurvive stomach and duodenal passage easily, while otherswill resist very well. This may be the cause of manymisinterpretations of the “normal“ indigenous Lactobacillusmicroflora of man as described in the literature even inrecent times, where even L. plantarum is mentioned as anautochthonous Lactobacillus species in the intestine (Ahrnéet al., 1998).

Some reports in the literature ignore the differencesof the intestinal microflora between infants on one hand,

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44 Reuter

and adults and elderly people on the other. In these agegroups, lactobacilli and bifidobacterial populations aresubstantially different in structure and quantities. Whilelactobacilli are changing only quantitatively, probably afterthey have randomly colonized immediately after birth, thedevelopment of the bifidobacteria microflora is influencedby nutritional factors also (e.g. in-breast fed or formula-fedinfants) or by the age of an individual.

In general, microbiological analyses of the humansmall intestine are difficult due to sampling problems. Manymethods were tested dating from the distant past (1922-1925) up to recent times (1960-1970) (Lerche and Reuter,1962; Reuter, 1975). The main problem seemed to beobtaining specimens correlating with the physiologicalstatus of the intestinal tract. Trials have been carried outduring abdominal surgery procedures or in post mortemcases for a long time. This way of sampling does not appearto be quite realistic in general, however. Prior to surgery,the ingestion of food has been interrupted for some days,and in this fasting period, the intestine was cleaned up,emptied, or even pre-treated with antibiotics or otherpharmaceuticals. In post mortem cases, the effect of clinicalpre-treatment or the time elapsing between death anddissection may have caused alterations of the microflora.Better results were achieved with the application oftransient capsules in different modifications, obtainingsamples during passage through the intestinal tract bydifferent mechanisms. Sampling with intubation andaspiration methods were other methods employed.Drainage sampling after ileostomy was sometimes used.Disadvantages exist with every method. Intubation methodsmay have been accompanied by an irritating effect causingchanges in the physiology of the intestine, andconsequently, in the microecology. Automatic capsules notconnected with an intubation system sometimes producedproblems such as recovery from the faeces at variabletimes after intake. Ileostomy-aspirates could have beeninfluenced by oxygen and bowel motility factors.

The microecological balance in the different habitatsof the intestine can have been disturbed considerably,however, depending on internal (intrinsic) and external(extrinsic) influences. Functional disturbances such asalterations of gastric acidity or bile secretion can influencethe microflora fundamentally as well as the administrationof pharmaceuticals with antimicrobial efficacy. Occasionally,usually suppressed components of the indigenousmicroflora can allow potential pathogens or toxinogenicstrains to colonize and to multiply thus causing diarrhoea,flatulence, or different variations of colitis (haemorrhagic,pseudomembranous etc.)

One way to re-establish the ecological balance of adisturbed habitat in the intestinal or urogenital tract, is toapply components of the indigenous microflora by oralintake or rectal or vaginal instillation. These procedureshave been applied for a very long time, but became morecommon again in the last decades of the past century asprobiotic cultures were propagated for that purpose world-wide (Reuter, 1997; Tannock, 1999a).

The selection of suitable species or strains as probioticcultures, however, is a very critical step (Bonaparte andReuter, 1997). The descriptions of strains used by

producers of probiotic cultures are often incomplete or evenfalse. The choice of strains has often been done at randomfrom culture plates. Occasionally, the selection of probioticstrains was determined preponderantly by technologicalreasons (e.g. viability, productivity, viability of the cultures)even if used in a pharmaceutical preparation (Reuter, 1969;Reuter et al., 2001). Probiotic cultures are often named asL. acidophilus, neglecting the fact that this termcharacterizes a group presently comprised of six separatespecies with few of them being relevant to humanautochthonous microflora (Fujisawa et al., 1996). L.fermentum was often reported, not recognising that thisspecies must be differentiated from other phenotypicallysimilar ones such as L. reuteri, L. oris, and L. vaginalis. L.fermentum has its natural habitats in food, especially indairy products, while the others are autochthonous speciesin humans. Bifidobacterium bifidum has been often citedas a component of probiotic food, disregarding the factthat this species requires the most strict anaerobicconditions for growth of all bifidobacteria of humans.Maintenance of viability of this species is not possible forthe required shelf life in probiotic foods (Reuter, 1990). B.longum has been preferred because of its less demandingrequirements for anaerobic conditions in foods and inpharmaceuticals (Dreher et al., 1991), taking into accountthat in many cases the species which was actually used,belonged to the related species B. animalis because of itshigher tolerance to acidic conditions. Furthermore, a falsedeclaration of strains of this last species was propagatedby creating a new species taxon, B. lactis, which must beconsidered non-valid (Cai et al., 2000). The choice of strainsas probiotic cultures should therefore be made morethoroughly with bifidobacteria than with lactobacilli.

The selection of strains out of the L. casei group is aspecial field of probiotic research (Tannock et al., 2000).Since L. casei and L. rhamnosus strains are transientorganisms in the intestine, their use is justified mainly bytechnological reasons. Further interpretations are given ina recent paper (Reuter et al., 2001). A recent report uponadhesion properties of novel probiotic strains was publishedby Oweland et al. (2001).

I. Composition of the Microflora of the Human SmallIntestine and the Fate of Cultures after Oral Intake

To provide representative sampling, different series ofinvestigations both from post mortem cases and fromsamples obtained with an automatic capsule system fromchildren 10-14 years of age as well as from adults mainlyin the 20-40 years age group, were performed. Samplesfrom faeces were investigated in parallel as far as possible(Lerche and Reuter, 1961, 1962; Reuter, 1961, 1963,1965b, 1975; Hirtzmann and Reuter, 1963; Hirtzmann,1965).

The selection of post mortem cases was made directlyby the investigator. Non-physiological influences could beminimized due to the selection of cases of sudden deathcaused by heart attack or traffic accidents . Samples weretaken by excision of ligated parts from the intestinecontaining ingesta, from the upper jejunum, the upper andthe lower ileum, the caecum, and the descending colon

Lactobacillus and Bifidobacterium 45

(Lerche and Reuter, 1962). Microbiological analyses wereperformed no later than 2 hours after sampling.

The automatic capsule used was about 2.4 cm inlength and 0.85 cm in diameter, with a weight of 4.1 g.Most test persons, including children, were able toincorporate the capsule without difficulties. Each capsuleopened and closed at defined times for about half an hourduring the passage through the intestinal tract. Thecapsule’s location in the intestinal tract during the openingstage was checked by clinical radiographs at the end ofthe opening phase. The functionality of the capsulesconcerning mechanisms and tightness was tested byHirtzmann using dyes and disinfection solutions(1963,1965) before and after their application.

The bacteriological analyses included a quantitativecultivation procedure with ten-fold dilution steps afterhomogenization of the samples, the application of a set ofpolyvalent media, supplemented blood agar, and variousselective media (Reuter, 1961; Reuter, 1963, 1985). Theaerobic as well as anaerobic microflora was analyzed, withspecies and biotype identification from multiple subcultures.The lactobacilli and bifidobacteria, and other importantgroups or species, could be identified by classical methodsaccording to the state of knowledge at that time (Lercheand Reuter, 1961, 1962; Reuter, 1963, 1964). Thequantitative identification of Bacteroides-species and ofveillonellae in correlation to bifidobacteria was possible,too. “coryneforms“ isolated at that time should be regardedas identical to peptococcaceae nowadays (Mitsuoka,1982). The lower range of cultivation was about log10 6.0/g for bifidobacteria and lactobacilli, with selective plateseven two log cycles lower.

Results of 9 Post Mortem Cases Selected from 30 inTotal (Series 1)

In 14 of 30 cases examined at autopsy, the small intestinewas overgrown by gram-negative bacteria or byenterococci. These cases were mainly based on septic andsevere chronic diseases or on elderly people suffering fromdifferent dysfunctions. These were not considered furtherfor judging the lactobacillus and bifidobacterial microfloraof the small intestine. Of the remaining 16 cases, 9 showeda remarkable Lactobacillus microflora in the small intestine.These cases consisted of middle-aged adults who weresudden or accidental death victims, without preceedinglong-term illnesses. Dissection was performed within 2 daysof death. The microbiological results of these casestherefore were considered as a plausible interpretation ofthe microecological situation in the upper intestine beforedeath (Table 1). In cases where ingesta was present, thej e j u n u m contained lactobacilli in considerable counts.The median value was log10 7.7/g. Predominant strainsbelonged to the species L. acidophilus and L. fermenti,according to the existing taxonomic system of that time.Their classification must be seen nowadays ascorresponding to the new species L. gasseri (= L.acidophilus, biotype I, formerly) and L. reuteri (= L. fermenti,biotype II b, formerly). L. salivarius appeared lessfrequently. Enterobacteria, micrococci, enterococci, yeastsand fungi occurred occasionally. E. coli and Bacteroidesstrains might have been detected through their retrogradespread from the ileum after death. The presence of anadditional strain of enterobacteria might have been causedby a transient strain.

The i l e u m contained enterococci more frequently,and bifidobacteria in single cases. Lactobacilli showed asimilar distribution as in the jejunum. In the large intestine(caecum/descending colon) enterococci and E. coli werepresent on a regular basis, accompanied by theautochthonous lactobacilli: L. gasseri, L. reuteri, and L.salivarius. Although in different frequency, some transientlactobacilli (L. casei, L. plantarum, L. buchneri) werepresent in all parts of the small intestine.

Results with the Automatic Capsule in Children, Aged6-12 Years (Series 2)

The intestinal microflora of 24 hospitalized children, 9 ofwhich were considered as physically healthy, yet most ofthem being mentally retarded, was analyzed. Data werereported by Hirtzmann and Reuter (1963). Repeatedsampling followed from 10 children selected out of thisgroup. In total, 47 tests were evaluated. On thirty eightoccassions, the capsule opened in the ileum, and 9 timesin the caecum. In most cases the passage time throughthe intestinal tract took between 2 and 3 days (Table 2).

In i l e a l samples, lactobacilli were the most frequentmicroorganisms, followed by E. coli, enterococci,bifidobacteria, and aerobic bacilli. An anaerobic lactic acidbacterium could be recognized and was determinedaccording to the existing taxonomic system of Bergey’sManual (1957) as Catenabacterium catenaforme. This

Table 1. Microecology (log10/g) in different habitats of the intestine of 9adults. Samples obtained from selected cadavers (≤ 2 days p.m., n = positivesamples) (according to Lerche and Reuter, 1962; Reuter, 1975)

46 Reuter

nonmotile variant, whereas strains from the rumen of cattleprincipally showed motility. Micrococci, yeasts and fungioccurred sporadically as well as bacteroides andcoryneforms (nowadays peptococcaceae) in the distalileum. Quantitatively, the bifidobacteria and lactobacillishowed the highest median values followed by those of“catenabacteria” and enterococci. The counts of E. coliwere low, and bacilli showed the lowest quantities. Thevariation in appearance and in counts of yeasts and fungimight have been caused by prior oral intake.

Samples from the c a e c u m qualitatively showed thepredominance of bifidobacteria, and bacteroides species,followed by E. coli, lactobacilli and enterococci. The countsof bacilli were reduced, veillonellae occurred sometimes.The median values of counts of bifidobacteria andenterococci were markedly increased compared with thoseof the ileum. Bacteroides populations were predominant.A selected group of 5 children tested continuously (5 and7 times) over a period of 3 to 5 weeks yielded informationabout the personal characteristics of the microflora in thesmall intestine of each individual. Definite strains oflactobacilli or bifidobacteria appeared regularly in one orthe other child, and, in addition, nearly in equal quantitiesduring the entire investigation period. In 3 children, bacillicould be isolated from the ileum on a regular basis; in theother two not at all. Also frequency and quantities ofenterococci appeared to be related to the individual host.In two children the “catenabacteria” were an obligatorypart of the individual microflora. The opportunisticoccurrence of other strains, such as some otherLactobacillus species, micrococci, yeasts and fungi mayhave been caused by oral intake with food just prior tosampling. The results have been reported in detailpreviously (Reuter, 1965a, 1975).

Table 2. Microecology (log10/g) in the ileum and caecum of 24 childrenaged 6-12 years. Samples obtained by automatic capsule method (accordingto Hirtzmann and Reuter, 1963; Reuter, 1965a, b).

Table 3. Microorganisms other than lactic acid bacteria from the small intestine and caecum of healthy adults under non- restricted diet. Samples obtained bythe automatic capsule method (n = number of positive samples, brackets = total number of samples).

species seemed to be a regular component of theindigenous microflora. It was later classified as L. ruminis(Sharpe, 1974; Sharpe and Dellaglio, 1977), but as a

Lactobacillus and Bifidobacterium 47

f a e c e s showed a nearly corresponding situation. Typicalcomponents were Bacteroides, “coryneforms“ andveillonellae with a regular appearance of bifidobacteria andE. coli. Enterococci and “catenabacteria” were morenumerous in the colon than in the faeces. That was alsothe case with lactobacilli (Reuter, 1965b).

The individual character of the microflora of eachsubject became obvious again during this series. Yeastscould be repeatedly detected in certain individuals indifferent parts of the intestinal tract. This was the case withbacilli and klebsiella strains in single cases, too. Somepeople had only lactobacilli in the small intestine, as didothers combined with E. coli (Table 3).

The distribution of lactobacilli divided intoautochthonous and transient species was reported in detail(Table 4). The species L. fermentum and L. acidophiluswith its special biotypes, which are now classified as L.reuteri and L. gasseri respectively, and in some cases L.salivarius, could be identified as autochthonous .This wasalso the case with “catenabacteria”. Some individualsshowed only L. reuteri regularly, others only“catenabacteria”. In some persons, the same strain wasdetected throughout the gut, from the stomach down tothe faecal sample.

Transient lactobacilli were mainly isolated in the faeces,a few in the ileum, and very few in the jejunum and stomach.This indicated that these bacteria will pass the smallintestine quickly and multiply in the colon for a limited time.The most frequently isolated transient strains were L.plantarum followed by L. casei, L. buchneri and L. brevis,

Table 4. Lactobacillus species from the intestine of healthy adults under nont- restricted diet. Samples obtained by the automatic capsule method and fromfaeces in parallel.

Results with the Automatic Capsule in 23 adults (Series3)

The subjects were healthy, 20 to 57 years old, and engagedin the production process of a company. Stool specimenswere investigated simultaneously with the contents of thecapsules shed in the faeces. The transfer of the capsulethrough the intestinal tract of adults was markedly slowerthan that in children, although the latter had beenhospitalized. Due to this fact, the capsules remained inthe stomach during their opening phases in numerouscases. Twenty one out of 45 stomach samples showed amicrobiological content even after a transfer of the capsulesthrough the intestine for 2-4 days (Reuter, 1965b). Theycontained lactobacilli and “catenabacteria” in about 20%of the cases, followed by bacilli and enterococci.Micrococci, yeasts and fungi occurred in five samples, andenterobacteria in two. Considering median values, thelactobacilli were present in relatively high quantities, bacillithe lowest.

Samples from the j e j u n u m contained predominantlyenterococci, qualitatively as well as quantitatively, followedby lactobacilli, yeasts and fungi. E. coli occurred in fivecases. I leal samples frequently contained lactobacilli, E.coli and enterococci. The median value was highest withE. coli. bifidobacteria, “catenabacteria”, yeasts and fungi,bacilli, and other enterobacteria occurred in about 20 to25% of the cases. Bacteroides strains occurred three timesand micrococci and clostridia two times each.

Samples from the c a e c u m , c o l o n and from the

48 Reuter

and the food species L. fermentum. All these species hadbeen recognized before as components of fermented foodsfrom which they were isolated regularly and in considerablenumbers (Reuter, 1965a).

Results with Cultures after Oral Intake (Series 4)

The fate of lyophilized cultures after daily oral intake forthree days was tested in a trial with automatic capsulesand with faecal sampling. Tested strains were lactobacilli,bifidobacteria, “catenabacteria” and E. coli (Table 5). Thecultures were administered as a single strain or incombinations of 2 to 4 strains. Test cultures were givenpreferably to persons which had been repeatedly provenfree from the occurrence of the particular strains in theirfaeces prior to the beginning of the experiment.

E. coli was re-isolated from the ileum in considerablenumbers in all 3 tested subjects. L. gasseri was re-isolatedfrom the jejunum and the ileum only 3 times in 12 trialsand, due to low numbers, only 4 times from the faeces.The identification of this strain was difficult as it could notbe separated easily from the autochthonous strains of this

species frequently present in lower counts in the testsubjects included in the trial. One L. gasseri mutant, whichhad been selected for streptomycin resistance, hadreduced resistance during the multiplication in the intestine.Thus, verification with antibiotic selective plates wasdifficult. The L. reuteri strain tested could not be re-isolated.That was also the case with dairy lactobacilli (L. bulgaricus,L. helveticus, and L. lactis). The data recording these re-isolations are shown in Table 5.

Summarizing the results of these four series mentionedabove, it can be stated that the stomach and the smallintestine are not free of microorganisms, but rather thatthey contain a lot of different strains in variable, and insome cases, considerable quantities. The appearance ofautochthonous as well as transient components of themicroflora in these habitats should be noted. This wasmentioned already in several papers, even in the classicalliterature, but it was questioned in about the same numberof publications. It is impossible to cite all the authorsengaged on that field. Some early references are cited inthe paper of Lerche and Reuter (1962).

It can be concluded that in the small intestine theremight exist a more defined individual character ofindigenous flora than in the colon. On the other hand, thesmall intestine’s microflora is influenced on a large scaleby transient microorganisms from the oral cavity and fromingested food.

In detail, the microflora of the small intestine consistsof enterococci and lactobacilli as predominating groupsinhabiting the jejunum as well as the ileum. E. coli,“catenabacteria” and bifidobacteria do appear in the distalileum, but not regularly. Strains of L. gasseri and L. reuterimust be regarded as autochthonous lactobacilli there.However, the L. gasseri strains generally isolated in all theseries corresponded to the strain which was deposited atATCC under No. 19992 and reported by Hansen (1968).This strain is not identical with the type strain of L. gasseri(ATCC 33323T) isolated by Gasser from the human vagina.The other frequently detected species was L. reuteri. Onestrain was chosen as the type strain and deposited as ATCC23272 (Hansen, 1968). This strain had been found regularlyin one young female test person throughout all of theintestinal regions. L. salivarius may be primarily regardedas a transient microorganism derived from the oral cavity,where it is an autochthonous species. True transientlactobacilli are, in sequence of frequency and quantity: L.plantarum, L. casei, L. buchneri, L. brevis, and L.fermentum (the food species). A compilation ofautochthonous and transient species of lactobacilli indifferent habitats of humans has been formulated in Tables6 and 7, which might be considered as orientating material.Other transient organisms included yeasts, bacilli,micrococci, enterobacteria and moulds. An interestingobservation seemed to be that in the caecum and colon,counts of “catenabacteria“ and enterococci appeared tobe increased along with those of lactobacilli, comparedwith the counts in faeces.The fact that stomach samples contained autochthonous

as well as transient strains has been described by otherauthors, too. The predominance of L. fermentum biotypesI-IV and of L. acidophilus biotype I (= L. gasseri) were

Table 5. Cultures re-isolated from different habitats of the intestine of healthyadults after oral intake for 3 days. Samples obtained with capsule methodand from faeces in parallel (log10 n)

* formerly Catenab. catenaforme, now L. ruminis, non-motile** streptomycin-resistent strain (1000µ/ml)+ instead of original E. coli (S- instead of R-shape)++ E. coli instead of other coliforms

Lactobacillus and Bifidobacterium 49

reported by Voronina and Lenzner (1968) and Bernhardt(1974). Bernhardt confirmed the presence of lactobacilli inpopulations up to 109/ml aspirate from the stomach with adirect intubation method.

Concerning the fate of test strains after oral intake, itcan be seen that E. coli persisted for some days inremarkable numbers in the upper intestine. This is inaccordance with the results of Shiner et al. (1963), whofound that the jejunal juice showed no bactericidal effectsagainst enterobacteria. On the other hand, the strains ofL. gasseri and “catenabacteria“ could not be re-isolatedregularly, only in certain subjects. Also the test strain of L.reuteri and L. salivarius failed to do so. L. salivarius wasmentioned in the literature as bile sensitive and beingreduced in count or even incapable of surviving in the smallintestine.

Transient lactobacilli, however, multiplied in thecaecum and colon for some days. The fact that the

Table 6. Autochthonous lactobacilli in different habitats of humans(according to Reuter, 1965a, b, 1975; Reuter et al., 2001).

Table 7. Transient lactobacilli in different habitats of humans (accordingto Reuter, 1965a, 1975).

Table 8. Bifidobacterium species. in the intestine of infants and adults(according to Lerche and Reuter, 1961; Reuter, 1963, 1971; Mitsuoka,1969, 1982).

thermophilic dairy lactobacilli like L. bulgaricus, L.helveticus, and L. lactis did not survive passage throughthe stomach and duodenum, seems to be very importantfor judging products containing these species as probiotics.

II. Bifidobacterium Species in the Intestine of HealthyInfants and Adults

Bifidobacteria have been known since Tissier (1900)described a species in infants, which was later namedLactobacillus bifidus by Orla-Jensen (1924) (Table 8). Sincethat time numerous papers were published concerning theecology and importance of bifidobacteria in the intestineof man, primarily in infants. But in many investigations, thedemonstration of bifidobacteria was performed bymicroscopy rather than by cultivation and identification ofsubcultures. Important progress was made in the 1960s.Paediatricians and microbiologists detected different typesof bifidobacteria in infants (e.g. Dehnert (1957) inGermany). But cultivation of this intestinal microflora wasperformed mostly as deep agar cultures. It was still difficultto obtain surface cultures on plates because of the strictanaerobic requirements of these microorganisms. Aremarkable improvement was achieved by using adequateanaerobic systems combined with surface plating (Reuter,1961). These systems enabled the culturing of thecomprehensive anaerobic microflora of faeces of adultson polyvalent plates (e.g. on blood agar base). Theseprocedures were later optimized by Mitsuoka’s techniquein Japan and made it possible to separate many differentvariants characterized by special features of oxygentolerance, nutritional requirements, and ecologicalspecificities.

50 Reuter

After culturing and subculturing of bifidobacteria onplates was possible, the description of this very complexpart of the microflora could start. This was performed withclassical phenotypical methods (e.g. by morphology,physiology, fermentation reactions and demonstration ofserological relationships) (Reuter, 1963). A new taxonomicsystem could be established by creating a genusBifidobacterium (Taxonomic Subcommittee forBifidobacteria) with the description of several new speciesbesides B. bifidum, which was the only existing species atthat time (Reuter, 1963, 1971). That was followed by an

increasing number of new species recognized in humansand other animals (Mitsuoka et al., 1969, 1984; Lauer andKandler, 1983; Scardovi, 1986). The new concept ofBifidobacterium taxonomy was summarized in specialchapters of Bergey’s Manual (1986) and in the Prokaryotes(1981) by Scardovi and Biavati et al. (1991). Thepredominating species in the intestine of humans are stillthose which were described at the first stage of taxonomicidentification by Reuter and Mitsuoka (Table 8).

Data about the ecology of the bifidobacteria in theintestine of individuals are still very rare because there may

Table 9. Structure of Bifidobacterium microflora and other related components in the faeces of healthy adults during a long term investigation period (≥log10=6.0/g) (according to Lerche and Reuter, 1961).

Lactobacillus and Bifidobacterium 51

exist an agglomeration of up to 4 or even 5 different speciesin one habitat. It was difficult to separate them by classicalbacteriological methods as no adequate selective agarmedia existed, which enabled the culturing of all speciesor strains of bifidobacteria qualitatively and quantitatively.Modern methods using PCR/RAPD or gene probes mayproduce more accuracy (McCartney et al., 1996; Tannock,1999b; O’Sullivan, 1999; Roy et al., 2000) but must besuitable for the identification of all species which may beexpected in the sample. Some recent papers showed somevagueness in that concern and came to conclusions, whichmight not correlate with results obtained by universalclassical methods e.g. Sghir et al. (2000).

Therefore, our data about composition and successionof bifidobacterial species in the intestine of humans shallbe referred to here. They were collected in parallel to thetaxonomic research. A comprehensive analysis of eachsurface culture by subculturing many isolates undermicroscopic control was the base of these investigations.The first paper was published in 1961 (Lerche and Reuter).The group of bifidobacteria was still named asLactobacillus. The different strains were separated asbiotypes. These results, combined with ecological data,are summarized in Table 9. The procedure was as follows:7 healthy test persons belonging to the staff of the institute,supplied stool samples regularly over a period of more than165 days and gave information about food consumptionhabits which might have influenced the successions ofstrains within the stool microflora. The results were reportedin a publication as direct counts (Lerche and Reuter, 1961).Transferring these counts into logarithms and simplifyingthe results by symbols, shows interesting relationshipsbetween different components individually distributed in testsubjects (Table 9). From that data it can be seen that ahealthy young female showed a very comprehensivebifidobacterial microflora. Representatives of B.adolescentis (4 variants) were regularly present in eachstool sample. This situation remained stable over the entireinvestigation period of more than 165 days. Samples fromthis subject also showed the presence of the specificbiotype of L. fermentum, later identified as L. reuteri,representing the type strain of this new species.Additionally, the presence of an anaerobic species of lacticacid bacteria became obvious and it was identified asCatenabacterium catenaforme due to the currenttaxonomic system and nomenclature at that time. Thisspecies seemed to be an autochthonous species of theintestinal tract of humans and was included in the genusLactobacillus as L. ruminis by comparative investigationsof a submitted strain. But this strain was not motile like theoriginal L. ruminis strains (Sharpe, 1974). It was designedas strain 194e = ATCC 25644 (Sharpe and Dellaglio, 1977).Another subject, 31 years of age, showed an abnormalcomposition of the intestinal gram-positive microflora. Nobifidobacteria beyond the level of log10 6.0/g of faeces couldbe isolated by the method of cultivation that was utilised.Instead of that a high count of Enterococcus faecium wasobtained from each sample. This situation remained stableduring the whole investigation period. The gram-negativeanaerobic count corresponded to that of the other testsubjects.

Another male subject of 52 years of age showed areduced quantity of bifidobacteria and of the anaerobicLactobacillus. That is in accordance with other observations(Mitsuoka, 1982; Gavini et al., 2001), where elderly peopleshowed a decrease of that kind of microflora.

The lack of counts in the Table does not necessarilymean that this species might not have been present. Itindicates that the counts may be below the detection level(e.g. log10 6.0/g.). The conclusion from the results in thisTable, in general, may be that each healthy adult personwill have and maintain its own fixed composition of thispart of the gram-positive anaerobic microflora over a longperiod of life. Inter-personal differences in the compositionof the bifidobacterial microflora were confirmed by theinvestigations of McCartney et al. (1996).

In general, the bifidobacterial microflora of adultsconsisted mainly of strains of the species B. adolescentiswith the biotypes a, b and c, to a lesser extent also type d,which was listed separately due to the dry and roughconsistency of colonies growing on blood agar plates. Asa biotype a strain was chosen as the type strain of thespecies B. adolescentis, the other biotypes b, c, d maycorrespond to other new species described later on byScardovi (1986). These relations are pointed out in Table 8,based on indications made by Mitsuoka (1982). This matteris not finished as some overlaps exist (Gavini et al., 2001).Another species often detected was B. longum with itsvariant a which could be found in adults and was separatedfrom variant b which was mostly isolated from infants. Thespecies B. bifidum appeared as variant a in stools of adults,too, but at lower counts than other Bifidobacterium species.Variant b of B. bifidum was the breast-fed-infant type ofthis species as described by Tissier (1900). Nevertheless,the dominant bifidobacterial species in infants were B.infantis, B. breve, B. parvulorum etc. (Reuter, 1963, 1971).The existence of biotypes (variants) within these speciesdemonstrates the adaptation to different habitats. This isof importance for the choice of strains for the use asprobiotic cultures (Reuter et al., 2001).

The differences within the ecological structure of thebifidobacterial microflora between infants and adults areobvious. Successions may occur during life since breast-fed or formula-fed infants may have been the starting basefor further development of this part of the microflora. Thesuccessions of bifidobacterial species within differentperiods of human life are not described very well, as onlyvery few long term investigations in the same individualshave been performed (e.g. by McCartney et al. (1996)).Several reports describing the structure of thebifidobacterial microflora in different age groups of humansexist. The results are based on separate groups ofindividuals, however. Also the number of subjects varieddue to experimental conditions.

Geographic specificities in the composition of thebifidobacterial microflora have to be considered, too. Themost comprehensive surveys had been performed byMitsuoka and his working group in long term investigationswith many reports in the literature including review articlesand monographs (e.g. Mitsuoka (1982, 1984, 1992).Recently, publications from the groups of Gavini et al.(2001) and Tannock et al. (1999b) support the knowledge

52 Reuter

about the composition and succession of the bifidobacterialmicroflora.

The unquestionable conclusion is that thebifidobacterial microflora in elderly people will be reducedquantitatively as well as qualitatively. This fact wasmentioned already by Orla-Jensen and by Mitsuoka in theirearly investigations and can also be seen from the data inTable 9. In general, it can be further stated that thebifidobacterial microflora of humans may contain up to 4or 5 different species or special strains continuously, andthat one or two are quantitatively predominant. B. longumand B. bifidum are not the predominating species, however.That is a very important fact to be considered in view ofthe selection of bifidobacterial strains for their use asprobiotics.

Acknowledgments

The investigations were supported by long term grants of“Deutsche Forschungsgemeinschaft” in the years 1960-1970. Many thanks are due for this support. Preparationof this manuscript was supported by PH.-D. doctorandsUrte Köpke and Marc Goldberg. Many thanks to them fortheir continuous engagement.

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