Biology of the Bifidobacteria · 138 POUPARD,HUSA Blaurock (11), Boventer (15), andTomarelli et al. (168, 169) did not accomplish the desired result. It wasnotuntilthemediumofTeplyand

BACTERIOLOGICAL REVIEWS, June 1973, p. 136-165Copyright i 1973 American Society for Microbiology

Vol. 37, No. 2Printed in U.S.A.

Biology of the BifidobacteriaJAMES A. POUPARD, INTISAR HUSAIN,1 AND ROBERT F. NORRIS

William Pepper Laboratory, Department of Pathology, Medical School, University of Pennsylvania,Philadelphia, Pennsylvania 19104

INTRODUCTION ...................... ............................... 136MORPHOLOGY ..................................................... 137Pleomorphism and Branching .................................................. 139Morphological Relationships with Other Organisms ........ .................... 141Ultrastructure ..................................................... 141

PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERISTICS ...... ........... 142Nutrition and Atmospheric Growth Requirements ......... ...................... 142

Nutrition ............... ...................................... 142Atmospheric growth requirements ..................... ....................... 143

Biochemical Characteristics .................................................... 144Carbohydrate metabolism .................................................... 144Polysaccharide formation ..................................................... 145Extracellular dextranase and intracellular a-1 a 6 glucosidase ...... .......... 145

Deoxyribonucleic Acid Base Composition .............. ......................... 145Cell Wall, Lipid, and Phospholipid Composition .......... ....................... 146

Cell wall composition ..................................................... 146Lipid and phospholipid composition ................. ......................... 147

CLASSIFICATION ..................................................... 148First Period ............... ...................................... 148Second Period ................. .................................... 151Identification 154

ECOLOGY ............. ........................................ 155Occurrence ............... ...................................... 155Origin and Mode of Transmission............................................... 156Interaction With Other Organisms............................................... 156Effects of Bifidobacteria on the Host ..................... ....................... 157

CONCLUDING REMARKS ..................................................... 158

INTRODUCTION

In much of the early literature, in retrospect,it is now apparent that many species ofBifidobacterium were all designated Lac-tobacillus bifidus. When the species identity ofsuch organisms is in doubt, therefore, the term"bifidobacteria" will be used in the presentpaper. However, in cases where the changewould significantly alter the original author'sobservation no change will be made in hisdesignation. Bifidobacteria were first describedin 1899 and 1900 by Tissier (166, 167) andnamed by him Bacillus bifidus communis orsimply B. bifidus. This organism was a gram-positive, curved, and often bifid rod which wasthe predominant organism in the stools ofbreast-fed infants. In bottle-fed infants, how-ever, gram-positive, straight, unbranched rodswere the predominant organisms and were iden-tified as B. acidophilus, the organism first

aPresent adress: Department of Biochemistry, Faculty ofMedicine, Aligarh Muslim University, Aligarh, Uttar PradeshIndia

isolated in 1900 by Moro (105) also from stoolsof breast-fed infants. For the next 50 yearsinvestigators were concerned with the organismin the human intestinal tract, with devisingculture media for isolation and maintenance ofthe organism on subculture, and with the signif-icance of the organism in the health of thenewborn. It was determined that the numbers ofbifidobacteria were fewer in the stools of bot-tle-fed infants and that, when weaned, infantsharbored bifidobacteria only in small numbers.It was shown that when an adult diet wasconsumed, the stools of the infant shifted to thegram-negative bacillary flora of the adult.

Scientific interest in the organisn vas pro-moted by clinical observations in Et )pe thatbreast-fed infants were apt to be less susceptibleto infections than bottle-fed babies and byspeculation that bifidobacteria in the colonmight play a role in the nurslings' resistance toinfection. During this period, however, pasteuri-zation of cow's milk was more widely employed,and measures to minimize bacterial contamina-tion during handling and distribution of milk

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BIOLOGY OF THE BIFIDOBACTERIA

were made more effective. Artificial formulaefor bottle feeding with cow's milk were alsogreatly improved. In economically advancedcountries, therefore, the bottle-fed infant nowhas much less exposure to bacterial infectionthan does his predecessor.Meanwhile the importance of maternal anti-

bodies transferred to the infant by nursing inpromoting passive resistance to both bacterialand viral infections was gradually appreciated.Continuing investigation of bifidobacteria

was also occasioned by scientific interest amongbacteriologists in the nature and classificationof this organism. Difficulties were soon ex-perienced in culturing organisms seen in thegram-stained preparations of stools. The cul-ture media in common use were unsatisfactoryfor isolation and maintenance of the organisms.For this reason uncertainty continued amonginvestigators about whether organisms desig-nated L. bifidus were identical in differentlaboratories and whether the organisms kept asstock strains were identical to those in nurs-lings' stools. Although there were dissenters,most workers classified the bifid organisms asbelonging to the genus Lactobacillus, but thetaxonomic relationship to the better studied L.acidophilus was not well established. The litera-ture up to 1950 has been amply reviewed byWeiss (187), Weiss and Rettger (188), Orla-Jensen et al. (118), Orla-Jensen (117), andOlsen (114).

In 1950, Norris et al. (112) reported thecomposition of a culture medium which waspartially defined chemically and which wassatisfactory for the primary isolation and labo-ratory maintenance of at least some strains ofbifidobacteria. Because of the suspicion that aningredient of breast milk might be essential forthe growth of some strains of bifidobacteria,Gybrgy (50) and Gyoirgy and Rose (55) addeddefatted breast milk to the Norris medium.They isolated a milk-requiring strain of theorganism and designated it L. bifidus var.pennsylvanicus. Since these reports, much morehas been learned about the structure, culturalrequirements, metabolism, and immunologicalcharacteristics of this organism. The number ofknown strains differing in part from one anotherhas greatly increased. Their relationship to thelactobacilli, corynebacteria, and actinomyces,furthermore, has not been clarified to such anextent as to engender widespread acceptance.This paper reviews the knowledge of bifido-

bacteria which has accumulated, especiallyduring the past two decades. Emphasis will beplaced on structure, nutritional requirements,

classification, and possible ecological signifi-cance.

MORPHOLOGYIn his original description of bifidobacteria,

Tissier (166) described curved rods and rodswith ends split to give the characteristic Y-shape which led to the designation of "bifid." Inaddition, bizarre small branches and bulbous orswollen ends were seen less frequently. He andMoro (105) concurred that the organisms weredifferent from L. acidophilus which was astraight rod or coccobacillus, sometimes occur-ring in chains, and which was more easilyisolated from stools in the presence of oxygen.

Various workers generally confirmed the ob-servations of Tissier on the structural variationsof bifidobacteria and their differences from L.acidophilus. These included Herter (65), BlUh-dorn (14), and Kendall and Haner (78). Othersexpressed doubt that bifidobacteria and L.acidophilus were different organisms and be-lieved that the latter likewise became pleo-morphic and bifid when exposed to certainenvironmental conditions. Supporting this posi-tion were Rodella (135), Roos (136), and Stitt(159). Cruickshank (20) found that bifid formswere rare in direct examination of stools and, onculture, that the appearance of branching wasoften the result of overlapping of organisms tocreate the illusion of branching. Weiss (187)agreed with this point of view and with theconclusion that branching was rare. He alsofavored the view that both L. acidophilus andbifidobacteria produce rudimentary branchingunder unfavorable conditions. In 1935 Eggerth(31) described the characteristics of gram-posi-tive rods isolated from the stools of bothbreast-fed and bottle-fed infants and foundthem to be similar to those described by Tissier.

Eggerth (31) noted an increase in branchedforms in 4- to 6-day-old cultures. In somestrains six to eight lateral branches, as well ascompound branching, were observed. He de-scribed also cultures which became so granularthat they had the appearance of a chain of close,irregular cocci and noted a decrease in meta-chromatic granules with age. Eggerth also de-scribed two types of bifidobacteria, group I andgroup II, based on certain biochemical charac-teristics.By this time it was apparent that a serious

problem to be solved was the inadequacy ofavailable culture media for the primary isola-tion and continued maintenance of bifidobac-teria. Attempts to improve media for this pur-pose by Weiss (187), Weiss and Rettger (189),

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138 POUPARD, HUSA

Blaurock (11), Boventer (15), and Tomarelli etal. (168, 169) did not accomplish the desiredresult. It was not until the medium of Teply andElvehjem (165) for folic acid assay was modi-fied, first by Tomarelli et al. (170) and then byNorris et al. (112), that bifidobacteria could beconsistently isolated from stools and body secre-tions and could be carried indefinitely as stockcultures in the laboratory. This medium (here-after called Norris medium) was selective,because it was inhibitory for other intestinalorganisms except for enterococci, probably be-cause of its acetate content. Its use thereforefacilitated the isolation of bifidobacteria in pureculture. The aforementioned addition of defat-ted breast milk to this medium by Gy6rgy et al.(54) was also used for primary isolation. At leastone strain of bifidobacteria requiring milk for itsgrowth was isolated. The composition of theNorris medium currently in use is given inTable 1.By using the new medium, Norris et al. (112)

TABLE 1. Composition of Norris mediuma

Component

Adenine ...............................Alanine ..............................p-Aminobenzoic acid ...................Ascorbic acid .........................Asparagine ............................Biotin .................................Calcium pantothenate ..................Cystine ...............................Ferrous sulfate (FeSO4 7H20) ..........Folic acid ..............................Guanine ...............................Lactose ...............................Magnesium sulfate (MgSO4.7H20).Manganese sulfate (MnSO4. H20) .......Nicotinic acid .........................N-Z case ..............................Potassium phosphate, diabasic(K2HPO4) ...........................

Pyridoxine hydrochloride ...............Riboflavine ............................Sodium acetate, anhydrous..............Sodium chloride .......................Sorbitan monooleate (Tween 80) ........Thiamine hydrochloride ................Tryptophan ...........................Uracil .................................Xanthine ..............................Human milkc ..........................

Quantitiesper liter

17.4 mg200.0 mg10.0 mgl.Og

100.0 mg5.0 qg

200.0 jig200.0 mg10.0 mg10.0 ,ug12.4 mg35.0g

200.0 mg6.7 mg

600.0 jig5.0g

2.5g1.2mg

200.0 jg25.0g10.0 mg0.5 ml

200.0 jg200.0 mg10.0 mg10.0 mg2%

IN, AND NORRIS

found that two morphological types of gram-positive rods were in infant stools. One was acurved rod, sometimes bifid, and occasionallybranched. It was microaerophilic and did notproduce gas in liquid medium. The second wasuniformly straight, unbranched, and often coc-coid. Long chains were sometimes observed.This organism did produce copious amounts ofgas and tolerated atmospheric oxygen. Becausethe two were frequently associated on primarycultures, the relationship of one to the otherposed a problem. When supposedly pure cul-tures of the bifid organism were transferred inthe laboratory, the straight-rod organisms occa-sionally appeared unexpectedly and sometimescompletely replaced the bifid organisms in liq-uid cultures. By use of a micromanipulator, asingle-cell clone of bifid organisms was estab-lished as a stock strain, the so-called Jacksonsingle-cell 8, by Norris et al. (112). After serialtransfers at 48-h intervals for several weeks,straight-rod organisms suddenly appeared inthe culture on plating. Because precautions hadbeen taken to prevent contamination, it washypothesized that the straight rods were derivedfrom the bifid forms by mutation. This hypoth-esis found support at a later date when two bifidstrains, which were observed by Williams et al.(195) for a period of time, acquired an ag-glutinating antigen characteristic of thestraight-rod strains and not found in bifids.Subsequently, these strains became aerobic,straight rods which no longer branched. Theseobservations suggested that the surface antigenchanged in advance of the morphologicalchange to straight rods. On the basis of thiswork, Norris et al. (112) proposed that thenaturally occurring bifidobacterium was acurved rod, often bifid, sometimes branched,and microaerophilic. It was designated L.bifidus (Tissier). The straight rod, unbranchedstrain, which was aerobic and which wasthought to be derived from L. bifidus by muta-tion, was designated L. parabifidus. It wasbelieved that bifid organisms described by theseworkers were the same as those described byTissier, but that he and others were unable tomaintain them as bifid and branched rods instock culture because of the lack of a suitableculture medium. They also resembled thosestrains described by Orla-Jensen (115), Orla-Jensen et al. (118), Blaurock (13), Boventer(15), and Weiss and Rettger (189) and classifiedby the latter authors as L. bifidus, type II, or L.parabifidus. The proposal of Norris et al. ineffect transposed the designations of Weiss andRettger (189).

In 1955 Gyllenberg (44) studied the bifid or

0Autoclaved for 10 min at 15 lb at 121 C afteradjustment to pH 6.8. For plates, add 17.4 g of agar(see references 50, 54, 111, 112, and 138).

'Ascorbic acid was added after autoclaving.c Used to enhance growth of some strains.

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branched form and the straight-rod form. Hedescribed the formation of straight rods fromcoccoid granules in the branched form. A cyclewas described in which the organism developedinto a highly branched form and into mycelialbranched filaments. Some mycelial cells wereobserved to swell and contain intensely stainedcoccoid granules which, it was believed, gaverise to the straight rods. The author did notobserve the conversion of short, unbranchedrods to branched or bifid organisms. Althoughsimilar observations were made on several otherstrains, none was a single-cell isolate, and thepossibility of contamination of stock strains atthe time of isolation was not eliminated.

In a later study, Gyllenberg (46) noted thatother reports described bifid bacteria abruptlychanging from microaerophilic branched orga-nisms to straight rods capable of aerobicgrowth. He suggested that there are two possi-ble explanations: (i) the straight rods must beconsidered variants or mutants of the branchedtype, or (ii) the straight rods are contaminantsof bifidobacteria cultures. Gyllenberg studiedthe cultural characteristics and the conditionswhich gave rise to nine straight-rod forms frombifid bacteria. It was discovered that the ninestrains were almost identical in most of theircharacteristics and that they differed from thebranched form in many ways. They resembledan authenticated Corynebacterium acnes.Investigations on the source of the C. acnes ledthe author to conclude that C. acnes of intes-tinal origin is sometimes a "latent contaminant"of bifidobacteria cultures and is capable, on re-peated transfers, of overgrowing and replacingbifidobacteria in liquid cultures. The authoremphasized, however, that the unbranchedvariants described by Norris et al. (112) were notC. acnes, but were probably lactobacilli.In view of the uncertainties, Norris (unpub-

lished data) continued the investigation of therelationship of unbranched to bifid organisms.Strain Jackson single-cell 8 has been main-tained in this laboratory in Norris medium withperiodic agar transfers for more than 22 years.For most of this time, characteristics of theorganism have remained stable. The appear-ance of aerobic, gas-producing, straight rods hasnot occurred spontaneously. Various alterationsof the physical and chemical environment havebeen applied to the organism without elicitingthe appearance of the straight-rod form. In thecase of a number of more recently isolatedstrains of bifidobacteria from breast-fed infants'stools, spontaneous transformation to straightrods has not been observed. Other authors,furthermore, were unable to confirm the deriva-

tion of the straight rods from bifid organisms bymutation. The preponderance of evidence atthis time, therefore, favors the explanation thatstraight rods of the Lactobacillus type whenfound with cultures of bifidobacteria are con-taminants rather than derivatives of these orga-nisms.

Pleomorphism and BranchingHayward et al. (63) noted that in unfavorable

culture media organisms were so greatlybranched that resemblance to rods was lost.Other organisms were unbranched but wereswollen and irregular. As the strains becameadapted to artificial culture by transfers, thebranched and swollen forms became less fre-quent until rods predominated. During theperiod before satisfactory culture media weredevised, highly branched and bizarre distor-tions of the organism were often observed.These changes usually occurred just prior todeath of the culture on serial transfer and weretherefore usually interpreted to be involutionforms of the organisms (122). Sundman andBjirksten (161) also considered bizarre swellingof the organisms as involution forms, and swel-ling of rods was sometimes so extreme as toresemble a bladder which measured as much as4 ;im in diameter with a crosslike structure onthe surface. The bladder-like forms were noteasy to detect in stained preparations, but wereclearly visible in wet mounts with phase-con-trast microscopy. Two types of agar were used,Norris agar and tomato agar (161), but theinvolution form was observed only when grownin tomato agar. The authors concluded that,although tomato agar permitted abundantgrowth, it appears to be nutritionally deficient.All of the strains tested produced these forms ontomato agar. The abnormalities of structureappeared after repeated subculture. Sundmanand Bjorksten also confirmed that althoughbifidobacteria have a tendency to show morpho-logical variation when grown in vitro, they aremostly rod-shaped in the natural habitat. Theysuggested as an explanation for this phenome-non that bifidobacteria probably have a morecomplicated pathway of cell wall synthesis thanmany organisms. As later studies indicated,there is justification for this conclusion (seePhysiological and Biochemical Characteristicssection). This study thus reveals the importanceof defining the composition of the culture mediaused when studying the structure of bifidobac-teria.The first definitive study of the actual cause

of pleomorphism in this organism was made byGlick et al. (41) in 1960 when they demon-

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strated the formation of bizarre forms of L.bifidus var. pennsylvanicus (B. bifidum) result-ing from a deficiency of an N-acetyl-aminesugar which is an essential growth requirementand a precursor for cell wall synthesis. It wasfound that by varying the concentration of thisprecursor branching could be eliminated. Thisobservation led the authors to the conclusionthat the bifid character of the organism mightbe a consequence of an inadequate supply ofthis factor.

In 1968 Kojima et al. (82) studied pleomor-phism of a bifidobacterium based on the obser-vations of Homma et al. (70) that certaincarbonates induce bifid formation. They con-firmed the work of Homma et al. and also notedthat similar effects are obtained with sodiumchloride, sodium sulfate, sodium nitrate, tri-basic sodium phosphate, sodium acetate, andchlorides of other univalent cations. This effectwas not observed with chlorides of Ca2 , Ba2 ,Mg2+, Mn2+, or Zn2+. They also noted thatCaCl2 or MgCl2 in combination with NaClreduced the pleomorphism-inducing effect ofthe latter. Observations of the effect of rham-nose and polyethylene glycol, which were usedto alter the osmolarity of the media, led theauthors to conclude that the pleomorphic ef-fects of a univalent cation could not be ex-plained solely on the basis of osmotic pressure.In a set of experiments they concluded that Na+caused a much greater increase in cell massthan in the number of viable bacteria on cul-ture, because the former increased by as muchas 25 times, whereas the latter increased by only1.5 times. In 1970 Kojima et al. (83) found thatthe calcium ion content in intact cells and in thecell wall preparations of the bifid form weresignificantly lower than those of the bacilloidform. They also found that the addition ofchelating agents to the culture medium inducedbifid formation. When CaCl2 was added to suchCa 2+-deprived cultures, the bifid form was re-versed to the bacilloid form. They found thatglucose and the total amount of sugars weremore abundant in the bifid form than in thebacilloid form. Methionine and phenylalaninewere observed in the bifid form but were un-dectable in the bacilloid form. The authorsconcluded that although the mechanism ofinduction of pleomorphism is not clarified, itappears that the presence of Ca2+ ions plays aprincipal role in its prevention. In the same yearKojima et al. (84) studied electron micrographsof bifidobacteria grown in medium enrichedwith NaCl to induce the branched form and incalcium-enriched medium to induce the bacil-

loid form. Cross walls were observed in thebacilloid form but not in the bifid form. Theynoted that these observations lend further sup-port to the indispensable role of Ca2+ ions forcytokinesis in bifidobacteria.Husain et al. (72) recently investigated a

mucoid variant of bifidobacteria which hasshown a stable, straight-rod or bifid morphologyover a long period of time. They observed thatwhen this organism was grown in a definedminimal medium the organism showed profusebranching. It was observed that, by the additionof four amino acids, alanine, aspartic acid,glutamic acid, and serine, the organism couldbe converted to its bacilloid form. Therefore, bymanipulating these amino acids, their strain ofbifidobacteria could be maintained indefinitelyin either a bifid or highly branched form.Previous workers (22, 176) have demonstrated,furthermore, the presence of these amino acidsin the cell walls of bifidobacteria. These obser-vations imply that these amino acids are re-quired to preserve the organism in the straightrod or bifid form. Their absence, however, hasnot been demonstrated in highly branched var-iations of the organism. Husain et al. alsoconfirmed the work of Kojima et al. (82) thatpleomorphism or branching of the organism wasinduced by sodium chloride. However, the re-versal of this effect by the addition of Ca2+ ions,as reported by Kojima et al. (84), was not ob-served by these authors. Three cell wall pre-cursors, N-acetyl-D-glucosamine, a- e-diamino-pimelic acid and muramic acid failed to in-hibit branching when added to the minimal me-dium. They concluded that the proposal ofKojima et al. (84), that branching in bifidobac-teria was principally due to its inability to formcross walls when grown in a medium deficientin Ca2+ ions, was an oversimplification of amore complex phenomenon. Reports such asthese have been influential in eliminating thedesignation of variants or subspecies solely onthe basis of morphological grounds.

In our laboratory, observations concerningthe morphology of bifidobacteria are in agree-ment with those of most workers quoted. Wehave found that in stools of infants the bifido-bacteria are curved, gram-positive rods whichare sometimes bifid. When cultured for as longas 22 years on the Norris medium, this structureis maintained, but when grown on other media,such as tomato agar and thiogylcollate,branched and bizarre forms are often observed.Most evidence at hand, therefore, indicates thatthe curved bifid rod phase of bifidobacteria canbe maintained in the laboratory for indefinite

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periods of time on a nutritionally optimal me-dium. The branched phase of the organism canalso be preserved for long periods of time in amedium which lacks certain amino acids. Theimplication of these observations is not thatbranching is necessarily an involution or degen-erative form of the organism, but that it is anadaptive reaction to a less than optimal nutri-tional environment.

Morphological Relationships with OtherOrganisms

Although bifidobacteria were included in thegenus Lactobacillus for many years, partlybecause of morphological resemblance to othermembers of this genus, branching when itoccurred was greater than that of lactobacilli.Reservations about the taxonomic position ofbifidobacteria were, therefore, engendered onmorphological grounds.Negroni and Fischer (110) compared the

structure of bifidobacteria with Actinomycesbovis. Their illustrations of the bifid andbranched phases show more pleomorphism thanin A. bovis and confirm previous reports on thepleomorphic characteristics of the former. Bifidsplitting of the bacillary ends, simple andcomplex branching, and swelling of the bacilliwere illustrated. It was concluded that the twoorganisms were similar but not morphologicallyidentical. Frank and Skinner (35) also com-pared these organisms and agreed that pleomor-phism is greater in bifidobacteria than in A.bovis. Simple branching and pleomorphism instrains of anaerobic diphtheroids were less fre-quent than in bifidobacteria. Hayward et al.(63) described the marked pleomorphism whichwas found in cultures of bifidobacteria soonafter isolation. Branched and unbranched rods,irregularly septate, were illustrated. Some werebifid. Some were swollen in the middle of thebacillus, and others had terminal swellings.Short, lateral branches asymmetrically placedalso were numerous. As the culture becameadapted to artificial media after repeated sub-culture, the pleomorphism became less frequentand the rod form predominated. The authorsconcluded that the organisms had morphologi-cal features in common with both actinomycesand corynebacteria, and they thought that theresemblance to corynebacteria was greater.Only in the case of well-adapted cultures wasthe resemblance to lactobacilli close. Sundmanet al. (162) compared the structure of strains ofbifidobacteria with representatives of the gen-era of Actinomyces, Butyribacterium, Coryne-bacterium, Lactobacillus, and Propionibac-

terium. On tomato agar their strains of bifido-bacteria showed the pleomorphic variation de-scribed by others. Marked branching and blad-der-like and protoplastic-like swellings were nu-merous. Reversion to the straight-rod form oc-curred regularly when such organisms weretransferred to a nutritionally more complex me-dium. Morphological similarities were notedamong all the groups studied, but relationshipof the bifidobacteria with any of them was notdetermined. Three of four morphological sub-types of bifidobacteria were thought to resembleButyribacterium most closely; the fourth, L.bifidus var. pennsylvanicus (B. bifidum), hadstructural features in common with Actinomy-ces. Beerens et al. (5) noted that the morphol-ogy of the bifidobacteria is not always charac-teristic and also noted the importance of bio-chemical and cultural properties for the inden-tification of these organisms.By light microscopy, therefore, investigators

are in agreement that the bifidobacteria havecertain morphological features which are notunique but which, taken together, are of aid indistinguishing these organisms from others. Oncertain media, notably tomato broth and agar, ahigh degree of branching, which is sometimescomplex, is observed. Swelling of the bodies ofthe organism and bladder-like globular swel-lings, which are often terminal, are observedless often. Septa, when present, are often infre-quently or irregularly placed, as if branchingmay occur in lieu of cell division. When theorganisms are transferred to more complexmedia, they revert to the straight or curved-rodform which is sometimes bifid. The observa-tions of Tissier, therefore, have been confirmedand expanded.

UltrastructureAlthough the morphology of bifidobacteria

has concerned many workers, very little atten-tion has been given to their ultrastructure. In1963 Overman and Pine (120) studied electronmicrographs of several species of Actinomycesand included one micrograph of L. bifidus var.pennsylvanicus (B. bifidum). This appears tobe the first published electron micrograph of asectioned organism of a member of the genusBifidobacterium. Of the organisms studied, nomembrane complexes were observed in L. bifi-dus var. pennsylvanicus or A. bovis. However,"dark bodies" were observed which were sugges-tive of bacteriophage. The authors believed thatthe lack of "membrane coils" in L. bifidus var.pennsylvanicus indicated that the organism is

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more primitive than L. acidophilus and that itis more closely related to A. bovis.Kojima et al. (84) published electron micro-

graphs of branched organisms and of a bacilloidform of bifidobacteria and noted a lack of crosswalls in the former. Because the former werecells grown in a Ca2+-deficient medium and thelatter were grown in a Ca2+-enriched medium,they believed that the electron micrographssupport their contention that Ca2+ ions areimportant in the formation of cross walls and inthe prevention of pleomorphism in bifidobac-teria.

Exterkate et al. (34) used electron micro-graphs to study the ultrastructure of lysozyme-treated L. bifidus var. pennsylvanicus (B.bifidum). They noted that in these cells thecytoplasmic membrane was much easier todistinguish because it was separated from theelectron-dense layer by a bright zone. It wasalso noted that incubation with lysozyme re-sulted in a complete disappearance of the cellwall of L. bifidus var. pennysIvanicus, but thatthe cell walls of the branched form of theorganism showed no changes after lysozymetreatment.For electron micrographs, B. bifidum (L.

bifidus var. pennsylvanicus) was grown in theNorris medium supplemented with breast milkfor 48 h. Organisms were fixed and embeddedby the method of Higgins (personal communica-tion). The method of fixation is similar to thatdescribed by Reyn et al. (134). Our procedurediffered from theirs in that the organisms werefixed in 3% glutaraldehyde, followed by aadi-tional fixation with Kellenberger osmium fixa-tive. For embedding, Epon 812 was used insteadof Vestopal. Grids were stained with uranylacetate and counterstained with lead citrate.Photomicrographs (Fig. 1) show bifid and

nonbifid forms. Although a nuclear area and adistinct cell wall are present, the character ofother intracellular structures is uncertain.

It has been demonstrated in the past that thecomposition of the growth medium greatly af-fects the structure of this organism. Before anydefinite conclusions can be drawn concerningthe ultrastructure of the organisms, therefore,additional comparative studies of bacillary,bifid, and branched phases of the organisms areneeded.

PHYSIOLOGICAL AND BIOCHEMICALCHARACTERISTICS

Nutrition and Atmospheric GrowthRequirements

Nutrition. Bifidobacteria are not a nutri-tionally homogeneous group, and nutritionally

different types of bifidobacteria have been re-ported (47, 48, 54, 62, 197). Prior to the studiesof Tomarelli et al. (168-170), very little infor-mation was available concerning the growthrequirements of these organisms. Norris et al.(112) described a semisynthetic medium, sup-plemented with most of the factors known tostimulate the growth of lactobacilli, whichwould support the growth of bifidobacteria inthe bifid phase. The growth stimulatory activityof each of the constituents of this medium wasinvestigated by Hassinen et al. (62) in order todetermine the minimal nutritional requirementsof four strains of this organism. It was found bythese workers that in a relatively simple me-dium, containing cysteine (or cystine), thesestrains utilized ammonium salts as a source ofnitrogen. This is, perhaps, the most strikingdifference in the nutritional requirements ofthese strains from most lactobacilli, which gen-erally have more complex nutritional require-ments. Of the B-group vitamins, only biotin andcalcium pantothenate were required; purineand pyrimidine compounds were not essential;and the cysteine (cystine) requirement was notreplaceable by methionine, homocysteine, orrelated compounds.A strain which showed only scant or un-

detectable growth in the regular synthetic me-dium (112), but which could be propagated eas-ily in the basal medium after the addition ofhuman milk, was isolated from the stools ofbreast-fed and bottle-fed infants and from thevaginal secretions of pregnant women (50, 61).It was named L, bifidus var. pennsylvanicus(54), the present-day B. bifidum.The essential factor in human milk, which

was lacking in cow's milk (bifidus factor), wassubsequently identified as N-acetyl-D-glucos-amine-containing saccharides. These growthfactors were shown to be used by this organismas a substrate for cell wall synthesis (41, 113).Bifidus factor and related subjects have beenstudied extensively by Gy6rgy (53), Gy6rgy andRose (56), Gyorgy et al. (57, 58), O'Brien et al.(113), Pope et al. (125), Rose and Gyorgy (138),and Zilliken et al. (198-201). Certain N-sub-stituted derivatives of D-glucosamine, e.g., N-benzoyl - D - glucosamine, N - carboethoxy - D -glucosamine, and N-caproyl-D-glucosamine,and 0- and N-f,-glycosides of N-acetyl-D-glucosamine also promote growth of this mi-croorganism (87, 139). When the first two com-pounds, radioactively labeled in the carbohy-drate moiety, were added to the culture me-dium, marked radioactivity was measured inthe muramic acid and D-glucosamine of thebacterial cell walls (86). These two growthfactors, representing synthetic derivatives of

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D-glucosamine, can be considered, therefore, toact as a source for the glucosamine unit which isessential for cell wall synthesis. Some of theseN-substituted glucosamine derivatives exhibita growth-promoting activity exceeding that ofthe naturally occurring N-acetyl-D-glucos-amine. A possible explanation of the greatergrowth-promoting activity of these syntheticderivatives was given by Veerkamp (172), basedon his experimental observations. It was consid-ered that the slower conversion of these com-pounds to glucosamine-6-phosphate, as com-pared with the less active N-acetyl-D-glucos-amine, and an inhibition of deamination of theresulting glucosamine-6-phosphate (to fructose-

A

(dCb

(n))

(d b

6-phosphate) by the 6-phosphate of these deriv-atives enabled the organism to use glucos-amine-6-phosphate more efficiently for cell wallmucopeptide synthesis. Later studies by Veer-kamp (171) also established that the slightgrowth-promoting activity of N-acetyl-D-glucosamine compared with other N-sub-situted glucosamines is especially caused by itsrapid conversion to fructose-6-phosphate, whichis for the most part degraded via acetyl phos-phate to acetate and lactate.Atmospheric growth requirements. Inves-

tigators have been concerned with the questionof whether bifidobacteria are strictly anaerobic.In studying branched and unbranched strains of

SIB

(ib)

FIG. 1. Electron micrographs of glutaralydehyde-osmium tetroxide-fixed, uranyl acetate-lead citrate-stainedB. bifidum. A, An unbranched phase; B, the bifid phase demonstrating the presence of a nuclear area (n),dense bodies (db), and a distinct cell wall (cw). x60,OOO.

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bifidobacteria, Norris et al. (112) noted differ-ences in their requirements for atmosphericCO2, and sensitivity to atmospheric 02, whenthey were grown on solid media or in brothtubes. Although Mayer et al. (98) claimed thatbifidobacteria could be grown under aerobicconditions, with concomitant production of cat-alase activity, it is now generally accepted thatbifidobacteria are strictly anaerobic and do notpossess catalase activity (30, 153, 154).

In a detailed study of the factors determiningthe degree of anaerobiosis of bifidobacteriastrains, de Vries and Stouthamer (181) deter-mined the degree of sensitivity of 12 bifidobac-teria strains to 02 by measuring the size of thezones of inhibition obtained when they were

grown in deep agar cultures under air and bymeasuring growth in aerated cultures. None ofthe strains tested grew on agar plates underaerobic conditions, thereby confirming the ob-servations of earlier workers (25, 112, 153).However, great differences in sensitivity tooxygen were found for different bifidobacteriastrains, as was evident from the size of the zones

of inhibition obtained.It was also shown that the principal reason for

anaerobiosis was different for different strains ofbifidobacteria. Some strains, which had smallzones of inhibition in agar stabs, appeared topossess a weak catalase activity. Hydrogenperoxide did not accumulate in the presence ofair or, if traces were formed, H202 was removedby catalase. It is also likely that reduced-formnicotinamide adenine dinucleotide oxidase ofthese strains did not form H202 at all. Theabsence of growth on agar medium probablyresulted from the fact that these strains grew

only below a certain oxidation-reduction poten-tial.

In the case of some strains of bifidobacteria,accumulation of H202 was found to be theprincipal reason for the requirement for anaero-

bic conditions. Hydrogen peroxide causes a

block in the fermentation pathway by inactiva-tion of fructose-6-phosphate phosphoketolase,which is a key enzyme in the carbohydratemetabolism of this organism.Other strains of Bifidobacterium, however,

which had the largest zones of inhibition in agar

medium did not accumulate H202. These were

found to ferment glucose only when cysteine or

ascorbic acid was added. The presence of 02 was

not lethal for these strains. It was concluded bythese workers (18) that 02 prevented growthand fermentation of these strains by establish-ing too high an oxidation-reduction potential. Itis obvious from their study that, even in a group

of such closely related microorganisms, the

principal reason for anaerobiosis may be differ-ent for different members of the same group.Dehnert (26) noted the effects of the culture

media when studying this organism and notedits strict anaerobic requirements. Kandler (75)in a comparative study of the lactobacilli alsonoted that the bifidobacteria were strict anaer-obes and, therefore, were different from most ofthe other organisms he studied.

Biochemical CharacteristicsCarbohydrate metabolism. Kuhn and

Tiedemann (85) gave some suggestions concern-ing the pathway of carbohydrate fermentationin bifidobacteria. They reported the formationof labeled acetate and lactate from [1-'4C]glucose. Aldolase activity was detected incell-free extracts of the organism. On the basisof these observations, they concluded that glu-cose is fermented via the glycolytic system andthat both acetate and lactate are formed viaphosphoenol pyruvate.More recently, a new pathway for the fermen-

tation of hexoses has been reported in bifidobac-teria by Scardovi and Trovatelli (147) by deVries et al. (178), and by de Vries and Stouth-amer (180). The pathway was elucidated bychemical analysis of end products, determina-tion of radioactive carbon distribution afterfermentation of [1- "C ]glucose, and assay ofenzymes in cellular extracts. A key feature ofthe pathway is the participation of fructose-6-phosphate phosphoketolase, an enzyme whichwas first discovered by Schramm et al. (152) inthe obligate aerobe Acetobacter xylinum andwhich has also been reported in Leuconostocmesenteroides (42, 71). Another unique aspectof this new fermentative pathway is that it isthe only one in which both transketolase andxylulose-5-phosphate-phosphoketolase play im-portant roles. Aldolase and glucose-6-phosphatedehydrogenase could not be detected in ex-tracts, which rules out the glycolytic system andhexosemonophosphate shunt, characteristicpathways for degradation of glucose in membersof the genus Lactobacillus.Thus, hexoses are converted to fructose-

6-phosphate which is cleaved by phosphoketo-lase to yield erythrose-4-phosphate and acetylphosphate. Erythrose-4-phosphate and fruc-tose-6-phosphate are converted to 2 mol ofpentose phosphate by the action of transaldo-lase and transketolase. Xylulose-5-phosphate isthen cleaved by phosphoketolase to yield acetylphosphate and glyceraldehyde-3-phosphate.Glyceralde-3-phosphate is converted to pyru-vate, which can either be reduced to lactate or

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cleaved, presumably by a phosphoroclastic re-action, to acetate and formate. Thus, in caseswhere all of the pyruvate was cleaved ratherthan reduced, no lactate was formed. Becausethis pathway does not occur in any other speciesof the genus Lactobacillus, de Vries and Stouth-amer (179), in agreement with some previousworkers, suggested that classification of thebifidobacteria in this genus is not justified. Thesame alternative route of glucose dissimulationhas been established in L. bifidus var.pennsylvanicus by Veerkamp (171). The elec-trophoretic behavior of fructose-6-phosphatephosphoketolase from the different members ofthe genus Bifidobacterium was studied by Scar-dovi et al. (146) in the search for an additionaltool for the speciation of this genus. They foundthat the electrophoretic mobility of this en-zyme, although not a useful characteristic forthe differentiation of species in the genus, wasclosely related to their ecology.Polysaccharide formation. Occurrence of

mucoid strains of bifidobacteria has been re-ported by several workers. A mucoid variant ofbifidobacteria was first described by Malyothand Bauer (91, 92). Later, Norris et al. (111)reported that when strains of bifidobacteriaisolated from the stools of breast-fed infantswere carried in their chemically defined me-dium (112) by periodic transfer, mucoid colo-nies occasionally appeared among the smoothparent colonies. The cultural requirements ofthe mucoid strains appeared to be the same asthose of the parent type, and differences inagglutination reactions between them were notdetected by Williams et al. (195). Although themucoid organisms appeared to be variants ofthe smooth parent type, their reversion to thenonmucoid type was not observed. Aqueoussolutions of the polysaccharide produced bythese strains were highly viscous. Reports on thecomposition of these extracellular polysaccha-rides have been few. Vogel (177) found that thecomponent sugars of the capsular polysaccha-ride of a strain of bifidobacteria were glucose,xylose, uronic acid, an unidentified pentose,and an unidentified hexose exhibiting an Rfvalue suggestive of a methylated sugar. Wang etal. (184) found that the extracellular polsac-charide of a mucoid but non-encapsulated vari-ant of bifidobacteria (Jackson M) was com-posed of four sugars: D-glucose, D-galactose,6-deoxy-L-talose, and D-galacturonic acid. Thispolysaccharide was named "Bifidan." It is rea-sonable to suppose that different strains ofbifidobacteria elaborate extracellular polysac-charides of differing chemical composition,which may be the reason for dissimilarities in

the composition of "Bifidan" and the polysac-charides studied by Vogel (177).

Colonies of the mucoid variant described byMalyoth and Bauer (91, 92), incubated ana-erobically on solid media, were said to behygroscopic and became watery on exposure toatmospheric conditions at room temperature.Norris et al. (111) presented evidence indicatingthat the extracellular mucoid substance pro-duced by their mucoid strain (Jackson M) was ahighly polymerized polysaccharide which un-derwent depolymerization with loss of relativeviscosity when exposed to atmospheric condi-tions. This viscosity-reducing activity wasfound by Wang et al. (185) to be proportional tothe concentration of ascorbic acid and wasenhanced by atmospheric oxygen. Hydroperoxyradicals were considered responsible for theviscosity reduction activity.

Extracellular dextranase and intracellulara-i - 6 glucosidase. Although several speciesof molds have been shown to produce extracel-lular dextranases, very few bacterial specieshave been reported to produce this type ofenzyme. Hehre and Sery (64) reported theproduction of extracellular dextranases bymembers of the genus Bacteroides, and Baileyand Clarke (1) gave an account of an extracellu-lar dextranase secreted by several strains of B.bifidum. Hydrolysis of dextran by the dextra-nase from the latter source was unusual in thatit did not liberate glucose or isomaltose, whichare the main products from the action of allother known dextranases on dextran. Instead,the enzyme hydrolyzed dextran, by randomcleavage of the a-1 - 6 glucosidic links, to amixture of isomaltotriose, isotetraose, isopen-taose, and higher isomalto dextrins. All ex-tracts, prepared from a rumen strain of bifido-bacteria grown on dextran, were shown to con-tain an a-1 - 6 glucosidase (2).

Deoxyribonucleic Acid Base CompositionThe mean base composition of the deoxyribo-

nucleic acid (DNA) of bacteria has been shownto have important taxonomic significance (66,140). Precise comparisons of DNA base com-positions have therefore assumed great im-portance.Werner and Seeliger (192) and Sebald et al.

(153) studied the guanine plus cytosine (G + C)contents of 28 strains of Bifidobacterium byusing a chemical method employing hydrolysisof purified DNA. These workers included arepresentative organism of each of the groupspreposed by Reuter (132). They found theaverage G + C values to be 60.1 ± 0.33% for

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Bifidobacterium and to be less than 50% forLactobacillus. This difference in values wasbelieved to constitute a valid method for distin-guishing between these genera and also fordifferentiating Bifidobacterium from Pro-pionibacterium, Catenabacterium, and Coryne-bacterium, which also had G + C values lowerthan that of Bifidobacterium. The relationshipof Bifidobacterium and Actinomyces was notclarified. It was believed that DNA base compo-sition, in addition to morphological and bio-chemical characteristics, supported the conten-tion that Bifidobacterium should be classifiedas an independent genus.

Gasser and Mandel (37), by using a chroma-tographic method, studied the DNA base com-position of the genus Lactobacillus and in-cluded five strains of Bifidobacterium. Withpresent-day nomenclature, these strains of bifi-dobacteria would be representative of B.infantis and B. breve. These strains were se-lected because they represented the mean andextreme values obtained in previous studies bythe hydrolysis method (153, 192). They foundthat the five strains of Bifidobacterium had anidentical base composition of 58% G + C whichwas higher than for Lactobacillus.However, the work of Scardovi et al. (150,

151) and Corciani et al. (19) on the DNAhomology relationships among species of thegenus clearly indicated that the genusBifidobacterium is not as genetically homogene-ous as might be deduced from the narrow rangeof the DNA base composition of its members.Genetic relatedness among the strains repre-senting several named and unnamed species ofthe genus Bifidobacterium was assessed bymeans of DNA-DNA hybridization in competi-tion experiments by using the technique ofJohnson and Ordal (73). Several geneticallydistinct groups were recognized among the vari-ous proposed species of Bifidobacterium. B.bifidum was, however, found to be clearlydistinct genetically from the other bifidobac-teria and, hence, its designation as a species wasvalidated at the genetic level (150).

Cell Wall, Lipid, and PhospholipidComposition

Cell wall composition. Biochemical compo-sition of bacterial cell walls is important in theclassification of gram-positive anaerobes (21,23, 128). The cell wall composition of eightstrains of bifidobacteria was examined by Cum-mins et al. (22). An unusual degree of variationwas found among the strains which, at first,were thought to be a single bacterial species.

Alanine, glutamic acid, and lysine were presentin fractions from all strains, as were glucos-amine and muramic acid. Either one or two, butnot more, of the amino acids serine, glycine,threonine, and aspartic acid were detected insignificant quantities. Hexoses in the cell wallalso varied. Rhamnose appeared to be presenteither in fairly large amounts or only in minutequantities. The acid-resistant peptide of lysineand aspartic acid, previously noted in cell wallhydrolysates from lactobacilli, was not presentin those strains of B. bifidum which containaspartic acid. This observation suggests thatthe cell walls are structurally different fromthose lactobacilli in which these peptides occur.Absence of arabinose and a-E-diaminopimelicacid, which are characteristically present in thecell walls of Corynebacterium, and differencesin the composition of cell hydrolysates fromthose of any groups examined inActinomycetales (23) led these workers to statethat "as far as cell wall composition is con-cerned, these eight strains do not correspond toany group of gram-positive organisms previ-ously examined."The composition of the cell wall of L. bifidus

var. pennsylvanicus (B. bifidum) was studiedby Veerkamp et al. (176). The cell wall muco-peptide was found to contain serine, alanine,aspartic acid, glutamic acid, and muramic acid.In addition, ornithine was identified as thebasic amino acid component. This observationwas considered significant because Cummins etal. (22) did not report its occurrence in theirexperiments. It was suggested, however, thatornithine could have been mistaken for lysine,for the two homologous amino acids do notseparate easily in most solvent systems used inpaper chromatographic methods. Rhamnose,glucose, and galactose were detected in largeamounts in the cell wall preparation. Phos-phorus and glycerol were also present.

Although the amino acid composition of themurein (peptidoglycan, mucopeptide) is sup-posed to be useful as a chemotaxonomic crite-rion (23), the determination of the qualitative orquantitative amino acid composition of the cellwall is no longer considered sufficient to charac-terize the type of murein unequivocally. It waspointed out by Kandler (76) that the aminoacid content of two strains may be identical, butthat the amino acid sequence, or even the typeof cross-linkage, may be quite different.

In their study of the amino acid sequence ofmurein (peptidoglycan) of six strains of bifido-bacteria, Kandler et al. (77) showed that thepeptidoglycan contained muramic acid, glucos-amine, alanine, glutamic acid, lysine, and

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glycine at a molar ratio of 1:1:2:1:1:1. Theanalysis of the peptides obtained by partial 'acidhydrolysis indicated that the amino acid se-quence of the tetrapeptide is identical to that ofmost bacteria (L-Ala-D-glu-L-lys-D-Ala). Glu-tamic acid was present as an amide. Glycinewas involved in the cross-linking of adjacentmucopeptides by forming a bridge between theE-amino group of lysine and the carboxyl groupof a C-terminal D-alanine. About 50% of theglycine is N terminal, indicating that only 50%of the possible cross-linkages are realized. Themurein of these strains of B. bifidum resemblesthe murein of staphylococci, but differs by thenumber of glycine molecules. Although a pen-tameric glycopeptide occurs in the murein ofstaphylococci, only one molecule of glycine isinvolved in the cross-linkage of the murein ofthe B. bifidum strains studied.

Reporting results of a survey of the distribu-tion of the various murein types within thelactobacilli and related organisms, Kandler (76)stated that all of the species of some groups(Thermobacterium, Pediococcus) containedonly one type of murein, but that the species ofother groups (Bacterium, Bifidobacterium,Leuconostoc) contain several types. The mureintypes of the various strains in the genusBifidobacterium were found to correlate verywell with the species of this organism which wasdefined by Reuter (132). Kandler thought thatthe murein type may be a valuable criterion forthe separation of species within the gendsBifidobacterium.The various murein types of several species of

Bifidobacterium have been' described in theliterature. Holzapfel et al. (69) studied theamino acid sequence of the ornithine- andlysine-containing mureins of six strains of B.globosum. They found that murein containedMurNAc, glycine NH1NAc, glutamine, alanine,and diamino acids in a molar ratio of1:1:1:5:1. No teichoic acid was found. Theyfound the tetrapeptides attached to the mu-ramic acid to be equal to other mureins: L-ala-nine-D-glutamine-L-lysine (or ornithine)-O-ala-nine and believed that the evidence indicatedthat 10 to 20% of the interpeptide bridges werenot cross-linked. Some of the peptide unitswere thought also to be incomplete.Koch et al. (80) studied the amino acid

sequence of the threonine- and serine-contain-ing murein of 18 strains of B. longum and onestrain of B. lactentis and found them to containmurine, glycine, glutamine, alanine, ornithine(or lysine), threonine, and serine in a molarratio of about 1: 1: 1: 4: 1: 1: 1. The peptidesubunits attached to the muramic acid were:

L-alan ine- D-glutam ine-L-ornithine- D-alanine,and the interpeptide bridge consisted of f-D-as-partyl-L-serine. They found 60% of the subunitsto be cross-linked. Teichoic acid was not found.They noted that all strains of other species ofBifidobacterium investigated contained differ-ent types of murein. The murein of B. bifidumcontained an Orn-Ser-Asp-type murein whichdifferentiated B. bifidum from the other bifido-bacteria.The structure of the cell wall peptidogylcan of

B. bifidum var. pennsylvanicus was studied indetail by Veerkamp (175). It appeared to con-tain equimolar amounts of N-acetyl-D-muramicacid, N-acetyl-D-glucosamine, D-isoglutamine,D-(iso)asparagine, D-serine, L-ornithine, L-ala-nine, and D-alanine. Analysis of peptides ob-tained by acid hydrolysis indicated that thetetrapeptide linked to muramic acid has thestructure: N-a-L-alanyl- y-D-isoglutamyl-L-orni-thyl-D-alanine. Seryl asparagine is involved inthe cross-linking of adjacent tetrapeptides byforming a bridge between the 5-amino group ofornithine and the carboxyl group of C-terminalD-alanine. About a quarter of the (iso)aspara-gine is N terminal, indicating that only 75% ofthe possible cross-linkages are realized. Thepeptidoglycan structure of L. bifidus var.pennsylvanicus (B. bifidum) differs, therefore,from that of other bifidobacteria and that oflactobacilli by the structure of this specificcross-linking depiptide.Lipid and phospholipid composition. A

comparative study of the lipid content of cell,membrane, and cytoplasmic preparations fromnormally grown cells of L. bifidus var. pennsyl-vanicus (B. bifidum) and cells in which cell wallsynthesis was inhibited by growth in a mediumlacking human milk was undertaken by Exter-kate and Veerkamp (33). The percentage oflipids in cells grown without human milk ex-ceeded that of normally grown cells. The lipidcontent of the membrane, under the two condi-tions of growth, was not significantly different.The carbohydrate content in whole cell, mem-brane, and cytoplasmic lipids was consider-ably decreased after cell wall inhibition. Thephosphorus content also showed a minor de-crease. These changes were not due to differ-ences in pH values in the media with or withouthuman milk at the time of harvesting. Eighteenphospholipids were detected. Diphosphatidylglycerol, phosphatidyl glycerol, and a poly-glycerol phospholipid, identified as glycero-phosphorylglyceroldiacylphosphatidyl glycerol,were the main phospholipids. Mono-, di-, andtriacyl-bis-(glycerophosphoryl) glycerol, alanylphosphatidyl glycerol, phosphatidic acid, and

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two lyso derivatives, glycerophosphoryl glyceroland diacylphosphatidyl glycerol, were detectedin smaller quantities. After cell wall inhibition,the amount of triacyl-bis-(glycerophosphoryl)glycerol was increased considerably. Phosphati-dyl glycerol, glycerophosphoryl glycerol, anddiacylphospatidyl glycerol (and its first lysoderivative) were decreased, but diphosphatidylglycerol remained constant. The decrease inphosphatidyl glycerol, and to a lesser extent theincrease in triacyl-bis- (glycerophosphoryl)glycerol, was a response upon exposure to amedium of lower acidity. Phospholipid compo-sition was found to be greatly affected by thetemperature at which the cells were grown. Anearly complete reversal between the poly-glycerol phospholipids was observed after cellwall inhibition when cells were grown at 37rather than at 23 C.

All glycolipids were galactolipids. Besidesmono-, di-, and tri-galactosyldiglyceride, a mo-noacyl and diacyl derivative of monogalactosyl-diglyceride and a monoacyl derivative of diga-lactosyldiglyceride were detected. Monogalac-tosyl- and digalactosyl-monoglyceride werepresent in small quantities. All galactolipidshad the pyranosyl configuration. A proline-con-taining lipid without phosphorus was also de-tected.

Analysis of fatty acid composition of L.bifidus var. pennsylvanicus (B. bifidum) wasmade on different lipid fractions isolated fromcells grown with or without human milk (173).No large differences were found in the fatty acidpattern between total, membrane, and cyto-plasmic lipids and glyco- and phospholipids.Major constituents were the normal even-num-bered saturated and monoenoic acids. The per-centages of lactic acid and branched fatty acidswere low. Fatty acid composition ofBifidobacterium and Lactobacillus was alsocompared (174).

Phospholipid composition of 10 strains ofbifidobacteria of human intestinal origin, twostrains of B. bifidum (as well as eight otherstrains of bifidobacteria), and nine strains ofLactobacillus were compared by Exterkate etal. (32). Included in the study were one straineach of B. asteroides, B. coryneforme, and B.indicum from honey bees and two strains frombovine rumen liquor, B. ruminale and B.globosum. Diphosphatidyl glycerol and phos-phatidyl glycerol were prsent in strains of bothgenera. All. Bifidobacterium strains containedas specific phospholipids a new polyglycerolphospholipid and its lyso derivatives earlierdetected in L. bifidus var. pennsylvanicus (33).Also lyso compounds of diphosphatidyl glycerol

and alanyl phosphatidyl glycerol were onlypresent in this genus in variable amounts. Lysylphosphatidyl glycerol was the only ninhydrin-positive phospholipid in seven Lactobacillusstrains. In L. delbrucki and L. helveticus it wasabsent and partially replaced by an unidenti-fied ninhydrin-negative phospholipid. The dif-ferences in phospholipid composition betweenbifidobacteria and lactobacilli, especially in thepolyglycerol phospholipids and the aminoacylphosphatidyl glycerol, may, therefore, beanother means of differentiating these two gen-era.

CLASSIFICATIONHistorically the identification of members of

the genus Bifidobacterium in the literature isoften difficult because many unrelated gram-positive rods, branched or unbranched, withoutspores, were assigned to the genus Bacillus andlater to the genus Lactobacillus. During the firsthalf of the present century, interest was cen-tered on those organisms found in man whichwere designated L. bifidus and L. acidophilus.Less attention was paid to the possible occur-rence of this genus or of related organisms else-where in nature until recent years. For thisreason, the historical review is divided into twoperiods: from 1899 to 1957 and from 1957 to thepresent.

First Period

The distinction between B. bifidus communisor B. bifidus and B. acidophilus, first describedand agreed to by Tissier and Moro (166, 167,105, 106), was generally accepted, but close bio-logical relationship between these organismswas also believed to exist. However, B. acido-philus was not assigned to the genus Lactobacil-lus, first characterized by Beijerinck in 1901 (4),unit 1920 when Holland (68) did so. The firstedition of Bergey's Manual of DeterminativeBacteriology (8) also classified the organism asLactobacillus acidophilus, and from that time onthis designation was in general use. The desig-nation B. bifidus, meanwhile, was not changedfor some years. The designation was changed tothat of Bacteroids bifidus in the third edition ofthe Manual of Tropical Medicine (1919) byCastellani and Chalmers (17), and this designa-tion was also the one given in the first throughthe fourth editions of Bergey's Manual (6-10).In the fifth edition (7) the organism was classi-fied as L. bifidus. Placing the organism in thisgenus was based on morphological grounds be-cause it was a gram-positive nonsporeformingbacillus and because its biochemical character-istics were consistent with those of the genus

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BIOLOGY OF, THE BIFIDOBACTERIA

Lactobacillus. In the United States, therefore,L. bifidus was the generally accepted designa-tion which, however, was not so widely acceptedin Europe. Orla-Jensen (116) in 1924 proposedthat this organism be placed in a new genus

Bifidobacterium. However, the designationBifidobacterium was not generally accepted. Inthe European literature the terms Bacillus or

Bacteroides bifidus, as well as L. bifidus, con-

tinued to be employed. Cruickshank (20) in1925 reported serological similarities between L.bifidus and L. acidophilus. These similaritieswere also noted a few years later by Weiss andRettger (188). Lehmann and Neumann (88)studied the organism in 1927 and classified it as

Bacterium bifidum. Two years later the name

Tissieria bifida was proposed by Pribram (129).Beginning in 1930, investigators began noting

the relationship of this organism to Ac-tinomyces. Vuillemin (182) in 1931 called theorganism Nocardia bifida; Nannizzi (109) latercalled it Actinomyces bifidus. Puntoni (130,131) maintained that L. bifidus and A. boviswere the same organism and presented morpho-logical observations, as well as serological stud-ies, to confirm this relationship.

In 1933-1934, Weiss (187) and Weiss andRettger (188, 190) studied various strains ofbifidobacteria isolated from human feces as wellas from other sources. They concluded that L.bifidus and L. acidophilus showed similar mor-

phological and cultural characteristics and thatfermentation patterns were not significantlydifferent. They also found that serological dif-ferences between the two organisms were notgreat. They concluded that L. bifidus was a

variant of L. acidophilus and should be classi-fied in the genus Lactobacillus.

In the following year (1935), Eggerth (31)published his work on the gram-positive non-

sporeforming anaerobic bacilli in human feces.He was one of the first investigators to realizethat organisms, isolated from human subjectsand classified as Bacteroides bifidus, were nothomogeneous. After studying isolates from 85stools of adults and breast-fed infants andtesting them for their ability to ferment 12carbohydrates, he concluded that these isolatescould be divided into two major groups, basedmainly on the fermentation of four key carbohy-drates. These were arabinose, xylose, melezi-tose, and mannose. On the basis of thesereactions he classified all organisms as Bacillusbifidus group I or II. All organisms isolated frominfants were group I, whereas most isolates fromadult stools were group II. Eggerth also ex-

pressed the opinion that these organisms shouldbe placed either in the genus Lactobacillus or in

a separate genus, possibly in the orderActinomycetales, but not in the genusBacteroides. The name Actinobacteriumbifidum was proposed by Puntoni (131) in 1937.Serological relationships between Bacillusbifidus and L. acidophilus were reinvestigatedin the years 1936-1939 by Blaurock (11, 12) andBoventer (15). These workers failed to demon-strate the serological relationship between thesetwo organisms, which had been reported byearlier investigators (20, 188).The Orla-Jensens and Winther (118) pub-

lished information which was not in agreementwith the results of Weiss and Rettger (187, 188).The former authors considered B. bifidus abranched organism and L. acidophilus andunbranched one and believed the former shouldbe classified as an Actinomycetales.

In 1938 Weiss and Rettger (189), havingstudied several of the Orla-Jensen strains, con-cluded that the organisms were unlike either L.acidophilus or B. bifidus isolated from nurs-lings' stools and were similar to B. bifidus, groupII, of Eggerth (31). Weiss and Rettger were stillconvinced that their L. bifidus was a variant ofL. acidophilus and was not identical with theorganism originally isolated by Tissier. Thelatter was therefore considered to be the "trueLactobacillus bifidus." Weiss and Rettger pro-posed that, since there appeared to be at leasttwo types of L. bifidus, the organisms should beclassified as L. bifidus, type I, which usuallybecomes aerobic and unbranched after primaryisolation, and L. bifidus, type II, or L.parabifidus, which usually remains anaerobicand branched. Several workers accepted theterm L. parabifidus, as described by Weiss andRettger, and in the sixth edition of Bergey'sManual of Determinative Bacteriology (16)both L. bifidus and L. parabifidus are listed.Also in 1938, Prevot (126) placed what he calledBifidobacterium bifidum (Tissier) Orla-Jensenin the family Actinomycetaceae, but in a genusdifferent from A. bovis. In 1940 Lewis andRettger (90) agreed with the conclusion ofOrla-Jensen et al. (118) that at least somestrains of L. bifidus were related to the ac-tinomycetes. However, in 1942 King and Rett-ger (79) reaffirmed the position of Weiss andRettger (189) that these organisms should beclassified as lactobacilli. Olsen (114) in 1949reviewed the literature and added his own obser-vations. He disagreed with classifying this orga-nism as Bacterium bifidum and believed thatthe organism should be classified in the genusCorynebacterium and should be called C.bifidum. In 1944 Negroni and Fisher (110)confirmed the relationship of the organism with

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the actinomyces and placed it in the genusCohnistreptothrix (C. bifidus), which was alsothe genus in which they classified A. bovis.

Norris et al. (112) in 1950 studied the mor-phology of bifidobacteria with a culture me-dium designed for this purpose. These workersdisagreed with Weiss and Rettger (189) in theuse of the term L. parabifidus to describe thebranched anaerobic form and L. bifidus todescribe a type that became unbranched andaerobic or microaerophilic. These workers pro-posed that the straight-rod type was a variant ofthe anaerobic type and that the term L. bifidusshould be used for the branched form, whereasthe term L. parabifidus should be used for theunbranched variant. Therefore L. parabifidus,as used by Weiss and Rettger, describes abranched organism and the term, as used byNorris et al., describes an unbranched bacillus.In 1953 Williams et al. (195) studied the anti-genic relationships of L. bifidus and L.parabifidus, as defined by Norris et al. (112),and included some strains of L. acidophilus.These authors found it difficult to prepare asatisfactory antiserum for L. parabifidus, butdid find a distinct antigenic component of theorganism and noted much serological variationamong most of the strains tested. They found noserological relationships between L. acidophiluswith L. bifidus or L. parabifidus, and theyconcluded that serologically there was justifica-tion for use of the three separate species desig-nations. In 1954 Gy6rgy et al. (54) described avariant of L. bifidus that required the additionof human milk to the medium to maintain theorganism after primary isolation. Gybrgy et al.(50) originally designated the organism L.bifidus var. penn. Because this designation wasnot acceptable by taxonomic standards, thedesignation was subsequently changed to L.bifidus var. pennsylvanicus (55). Morpholo-gically L. bifidus and L. bifidus var.pennsylvanicus were indistinguishable. It wasnoted that the milk-dependent strain couldconvert to the regular L. bifidus type, butattempts to make the regular L. bifidus milkdependent failed. These authors suggested thepossibility that L. bifidus var. pennsylvanicuscould become the regular L. bifidus, which inturn could become the unbranched L.parabifidus.

Frank and Skinner (35) restudied the mor-phological relationship of L. bifidus and A.bovis. They concluded that, based on severalmorphological pecularities such as primitivebranching and similar appearance on gram-stained preparations when grown on severalmedia, these two organisms were similar and

were related at least at the genus level. Theythought that the evidence justified designatingthe organism Actinomyces bifidus.

Pine and Howell (124), in an attempt to moreclearly define the relationship of the bifidobac-teria to the Actinomyces, compared the bio-chemical and physiological characteristics ofseveral Actinomyces species with four isolates ofbifidobacteria. They found the sugar of choicefor growth of their Actinomyces species to beglucose or maltose, whereas lactose or maltoseproduced optimal growth of the bifidobacteria.None of the bifidobacteria strains reduced ni-trate to nitrate, whereas the Actinomyces didpossess this ability. It was also noted that thebifidobacteria fermented 50 to 89% of a 1%glucose medium, but the Actinomyces fer-mented an average of 34 to 59% of the glucose.Both groups of organisms produced the samefermentation products from glucose (lactic,acetic, formic, and succinic acids). Althoughthe Actinomyces strains predominantly formedlactic acid with small amounts of acetic, formic,and succinic acids, the strains of bifidobacteriaformed approximately equal amounts of lacticand acetic acids. The authors concluded thatthe last point suggested a major metabolicdifference between Actinomyces and the bifido-bacteria.

In evaluating the taxonomy of L. bifidus in1955, Gyllenberg (45) stated that from thedefinition of the variant concept outlined bySmith et al. (157) the designation of L.parabifidus, proposed by Norris et al. (112) andWeiss and Rettger (189), is invalid because ofthe variability of the parent strain. Gyllenbergalso noted that, unlike L. parabifidus, L. bifidusvar. pennsylvanicus could be considered a truevariant of L. bifidus and fulfilled the require-ments of Smith et al. (157) for the variantconcept.To summarize, it was first thought that L.

bifidus and L. acidophilus were closely enoughrelated to justify their inclusion in the samegenus. During much of the first period, tech-nology was comparatively primitive, and meth-ods of analysis were generally limited to the useof morphological observations and simple fer-mentation patterns. The results of studies bythese methods did not reveal morphological andbiochemical characteristics which were so dis-tinctive as to convince workers that L. bifidusbelonged in one genus or another. In the UnitedStates, for lack of definitive criteria, however,the organisms were regarded by the majority ofworkers as belonging to the genus Lactobacillus.In chronological order the various proposedgeneric designations of the organisms are as

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follows: Bacillus bifidus communis, Bacillusbifidus, Bacteroides bifidus, Lactobacillus bifi-dus, Bifidobacterium bifidum, Bacterium bifi-dum, Tissieria bifida, Actinomyces befidus,Actinobacterium bifidum, Corynebacterium bi-fidum, and Cohnistreptothrix bifidus. Althoughthe genus Bifidobacterium was originally pro-posed by Orla-Jensen (116) in 1924, it has onlyrecently gained general acceptance. The genusBifidobacterium will be recognized as an inde-pendent genus in the eighth edition of Bergey'sManual of Determinative Bacteriology.

Second PeriodPrior to 1957 L. bifidus var. pennsylvanicus

was the only generally accepted additional spe-cies or variant of bifidobacteria. In 1957 Deh-nert (25) presented a scheme for the differentia-tion of five groups of bifidobacteria basedmainly on the fermentation of 24 carbohydrates.Species were not designated, but the variousgroups were assigned a number. L. bifidus var.pennsylvanicus was placed in Dehnert's groupII. In an effort to clarify the relationship of thebifidobacteria, recently proposed taxonomicschema are tabulated in Table 2. For complete-ness Eggerth's groups I and II are also listed,although the relationship to them of recentlydescribed species and types is often uncertain.It is seen that Eggerth's group I includes Deh-nert's groups I through IV, and Eggerth's groupII corresponds with Dehnert's group V. Thework initiated by Dehnert was the first torecognize the existence of multiple biotypes ofBifidobacterium and was the basis of laterstudies which eventually led to the recognitionof species and subspecies. In the same yearCummins et al. (22) examined the cell wallcomposition of several strains of bifidobacteriaand concluded that these organisms differedfrom all previously tested gram-positive orga-nisms. The taxonomic classification of bifido-bacteria was therefore open to question and thesubject of renewed investigation.Sundman et al. (162) in a comparative study

of several organisms concluded that the bifido-bacteria in general were most closely related toButyribacterium. However, they believed thatL. bifidus var. pennsylvanicus closely resem-bled an Actinomyces. In 1961 Slack et al. (155)compared the serological grouping of bifidobac-teria with Actinomyces and Corynebacteria, byusing fluorescent antibody techniques, andfound no relationship between the bifidobac-teria and the other organisms. Lerche andReuter (89) in the same year studied morpho-logical and biochemical variants of the anaero-

bic lactobacilli and divided them into fourgroups, one with four variants. However, noattempt was made to designate species.

In 1963 Reuter (132) did biochemical andserological tests on isolates from 136 stools of 38infants and compared his results with those ofDehnert (25, 27). From a study of these strainsand of strains previously isolated from adults,Reuter devised the following scheme for iden-tification. If a gram-positive anaerobic bacillusresembled lactobacilli except for morphologicvariability, then the character of acids producedfrom glucose was examined. If the ratio of lacticto acetic acid was two to one, the strain wastested for its ability to ferment 11 additionalcarbohydrates. On this basis all strains wereplaced into one of ten groups (Table 1). Group Ihad four biotypes; groups II and III had two;and the remaining groups had no additionalbiotypes. After evaluating his data, he con-cluded that these organisms should be classifiedwithin the tribe Lactobacilleae, of the familyLactobacillaceae, and in the genus Bifidobac-terium. He concluded that the genusBifidobacterium should be divided into eightspecies and several species variants. He recog-nized the following species of Bifidobacterium:bifidum a and b, infantis, pavulorum a and b,breve a and b, lactentis, adolescentis a, b, c,and d, and longum a and b. The classification ofDehnert (25) was thus expanded by Reuter(132) with the designation of eight species and anumber of variants on the basis of carbohydratefermentation. This precedent of designatingspecies on the basis of carbohydrate fermenta-tion was to lead to recognition of additionalspecies and to increased reliance on the patternof carbohydrate fermentation for species desig-nation. Many of these strains were character-ized and deposited in the American Type Cul-ture Collection by Reuter (133).Between the years 1965 and 1969 biochemical

studies were expanded to include the pathwaysof carbohydrate fermentation. Scardovi andTrovatelli (147) and de Vries et al. (178, 180)discovered a new pathway for the fermentationof hexoses in bifidobacteria, previously referredto, which does not occur in any species of thegenus Lactobacillus. Veerkamp (17) demon-strated a similar pathway in L. bifidus var.pennsylvanicus. Many of the earlier classifica-tion schemes were reexamined by the originalauthors (28).

Also during these years, a study of the DNAbase composition by Sebald et al. (153) andGasser and Mandel (37) confirmed the earlierwork of Werner and Seeliger (192) that mem-

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TABLE 2. Comparison of the major classification schemes of the genus Bifidobacteriuma

Eggerth, Dehnert, R Scardovi et al., Holdeman and Moore,1935 1957 ete,193-64 Misok,1969 1971 11972(31) (25) j 12 1)(150) j(67)

I

II

I, II

III

IV

III

V

a SS, Subspecies.

bifidum a, IIIabifidum b, IIIbinfantis V

liberorum IXlactentis Xbreve a, VIIbreve b, VIII

parvulorum a, IVparvulorum b, VI

adolescentis Iaadolescentis Ibadolescentis Icadolescentis Idlongum alongum blongum Ilalongum IIb

bifidum abifidum binfantis ainfantis bliberorumlactentisbreve abreve bbreve cparvulorum aparvulorum b

adolescentis aadolescentis badolescentis cadolescentis dlongum var. longum alongum var. longum blongum var. animalis alongum var. animalis bpseudolongum apseudolongum bpseudolongum cpseudolongum dthermophilum athermophilum bthermophilum cthermophilum d

bifidum

infantis

breve

adolescentis

longum SS

pseudolongum

thermophilumruminale

asteroides"dentium""angulatum""catenulatum"suisglobosumindicumcoryneforme

bifidum

infantis SS infantisinfantis SS liberoruminfantis SS lactentisinfantis-otherbreve

adolescentis Aadolescentis Badolescentis Cadolescentis Dlongum SS longum

pseudolongum

thermophilum

asteroides"dentium"

cornutumeriksonii

bers of the genus Bifidobacterium were nothomogeneous with members of the genus Lac-tobacillus or other related genera. Exterkateand Veerkamp (33) noted that bifidobacteriaand lactobacilli also differed in their phos-pholipid composition. Snyder et al. (158) reex-amined the serological relationships of bifido-bacteria and members of Actinomyces andNocardia together with other related genera.They confirmed the earlier observations ofSlack et al. (155) that these organisms are notserologically related to the Actinomyces, Co-rynebacterium, or any other of the gram-posi-tive filamentous bacteria tested. Bifidobac-terium thus appears to be a serological entity.Werner (191) and Werner and Seeliger (193)

confirmed and simplified Dehnert's classifica-tion by singling our five carbohydrates as beingsufficient for identification of the five groups:arabinose, xylose, mannose, mannitol, andsorbitol.The next major classification scheme was

presented by Mitsuoka (100) in 1969. Fourhundred and eighty-three strains of bifidobac-teria, isolated from human as well as nonhumansources, were studied and classified on the basisof physiological and biochemical characteristics(Table 2). Mitsuoka also compared his resultswith those obtained by Reuter (132). The spe-cies designations originated by Reuter wereretained by Mitsuoka, who increased the num-ber of variants. In addition, he described two

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new species: B. pseudolongum and B. thermo-philum. It was noted in this study that nonhu-man were clearly differentiated from humanstrains by their temperature requirements (abil-ity to grow at 46.5 C) and carbohydrate fermen-tation patterns. As a consequence, the 15 spe-cies and variants given by Reuter were in-creased to 27. Unlike the three previously men-tioned classifications, the Mitsuoka schemeincluded animal strains.Pine (123) in 1970, evaluating the serological

relationships of the family Actinomycetaceae,agreed with Slack et al. (155) and others that nosignificant cross-reactions exist between bifido-bacteria and any of the other organisms tested.In this same year Koch et al. (81) studied themurein composition of the cell walls of B.bifidum and demonstrated a particular type ofmurein (Orn-Ser-Asp type) which was typicalfor B. bifidum and which was not found in anyother species of Bifidobacterium.By a different approach Kandler (76) in 1970

studied the murein types in the cell walls ofBifidobacterium and found agreement with thespecies proposed by Reuter (132). Kandler sug-gested, therefore, that murein characterizationmay be a valuable criterion for the separation ofspecies within the genus Bifidobacterium. Dif-fering phospholipid composition of severalstrains of Bifidobacterium led Exterkate et al.(32) to propose that these differences can alsobe employed in classifying members of genusBifidobacterium.As can be seen from the schemes presented so

far, although other factors are taken into con-sideration, the use of carbohydrate fermenta-tion patterns remains the main basis for speciesdesignation. Scardovi et al. (150) noted thetaxonomic uncertainties created by such a sys-tem and noted that further separation of thespecies into subspecies or biotypes would onlyadd to the confusion. In a previous reportScardovi et al. (146) described three types ofelectrophoretic behavior of fructose-6-phos-phate phosphoketolase in various strains ofBifidobacterium. In this study it was found thatthe R. of the enzyme did not correspond withthe name of the species, but with the host of theorganism. This observation suggested divergentevolutionary pathways in varying habitats ofBifidobacterium. In addition, by DNA-DNAhybridization competition studies, Scardovi etal. (145, 151) and Crociani et al. (19) reportedthe genetic relatedness among some proposedspecies of Bifidobacterium from the rumen ofcattle and the intestines of honey bees. Scardoviet al. (150) in 1971 presented a summary chartof the recognized species and biotypes of

Bifidobacterium along with the biochemicaland physiological characteristics of each type.This chart included all the species and biotypesmentioned so far, and the following additionalspecies were included: B. asteroides, B. suis, B.ruminale, B. globosum, B. indicum, and B.coryneforme, for a total of 34 species andbiotypes. The additional species were of nonhu-man origin. B. ruminale and B. globosum werefirst described by Scardovi et al. (149) in 1969and were isolated from the bovine rumen. B.asteroides, B. coryneforme, and B. indicumfrom the alimentary tract of honey bees werefirst described by Scardovi and Trovatelli (148)and were possibly related to some of the orga-nisms described earlier by White (194). B. suiswas isolated by Matteuzzi et al. (96) in 1971from the feces of pigs. Scardovi et al. (150)noted that B. bifidum had a distinct fermenta-tion pattern in contrast to other species whichhad patterns resembling each other. They stud-ied DNA homology relationships of 179 strainsof Bifidobacterium isolated from man, bovinerumen, and other animal sources. These work-ers found all of the strains of B. bifidum to behomologous and also found B. bifidum and B.bifidum var. pennsylvanicus to be homologouswith each other. Little similarity was foundbetween B. bifidum and other species of Bifido-bacterium. The authors expressed the opinionthat the validity of this species is well estab-lished. They proposed also that B. infantis, B.liberorum, and B. lactentis be merged into thesingle species B. infantis on the basis of theirhomology studies and their evaluation of thefermentation patterns of these three species.Likewise, they suggested that B. breve and B.parvulorum be merged into a single species, B.breve. They also found B. rumenale and B.thermophilum to be similar. They noted simi-larities between B. Pseudolongum and B. globo-sum, but they did not feel that they had enoughevidence to recommend that these species bemerged. Several of the previous biotypes as-sociated with these species were eliminatedfrom their classification (Table 2). The possibil-ity of the existence of three additional species,"dentium," "catenulatum," and "angulatum,"was noted, but the authors thought that moreevidence was needed to establish species statusof these organisms.DNA homology patterns, as employed by

Scardovi et al. (150), appear to aid materially inidentifying species, This methodology is a sig-nificant advance in determinative bacteriologyand should help resolve much of the confusionpreviously encountered when species differen-tiation of Bifidobacterium was made principally

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on carbohydrate fermentation patterns.The latest taxonomic scheme for the genus

Bifidobacterium is presented in the 1972 editionof the Anaerobic Laboratory Manual by Holde-man and Moore (67). This scheme is basedmainly on the proposals made by Scardovi et al.(150) with the following modifications. B.infantis is divided into three subspecies:subspecies infantis, subspecies liberorum, andsubspecies lactentis. In addition, a fourth cate-gory, "B. infantis-other," is included to giverecognition to subspecies intermediate to theirrecognized subspecies. B. adolescentis is di-vided in groups A, B, C, and D on the basis ofthe work of Reuter (132). In this scheme, severalnonhuman species designated by Scardovi et al.(150) are not included (Table 2). The species"dentium" is included as a separate category.In addition to the species previously mentioned,two additional species are recognized: B.cornutum and B. eriksonii.Bifidobacterium cornutum was first de-

scribed by Distaso (29) in 1912 as Bacilluscornutus. This was apparently the same orga-nism described 7 years later by Casstellani andChalmers (17) as Bacteroides corntus. Prevot(126) in 1938 was the first to classify thisorganism as a Bifidobacterium and assigned thename B. cornutum. This organism is found inthe mouth and intestines of man and is fre-quently found in the horse. B. eriksonii was firstdescribed by Georg et al. (38) in 1964. Theseworkers isolated two organisms, one fromhuman pleural fluid and the other from a lungabscess, and designated them as strains X407and X573. The following year Georg et al. (39)designated these organisms Actinomyces eriko-sonii. Holdeman and Moore (67) placed thisorganism in the genus Bifidobacterium in 1972.

In summary, the major classification schemeshave been presented here and summarized intable form. It can be seen from Table 2 that theinitial designations presented by Eggerth (31)and Dehnert (25) consisted of a number nota-tion system only. The Dehnert classificationwas the first to divide the group of bifidobac-teria into several groups on the basis of carbohy-drate fermentation. Reuter (132) expanded thisclassification and gave actual species designa-tion to many of the organisms. A period ofnaming additional species followed until thework of Scardovi et al. (150), in which DNAhomology studies supported the elimination ofsome of these designations and made one of thefirst serious attempts to place the taxonomicclassification of the bifidobacteria on a moreconcrete basis. This was followed by the latestscheme presented by Holdeman and Moore (67)

which demonstrates the trend of decreasing thenumber of species. It is our opinion that thework of Scardovi is a significant advance in themethodology for the classification of bifidobac-teria and that no new species should be ac-cepted solely on the basis of a unique carbohy-drate fermentation pattern.

IdentificationDefinitive identification of Bifidobacterium

in the routine laboratory is difficult to accom-plish. Although the usual biochemical and mor-phological methods can be employed for atentative identification, the use of a methodthat identifies the fermentation product is nec-essary to render a final identification. Themethod for differentiating Bifidobacteriumfrom other anaerobes, as described in the Ana-erobic Laboratory Manual (67), is currently themost complete method available. The methodrelies on a gas chromatography analysis of thefermentation products of glucose for a finalidentification, which limits its use to laborato-ries equipped with this technique. However,laboratories without this technique can stillobtain reliable information by depending onmorphology and the usual biochemical meth-ods. It is apparent that members of the genusBifidobacterium can be confused with severalrelated organisms. On primary isolation or insmears of feces the organism cannot always bedifferentiated from members of the Ac-tinomyces, Corynebacterium, Lactobacillus,Propionibacterium, Eubacterium, Butyribac-terium, Ramibacterium, Cillobacterium, andClostridium. However, when the organisms aresubcultured onto media, such as the one de-scribed by Norris et al. (112), bifidobacteriademonstrate typical bifid morphology whichenables the experienced observer to eliminatealmost all of the related gram-positive anaero-bic organisms. Most strains of Bifidobacteriumproduce easily distinguishable porcelain whitecolonies on this medium which further aids intheir differentiation from related genera. Al-most all of the biochemical tests used for thedifferentiation of Bifidobacterium from relatedorganism are negative or nonreactive. Until awider spectrum of biochemical characteristics isdevised, the identification of Bifidobacteriumwill remain difficult. One of the more practicalapproaches to a tentative differentiation ofbifidobacteria from related anaerobic gram-positive nonsporeforming bacilli is the schemepresented by Moore and Holdeman (104). Thismethod relies on a morphological scheme ini-tially limited to the possibilities within the

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group of organisms mentioned above. Theseauthors place Bifidobacterium in the samemorphological group as Eubacterium, Lac-tobacillus, and Cillobacterium. Members ofClostridium are eliminated by testing for leci-thinase activity. Lack of catalase activity dif-ferentiates Bifidobacterium from Corynebac-terium and Propiniobacterium. The differentia-tion among members of the actinomyces is moredifficult but can be made in general by thethicker cell wall and more rapid growth ofBifidobacterium. By employing methods suchas these, one can usually identify the typicalBifidobacterium and rely on reference labora-tories for the final identification of the moreunusual strains that may be encountered. Itis hoped that in the future more definitive andpractical biochemical methods will be developedto make possible an accurate identification ofmembers of the genus Bifidobacterium.

ECOLOGY

OccurrenceSoon after Tissier described B. bifidus in the

stools of breast-fed infants, it was recognizedthat the organisms became predominant in thestools between days 4 and 7 after birth. Al-though L. acidophilus was the most numerousorganism in the stools of bottle-fed infants,bifidobacteria were also found in smaller num-bers. It was believed that bifidobacteria werepresent in small numbers in stools of adults, butthey were difficult to isolate because of the lackof a suitable culture medium or method ofreducing oxygen tension. For the next halfcentury little progress was made in extendingthese observations. When more selective culturemedia became available, interest in the occur-rence and distribution of bifidobacteria wasrevived.Mata et al. (93) reported the average figures

for the incidence of bifidobacteria in the feces ofbreast-fed infants to be approximately 1011, inweanlings 1010, and in adults 10' organisms perg (wet weight). The figures expressed by theseauthors are in general agreement with thoseexpressed earlier by Gyllenberg and Roine (49),Smith and Crabb (156), Zubrzycki and Spauld-ing (202), Weijers and van de Kamer (186), andWerner (191). They are somewhat higher thanthose reported by Kalser et al. (74), Werner andSeeliger (192), Mossel (107), and Gorbach et al.(43). In a study of intestinal flora in a rural areaof Guatemala, Mata and Urrutia (94) reportedthat bifidobacteria appeared on the first day oflife in only a few infants. One-third of the babiesthey studied had these bacteria on the second

day of life. By the end of the first week allinfants had them in concentrations rangingfrom 101 to 1011 organisms per g of feces. Theseworkers also reported that Bacteroides andVeillonellae were not frequently found in thestools of breast-fed neonates, although theirconcentration ranged from 108 to 1011 whenfound. When 12 breast-fed infants were studiedthroughout the first year of life, bifidobacteriacontinued to be the most numerous bacterium,amounting to 1010 to 1011 organisms per g offeces. With food supplementation the anaerobicgram-negative bacilli became more numerousand eventually outnumbered other bacterialgroups. Mata and Urrutia (94) summarizedtheir data by stating that nearly 100%6 of allbacteria cultured from the stools of breast-fedinfants were bifidobacteria. During weaningthere was a decrease by 1 log and a proliferationof Bacteroides. In adults, Bacteroides outnum-bered all other groups. Although this study wasa valuable aid in estimating the relative occur-rence of various groups of intestinal organismsand their variation with age and other factors,no attempt was made at a finer classification ofmembers of the genus Bifidobacterium. Theseorganisms were simply referred to as the bifido-bacteria group. There is a definite need for suchinformation based upon one of the recent clas-sification schemes.

Miller (99) reported on the fecal flora of sevenEskimo children. He reported B. adolescentis inconcentration of 108 to 1011 per g in all thespecimens studied and noted that in one casemembers of the genus Bifidobacterium ex-ceeded those of the Bacteroides group. In astudy of the intestinal flora of adults, Moore etal. (103) reported that in some cases members ofBifidobacterium outnumbered Bacteroides,thus confirming the observations of some earlierworkers on this point. Significant differences inincidence of bifidobacteria among individualsand possibly among certain restricted popula-tions are evident.An investigation of variation in the incidence

of bifidobacteria among newborns was carriedout in our laboratory (unpublished data). In thecourse of investigations, we noted fewer bifido-bacteria in newborns in a large, urban univer-sity hospital than in a suburban hospital in thesame area. In the past, bifidobacteria couldreadily be cultured from the stools of breast-fedinfants at the Hospital of the University ofPennsylvania. Recently, difficulty was ex-perienced in culturing these organisms from thestools of infants in the nursery. Gram-stainedspreads of feces of breast-fed infants, 3 to 4 daysold, were examined. Of 61 breast-fed infants

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studied, only 20% had a significant number ofgram-positive nonsporeforming bacilli. A simi-lar study was conducted at a suburban hospital.By contrast, two-thirds of 21 breast-fed babieshad significant quantities of these organisms.An explanation has not been found, althoughsome unidentified environmental factor is prob-ably responsible for the difference in incidence.Experiments such as these indicate that, al-though there is general agreement on the overalloccurrence of the bifidobacteria, there may besignificant variation among various groups

within a particular population.Dehnert (25, 27) reported that one biochemi-

cal type, Dehnert's group IV (B. infantis), pre-

dominates in the stools of breast-fed infants.Seeliger and Werner (154) reported that Deh-nert's group III (a subspecies of B. infantis) was

predominant in Bonn (West Germany), al-though group IV was also present. Petuely andLinder (121) noted the predominance of Deh-nert's group IV in the stools of breast-fed in-fants. Werner (191) also reported the prevalenceof Dehnert's group IV in breast-fed infants andstated that group IV has been found only in thestools of breast-fed infants or of infants fedmaternal milk together with cow's milk. Heconfirmed the fact that this group has not beenfound in the stools of adults. Werner alsoreported that Dehnert's group I (B. bifidum),III (B. breve), and V (B. adolescentis and B.longum) were found in adult stools and notedthe presence of groups III and V in the humanappendix. Werner concluded that cooperativeresearch in different parts of the world is neededto widen our knowledge about the global ecologyof different species of Bifidobacterium andparticularly of the source and mode of transmis-sion of those in Dehnert's group IV.

Origin and Mode of TransmissionSeveral proposals have been made concerning

the source of initial colonization of the newbornwith bifidobacteria. Some early workers re-

ported the isolation of bifidobacteria from thevagina: Blaurock (13), Roufogalis (141-143),and Montagna and Cataldi (101). Harrison etal. (61) studied the incidence of bifidobacteriain the vaginas of normal nonpregnant women

and in women in the second and third trimes-ters of pregnancy. They noted a significantincrease in incidence of the organism at term.Although these workers found similar culturalcharacteristics between the vaginal strains iso-lated from the stools of breast-fed infants, theywere not successful in conclusively demonstrat-ing the antigenic relationship between these two

groups of organisms. They suggested that theoccurrence of bifidobacteria in the stools ofadults makes fecal contamination a possiblesource, either directly at the time of delivery orindirectly from other parts of the mother's bodyafter birth.

Cataldi and Muller (18) expressed the viewthat colostrum and the skin of the breast wereprobable sources of bifidobacterium, particu-larly for infants born by Caesarean section.They reported the recovery of the organism fromcolostrum in 22% of the cases and also reportedrecovering the organism in 14% of the cases fromthe skin of the maternal breast immediatelyafter delivery. Wyatt and Mata (196) found anaverage of 3 x 10' bacteria per ml in colostrum.The presence of lactobacilli was demonstratedin 17 of 51 samples of colostrum by them, butbifidobacteria were not specifically identified.Stolte (160) doubted that the infant acquiredthe organism by swallowing, because she couldnot demonstrate the presence of bifidobacteriain the mouths of newborn infants nor in themilk or about the breasts of the mother. Shesurmised that the portal of entry was the anus;however, the conclusion rests on the assumptionthat failure to isolate the organism from themouth eliminates swallowing of the organism asa means of colonization.

It should be noted that (i) none of thesestudies has definitely determined the mainsource of colonization of the infant bowel withbifidobacteria; (ii) the identity of the infant'sstrain with that of the mother has not beenestablished; and (iii) these studies were con-ducted prior to the proposal and acceptance ofthe current classification scheme presented inthe previous section. With the use of morerefined methods of isolation and of classifica-tion, it is hoped that a better understanding ofthe pathways of colonization of the infantintestine with bifidobacteria will be obtained inthe future.

Interaction with Other OrganismsIn an attempt to explain the significance of

bifidobacteria in humans, several investigatorsstudied the effects of this organism on othermicroorganisms. One of the first reports wasthat of Rose and Gyorgy (137). These workersreported an inhibitory effect of L. bifidus var.pennsylvanicus (B. bifidum) on other strains ofbifidobacteria and on E. coli. Whether bifido-bacteria in the intestine of the infant canlikewise inhibit other bacteria has not beendetermined.Wagner and Starr (183) employed germfree

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mice in the study of the interaction of bifidobac-teria and Salmonella typhimurium. Colonies ofanimals mono-associated with either S.typhimurium or bifidobacteria in the intestinewere first established. When mice mono-

associated with bifidobacteria were pooled withmice mono-associated with the S. typhimuriumgroup, the former group rapidly picked up theS. typhimurium. A di-associated state was thusestablished. However, there was a precipitousdrop in the bifidobacteria population of thedi-associated mice. In the mice from the origi-nal Salmonella population there was a low-leveluptake of bifidobacteria, but after 14 daysbifidobacteria were no longer recovered fromthese mice. There was no reduction in thenumber of the original Salmonella population.Although these workers were the first to usegermfree animals in studying the interaction ofbifidobacteria with other bacteria in vivo, theirfindings are not conclusive in determining thatbifidobacteria in the colon protect the animalagainst infection from intestinal pathogens.Mata et al. (95) studied Shigella infections in

breast-fed Guatemalan neonates. Of 210 babiesborn in a village, 109 were breast-fed. Only fourof these infants developed Shigella infections, a

rate which was significantly less than the infec-tion rate of the total population. Bifidobacteriawere the predominant organism in the flora ofthe breast-fed babies. Of the four Shigella-infected infants, one was exclusively breast-fedand had a transient and a synptomatic infec-tion. The infection was asymptomatic, butpersistent, in two children receiving food sup-plementation. This infection was associatedwith severe diarrhea. These authors concludedthat the intestinal microflora in breast-fed chil-dren favors resistence to Shigella infection or

elimination of the agent when it is acquired.Mata and Urrutia (94) also studied entero-pathogenic E. coli in breast-fed infants from a

rural area of Guatemala. The authors noted no

infections with enteropathogenic E. coli in thebreast-fed neonates and concluded that thepredominance of bifidobacteria was responsiblefor the low incidence of enteropathogenic E.coli.Haenel (59) noted a reduction or absence of

putrefactive bacteria such as Bacteroides, Veil-lonella, Clostridium, Proteus, and others in thestools of breast-fed infants. He attributed theinhibition of these organisms to unknown prop-erties of human milk as well as to certainenvironmental conditions. The possibility ofbifidobacteria playing an important role ininhibition was not pursued.

The viral inhibitory effect of bifidobacteriahas been given little attention. Mayer (97) haspresented some evidence for the inhibition ofenteric viruses by bifidobacteria. He concludedthat the disappearance of the bifidobacteriafrom the intestine is an indication that theinfant has become more susceptible to entericviral infections. Tassovatz and Kragouyevitch(164) and Damjanovic and Radulovic (24) havealso presented data to support this contention.However, these experiments do not appear to beconclusive. Several experiments were con-ducted in our laboratory (unpublished data) todetermine whether bifidobacteria, or one oftheir metabolites, produce interferon or someother antiviral substance. Monolayers of HeLacells, known to be capable of producing inter-feron, were exposed to filtrates of broth cul-tures, adjusted to pH 7.0, of several strains ofbifidobacteria. After overnight exposure ofthese monolayers to appropriate dilutions of thefiltrates, the monolayers were challenged with adilution of vesicular stomatitis virus (VSV) thatproduces a 50% cytopathogenic effect in 24 h.The experiment was attempted several timeswith VSV and the Mahoney strain of polio virus.No significant reduction in cytopathogenic ef-fect was observed in any of these attempts.

Several attempts were made to demonstrateantiviral properties of a polysaccharide pro-duced by the Jackson mucoid strain of bifido-bacteria. Both purified and crude preparationof this polysaccharide were injected intrave-nously twice a day for 3 days into rabbits. Thesera of these rabbits failed to show any antiviralactivity when tested in the HeLa-VSV systemdescribed above.

In summary, evidence has been presentedthat strains of bifidobacteria may exert aninhibiting effect on one another and on otherbacteria both in vitro and in vivo. The produc-tion of organic acids by the organism increasesthe hydrogen ion concentration in the growthmedium. The increased acidity is probablyinhibitory to some other bacteria. The organicacids may also exert a direct, toxic effect onother bacteria. Gydrgy expressed this conclu-sion in several reports and suggested that fer-mentation products of bifidobacteria, lactate,acetate, and formate, may be directly toxic forother bacteria (51, 52, 55).

Effects of Bifidobacteria on the HostBarbero et al. (3) studied the physiological

effects of the intestinal flora on human infants.These authors confirmed the well-publicizedfact that stools of breast-fed infants are more

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acidic than are the stools of formula-fed infants.Stools of 236 infants fed exclusively eitherhuman milk or cow's milk (or one of its deriva-tives) were studied. Some babies from eachgroup were intubated to study conditions atvarious locations in the intestinal tract. Theduodenum was found to be relatively sterile.Bacteria were found in the ileum, and markedbacterial proliferation was noted in the cecum.The pH was highest in the cecum and tended tofall in the distal portion of the colon. Theaverage pH in the stools of breast-fed infantswas 5.5, and in formula-fed infants the averagewas 6.7. Barbero et al. concluded that low pH inbreast-fed infants was due to the greateramounts of acid produced by bifidobacteria.These authors also found reducing sugars invarying amounts throughout the intestine. Alow pH of the stools appears to be the only effectwhich, at this time, can be attributed to thepresence of large numbers of bifidobacteria inthe colon.Muting et al. (108) induced a bifidobacteria

intestinal flora in 20 patients with liver diseaseand noted a significant decrease in blood am-monia, free serum phenol, and free aminonitrogen. A decrease of free phenols and freeamino acids in the urine was also noted. Theseauthors concluded that bifidobacteria signifi-cantly contributed to these physiologicalchanges and aided in the recovery of the pa-tients.

Gall (36) noted that the intestinal bacteriahave been reported to affect natural resistenceto infection and have been implicated in themetabolism of cancer, serum proteins, choles-terol, hormones, vitamins, and the incidence ofcaries. Her study of the anaerobic intestinalflora indicated that (i) many of the majorprocesses associated with digestion, includingthe metabolism of certain carbohydrates, fats,and proteins, and the production of certain Bvitamins, can be carried out by these orga-nisms; (ii) that symbiotic relationships may beimportant in maintaining the normal balance ofmicroflora in the gut; and (iii) that organismsmay exert a deleterious effect on the host by theproduction of cholesterol or by the formation oftoxic amines by the decarboxylation of certainamino acids.Savage (144), in summarizing the recent infor-

mation on colonization of the gastrointestinalmucosa by bacteria, concluded that interactionbetween bacteria and the mucosa may be im-portant in mammalian physiology and in theresistence of animals to certain infectious dis-eases. Donaldson (30) and Smith and Crabb(156) have made preliminary studies on the

physiology and development of the intestinalflora in man and animals, but there has been nospecific study of this nature on bifidobacteria.

It is evident from this discussion that thesignificance of bifidobacteria in the intestinaltract of man has not been defined. It appearsthat techniques with gnotobiotic animals, suchas those employed by Syed et al. (163) andGibbons et al. (40), will be useful in studyingthe physiological effects of bifidobacteria on thehost. Difficulties in establishing pure cultures ofbificobacteria and other anaerobes in the intes-tine were experienced by these authors, how-ever, and have not yet been resolved. Studies onthe effects of the bifidobacteria on the hostoutside of the intestinal tract has received littleattention. Harrison and Hansen (60) have pre-sented the possibility of some strains of bifido-bacteria as secondary invaders in livers ofturkeys, and B. eriksonii has been isolated frompleural fluid and a lung abscess (38). Thus, theorganism appears to have possible pathogenicsignificance when isolated from sources otherthan the intestinal tract.

CONCLUDING REMARKSThe occurrence of bifidobacteria as the pre-

dominant organism in the stools of breast-fedinfants was intriguing to investigators who haddifficulting in isolating and maintaining theorganisms in the laboratory. Effects at classifi-cation were impeded until the availability ofnutritionally more appropriate culture mediapermitted more detailed studies. At present, theacceptance of the distinct genusBifidobacterium appears to be justifiable onmorphological, biochemical, and serologicalgrounds. A number of species in this genus arenow recognized, and it seems likely that morewill be identified in the future.The peculiar, if not unique, pleomorphic

features of bifidobacteria have been studied buthave not been adequately explained on bio-chemical grounds. Structural features seen bylight microscopy have not been examined exten-sively by electron microscopy. Future research,it is hoped, will elucidate the biochemicalpathways which are responsible, on the onehand, for keeping the organism in the curved-rod bifid phase and, on the other hand, formaintaining it in the compound-branchingphase. The relationship of branching to a possi-ble life cycle in the organism and to adaptationto an unfavorable environment needs furtherstudy. An understanding of the mechanism ofbranching in bifidobacteria might aid in eluci-dating branching in other microorganisms andalso in higher forms of life.

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The ecological significance of bifidobacteriain infants has attracted attention, but in spiteof considerable research the organisms have notbeen shown to elaborate antimicrobial sub-stances. It is possible, in the light of empiricalobservations on the better health of breast-fedinfants, that bifidobacterium aids in the betternutrition of infants and, thus, indirectly con-tributes to a greater resistance to infection. Asbetter techniques for study are devised, moredefinitive information on the importance of thebifidobacteria as part of the intestinal flora willbe obtained. Now that it appears that bifido-bacteria-like organisms occur elsewhere in na-ture, more research on related organisms shouldprove to be scientifically productive and shouldexpand knowledge of the ecological significanceof this organism in nature.

ACKNOWLEDGMENTSWe express our appreciation to Mollie Gallagher for

her assistance in compiling the manuscript, to W. G.Hutchinson (Department of Biology, University ofPennsylvania) and Josef Weibel for preparing theelectron micrographs, to M. L. Higgins (TempleUniversity School of Medicine) for advice in methodsof fixation and embedding, and to Ruth Wright fortyping the manuscript.

LITERATURE CITED1. Bailey, R. W., and R. T. J. Clarke. 1959. A

bacterial dextranase. Biochemistry 72:49-54.2. Bailey, R. W., and A. M. Roberton. 1962. Car-

bohydrases of a rumen strain of Lactobacillusbifidus. w.;The intracellular a-1i 6-glucosi-dase (isomaltodextrinase). Biochem. J.82:272-277.

3. Barbero, G. J., G. Runge, D. Fischer, M. N.Crawford, F. E. Torres, and P. Gybrgy. 1952.Investigations on the bacterial flora, pH, andsugar content in the intestinal tract of infants.J. Pediat. 40:152-163.

4. Beijerinck, M. W. 1901. Sur les ferments lac-tiques de l'industrie. Arch. Neerl. Sci. ExactesNatur. Ser. 2. 6:212-243.

5. Beerens, H., A Gerard, and J. Guillaume. 1957.Etude de 30 souches de Bifidibacterium bifi-dum (Lactobacillus bifidus) characterisationd'une variet6 buccale comparaison avec lessouches d'origine fecale. Ann. Inst. Pasteur(Lille) 9:77-85.

6. Bergey, D. H., R. S. Breed, B. W. Hammer, F.M. Huntoon, E. G. D. Murray, and F. C.Harrison. 1934. Bergey's manual of deter-minative bacteriology, 4th ed. The Williams& Wilkins Co., Baltimore.

7. Bergey, D. H., R. S. Breed, E. G. D. Murray, andA. P. Hitchens. 1939. Bergey's manual ofdeterminative bacteriology, 5th ed. The Wil-liams & Wilkins Co., Baltimore.

8. Bergey, D. H., F. C. Harrison, R. S. Breed, B. W.Hammer, and F. M. Huntoon. 1923. Bergey's

manual of determinative bacteriology, 1st.ed. The Williams & Wilkins Co., Baltimore.

9. Bergey, D. H., F. C. Harrison, R. S. Breed, B. W.Hammer, and F. M. Huntoon. 1925. Bergey'smanual of determinative bacteriology, 2nded. The Williams & Wilkins Co., Baltimore.

10. Bergey, D. H., F. C. Harrison, R. S. Breed, B. W.Hammer, and F. M. Huntoon, 1930. Bergey'smanual of determinative bacteriology, 3rded. The Williams & Wilkins Co., Baltimore.

11. Blaurock, G. 1937. Zur Physiologie der Bifidus-bakterien. Monatsschr. Kinderheilk.68:304-309.

12. Blaurock, G. 1939. Bifiduszuchtung auf zystin-haltigen Nahrboden. Zentralbl. Bakteriol. Pa-rasitenk. Infektionskr. Hyg. Abt. Orig. 144:75-79.

13. Blaurock, G. 1940. Zuchtung von Bifidusbakte-rien aus der Vaginalflora. Deut. Med. Wo-chenschr. 66:1133.

14. Bliuhdorn, K. 1910. Untersuchugen uiber denBacillus bifidus communis und den so gen.Bacillus acidophilus (Streptobacillus fae-calis). Jahrb. Kinderheilk. 72:693-704.

15. Boventer, K. 1938, Untersuchungen fiber dasBacterium bifidum. Zentralbl. Bakteriol. Pa-rasitenk. Infektionskr. Hyg. Abt. Orig. 142:419-430.

16. Breed, R. S., E. G. D. Murray, and A. P.Hitchens. 1948. Bergey's manual of deter-minative bacteriology, 6th ed., The Williams& Wilkins Co., Baltimore.

17. Castellani, A., and A. J. Chalmers. 1919. Ma-nual of tropical medicine, 3rd ed. WilliamWood and Co., New York.

18. Cataldi, M. S., and A. Muller. 1947. Microfloradel calostro y de la superflcie cutauiea del senomaterno en relaci6n con la del tubo digestivodel recien nacido. Dfa. Med. 19:473-475.

19. Crociani, F., V. Scardovi, and L. D. Trovatelli.1970. Mannitol fermenting bifids from rumenand their DNA homology relationships. Ann.Microbiol. 20:99-106.

20. Cruickshank, J. 1925. Bacillus bifidus: its cha-racters and isolation from the intestine ofinfants. J. Hyg. 24:241-254.

21. Cummins, C. S. 1970. Cell wall composition inthe classification of gram positive anaerobes.Int. J. Syst. Bacteriol. 20:413-419.

22. Cummins, C. S., 0. M. Glendenning, and H.Harris. 1957. Composition of the cell wall ofLactobacillus bifidus. Nature (London)180:337-338.

23. Cummins, C. S., and H. Harris. 1956. Thechemical composition of the cell wall in somegram positive bacteria and its possible valueas a taxonomic character. J. Gen. Microbiol.14:583-600.

24. Damjanovic, V., and D. Radulovic. 1967. Sur-vival of Lactobacillus bifidus after freeze-dry-ing. Cryobiology 4:30-32.

25. Dehnert, J. 1957. Untersuchung fiber die gram-positive Stuhlflora des Brustmilchkindes.Zentralbl. Bakteriol. Parasitenk. Infektionskr.Hyg. Abt. Orig. 169:66-79.

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POUPARD, HUSAIN, AND NORIS

26. Dehnert, J. 1960. Betrachtungen zum Bifidum-problem. Zentralbl Bakteriol. Parasitenk. In-fectionskr. Hyg. Abt. Orig. 179:490-498.

27. Dehnert, J. 1961. Ober die Bedeutung der Inte-stinalbesiedlung beim Menschen. Bakteri-ologische Untersuchungen als Beitrag zumBifidumproblem. Dissertation, University ofHeidelberg, Heidelberg, West Germany.

28. Dehnert, J. 1965. Zur Systematik der Bifidum-Gruppe. Int. Mikrobkolog. Symp. Berlin, Er-nahrungsforsch. 10:465-471.

29. Distaso, A. 1912. Contribution 'a l'tude surl'intoxication intestinale. Zentralbl. Bakte-riol. Parasitenk. Infektionskr. Hyg. Abt. Orig.62:433-468.

30. Donaldson, R. M. 1964. Normal bacterial popu-lations of the intestine and their relation tointestinal function. N. Engl. J. Med.270:938-945, 994-1001, 1050-1056.

31. Eggerth, A. H. 1935. The gram-positive non-spore-bearing anaerobic bacilli of humanfeces. J. Bacteriol. 30:277-299.

32. Exterkate, F. A., B. J. Otten, H. W. Wassenberg,and J. H. Veerkamp. 1971. Comparison of thephospholipid composition of Bifidobacteriumand Lactobacillus strains. J. Bacteriol.106:824-829.

33. Exterkate, F. A., and J. H. Veerkamp. 1969.Biochemical changes in Bifidobacterium bifi-dum var. pennsylvanicus after cell wall inhibi-tion. I. Composition of lipids. Biochim. Bio-phys. Acta 176:65-77.

34. Exterkate, F. A., G. F. J. Vrensen, and J. H.Veerkamp. 1970. Biochemical changes in Bifi-dobacterium bifidum var. pennsylvanicusafter cell wall inhibition. III. Morphologicalstructure and osmotic properties of the proto-plasts and membrane composition. Biochim.Biophys. Acta 219:141-154.

35. Frank, H. A., and C. E. Skinner. 1954. Therelationship between Actinomyces bovis andLactobacillus bifidus. Mycologia 46:728-735.

36. Gall, L. S. 1970. Normal' fecal flora of man.Amer. J. Clin. Nutr. 23:1457-1465.

37. Gasser, F., and M. Mandel. 1968. Deoxyribonu-cleic acid base composition of the genus Lac-tobacillus. J. Bacteriol. 96:580-588.

38. Georg, L. K., G. W. Robertstad, and S. A.Brinkman. 1964. Identification of species ofActinomyces. J. Bacteriol. 88:477-490.

39. Georg, L. K., G. W. Robertstad, S. A. Brinkman,and M. D. Hicklin. 1965. A new pathogenicanaerobic Actinomyces species. J. Infect. Dis.115:88-99.

40. Gibbons, R. J., S. S. Socransky, and B. Kapsi-malis. 1964. Establishment of human indige-nous bacteria in germ-free mice. J. Bacteriol.88:1316-1323.

41. Glick, M. C., T. Sall, F. Zilliken, and S. Mudd.1960. Morphological changes of Lactobacillusbifidus var. pennsylvanicus produced by acell-wall precursor.' Biochim. Biophys. Acta37:361-363.

42. Goldberg, M. L., and E. Racker. 1962. Forma-

tion and isolation of a glycolaldehyde-phos-phoketolase intermediate. J. Biol. Chem.237:3841-3842.

43. Gorbach, S. L., L. Nahas, P. I. Lerner, and L.Weinstein. 1967. Studies of intestinal micro-flora. I. Effects of diet, age and periodicsampling on numbers of fecal microorganismsin man. Gastroenterology 53:845-855.

44. Gyllenberg, H. G. 1955. The development of the'straight rod type' of Lactobacillus bifidus. J.Gen. Microbiol. 13:394-397.

45. Gyllenberg, H. H. G. 1955. A note on thetaxonomy of Lactobacillus bifidus. Int. Bull.Bacteriol. Nomencl. Taxon. 5:161-164.

46. Gyllenberg, H. 1958. Specific contaminants incultures of bifid bacteria (Lactobacillusbifidus). Acta Pathol. Microbiol. Scand.44:293-298.

47. Gyllenberg, H., and G. Carlberg. 1958. Thedominance of a specific nutritional type ofLactobacillus bifidus in breast-fed infants.Acta Pathol. Microbiol. Scand. 42:380-384.

48. Gyllenberg, H., and G. Carlberg. 1958. Thenutritional characteristics of the bifid bacteria(Lactobacillus bifidus) of infants. Acta Pa-thol. Microbiol. Scand. 44:287-292.

49. Gyllenberg, H., and P. Ronie. 1957. The value ofcolony counts in evaluating the abundance ofLactobacillus bifidus in infant feces. ActaPathol. Microbiol. Scand. 41:144-155.

50. Gybrgy, P. 1953. A hitherto unrecognized bio-chemical difference between human milk andcow's milk. Pediatrics 11:98-108.

51. Gyorgy, P. 1957. Trends and advances in infantnutrition. W. Va. Med. J. 53:1-24.

52. Gydrgy, P. 1957. Development of intestinal florain the breast-fed infant. Mod. Probl. Paediat.2:1-12.

53. Gyorgy, P. 1958. N-containing saccharides inhuman milk. Ciba Found. Symp. Chem. Biol.Mucopolysaccharides, p. 140-154.

54. Gyorgy, P., R. F. Norris, and C. S. Rose. 1954.Bifidus factor I. A variant of Lactobacillusbifidus requiring a special growth factor. Arch.Biochem. Biophys. 48:193-201.

55. Gyorgy, P., and C. S. Rose. 1955. Furtherobservations on metabolic requirements ofLactobacillus bifidus var. pennysIvanicus. J.Bacteriol. 69:483-490.

56. Gyorgy, P., and C. S. Rose. 1955. Microbiologi-cal studies on growth factor for L. bifidus var.pennsylvanicus. Proc. Soc. Exp. Biol. Med.90:219-223.

57. Gydrgy, P., C. S. Rose, and G. F. Springer. 1954.Enzymatic inactivation of bifidus factor andblood group substances. J. Lab. Clin. Med.43:543-552.

58. Gybrgy, P., L. M. Tocantins, F. R. Miller, and A.Weiss. 1954. Hematopoiesis, blood group sub-stance, and bifidus factor. Trans. Ass. Amer.Physicians Philadelphia 67:117-122.

59. Haenel, H. 1970. Human normal and abnormalgastrointestinal flora. Amer. J. Clin. Nutr.23:1433-1439.

BACTERIOL. REV.160


http://mm

br.asm.org/

Dow

nloaded from



60. Harrison, A. P., Jr., and P. A. Hansen. 1954.Characterization of the Lactobacteriaceaeencountered as secondary invaders in black-head liver lesions of turkeys. Zentralbl. Bak-teriol. Parasitenk. Infektionskr. Hyg. Abt.Orig. 161:125-137.

61. Harrison, W., R. C. Stahl, J. Magavran, M.Sanders, R. F. Norris, and P. Gybrgy. 1953.The incidence of Lactobacillus bifidus in vagi-nal secretions of pregnant and nonpregnantwomen. Amer. J. Obstet. Gynecol.65:352-357.

62. Hassinen, J. B., G. T. Durbin, R. M. Tomarelli,and F. W. Bernhart. 1951. The minimal nutri-tional requirements of Lactobacillus bifidus.J. Bacteriol. 62:771-777.

63. Hayward, A. C., C. M. F. Hale, and K. A. Bisset.1955. The morphology and relationships ofLactobacillus bifidus. J. Gen. Microbiol.13:292-294.

64. Hehre, E. J., and T. W. Sery. 1952. Dextran-splitting anaerobic bacteria from the humanintestine. J. Bacteriol. 63:424-426.

65. Herter, C. A. 1907. Bacterial infections of thedigestive tract, p. 41-45. MacMillan Co., NewYork.

66. Hill, L. R. 1966. An index to deoxyribonucleicacid base compositions of bacterial species. J.Gen. Microbiol. 44:419-437.

67. Holdeman, L. V., and W. E. C. Moore. 1972.Anaerobe laboratory manual. Virginia Poly-technic Institute and State University,Blacksburg.

68. Holland, D. F. 1920. Generic index of the com-moner forms of bacteria. J. Bacteriol.5:215-229.

69. Holzapfel, W., V. Scardovi, and 0. Kandler.1969. Die Aminosguresequenz des Ornithinund Lysin enthaltenden Mureins einigerStamme von Lactobacillus bifidus aus demPansen. Z. Naturforsch. 24B:1524-1528.

70. Homma, N., J. Ota, and S. Arai. 1960. Variouscarbonates affecting the branching of Lac-tobacillus bifidus. Jap. J. Pediat.13:1274-1277.

71. Hurwitz, J. 1958. Pentose phosphate cleavage byLeuconostoc mesenteroides. Biochim. Bio-phys. Acta 28:599-602.

72. Husain, I., J. A. Poupard, and R. F. Norris. 1972.Influence of nutrition on the morphology of astrain of Bifidobacterium bifidum. J. Bacte-riol. 111:841-844.

73. Johnson, J. L., and E. L. Ordal. 1968. Deoxyri-bonucleic acid homology in bacterial taxon-omy: effect of incubation temperature on reac-tion specificity. J. Bacteriol. 95:893-900.

74. Kalser, M. H., R. Cohen, I. Arteaga, E. Yawn, L.Mayoral, W. Hoffert, and D. Frazier. 1966.Normal viral and bacterial flora of the humansmall and large intestine. N. Engl. J. Med.274:500-505.

75. Kandler, 0. 1967. Taxonomie und tech-nologische Bedeutung der Gettung Lac-tobacillus Beijerinck. Zentralbl. Bakteriol.

Parasitenk Infektionskr. Hyg. Abt. Suppl.2:139-164.

76. Kandler, 0. 1970. Amino acid sequence of themurein and taxonomy of the genera Lac-tobacillus, Bifidobacterium, Leuconostoc, andPediococcus. Int. J. Syst. Bacteriol.20:491-507.

77. Kandler, O., D. Koch, and K. H. Schleifer. 1968.Die Aminosduresequenz eines glycinhaltigenMureins einiger Stamme von Lactobacillusbifidus. Arch. Mikrobiol. 61:181-186.

78. Kendall, A. I., and R. C. Haner. 1924. Bacillusbifidus. Studies in bacterial metabolism. J.Infect. Dis. 35:77-88.

79. King, J. W., and L. F. Rettger. 1942. Gram-posi-tive non-sporulating anaerobic rod-shapedbacteria of the intestinal tract. III. Intra- andextra-group relationships. J. Bacteriol.44:301-316.

80. Koch, D., K. H. Schleifer, and 0. Kandler. 1970.Die Aminosauresequenz des Threonin undSerin enthaltenden Mureins von Bifidobacte-rium longum Reuter. Arch. Mikrobiol.74:315-325.

81. Koch, D., K. H. Schleifer, and 0. Kandler. 1970.Die Aminosauresequenz des Serin undAsparaginsaure enthaltenden Mureins von Bi-fidobacterium bifidum Orla-Jensen. Z. Natur-forsch. 25B:1294-1301.

82. Kojima, M., S. Suda, S. Hotta, and K. Hamada.1968. Induction of pleomorphism in Lac-tobacillus bifidus. J. Bacteriol. 95:710-711.

83. Kojima, M., S. Suda, S. Hotta, and K. Hamada.1970. Induction of pleomorphy and calciumion deficiency in Lactobacillus bifidus. J.Bacteriol. 102:217-220.

84. Kojima, M., S. Suda, S. Hotta, K. Hamada, andA. Suganuma. 1970. Necessity of calcium ionfor cell division in Lactobacillus bifidus. J.Bacteriol. 104:1010-1013.

85. Kuhn, R., and H. Tiedemann. 1953. Zum staff-wechsel des Lactobacillus bifidus, die Umset-zung von radioactive "C-1-glucose. Z. Natur-forsch. 86:428-436.

86. Lambert, R., Y. Saito, and J. H. Veerkamp.1965. Incorporation of labeled derivatives of2-deoxy-2-amino-D-glucose into the cell wallsof Lactobacillus bifidus var. pennsylvanicus.Arch. Biochem. Biophys. 110:341-344.

87. Lambert, R., and F. Zilliken. 1965. Novel growthfactors for Lactobacillus bifidus var. pennsyl-vanicus. Arch. Biochem. Biophys.110:544-550.

88. Lehmann, K. B., and R. 0. Neumann. 1927.Bakteriologie insbesondere BakteriologischeDiagnostik, Chapter 7. Allgemeine und spe-zielle Bakteriologie, vol. 2.

89. Lerche, M., and G. Reuter. 1961. Isolierung undDifferenzierung anaerober Lactobacilleae ausdem Darm erwachsener Menschen. Zentralbl.Bakteriol. Parasitenk. Infektionskr. Hyg. Abt.Org. 182:324-356.

90. Lewis, K. H., and L. F. Rettger. 1940. Non-sporulating anaerobic bacteria of the intesti-

VOL. 37, 1973 161


http://mm

br.asm.org/

Dow

nloaded from


162 POUPARD, HUSA

nal tract. J. Bacteriol. 40:287-307.91. Malyoth, G., and A. Bauer. 1950. Beobachtun-

gen am Bacterium bifidum. Z. Kinderheilk.68:358-367.

92. Malyoth, G., and A. Bauer. 1951. Die Darstel-lung der Schleimkapsel bei Mucoidstimmendes Bact. bifidum mittels Immunserum inPhasenkontrast-mikroskop. Z. Biol. 104:404-408.

93. Mata, L. J., C. Carrillo, and E. Villatoro. 1969.Fecal microflora in healthy persons in a prein-dustrial region. Appl. Microbiol. 17:596-602.

94. Mata, L. J., and J. J. Urrutia. 1971. Intestinalcolonization of breast-fed children in a ruralarea of the low socioeconomic level. In B.Tennat (ed.), Neonatal enteric infectioncaused by E. coli. Ann. N.Y. Acad. Sci.176:93-108.

95. Mata, L. J., J. J. Urrutia, B. Garcia, R. Fer-nandez, and M. Bbhar. 1969. Shigella infec-tion in breast-fed Guatemalan Indian neon-ates. Amer. J. Dis. Child. 117:142-146.

96. Matteuzzi, D., F. Crociani, G. Zani, and L. D.Trovatelli. 1971. Bifidobacterium suis n. sp.: anew species of the genus Bifidobacteriumisolated from pig feces. Allg. Mikrobiol.11:387-395.

97. Mayer, J. B. 1965. Viren und Darmflora. Pae-diat. Paedol. 1:131-137.

98. Mayer, J. B., D. Torzewski, and J. Dittman.1964. Zum Stoffwechsel des Bacterium bifi-dum (Lactobacillus bifidus). IV. Katalse-activitft aerober und anaerober Kulturen. Z.Kinderheilk. 91:222-227.

99. Miller, L. B. 1969. Fecal flora of Eskimo chil-dren. Bacteriol. Proc., p. 103.

100. Mitsuoka, T. 1969. Vergleichende Untersuchun-gen fiber die Bifidobakterien aus dem Verdau-ungstrakt von Menschen und Tieren. Zen-tralbl. Bakteriol. Parasitenk. Infektionskr.Hyg. Abt. Orig. 210:52-64.

101. Montagna, C. P., and M. S. Cataldi. 1944. Sobrela presencia de Lactobacillus bifidus en elmeconio y en el ambiente vaginal de la madre.Rev. Ass. Argent. Dietrol. 2:47-50.

102. Moore, W. E. C., E. P. Cato, and L. V. Holde-man. 1969. Anaerobic bacteria of the gastroin-testinal flora and their occurrence in clinicalinfections. J. Infect. Dis. 119:641-649.

103. Moore, W. E. C., E. P. Cato, and L. V. Holde-man. 1972. Quantitation and speciation of thenormal human fecal flora. Abstr. Annu. Meet.Amer. Soc. Microbiol., p. 80.

104. Moore, W. E. C., and L. V. Holdeman. 1970.Gram-positive nonspore-forming anaerobicbacilli, p. 290-295. In J. E. Blair, E. H.Lennette, and J. P. Truant (ed.), Manual ofclinical microbiology. American Society forMicrobiol., Bethesda, Md.

105. Moro, E. 1900. Uber die nach Gram farbbarenBacillen des Sauglingsstuhles. Wien. Klin.Wochenschr. 13:114-115.

106. Moro, E. 1905. Morphologische und biologischeUntersuchungen fiber die Darmbakterien des

IN, AND NORRIS BACTERIOL. REV.

Sauglings. Jahrb. Kinderheilk. 61:687-734.107. Mossel, D. A. A. 1958. The suitability of bifidi-

bacteria as part of a more extended bacterialassociation, indicating faecal contaminationof foods. 7th Int. Congr. Mikrobiol. Stock-holm, Sweden. Abst. Papers, p. 440.

108. Muting, D., W. Eschrich, and J. B. Mayer. 1968.The effect of Bacterium bifidum on intestinalbacterial flora and toxic protein metabolites inchronic liver disease. Amer. J. Proctol.19:336-342.

109. Nannizzi, A. 1934. Repertio sistematico deimiceti dell'uomo e degli animali. G. Pollacci(ed.), Tratto di Micopathologia Umana(Sienna) 4:13.

110. Negroni, P., and I. Fischer. 1944. Estudio sobreel Lactobacillus bifidus (Tissier) Kulp y Rett-ger. Rev. Soc. Argent. Biol. 20:313-327.

111. Norris, R. F., M. DeSipin, F. W. Zilliken, T. S.Harvey, and P. Gyorgy. 1954. Occurrence ofmucoid variants of Lactobacillus bifidus. De-monstration of extracellular and intracellularpolysaccharide. J. Bacteriol. 67:159-166.

112. Norris, R. F., T. Flanders, R. M. Tomarelli, andP. Gyorgy. 1950. The isolation and cultivationof Lactobacillus bifidus. A comparison ofbranched and unbranched strains. J. Bacte-riol. 60:681-696.

113. O'Brien, P. J., M. C. Glick, and F. Zilliken.1960. Acidic amino-sugars from bacterial. In-corporation of [1- '4C j-a, #-methyl-N-acet-yl-D-glucosaminide into muramic acid. Bio-chim. Biophys. Acta 37:357-360.

114. Olsen, E. 1949. Studies on the intestinal flora ofinfants. Ejnar Munksgaard, Copenhagen.

115 Orla-Jensen, S. 1919. The lactic acid bacteria. C.R. Acad. Sci. Copenhague.

116. Orla-Jensen, S. 1924. La classification des bacte-ries lactiques. Lait 4:468-474.

117. Orla-Jensen, S. 1943. The lactic acid bacteria.Mem. Acad. Roy. Sci. Danemark Sect. Sci. 8ser. 5:81-197.

118. Orla-Jensen, S., A. D. Orla-Jensen, and 0.Winther. 1936. Bacterium bifidum und Ther-mobacterium intestinale. Zentralbl. Bakteri-ol. Parasitenk. II. 93:321-343.

119. Outline of clinical methods in anaerobic bacteri-ology, 2nd rev. 1970. Virginia PolytechnicInstitute Anaerobe Laboratory, Blacksburg.

120. Overman, J. R., and L. Pine. 1963. Electronmicroscopy of cytoplasmic structures in facul-tative and anaerobic Actinomyces. J. Bacte-riol. 86:656-665.

121. Petuely, F., and G. Lindner. 1965. KritischeUntersuchung fiber die Darmflora. III. Mit-teilung: Bewertung quantitativer Zuchtungs-methoden. Die Darmflora des Brustkindes.Zentralbl. Bakteriol. Parasitenk. Hyg. Abt.Orig. 195:347-384.

122. Petuely, F., and V. Lynau. 1954. Ein einfacher,vollsynthetischer Optimalniihrboden fur denLactobacillus bifidus. (Uber die Bedeutungder Ascorbinsaure fur das Wachstum des Lac-tobacillus bifidus.) Biochem. Z. 326:62-78.

Ul


http://mm

br.asm.org/

Dow

nloaded from



123. Pine, L. 1970. Classification and phylogeneticrelationship of microaerophilic Ac-tinomycetes. Int. J. Syst. Bacteriol.20:445-474.

124. Pine, L., and A. Howell, Jr. 1956. Comparison ofphysiological & biochemical characters of Ac-tinomyces spp. with those of Lactobacillusbifidus. J. Gen. Microbiol. 15:428-445.

125. Pope, S., R. M. Tomarelli, and P. Gyorgy. 1956.Bifidus factor. Isolation of ethyl N-acetyl-ft-D-glucosaminide from Aspergillus fermenta-tions. Arch. Biochem. Biophys. 68:362-366.

126. Prevot, A. R. 1938. Atudes de systematiquebacterienne. III. Invalidite du genre Bacte-roides Castellani et Chalmers demembrementet reclassification. Ann. Inst. Pasteur60:285-307.

127. Prevot, A. R. 1966. Class of actinomycetales, p.344-365. Manual for the classification anddetermination of anaerobic bacteria. Lea &Febiger, Philadelphia.

128. Prevot, A. R. 1970. Importance of the biochemi-cal composition of the cell wall as a taxonomiccharacteristic. Int. J. Syst. Bacteriol.20:539-540.

129. Pribram, E. 1929. A contribution to the classifi-cation of microorganisms. J. Bacteriol.18:361-394.

130. Puntoni, V. 1936. Sui rapporti fra gli at-tinomiceti anaerobi tipo "Wolff-Israel" ed il"Bacterium bifidum." Rend. Acad. Sci. Fis.Mat. (Roma) 23:274-276.

131. Puntoni, V. 1937. Sulle relazioni fra il b. bifidogli attinomiceti anaerobi typo Wolff-Israel.Ann. Ig. Sper. 47:157-168.

132. Reuter, G. 1964. Vergleichende Untersuchungenuiber die Bifidus-Flora in Sauglings-und Er-wachsenenstuhl. Zentralbl. Bakteriol. Parasi-tenk. Hyg. Abt. Orig. 191:486-507.

133. Reuter, G. 1971. Designation of type strains forBifidobacterium species. Int. J. Syst. Bacte-riol. 21:273-275.

134. Reyn, A., A. Birch-Andersen, and S. P. LaPage.1966. A electron microscope study of thinsections of Haemophilus vaginalis (Gardnerand Dukes) and some possibly related species.Can. J. Microbiol. 12:1125-1136.

135. Rodella, A. 1908. Magencarcinom und Milch-saurebacillen (Boas-Opplerscher Bacillus,Bacillus gastrophilus und Bacterium gastro-philum Lehmann-Neumann, Bacillus acido-philus und Bac. bifidus communis) Centralbl.Bakteriol. I. Abt. Orig. 47:445-466.

136. Roos, C. 1927. Cultural characteristics (variationof Bacillus acidophilus). J. Lab. Clin. Med.12:1053-1058.

137. Rose, C. S., and P. Gyorgy. 1955. Observationson bacterial interaction. Proc. Soc. Exp. Biol.Med. 89:23-26.

138. Rose, C. S., and P. Gyorgy. 1956. Reaction ofenzymes of Lactobacillus bifidus var. pennsyl-vanicus with bifidus factor: effect of monosac-charides. Proc. Soc. Exp. Biol. Med. 93:58-61.

139. Rose, C. S., R. Kuhn, F. Zilliken, and P. Gyorgy.

1954. Bifidus factor. V. The activity of a- and,-methyl-N-acetyl-D-glucosaminides. Arch.Biochem. Biophys. 49:123-129.

140. Rosypal, S., and A. Rosypalova. 1966. Genetic,phylologenetic and taxonomic relationshipsamong bacteria as determined by their deoxy-ribonucleic acid base composition. Folia Biol.(Prague) 14:1-91.

141. Roufogalis, S. 1940. Das Bacterium bifidum.Klin. Wochenschr. 19:598-599.

142. Roufogalis, S. 1940. Untersuchungen uiber dieBesiedelung des Neugeborenendarmes mitdem Bacterium bifidum. Z. Hyg. InfektionskrMed. Mikrobiol. Immunol. Virol. 123:195-217.

143. Roufogalis, S. 1941. Untersuchungen tiber dasHerkommen ds Bacterium bifidum bei derWerdenden Mutter. Arch. Gynaekol.171:459-470.

144. Savage, D. C. 1970. Associations of indigenousmicroorganisms with gastrointestinal mucosalepithelia. Amer. J. Clin. Nutr. 23:1495-1501.

145. Scardovi, V. 1964. Studies in rumen bacteriol-ogy. IV. The formation of acetate throughphosphorolytic cleavage of fructose-6-phos-phate in a group of branched gram-positivebacteria from sheep rumen. Ann. Microbiol.14:189-198.

146. Scardovi, V., B. Sgorbati, and G. Zani. 1971.Starch gel electrophoresis of fructose-6-phos-phate phosphoketolase in the genus Bifidobac-terium. J. Bacteriol. 106:1036-1039.

147. Scardovi, V., and L. D. Trovatelli. 1965. Thefructose-6-phosphate shunt as a peculiar pat-tern of hexose degradation in the genus Bifido-bacterium. Ann. Microbiol. Enzymol.15:19-29.

148. Scardovi, V., and L. D. Trovatelli. 1969. Newspecies of bifid bacteria from Apis mellifica L.and Apis indica F. A contribution to thetaxonomy and biochemistry of the genus Bifi-dobacterium. Zentralbl. Bakteriol. Parasi-tenk. Infektionskr. Hyg. Abt. Orig. 123:64-88.

149. Scardovi, V., L. D. Trovatelli, F. Crociani, andB. Sgorbati. 1969. Bifid bacteria in bovinerumen. Arch. Mikrobiol. 68:278-294.

150. Scardovi, V., L. D. Trovatelli, G. Zani, F.Crociani, and D. Metteuzzi. 1971. Deoxyribo-nucleic acid homology relationships amongspecies of the genus Bifidobacterium. Int. J.Syst. Bacteriol. 21:276-294.

151. Scardovi, V., G. Zani, and L. D. Trovatelli. 1970.Deoxyribonucleic acid homology among thespecies of the genus Bifidobacterium isolatedfrom animals. Arch. Mikrobiol. 72:318-325.

152. Schramm, M., V. Klybal, and E. Racker. 1958.Phosphorolytic cleavage of fructose-6-phos-phate by fructose-6-phosphate phosphoketo-lase from Acetobacter xylinum. J. Biol.Chem. 233:1283-1288.

153. Sebald, M., F. Gasser, and H. Werner. 1965.Teneru GC% et classification. Application augroupe des bifidobacteries et a quelques genresvoisins. Ann. Inst. Pasteur 109:251-269.

154. Seeliger, H. P. R., and H. Werner. 1963. Re-

VOL. 37, 1973 163


http://mm

br.asm.org/

Dow

nloaded from



cherches qualitatives et quantitatives sur laflore intestinale de l'homme. Ann. Inst. Pas-teur 105:911-936.

155. Slack J. M., A. Winger, and D. W. Moore, Jr.1961. Serological grouping of actinomyces bymeans of fluorescent antibodies. J. Bacteriol.82:54-65.

156. Smith, H. W., and W. E. Crabb. 1961. Thefaecal bacterial flora of animals and man: itsdevelopment in the young. J. Pathol. Bacte-riol. 82:53-66.

157. Smith, N. R., R. E. Gorden, and F. E. Clark.1952. Aerobic spore-forming bacteria, p. 5-7.U.S. Dep. Agr.

158. Snyder, M. L., M. S. Slawson, W. Bullock, andR. B. Parker. 1967. Studies on oral filamen-tous bacteria. II. Serological relationshipswithin the genera Actinomyces, Nocardia, Bac-terionema, and Leptotrichia. J. Infect. Dis.117:341-345.

159. Stitt, E. R. 1927. Practical bacteriology, bloodwork and animal parasitology, p. 185-186. P.Blakiston's Son & Co., Philadelphia.

160. Stolte, G. 1940. Versuche zur Feststellung, wiedas Bacterium bifidum (Tissier) in den Darm-tractus des Sauglings gelangt. Monatsschr.Kinderheilk. 83:83-94.

161. Sundman, V., and K. Bjorksten, 1958. Theglobular involution forms of the bifid bacteria.J. Gen. Microbiol. 19:491-496.

162. Sundman, V. K. Bjorksten, and H. G. Gyllen-berg. 1959. Morphology of the bifid bacteria(organisms previously incorrectly designatedLactobacillus bifidus) and some related gen-

era. J. Gen. Microbiol. 21:371-384.163. Syed, S. A., G. D. Abrams, and R. Freter. 1970.

Efficiency of various intestinal bacteria inassuming normal functions of enteric floraafter association with germ-free mice. Infect.Immunity 2:376-386.

164. Tassovatz, B., and D. Kragouyevitch. 1964.L'entero-colite aigue bacterienne chez le nour-rison essai de traitement par le bacille bifidus.Ann. Pediat. 22:291-297.

165. Teply, L. J., and C. A. Elvehjem. 1945. Thetitrimetric determination of "Lactobacilluscasei factor" and "folic acid." J. Biol. Chem.157:303-309.

166. Tissier, M. H. 1889. La reaction chromophiled'Escherichel et le Bacterium coli. C. R. Acad.Sci. 51:943-945.

167. Tissier, H. 1900. Recherches sur la flore intes-tinale des nourrissons (etat normal et pa-

thologique). Paris Thbses: 1-253.168. Tomarelli, R. M., R. F. Norris, and P. Gyorgy.

1949. Inability of vitamin B,2 to replace thedesoxyriboside requirement of a Lactobacillusbifidus. J. Biol. Chem. 179:485-86.

169. Tomarelli, R. M., R. F. Norris, P. Gyorgy, J. B.Hassinen, and F. W. Bernhart. 1949. Thenutrition of variants of Lactobacillus bifidus.J. Biol. Chem. 181:879-888.

170. Tomarelli, R. M., R. F. Norris, C. S. Rose, andP. Gy6rgy. 1950. The effect of fatty acids on

the growth of strains of Lactobacillus bifidus.J. Biol. Chem. 187:197-204.

171. Veerkamp, J. H. 1969. Catabolism of glucose andderivatives of 2-deoxy-2-amino-glucose in Bi-fidobacterium bifidum var. pennsylvanicus.Arch. Biochem. Biophys. 129:257-263.

172. Veerkamp, J. H. 1969. Uptake and metabolismof derivatives of 2-deoxy-2-amino-D-glucose inBifidobacterium bifidum var. pennsylvanicus.Arch. Biochem. Biophys. 129:248-256.

173. Veerkamp, J. H. 1970. Biochemical changes inBifidobacterium bifidum var. pennsylvanicusafter cell wall inhibition. II. Fatty acid compo-sition. Biochim. Biophys. Acta 210:267-275.

174. Veerkamp, J. H. 1971. Fatty acid composition ofBifidobacterium and Lactobacillus strains. J.Bacteriol. 108:861-867.

175. Veerkamp, J. H. 1971. The structure of the cellwall peptidoglycan of Bifidobacteriumbifidum var. pennsylvanicus. Arch. Biochem.Biophys. 143:204-211.

176. Veerkamp, J. H., R. Lambert, and Y. Saito.1965. The composition of the cell wall ofLactobacillus bifidus var. pennsylvanicus.Arch. Biochem. Biophys. 112:120-125.

177. Vogel, R. 1952. Untersuchungen zur Biologie vonBacterium bifidum. Inaugural dissertation,Ludwig-Maximillians-Universitat zur Min-chen, Munich, W. Germany.

178. Vries, W. de, Sj. J. Gerbrandy, and A. H.Stouthamer. 1967. Carbohydrate metabolismin Bifidobacterium bifidum. Biochim. Bio-phys. Acta 136:415-425.

179. Vries, W. de, and A. H. Stouthamer. 1967.Pathway of glucose fermentation in relation tothe taxonomy of bifidobacteria. J. Bacteriol.93:574-576.

180. Vries, W. de, and A. H. Stouthamer. 1968.Fermentation of glucose lactose, galactose,mannitol, and xylose by bifidobacteria. J.Bacteriol. 96:472-478.

181. Vries, W. de, and A. H. Stouthamer. 1969.Factors determining the degree of anaerobiosisof Bifidobacterium strains. Arch. Mikrobiol.65:275-287.

182. Vuillemin P. 1931. Les champignons parasites etles mycoses de l'homme. Lechevalier, Paris.

183. Wagner, M., and T. J. Starr. 1969. Microbicinteractions in. the gnotobiotic mouse, p.389-398. In E. A. Mirand and N. Bach (ed.),Advances in experimental medicine and biol-ogy, vol. 5. Germ-free biology. Plenum Press,New York.

184. Wang, M., E. Steers, and R. F. Norris. 1963.Extracellular polysaccharide of mucoid Lac-tobacillus bifidus. J. Bacteriol. 86:898-903.

185. Wang, M. M., K. C. Tsou, and R. F. Norris.1969. The depolymerization of "bifidan," apolysaccharide of Lactobacillus bifidus, byascorbic acid. Arch. Biochem. Biophys.131:513-520.

186. Weijers, H. A., and J. H. van de Kamer. 1965.Causes of diarrhea in disturbed digestion.Nutr. Dieta 7:233-242.

BACTERIOL. REV.164


http://mm

br.asm.org/

Dow

nloaded from



187. Weiss, J. E. 1933. Lactobacillus bifidus Tissierand its biological position in the group ofaciduric organisms. Dissertation, Yale Univer-sity, New Haven, Conn.

188. Weiss, J. E., and L. F. Rettger. 1934. Lactobacil-lus bifidus. J. Bacteriol. 28:501-521.

189. Weiss, J. E., and L. F. Rettger. 1938. Taxonomicrelationships of Lactobacillus bifidus (B.bifidus Tissier) and Bacteroides bifidus. J.Infect. Dis. 62:115-120.

190. Weiss, J. E., and L. F. Rettger. 1938. Taxonomicrelationship of Lactobacillus bifidus [Bacillusbifidus (Tissier) I and Bacteroides bifidus (Eg-gerth). J. Bacteriol. 35:17-18.

191. Werner, H. 1966. The gram positive nonsporinganaerobic bacteria of the human intestinewith particular reference to theCorynebacteria and Bifidobacteria. J. Appl.Bacteriol. 29:138-146.

192. Werner, H., and H. P. R. Seeliger. 1963. DieDiagnostik der Bifidusbacterien und ihre Ab-grenzung gegen morphologische ahnlicheKeime. Aerztl. Lab. 11:357-367.

193. Werner, H., and H. P. R. Seeliger. 1964. Verglei-chende Untersuchungen an Bifidus-Stammenverschiedener Herkunft. Pathol. Microbiol.(Basel) 27:202-215.

194. White, P. B. 1921. The normal bacterial flora ofthe bee. J. Pathol. Bacteriol. 24:64-78.

195. Williams, N. B., R. F. Norris, and P. Gyorgy.1953. Antigenic and cultural relationships of

Lactobacillus bifidus and Lactobacillusparabifidus. J. Infect. Dis. 92:121-131.

196. Wyatt, R. G., and L. J. Mata. 1969. Bacteria incolostrum and milk of Guatemalan Indianwomen. J. Trop. Pediat. 15:159-162.

197. Yoshioka, M., S. Yoshioka, Z. Tamura, and K.Ohta. 1968. Growth responses of Bifidobacte-rium bifidum to coenzyme A, its precursors andcarrot extract. Jap. J. Microbiol. 12:395-402.

198. Zilliken, F., G. A. Braun, and P. Gyorgy. 1956."Gynaminic acid" and other naturally occur-ring forms of N-acetylneuraminic acid. Arch.Biochem. Biophys. 63:394-402.

199. Zilliken, F., C. S. Rose, G. A. Braun, and P.Gyorgy. 1955. Preparation of alkyl N-acetyl-a-and-,B-D-glucosaminides and their microbio-logical activity for Lactobacillus bifidus var.Penn. Arch. Biochem. Biophys. 54:392-397.

200. Zilliken, F., P. N. Smith, C. S. Rose, and P.Gyorgy. 1954. Enzymatic synthesis of a growthfactor for Lactobacillus bifidus var. Penn. J.Biol. Chem. 208:299-305.

201. Zilliken, F., P. N. Smith, C. S. Rose, and P.Gyorgy. 1955. Synthesis of 4-O-fl-D-galac-topyranosyl-N-acetyl-D-glucosamine by in-tact cells of Lactobacillus bifidus varpennsylvanicus. J. Biol. Chem. 217:79-82.

202. Zubrzycki, L., and E. H. Spaulding. 1962. Stud-ies on the stability of the normal human fecalflora. J. Bacteriol. 83:968-974.

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Biology of the Bifidobacteria · 138 POUPARD,HUSA Blaurock (11), Boventer (15), andTomarelli et al. (168, 169) did not accomplish the desired result. It wasnotuntilthemediumofTeplyand