JOURNAL OF BACTERIOLOGYVol. 87, No. 1, pp. 162-170 January, 1964Copyright © 1964 by the American Society for Microbiology

Printed in U.S.A.

THE GENUS VEILLONELLA

I. GENERAL CULTURAL, ECOLOGICAL, AND BIOCHEMICAL CONSIDERATIONS

M. ROGOSALaboratory of Microbiology, National Institute of Dental Research, National Institutes of Health,

Bethesda, Maryland

Received for publication 10 October 1963

ABSTRACT

ROGOSA, M. (National Institutes of Health,Bethesda, Md.). The genus Veillonella. I. Generalcultural, ecological, and biochemical considera-tions. J. Bacteriol. 87:162-170. 1964.-Argumentsare presented for excluding Veillonella discoides,V. reniformis, V. orbiculus, and V. vulvovaginitidisfrom the genus, and for restricting it to aerogenicorganisms such as V. parvula and V. alcalescens.The genus Veillonella thus would comprise specieswhich are anaerobic and nonmotile; are small,spherical, gram-negative cocci appearing as pairs,masses, and short chains; and are cytochrome-oxidase- and benzidine-negative. Veillonella wouldbe further characterized in that glucose or anyother carbohydrate is not fermented; indole isnot produced; gelatin is not liquefied; nitrate isreduced; H2S is produced; propionic and aceticacids, C02, and H2 are produced from lactateduring growth; and pyruvic, oxaloacetic, malic,fumaric, and succinic acids are metabolized byresting cells, but citric, isocitric, and malonicacids are not. In addition to the above, a numberof cultural, ecological, and biochemical character-istics are described. At present, V. parvula (thetype species) and V. alcalescens would be retainedas valid species. Errors in the descriptions of V.parvula and V. alcalescens are corrected byamended statements. These species are differ-entiated serologically. Also, V. alcalescens differsfrom V. parvula in having an absolute require-ment for putrescine or cadaverine and in decom-posing H202.

The entire comment on the genus Veillonellaby Topley and Wilson (Wilson and Miles, 1955)is restricted to the following short paragraph:"We should perhaps refer to a group of anaerobicgram-negative cocci found mainly in the mouthand intestine of man and animals to which thegeneric name Veillonella was given by Prevotafter Veillon and Zuber (1898) who described thefirst species. The organisms are small spherical

cocci about 0.3 microns in diameter, usuallyarranged in pairs. They grow under anaerobicconditions on the usual media, having limitedfermentative powers, and appear to be harmlesssaprophytes, though they may at times invadethe blood stream after operations on the mouth(see McEntegart and Porterfield, 1949). Twospecies are recognized-Veillonella parvula andVeillonella gazogenes. It is very doubtful whetherthese organisms, some of which are gram-positive,form a homogeneous group, and we should forthe present not regard Veillonella as a validgenus."

Reviews of the earlier literature are availablein papers by Hall and Howitt (1925), Branham(1927, 1928), and Pr6vot (1933). The scheme ofPr6vot (1933) was followed by the 5th and 6theditions of Bergey's Manual (Bergey et al., 1939;Breed, Murray, and Hitchens, 1948), whichplaced the genus Veillonella in the family Neis-seriaceae and named two species, V. parvula andV. gazogenes. Both species were described asaerogenic; the gases produced by V. parvulawere mentioned as C02, H2, and H2S. V. gazo-genes was differentiated from V. parvula asfollows. "Distinctive characters: Differs fromVeilloneUa parvula in that it does not fermentsugars, does not produce H2S nor indole, is nothemolytic, does not produce nitrites from nitrates,and does not develop fetid odors."Foubert and Douglas (1948a, b) described an

oral organism, otherwise having the character-istics of a Veillonella species (Bergey et al., 1939;Breed et al., 1948), as gram-positive in the earlylogarithmic growth phase. For this reason, theyallocated the organism to the genus Micrococcusand named a new species, M. lactilyticus. Lang-ford, Faber, and Pelezar (1950) contested thisconclusion and insisted that the Veillonellaorganisms isolated from the human mouth weregram-negative diplococci. In a general study of

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anaerobic cocci, Hare et al. (1952) recognizedtheir group V as members of the genus Veillonella,as described in Bergey's Manual (Bergey et al.,1939; Breed et al., 1948).The 7th edition of Bergey's Manual (Breed,

Murray, and Smith, 1957) recognizes four newspecies, and introduces a new heterogeneity bycreating two divisions in the genus; three of thespecies are described as producing gas in culturemedia, and three species as not producing gas.In addition, four of the species are incompletelydescribed, unavailable for study, or based on theexamination of single strains isolated infrequentlybefore modern techniques were available.The evidence to be presented will show that,

in all probability, the descriptions of V. parvulaand V. alcalescens (V. gazogenes) in the 5th, 6th,and 7th editions of Bergey's Manual and that ofPr6vot (1933) are erroneous, particularly inrespect to the pertinent differentiating char-acteristics, i.e., the fermentation of sugars, in-dole and H2S production, and nitrate reduction.

Except for some fragmentary studies of smallnumbers of strains from human sources (Halland Howitt, 1925; Branham, 1928), no systematicserological study of the genus has been conducted.A preliminary report on serological and otherstudies of the genus Veillonella was presentedby Rogosa, Hampp, and MacKintosh (1961).The present paper and later reports will describemore extensive serological, biochemical, andcultural experiments with significant numbers ofstrains from man, the rat, the hamster, and therabbit, in an attempt to contribute toward abetter understanding of the genus.

MATERIALS AND METHODS

Source of cultures. The first experiments wereperformed with organisms isolated from thehuman mouth by Langford et al. (1950), andalso with the reference cultures listed by them.The cultures were revived from the lyophilizedstate in V16 or V17 medium (described below),and were replated repeatedly for purity in theV15 medium and under the conditions describedby Rogosa (1956) and Rogosa et al. (1958).Later, more isolates from the human mouth andalso from bacteremias after certain dental operat-ing procedures (Rogosa et al., 1960) were in-cluded. In all, 38 human strains were studied.

Samples from the rat, rabbit, guinea pig, andhamster were obtained by swabbing the oral

cavity of 25 animals of each species. The swabswere placed into tubes of fresh V17 broth con-taining either vancomycin (7.5 ,ug/ml) or strep-tomycin (5 ,ug/ml), then diluted 1:100 and1:1000, and pour-plated in V15 medium con-taining either of the above antibiotics. Plateswere incubated 3 days at 36 C in an atmosphereof 95% N2 and 5% CO2. Isolates from the rat(19), from the hamster (24), from the guineapig (11), and from the rabbit (15) remained viablethroughout this study and comprised only 1isolate per animal.

MIaintenance of cultures. All cultures werelyophilized from skim-milk suspensions, andremained viable at 5 C for at least 5 years. Theywere also grown in medium V22A consisting ofTrypticase (BBL), 1 %; yeast extract (BBL),0.5%; 85% lactic acid, 1 %; sodium thioglycolate,0.075%; Tween 80, 0.01%; agar, 0.1 %; adjustedwith solid K2CO3 to pH 6.6. This medium wasdispensed in 14-ml amounts into screw-cappedtubes (15 by 125 mm) and autoclaved at a pres-sure of 15 psi for 15 min. Each tube was inocu-lated with ca. 2.5 ml of culture, incubated at 36 Cfor 24 to 48 hr, and then stored at 5 C. Transfersof stock cultures were made monthly.

Media. All media were routinely boiled andcooled to an appropriate temperature immedi-ately before use. The V15 medium was the solidagar medium used as a selective medium fordirect plating of specimens by Rogosa (1956)and Rogosa et al. (1958). The V16 medium was amodification of V15 medium only in the reduc-tion of the agar content to 0.1%. The V17 me-dium was simply a broth medium with agar andbasic fuchsin omitted, but otherwise having thesame composition as the V15 medium. The V23medium consisted of Trypticase, 1 %; yeastextract, 0.5%; sodium thioglycolate, 0.075%;Tween 80, 0.1%; 85% lactic acid, 1%; adjustedwith solid K2CO3 while stirring, to pH 6.5 to 6.6.

Carbohydrate fermentation tests. Fermentationof carbohydrates was tested in V17 mediummodified by the omission of sodium lactate.Arabinose, galactose, fructose, maltose, mannose,rhamnose, sorbose, and xylose were sterilized byfiltration and added aseptically. Inulin wasalways autoclaved in the medium. Adonitol,amygdalin, cellobiose, dulcitol, glucose, glycerol,inositol, lactose, mannitol, melibiose, melezitose,a - methyl - D - glucoside, a - methyl - D-mannoside,raffinose, salicin. sorhitol, sucrose, and trehalose

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were sterilized by filtration. Separate portionswere also sterilized by autoclaving. The finalconcentrations of carbohydrates were 0.5 to 2%in different experiments. Tubed media were

arranged in racks allowing direct contact ofsteam with each tube, and were autoclaved byquickly raising the temperature to 121 C and byimmediately beginning the exhaust cycle. Cellsfrom 24- to 48-hr V17 broth cultures were washedtwice in freshly sterilized distilled water con-

taining either 0.01 % Na2S -9H20 or 0.0125%sodium thioglycolate, and diluted to the originalvolume; 1-drop inocula were then made. Inocu-lated tubes of basal medium without addedcarbohydrate were always included. Anaerobiosiswas effected within 10 min by repeated evacua-

tion and flushing with 95% N2 plus 5% CO2(at least four times) of McIntosh and Fildesaluminum jars. Slight positive pressure (100mm of Hg) was maintained, and no results were

accepted as valid unless positive pressure was

still present after the usual 2-week incubationat 36 C. The pH of all tube contents was deter-mined electrometrically.

Biochemical tests. Glucose was estimated bymeans of a coupled enzyme system involvingglucose oxidase and peroxidase (Keilin andHartree, 1948; Keston, 1956; Teller, 1956). The"glucostat" reagents supplied by the Worthing-ton Biochemical Corp. (Freehold, N.J.) were

used, and the determinations were accurate tod3%.Tests for indole were performed in V17 me-

dium by the Kovacs method described in theM1anual of Microbiological Methods (Society ofAmerican Bacteriologists, 1957), and were re-

peated many times in 107 cultures during growthperiods of 6 hr to 2 weeks. Positive reactionswere detected by the immediate red-color de-velopment when drops of reagent were introducedat the surface of cultures. Delayed reactions inwhich brown or greenish-brown colors occur are

not caused by the presence of indole but are

the result of nonspecific reactions and decomposi-tion of the reagent (Society of American Bac-teriologists, 1957).

"Catalase" tests were done repeatedly on

washed cells from V17 broth, from growth on

anaerobic streak plates, and from colonies inpour-plates; 5%7, H202 freshly diluted from re-

frigerated 30% H202 was used, and special care

was taken to avoid mixing peroxide and cellswith anv metal objects. These tests were done

immediately after removing cultures or cellsfrom an anaerobic environment, and also afterat least 1-hr exposure in the air.The benzidine test, using both the benzidine

base and the dihydrochloride for the presumptivedetection of iron porphyrin compounds, wasgenerally done as described by Deibel and Evans(1960) on plate cultures and cell suspensions frombroth cultures. Since the benzidine base is tentimes more sensitive than the hydrochloridesalt, cell suspensions were washed three times indistilled water to remove interfering components(e.g., excess Fe and Cu), and were resuspendedin 0.5 ml of the usual 5% H202 .H2S production was detected by the blackening

of cultures grown in V17 and V23 broth mediasupplemented with 0.5 g per liter of ferric am-monium citrate as an internal indicator. Thiscompound was nontoxic. Tests were performedunder a variety of conditions and medium supple-mentations with 26 sulfur-containing compounds.

Nitrate reduction was determined in V17 brothcontaining 0.1% KNO3 and adjusted to pH 7.5.Cultures were grown in this medium in the pres-ence and absence of thioglycolate, and nitritewas detected with the reagents and the spot-plate technique described by Rogosa (1961).

Tests for oxidase enzyme were conducted byflooding the growth on V15 agar plates with 1 mlof a 1% solution of dimethyl-p-phenylenediaminehydrochloride or the oxalate salt, and were ob-served for a period of 10 min for the usual colorchanges from pink to black.The ability of resting cells to metabolize a

number of compounds with the production ofC02, H2, or both, was tested by Warburgmanometry at 37 C in an atmosphere of N2 or95% N2 plus 5% C02. Cultures were grown 1day or less in V23 lactate broth. The cells werewashed once in distilled water, and resuspendedin distilled water to contain ca. 20 mg (dryweight) per ml. The cells were kept cold duringcentrifugation and washing procedures, gassedwith the above mixture in all resuspending opera-tions, qu:ckly introduced into the flasks alreadycontaining all other components, and gassedimmediately while shaking for 10 min. The maincompartment of each flask contained 1.0 ml of0.05 M potassium phosphate buffer (pH 6.5),0.3 ml of water, and 1.0 ml of the cell suspension.From a side arm was tipped 0.2 ml (20 ,UM) of theNa or K salts of each of the following acids:pyruvic, oxaloacetic, L(-)-malic, fumaric,

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succinic, isocitric, citric, malonic, L( +)-tartaric,and formic. Lactic acid was used as the Li, Na,or K salt.

Propionic and acetic acids were detected byCelite chromatography essentially as describedby Swim and Utter (1957). Also, ether extractsof the fatty acids were analyzed by means of aBeckman GC-2A gas chromatograph and re-corder. The copper column was packed with 15%polydiethylene glycol adipate (Hunter, Ortegren,and Pence, 1960) on Chromosorb W (60 to 80mesh; Johns-Manville Co., New York, N.Y.).Helium at 30 psi was the carrier gas, and thetemperature was 130 C. CO2 and H2 were deter-mined by differential manometry, in which 0.1ml of 50% KOH was injected at the end of thegrowth period through a rubber cap sealed onone of the side bulbs. The flasks were then shakenuntil equilibrated.

RESULTS

Occurrence and frequency distribution. Inhundreds of human salivary samplings, and inequally large numbers of oral specimens fromthe rat, the hamster, the rabbit, and the guineapig, there has never been an instance in whichVeillonella organisms were not recoverable inthe selective media of Rogosa (1956) and Rogosaet al. (1958). However, only one strain from 1 of78 mice was isolated, despite the use of enrich-ment cultures of combined fecal and oral ma-terial in V16 medium with and without vanco-mycin, streptomycin, azide, or cyanide. In thepresent study, confirming Douglas' (1950) ob-servation, the Veillonella organisms were presentin human saliva in nearly the same order ofmagnitude as the streptococci, the single largestgroup of organisms. In the rat, also, the veil-lonellae constituted 9% of the measurable totalorganisms and were half a numerous as the strep-tococci.

Morphology. The organisms were small spheri-cal cocci, generally 0.3 to 0.4 , in dilameter, butoccasionally smaller. Although Langford et al.(1950) contended that the basic cellular arrange-ment was the diplococcus, in wet mounts orgram-stained preparations from liquid media,the cells occurred as diplococci, irregular masses,short chains, or combinations of these forms.This agrees with descriptions of Pr6vot (1933).Some strains occasionally did not counter-stainwell, and were barely visible at a magnificationof 1,350 X. Cells from fluid media were gram-

negative in thin smears by a variety of gram-stain modifications. Motility was never de-tected.

Colonies in uncrowded poured-agar plateswere generally 1 to 3 mm in their greatest dimen-sion, and were smooth; entire; lens-, diamond-,or heart-shaped; opaque; grayish white; andbutyrous or soft to the touch.

Temperature relations. Growth was good inthe range of 30 to 37 C. At 40 C, all 107 strainsgrew through one transfer, but 42% of themfailed to grow subsequently. There was no growthof any strain in the second serial passage at 45 C,and only infrequently (16%) did growth occurin the first transfer. At 24 C, however, all culturesgrew well through serial transfers, althoughsometimes marked turbidity or other evidence ofgrowth developed only after 7 days of incubation.There was no growth at 18 C, and no organismsurvived heating at 60 C for 30 min.

General cultural and physiological charac-teristics. Continued aerobic growth occurredonly occasionally when such massive inocula as5 to 10% of the total culture were employed. Inmedia containing reducing agents such as ascorbicacid (0.05%), thioglycolate (0.1%), or cyanide(0.01%), growth often occurred in freshly madeor boiled medium without further anaerobicprecautions, particularly if the surface area-volume ratio was small, and if the medium wasdispensed in air-tight containers. There was nogrowth on the surface of Trypticase Soy Agar(BBL) slants inoculated with either a loop or adrop of broth culture and incubated in air.

All strains were cytochrome oxidase- andbenzidine-negative. Indole was not produced,and gelatin was not liquefied. Nitrate was reducedto nitrite or further-reduced products. H2S wasproduced in V17, V23, or in a defined medium tobe described elsewhere, from reduced glutathione(1.65 x 10-3 M), cysteine (8.25 X 10-4 M), orboth, and from KSCN (1.65 X 10-3 M).

Carbohydrates were not fermented by any ofthe 107 strains of Veillonella. Litmus and anumber of sulfonphthalein indicators, particularlyphenol red, bromothymol blue, bromocresolpurple, and chlorophenol red, were reduced totheir leuco state. Therefore, electrometric pHdeterminations were made, with results frominoculated tubes without added carbohydratealways included. Such media initially adjustedto pH 7.5 and incubated anaerobically in 95%N2 plus 5% CO2 or 95% H2 plus 5% CO2 gen-

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erally equilibrated at pH 7.2. If inoculated mediawithout added carbohydrates contained sufficientlactate, hypoxanthine, and other fermentablesubstrates derived from enzymatic digests ofcasein and yeast extract, the pH value fell to6.8 as the medium became saturated with theCO2 produced. It is only at initial acidic pHvalues, as demonstrated by Douglas (1950), thata rise in pH takes place.

Because V. parvula is described as fermentingglucose by Prevot (1933) and in the 5th, 6th,and 7th editions of Bergey's Manual, 107 strainsof Veillonella, including a strain of V. parvulastrain Te3 originally from Pr6vot, were testedfor this property by means of the glucose oxidasetechnique described earlier. Glucose was notutilized by any of the strains. The mean residualglucose concentration after 96 hr at 36 C was99.2 ±t- 3% of the initial 0.0096 M concentration.The effect of CO2 in the initiation of growth of

50 representative strains was tested by inoculat-ing a drop of culture from V23 broth into twotubes of the same medium. To one duplicate setof plugs were added 3 drops of 50% pyrogallicacid and 3 drops of 10% KOH, and to the otherset were added 3 drops of pyrogallic acid and 3drops of freshly prepared 10% K2CO3. Rubberstoppers were quickly inserted. Only six strainswere not limited in growth in a C02-free atmos-phere after 16 hr of incubation. In the absenceof CO2, 83% of the cultures had a mean opticaldensity (OD) of 0.08. This contrasts with thecultures grown in C02, 77% of which had anOD > 1.0 after 1 day, and none of which had anOD < 0.82. Although most strains eventuallygrew to some extent in the absence of CO2, 20%of the cultures did not grow, or grew very sparsely(OD < 0.10), even after long incubation.After 16 hr at 36 C in an atmosphere of 95%

N2 plus 5% C02, growth was absent at pHvalues < 5.5, barely observable at pH 6.0, goodfrom pH > 6.0 to 6.5, maximal from pH 7.0 to8.5, and suddenly ceased between pH 8.5 and9.0. The initial pH values supporting growth insimilar media were generally higher than Douglas(1950) reported for one strain (>5.5 as comparedwith 4.8). When the fermentation of lactate tookplace in an initially acidic environment (pH6.6), the pH value increased to 7.5. At slightlyalkaline initial pH values, the resultant pHalways decreased (from pH 8.0 to pH 6.9 to 7.6).The Veillonella strains were markedly re-

sistant to Tween 80. With 10% Tween 80, the

medium tended to separate into distinct layers,indicating obvious physical changes; yet, moststrains were able to grow. Further experimentswith six detergents were performed with V23medium without Tween 80. Heavy precipitatesand cloudiness were characteristic of media con-taining the higher concentrations of Triton Gr-5(Rohm & Haas, Inc., Philadelphia, Pa.), Glim(B. T. Babbitt, Inc., New York, N.Y.), or so-dium dodecylsulfate. Nevertheless, growth wasevident in such nonionic substances as TritonX-100 and Glim at the surprisingly low dilutionof 1:250; in trithiosulfonate at 1: 1,000; and evenin the more highly ionized Aerosol OT (FisherScientific Co., Pittsburgh, Pa.) and sodium do-decylsulfate, where the antibacterial effect maybe due only partially to decreased surface tension,at dilutions of 1:26,666 or less.The Veillonella were inhibited by 1% NaCl in

V23 medium, and failed to grow in 4% NaClmedia. The resistance of these organisms to bilewas often not as great as was anticipated, evenin the case of those strains isolated from theintestinal tract. Although all strains grew in 5%bile, growth was often markedly delayed. Of 18representative strains tested, 7 did not grow in10% bile media, 11 in 20%, and 16 in 30%; only3 strains grew weakly in 40% bile after extendedincubation. Also, no growth occurred in thepresence of potassium tellurite (10 mg per liter),2,3,5-triphenyl tetrazolium chloride was notreduced, and certain dyes (0.0002%) were vari-ably inhibiting. Crystal violet in V23 mediumprevented the growth of 9 of the 18 strains after1 day of incubation; after 96 hr, 1 strain still didnot grow. Although growth generally occurredwith relatively long incubation, at 96 hr cellcrops were usually only equal to, or less than,control growth after 24 hr or less. Brilliant green,although in most cases considerably less inhibit-ing than crystal violet, prevented the growth of3 of the 18 strains in Sims and Snyder's (1958)selective medium, and 8 of 18 in V23 mediumafter 24 hr at 36 C. Because the dye was reducedto the leuco compound in V23 medium, it appearsthat the leuco compound was more toxic than itscolored counterpart. However, when growthwithout lag occurred in brilliant green media,it was as good as in control media without dye.But even with extended incubation, 3 of 18 and2 of 18 strains failed to grow in Sims and Snyder'sand V23 medium, respectively.

Biochemical characteristics. In the present work,

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the only significant fermentation products de-tectable in the fermentation of lactate by growingcultures were propionic acid, acetic acid, C02,and H2. This agrees with previous observationsby Johns (1951b) and Foubert and Douglas(1948b).We wished to determine whether resting cells

of our strains would utilize pyruvate, oxaloace-tate, malate, fumarate, and succinate, as reportedby Johns (1951a, b) for a single strain isolatedfrom the rumen of the sheep and designated byhim as V. gazogenes (V. alcalescens). It was indeedfound that all strains, regardless of animal source,serological group, nutritional behavior, and lackor possession of a peroxide-decomposing enzyme,would metabolize these compounds in an atmos-phere of N2. Compounds not attacked wereL( +)-tartaric acid (by unadapted cells) andformic acid, as Johns (1951b) showed. In addi-tion, citric, isocitric, and malonic acids were notattacked. Although succinic acid was dissimilatedby resting cells with the production of propionicacid and C02, all strains were incapable ofgrowth in succinate media. This phenomenonwill be discussed more fully elsewhere.

Lactic acid was fermented poorly or not at allby resting cells, and Johns' (1951b) finding thatbicarbonate and CO2 stimulated the attack onlactate was confirmed with all our strains. How-ever, in Johns' (1951b) experiments, and alsoin ours, the rate and extent of lactate fermenta-

140_

120_

100 _

800u

MINUTES

FIG. 1. Aerobic respiration of Veillonella on

lactate typified by the data obtained with V. alcales-cens BL-78.

+

0 I'Jf

5 10 15 20 25 30 35 40 45 50 55 60MINUTES

FIG. 2. Typical aerobic dissimulation of oxalo-acetate by Veillonella sp. Z24B.

tion was still highly limited. In the best instance,only 6 tmoles of an initial 20 ,moles of lacticacid disappeared after 2 hr. It appears that thereprobably are more important factors thanbicarbonate and CO2 in the fermentation oflactate. This problem is currently under study.

Surprisingly, two compounds were subject toaerobic attack. Resting cells respired on lactatewith a quick net uptake of 02. The data for atypical experiment are shown in Fig. 1. Such aphenomenon was observed with another lactate-fermenting anaerobe, Butyribacterium rettgeri(C. L. Wittenberger, personal communication).The Veillonella species also metabolized oxalo-acetic acid aerobically, with the production of CO2and H2 in a molar ratio approaching 1:1 (meanof four experiments). Other products have notyet been studied. The results of a typical experi-ment are depicted in Fig. 2.

DISCUSSION

It was not always easy to obtain pure culturesof Veillonella. Common contaminations ofVeillonella strains from rodent specimens wereshort, coccoidal, gram-negative rods growing in athin, spreading, almost invisible film on isolationplates. Like the veillonellae, these rods wereresistant to vancomycin, but streptomycin and2.5% agar media often inhibited spreading, so

that, with patience and repeated purificationprocedures, pure cultures of Veillonella were

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obtained. We have seen that pure cultures ofVeillonella do not utilize glucose or any othercarbohydrates. However, in some fresh isolatesof apparently pure cultures of Veillonella, glucoserapidly disappeared; but, on careful examination,it was discovered that these were always con-taminated with glucose-utilizing organisms suchas streptococci, diphtheroids, Bacteroides, andProteus. After purification of the original isola-tion, however, carbohydrates were not fermented.

Berger (1960) was not sure of the significanceof weak, delayed, benzidine reactions. Suchdelayed results are highly doubtful becausethe peroxidase-H202 complex has an extremelyrapid reaction rate (Chance, 1949a, b), and Deibeland Evans (1960) showed that the benzidinebase, as contrasted with the chloride salt, gaverise to false-positive weak reactions. The Veil-lonella species gave us a benzidine-negativereaction. Berger (1961) described the Veillonellagenus as the "anaerobic, oxidase-negativespecies." We agree with this description, andthis plus the negative benzidine reaction serve,among other things, to differentiate the genusVeillonella from the genus Neisseria.Deibel and Evans (1960) showed that an

organism may decompose peroxide to water and02 (a catalase?), and may not have detectableiron porphyrin compounds; Delwiche and John-ston (1962) confirmed the existence of a nonheme,peroxide-decomposing enzyme. V. alcalescensand certain animal strains appear to be in thiscategory.Of the species described in the 7th edition of

Bergey's Manual, V. discoides, V. reniformis, andV. orbiculus have disc- or kidney-shaped cellstwo to five times greater in diameter than thespherical cells of V. alcalescens and V. parvula,the type species. The metabolism of V. discoides,V. reniformis, and V. orbiculus seems distinc-tively different from that of the aerogenic organ-isms, V. parvula and V. alcalescens, because theother three species are described as not producinggas. Another organism, V. vulvovaginitidis (Breedet al., 1957), although described as having spheri-cal cells three times larger than those of the typespecies, liquefies gelatin, and does not producegas. V. vulvovaginitidis was originally designatedas N. vulvovaginitis by Reynes (1947) on thebasis of a single reported isolation. The observa-tion by Langford et al. (1950) that this singlestrain is a long-chained, gram-negative strepto-Poccus, occurring often in chains of ten or more

cells, was confirmed by us from studies of theonly available strain (247-D) received fromPr6vot. A rereading of Reynes' (1947) short noteleaves an impression consistent with the judg-ment that this organism is a streptococcus. Thus,there are basic differences in morphology andmetabolism between these four organisms andV. parvula and V. alcalescens. It is thereforesuggested that V. discoides, V. reniformis, V.orbiculus, and V. vulvovaginitidis be excludedfrom the genus Veillonella.The Veillonella strains responded to poly-

myxin B, an agent considered to be specificagainst gram-negative species, as would beexpected of typical gram-negative organisms;in this respect as well as in the responses tovancomycin and novobiocin, the organisms re-sembled the seven nonpathogenic strains ofNeisseria tested (Fitzgerald, Parramore, andMacKintosh, 1959). Colonies were not formedin media containing 0.25% phenethyl alcohol;this is also generally characteristic of gram-negative bacteria (Lilly and Brewer, 1953;Berrah and Konetzka, 1962). In addition, lipo-polysaceharide fractions with properties char-acteristic of endotoxins from gram-negativebacteria were extracted from Veillonella cultures(Mergenhagen, Hampp, and Scherp, 1961). This,and direct results from gram-stained smears,reinforce the view that the genus Veillonella isgram-negative. M. lactilyticus strains T9 and T13(Foubert and Douglas, 1948a, b) have behavedlike Veillonella organisms in these and otherrespects, and it appears, therefore, that thename M. lactilyticus is not justified.The results presented here are not in complete

agreement with Pr6vot (1933) or with Bergey'sManual, because V. parvula is described asfermenting glucose and certain other sugars andas producing indole, whereas V. alcalescens isdescribed as not producing H2S and as not reduc-ing nitrate. On the other hand, the present resultsgenerally agree with those of Langford et a].(1950), Foubert and Douglas (1948a, b), Hareet al. (1952), Berger (1960), and Sims (1960). Inaddition, the present negative sugar reactionsagree with those obtained by Hall and Howitt(1925) and the description for M. gazogenesalcalescens anaerobius by Lewkowicz (1901).Prevot (1933) and Bergey's Manual state thatV. parvula is identical with Staphylococcusparvulus (Veillon and Zuber, 1898). The evidencefor this is a statement by Pr6vot (1933) that one

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CHARACTERISTICS OF GENUS VEILLONELLA

strain was authenticated by Veillon. However,it appears that Veillon and Zuber's (1898) strainswere not extant, and the only differentiatinginformation then available was that the originalorganism was a small gram-negative coccus,appearing as masses and diplococci, and produc-ing gas. These limited criteria apply to eitherV. parvula (S. parvulus; Veillon and Zuber,1898), or V. alcalescens (M. gazogenes alcalescensanaerobius; Lewkowicz, 1901).From the foregoing evidence, it is not possible

to differentiate between V. parvula and V.alcalescens. Furthermore, since the describednegative sugar reactions of V. alcalescens (Pr6vot,1933; Bergey et al., 1939; Breed et al., 1948,1957) may be considered correct, investigatorsgenerally designated any Veillonella isolate asV. alcalescens because their organisms possessedat least the important property of not fermentingcarbohydrates, even if their strains did not other-wise conform to previously published descrip-tions. This situation seems discouraging, but, ifwe accept the present behavior of Pr6vot'soriginal strains of V. parvula Te3, V. alcalescens259, and many others identical with them, thedescription of the genus should be presentlyamended to include only the two species, V.parvula and V. alcalescens, as was done in the 6thedition of Bergey's Manual. In addition, thespecies descriptions (Breed et al., 1957) shouldbe amended in the following specific respects:V. parvula, (i) carbohydrates are not fermented,(ii) indole is not produced; V. alcalescens, (i)H2S is produced, (ii) nitrate is reduced.

Such amended descriptions of the genus and ofthe species would still not permit the differentia-tion of V. parvula and V. alcalescens. However,unpublished but completed experiments, tooextensive to report here, indicate that the speciesof human origin are distinguishable in the follow-ing respects: (i) each species has distinctivenoncrossing agglutinogens; (ii) V. alcalescenspossesses a peroxide-decomposing enzyme notpresent in V. parvula; and (iii) V. alcalescens hasan absolute growth requirement for putrescineor cadaverine in a nutritionally defined medium.

ACKNOWLEDGMENTS

The author gratefully acknowledges the in-valuable assistance and cooperation of manyindividuals who have been engaged in this worksince 1954. Particular thanks are owing to the

highly qualified skills of M. Elizabeth MacKin-tosh, Alfred J. Beaman, and Ferial S. Bishop.

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