APPLIED MIcRomoLoGy, July 1969, p. 80-87Copyright @)_1969 American Society for Microbiology

Vol. 18, No. 1Printed In U.S.A.

Identification of Human Strains ofActinomyces viscosus

MARY A. GERENCSER AND JOHN M. SLACKDepartment of Microbiology, Medical Center, West Virginia University, Morgantown, West Virginia, 26506

Received for publication 1 April 1969

Catalase-positive actinomycetes which closely resemble the "hamster organism"described by Howell have been isolated from dental calculus and other humansources. These cultures could not be distinguished from the hamster strains on thebasis of morphology, oxygen requirements, biochemical reactions, or cell wall com-position. These human isolates have been classified with the hamster strains asActinomyces viscosus. The strains from hamster and human sources fell into twoserotypes. Serotype 1 contains the hamster strains plus one strain of unknown origin,whereas serotype 2 contains all of the human strains.

A gram-positive, catalase-positive, filamentousorganism isolated from subgingival plaque inhamsters was described by Howell (5) and Howelland Jordan (6). The association of this organismwith spontaneous periodontal disease in hamstersand its ability to produce the disease experi-mentally was demonstrated by Jordan and Keyes(9). Howell et al. (7) classified the "hamsterorganism" as Odontomyces viscosus, indicatingthat it differs considerably from Nocardia andRothia, and, although it closely resembles Actino-myces naeslundii, it differs by being aerobic andcatalase-positive. The "Subgroup on Taxonomyof the Microaerophilic Actinomycetes" recom-mended that the generic description of Actino-myces be broadened to include catalase-positiveorganisms. Therefore, Georg, Pine, and Gerenc-ser (2a) have proposed that 0. viscosus be re-named Actinomyces viscosus.The isolation of catalase-positive bacteria

similar to the "hamster organism" from thehuman mouth was reported by Gerencser andSlack (Int. Ass. Dental Res., p. 45, 1967). Also,Snyder et al. (13) observed this organism inhuman plaque material by using the indirectfluorescent-antibody (FA) technique.

This report compares the morphology, bio-chemical reactions, and serology of 19 strains ofcatalase-positive actinomycetes isolated fromhuman sources with hamster strains of A. viscosusand with human strains of A. naeslundii.

MATERIALS AND METHODSCultures. The human and hamster isolates and

strains of A. naeslundii used in this study are listed inTable 1. As the human and hamster strains are essen-tially identical, they are both referred to as A. viscosusin this report.

Morphology. The cultures were streaked on BBLBrain Heart Infusion Agar (BHIA) and were incu-bated aerobically with CO2 (candle jar) at 35 C.Microcolonies were observed after 24 and 48 hr ofincubation at a magnification of 100 to 400X.Mature colonies were observed with a stereoscopicmicroscope at magnifications of 25 to 40X after 7 to14 days. The type of growth produced in broth wasobserved in Fluid Thioglycollate Medium (Difco),Brain Heart Infusion Broth (BBL), and TrypticaseSoy Broth (BBL). Gram stains and dark-field prepara-tions were made from both liquid and solid media.

Determination of oxygen requirements. The abilityof the cultures to grow aerobically, aerobically withC02, anaerobically, and anaerobically with CO2 wasdetermined by the technique described by Gerencserand Slack (3).

Biochemical tests. Tests for catalase, indole, nitratereduction, methyl red reaction, acetoin, starch hydrol-ysis, and gelatin hydrolysis were done by the pro-cedures recommended by the"Subgroup on Taxonomyof the Microaerophilic Actinomycetes" (11).

Carbohydrate fermentation tests were done withthree basal media: Actinomyces Fermentation Broth(BBL), a thioglycollate fermentation base, and ameat extract-peptone fermentation base. The thio-glycollate fermentation base was prepared by adding2 g of yeast extract (glucose-free) and 2.0 ml of 1.0%aqueous bromocresol purple to each liter of Thio-glycollate Medium without dextrose or indicator(Difco). The meat extract-peptone broth with An-drade's indicator was prepared as described by Brownet al. (1). Each basal medium was tubed in 9.0-mlamounts and sterilized by autoclaving. Filter-sterilizedcarbohydrates were added to a final concentration of1.0%, except starch, salicin, and inulin which wereused at 0.5%. These tests were incubated at 35 C asfollows: Actinomyces broth with sodium carbonate-phosphate buffer seals (11), the meat extract broth ina candle jar, and the thioglycollate broth aerobically.

Esculin hydrolysis was determined by use of asemisolid medium made by adding 1.0 g of esculin

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TABLE 1. Laboratory number and source of cultures of A.viscosus and A. naeslundii

W.V.U. no. Received from Origina designation ClinicaJl smrce

A. viscosus220 Institut Pasteur, Paris A. bovis 1488 Human pyorrheae229 Institut Pasteur, Paris A. baudetii Unknown371 P. Negroni, Argentina A. discofoliatus 112, Human440 L. Georg, Atlanta, Ga. CDC X601; Howell HS-2 Hamster443 L. Georg, Atlanta, Ga. CDC X602; Howell HS-36 Hamster472 L. Georg, Atlanta, Ga. CDC W1020 Human sputum473 L. Georg, Atlanta, Ga. CDC W863 Human wound474 L. Georg, Atlanta, Ga. CDC W1053 Human475 L. Georg, Atlanta, Ga. CDC W872 Human sputum626 C. Cummins, Blacksburg, Va. VPI 3426 Human mouth627 C. Cummings, Blksburg, Va. VPI 3428 Human mouth745 ATCC 15987 (Type strain) Howell T 6 Hamster385 W.V.U. isolate Human dental calculus398B W.V.U. isolate Human dental calculus417 W.V.U. isolate Human dental calculus483 W.V.U. isolate Human dental calculus505 W.V.U. isolate Human dental calculus510 W.V.U. isolate Human dental calculus533 W.V.U. isolate Human dental calculus548 W.V.U. isolate Human dental calculus583 W.V.U. isolate Human dental calculus606 W.V.U. isolate Human dental calculus630 W.V.U. isolate Human dental calculus

A. naeslundii45 ATCC 12104 (Cotype strain) NIH 279 Human sinus398A W.V.U. isolate Human dental calculus509 W.V.U. isolate Human dental calculus550 W.V.U. isolate Human dental calculus631 W.V.U. isolate Human dental calculus

and 1.0 g of agar to each liter of Heart Infusion Broth(Difco). ibe medium was adjusted to p1H 7.0, dis-pensed in 8.0-ml amounts, and s i by autoclav-ing. The i ated medium was incubated for 7days. The development of a brownish-a colorupon addition of a few drops of 1% ferric citrate indi-cated hydrolysis.

For the urease test, 1OX concentrated Urea Broth(Difco) was and filter-sterilized Then 1.0ml of the urea broth was added to 8.0 ml of auto-claved thiogbrcollate femtation base without indi-cator. The inoculated medium waasiuted for 7days. Since the indicator may be reduced, a drop ofphenol red was added just before reading the test.To detect hydrogen sulfide production, lead acetate

paper strips were suspended over inoulated slants ofseveral different media. The media used were: DifcoTriple Sugar Iron Agar (CM3), BRA, BHIA contain-ing 0.3 g of sodium thiosulfate per liter, and LeadAcetate Agar (Difco). All media Wee tubed withdeep butts and short slants and inoculated by streak-ing the slant and stabbing the butt Hydrogen sulfideproduction was m ined after 7 days of incubation.

In addition to testing for nitrate reduction, nitritereduction tests were done by using Trypticase SoyBroth containing 0.01% potassium nitrite (autoclavedin the m ium). Tests were read after 7 days of incu-bation by use of standard reagents to indicate theprence of nitrite.

The inoculum for all of the above biochemicaltests was 0.1 ml of a well-mixed, 24- to 48-hr BrainHeart Infusion broth culture. All except the fermenta-tion test were incubated aerobically with C02 (candlejar) at 35 C.

Cell waD mlys. The cell wall composition of onehaoster isolate and four human isolates was deter-mined for us by Cecil Cu i Anaerobe Labora-tory, Virginia Polytechnic Insun, Blaksburg Va.

Serlogic stulies. Antiserum was prepared andconjugated with fluorescein isothioyanate for A.vicoasus W.V.U. strain 745 (ATCC 15987), for humanstrains W.V.U. 371 (ATCC 19246), 398B, and 220,and for A. naeund W.V.U. strain 45 (ATCC 12104).The methods used for preparing and conjugating theserum, determining the diagnostic titers of the conju-gates, and staining the FA slides were d ribed bySlack et al. (12). The fluorescein to protein ratios ofthe conjugates wer determined by the Nehod ofWells et al. (14). Conjugated antisera to other speciesof Actinomyces and to Arachniapropnzca (Actinomy-ces propwmicus, renamed by Pine and Georg, 1969, Int.J. Syst. Bacteriol., in press), R. dentocariosa (1, 2),Corynebacterium pyogenes, C. acnes, and Bacterionemamatruchotii (4) were also tested.

RESULTSMorphology. The cellular and colonial mor-

phology of hamster strains of A. viscosus and

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FIG. 1. Gram stain of 72-hr growth in Brain Heart Infusion broth, showing branchingfilaments. X 900.FIG. 2. Gram stain of 72-hr growth in Brain Heart Infusion broth, showing diphtheroidal rods. X 900.FIG. 3. Unstained, filamentous, 24-hr microcolony growing on BHIA. X 500.FIG. 4. Unstained, nonfilamentous or smooth, 24-hr microcolony growing on BHIA. X 580.FIG. 5. Mature, smooth, 14-day colony growing on BHIA. X 22.FIG. 6. Mature, 14-day colony with concentric rings growing on BHIA. X 15.

A. naeslundi have been described in detail byHowell and co-workers (5, 7, 8). Our resultswith three hamster strains and five A. naeslundiicultures agree closely with these descriptions;thus, only the human isolates will be describedhere.

All 19 human strains were gram-positive rodswith diphtheroidal cells and filaments (Fig. 1and 2). The shorter rods showed X, Y, and Vforms and some palisading. Branching was

often seen in the filaments which also frequentlyhad swollen, rounded, or clavate ends. Themorphology of the human isolates was indis-tinguishable from that of the hamster strainsand very similar to that of A. naeslundii.

Microcolony morphology. Microcolonies of thehuman isolates had a dense center composedprimarily of diphtheroidal rods with medium-length filaments showing angular branching ex-tending from the center (Fig. 3), which closely

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TABLE 2. Biochemical reactions of hamster andhuman strains of A. viscosus and of A. naeslindii

A. iscsus A.

Determination _ at-

Tr2 Human Total' siYii

Catalase....3./.. . 13/19/19 22/22 0/5Indole ............. 0/3 0/19 0/22 0/5Nitrate.............. 3/3 18/19 21/22 4/5Methyl red. 3/3 18/19 21/22 5/5Voges-Proskauer .... 0/3 0/19 0/22 0/5Urease.............. 0/3 0/19 0/22 0/5H1S onBHIA.0/3 0/19 0/22 0/5TSI... 3/3 19/19 22/22 5/5

Esculin hydrolysis... 3/3 18/19 21/22 5/5Starch hydrolysis 3/3 14/19 17/22 2/5Gelatin hydrolysis. 0/3 0/19 0/22 0/5Arabinosec . 0/3 0/19 0/22 0/5Xylose .............. 0/3 0/19 0/22 0/5Glucose... 3/3 19/19 22/22 5/5Cellobiose........... 0/3 4/19 4/22 1/5Lactose ............. 3/3 13/19 16/22 4/5Melibiose ........... 3/3 14/19 17/22 5/5Sucrose ... . 3/3 19/19 22/22 5/5Raffinose ............ 3/3 18/19 21/22 5/5Inulin.3/3 16/19 19/22 1/5Glycogen............ 0/3 2/19 2/22 0/5Glycerol ............ 2/3 18/19 20/22 5/5Inositol... 3/3 15/19 18/22 5/5Mannitol............ 0/3 0/19 0/22 0/5Salicin .............. 3/3 13/19 16/22 5/5Starch.3/3 18/19 21/22 5/5Trehalose.. 2/3 13/19 15/22 5/5

'Strain 229 of unknown origin is not included.b Number strains positive/number strains

tested.¢ Thioglycollate fermentation base was used,

and when carbohydrates were fermented acid butno gas was produced.

resembled the colonies produced by the hamsterstrains and by A. di. In some strains thisfilamentous fringe was very short. All of thecultures isolated in this laboratory produced fila-mentous microcolonies when first isolated. How-ever, smooth microcolonies were observed afterthe strains had been aintained in the labora-tory, and such colonies were also seen in somestrains sent to us from other laboratories. Thesmooth microcolonies were circular, with ahighly granular surface and an entire or irregularedge (Fig. 4). Some of the colonies on a platemight show short filaments at one point or onone side of the colony. The smooth coloniessometimes had a small, optically dark center andresembled colonies of A. bovis.Mature colony morphology. After 7 days of

incubation, the mature colonies were usuallycircular, raised to convex, smooth, entire, cream-

white, glistening, opaque, and soft to mucoid(Fig. 5). They varied mainly in elevation andsurface topography. Many colonies showedvarious radial or concentric striations (Fig. 6)with shallow central depressions, but the centralpits described by Howell (5) were rarely ob-served.Growth in broth. In Brain Heart Infusion

broth and Trypticase Soy Broth, the A. viscosusstrains grew diffusely and produced a moderateto heavy sediment, which was usually quiteviscous. In thioglycollate broth, the growth wasfinely granular to dispersed and generally washeavier in the upper portion of the tube. Growthof A. naesluni strains was similar except thatthe sediment produced was not viscous.Oxygen requireents. The A. viscosus cultures

grew well aerobically, aerobically plus GO2,and anaerobically plus O2. Although mostcultures grew best aerobically with C02, thedifferences in growth were small. Growth an-aerobically without CO2 was usually less abun-dant. The five A. naesludii cultures gave similarresults but did not show a preference for aerobicgrowth.

Biochemical test. The results of the biochemi-cal and fermentation tests are shown in Table 2.All A. viscosus cultures were catalase-positive.The cultures were tested for catalae by floodingthe slants used in the oxygen requirement testswith H202 immediately after removal of the var-ious seals. All cultures were strongly catalase-positive on aerobically grown slants. Ofthe 23, 14were only weakly positive or were negative on an-aerobically grown ants. All A. naeslwud cul-tures were catalase-negative under all growthconditions.

All of the A. viscosus cultures were indole-negative, acetoin-negative, urease-negative, anddid not hydrolyze gelatin. They were methyl red-positive and hydrolyzed esculin. Most strainsalso hydrolyzed starch. The reactions of A.naes in these tests were essentially the sameas those of A. viscosus, but a smaller percentagehydrolyzed starch.

In Indole-Nitrite medium (BBL), 22 of the23 A. viscosus cultures and 4 of the 5 A. naeslwzdicultures reduced nitrate to nitrite. They did notreduce the nitrite formed. The ability of some ofthese cultures to reduce nitrite was further testedin Trypticase Soy Broth containing 0.01%KNO2. In this medium, the two hamster strainstested consistently failed to reduce nitrite. Ofthe 10 human isolates tested, 4 reduced nitrite,3 did not reduce nitrite, and 3 gave variableresults in repeated tests. One of the two A.naesluwdUi cultures tested reduced nitrite.The results of tests for hydrogen sulfide pro-

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TABLE 3. Comparison offermentation reactions offour strains of A. viscosus andone strain of A. naeslundiiin three basal media

CarbohydratesStrain Basemediumb- Starch

CeHobiose Lactose Glycogen Glycerol Salicin

A. viscosus220 AFB - _ _ - _ _

TFB - A14 lA A7 AiMEP - A14 A. A7 A7 A7

371 AFB - A7 -_ _TFB - A7 A7 AMEP - A7 - A7 A7 A7

398B AFB - _ _ _ _ -TFB - - A A7 A7MEP A14 A7 A7 A7 A7 A7

745 AFB _ A7 _ _ _TFB A14 A7 _ A14 A14 A7MEP A14 A7 A14 A14 A7 A7

A. naeslundii45 AFB _ A14 - A7

TFB A7 A7 A7 A14MEP A14 A7 A14 A7 A7 A7

a Acid was produced from glucose and raffinose but not from xylose or mannitol in all three media.b AFB, Actinomyces Fermentation Broth (BBL); TFB, thioglycollate fermentation base; MEP,

meat extract-peptone with Andrade's indicator.c A7, acid in 7 days; A14, acid in 8 to 14 days; -, no acid in 14 days.

duction on TSI and BHIA are given in Table 2.All strains of A. viscosus and the five A. naeslundiicultures produced H2S, as indicated by a black-ening of the lead acetate papers, from TSI butnot from BHIA. In an attempt to explain thisdifference, BHIA supplemented with sodiumthiosulfate and Lead Acetate Agar (Difco) weretested. All strains grew well and produced hy-drogen sulfide on the supplemented BHIA.Growth was poor on the Lead Acetate Agar,but all strains which grew also produced hy-drogen sulfide. None of the strains blackenedthe indicator in the medium in either LeadAcetate Agar or TSI. These tests show that A.viscosus and A. naeslundii produced hydrogensulfide when the medium contained an adequatesulfur source and when a sensitive indicatorwas used. Several strains of other species ofActinomyces were tested, and all produced H2Sfrom TSI but not from BHIA. This test doesnot appear to be of differential value for specia-tion of Actinomyces.The A. viscosus strains did not grow well

enough in Actinomyces Fermentation Broth(BBL) to give reliable fermentation tests. There-fore, carbohydrate fermentation tests were donein the thioglycollate fermentation base medium,which supported good growth and gave repro-ducible results (Table 2) in repeated tests. Inthis medium, both hamster and human cultures

produced acid without gas from glucose, sucrose,raffinose, glycerol, and starch. The majority ofstrains also produced acid from lactose, melibiose,inulin, inositol, salicin, and trehalose. A fewstrains fermented cellobiose. They did notferment arabinose, xylose, glycogen, or mannitol.When grown in the thioglycollate broth base

medium, the five A. naeslundii cultures producedthe same fermentation pattern as did A. viscosus(Table 2), including acid production from glyceroland starch. When grown in Actinomyces Fer-mentation Broth, A. naeslundii grew well but didnot ferment glycerol, and starch fermentationwas strain-variable. Thus, the ability to supportgrowth does not explain the difference in thesefermentation results. The fermentation of glycerolby A. naeslundii was unexpected as it and otherspecies of Actinomyces were thought not toferment this carbohydrate. Thirteen strains ofA. israelii were then tested for glycerol fermen-tation in both media, but they were entirely nega-tive. The other species have not as yet beentested.A meat extract-peptone base medium has been

recommended for fermentation tests with R.dentocariosa (1); thus, it was tested with eightA. viscosus and one A. naeslundU cultures. Inaddition to some scattered differences with in-dividual strains, there was one consistent changein the fermentation pattern. Seven A. viscosus

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TABLE 4. FA reactions with A. viscosus and A.naeslundi antisera

l bes~~MIt dilution ofIa n o n~~~Of givng POsitive

florescence4s

Cultures tested Serum dilution A A.

74 220 371 398B ds!di45C

A. viscosus, Undiluted 0 0 0 1 1hamster, 4 10 0 0 0 0 0strains 20 0 0 1 0 0

40 1 0 0 0 080 3 0 0 0 0

A. viscosus, Undiluted 8 0 0 0 3human, 19 10 7 0 0 0 5strains 20 3 4 1 1 6

40 1 8 3 5 580 0 7 15 13 0

A. naeslundii, 5 Undiluted 2 0 3 5 0strains 10 0 0 0 0 0

20 3 2 2 0 040 0 0 0 0 180 00 O O° 4

a Fluorescein to protein ratios for antisera tostrains 745, 220, 371, 398B, and 45 are 36, 17, 22,24, 16 pg/mg, respectively.

i Strain 745 (ATCC 15987) is of hamster origin;strains 220, 371 (ATCC 19246), and 398B are ofhuman origin.

¢ Strain 45 is ATCC 12104 of human origin.d Includes one strain (229) of unknown origin.

cultures and the A. naeslidi culture fermentedglycogen. The results of certain fer tatests with five strains in these three media areshown in Table 3, with every strain showing avariation in results. This emphasizes the pointthat, when comparing fermentation resultsamong the Actinomyces, the basal medium mustbe considered and it is best if standardizedmethods are used.

Cell wai comnpodti The four human isolates(398B, 473, 626, 627) examined had the same cellwall composition. The major amino acids werealanine, glutamic acid, lysine, and ornithine.The sugars present were gaLactose, glucose,mannose (trace), and rhanose. The singlehamster strain (443) gave the same results, exceptgalactose was present in only trace amounts.

Serology. The serological reactions of the A.viscosus strains with the FA technique are shownin Table 4. The three strains of hamster originstained to titer with the antiserum (745) againsta hamster strain but did not stain with any of thethree anusera against human isolates (220, 371,398B). Strain 229, which is of unknown origin,stained at 1:40 with the hamster antiserum andshowed low titer cross-reaction with two of the

human sera. The 19 human isolates showedvariations in staining titer with the three antiserato human strains, but they all stained to titerwith at least one of the sera. The human isolatesalso showed considerable cross-staining withthe haster antiserum. This cross-staining wasusually at low titers, but three strains stained at1:20 and one stained at the diagnostic titer ofthe serum (1:40).These results showed a one-way cross-reaction

between the hamster and human isolates. Sorp-tion studies showed that, when ha antiserum745 was sorbed with a human strain, cross-staining to all the human isolates was removed(Table 5). The three hamster isolates and strain229 continued to stain with 745 serum sorbedwith 371, 398B, or 220. Therefore, the strainsfall into serotypes 1 and 2. Serotype 1 containsall of the hamster isolates plus strain 229. (Onreceipt, this strain was labeled Actinobacteriwnbaudetii, but the source was not given. All of thestrains of this species which we can find re-ferred to in the literature were isolated from catsor dogs so that this strain is possibly of animalorigin.) Serotype 2 contains all of the humanisolates.

Cro-reactiowas with A. naed..dL In theircross-reactions with A naeslWi, the hamsterand human isolates showed a pattern simila tothat seen in their cross-reactions with each other.The hamster isolates did not cross-react withA. naesiwad antiserum, but A esudii culturesall cross-reacted with the 745 antiserum, three ofthe five at a titer of 1:20. The human isolates allshowed cross-reactions with the A. naeslWantiserum, five at the diagnostic titer of 1:40.In this case, the cross-reaction was reciprocalsince all five A naeslisl cultures reacted withthe three A. viscosus sera, usually at low titer.

Sorption of hamster 745 antiserum with A.sln&i 45 (Table 5) removed all cross-reactions

with A. naeslwdi and also removed the cross-reactions with the human A. viscosus strains.

TABLE 5. FA reactions with sorbed antisera

FA reaction with cels of

Antiserum Sorbed withIWfl cells of A. viscesus A. iscaesms A.unae-(hamster) (humasn) sinudii

745 371 (human) 45

745 371 4+b 2+745 45 4+ -371 45 - 4+45 371 - 4+

a Antisera tested at a 1:2 dilution.Intensity of staining was graded from 0 to 4+;= less than 2+ intensity.

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Sorption of 745 antiserum with human A.viscosus 371 cells removed all of the cross-reac-tions to human A. viscosus isolates and reducedthe A. naeslundii staining to a 1 to 2+ intensity ata 1:2 serum dilution. The three hamster strainsand strain 229 behaved like strain 745 (Table 5),whereas all of the human strains behaved likestrain 371.

Reactions of other species and genera with A.viscosus antisera. It has been previously reported(12) that 14 of 64 strains of A. israelii reactedwith pooled A. viscosus antisera. However, withA. viscosus 745 and 371 antisera all the otherorganisms tested to date were negative. Theseinclude A. bovis (seven strains), A. odontolyticus(one strain), A. eriksonii (three strains), A.propionica (three strains), R. dentocariosa (ninestrains), B. matruchotii (five strains), C. pyogenes(two strains), C. acnes (five strains), Ramibac-teriwn pleuriticum (one strain).

Reactions of A. viscosus cultures with otherantisera. None of the A. viscosus cultures stainedat the diagnostic titer of the antiserum to A.israelii, A. bovis, A. odontolyticus, A. eriksonii,A. propionica, R. dentocariosa, B. matruchotil,C. pyrogenes, or C. acnes. However, some A.viscosus strains did stain with some of the un-diluted antisera, including 15 by A. propionicatype 2, 7 by R. dentocariosa, and 6 by A. israelfitype 1.

DISCUSSIONIn this laboratory, a specific FA antiserum was

prepared for the "hamster organism" (5) andused in the direct examination of samples ofhuman dental calculus. FA-positive organismswere observed in 10 of 12 specimens. Eight iso-lates from these specimens, 3 from other dentalcalculus specimens, and 8 from other sourcesprovided the 19 isolates from humans used inthis study. These human isolates could not bedistinguished from the three hamster isolates onthe basis of morphology, oxygen requirements,biochemical reactions, or cell wall compositionand thus were considered to be the same species,namely, A. viscosus.With FA techniques, the hamster and human

strains could be divided into serotypes 1 and 2.There was a one way cross-reaction betweenthese serotypes which was readily removed bysorption to provide type-specific antisera. Thethree hamster isolates and one strain of unknownorigin were placed in serotype 1, and all of thehuman isolates were placed in serotype 2. It islikely that as more strains are studied there willbe additional serotypes but that the serotype willnot continue to correlate with the habitat of theisolate.

A. viscosus has been demonstrated to producenatural and experimental periodontal diseasein hamsters (9); however, no such relationshiphas been shown in man even though, as indicatedabove, the organism is a common inhabitant ofthe human oral cavity. It has also been isolatedfrom clinical material outside of the oral cavity(Table 1); thus, it is important that the diagnosticlaboratory be able to differentiate and identifythis organism. Identification may be made on thebasis of the combined reactions described in thisreport, although initially cultures of A. viscosusmust be differentiated from species of Actino-myces, Arachnia, Nocardia, Rothia, and Coryne-bacterium.

A. viscosus is catalase-positive, which separatesit from the other species of Actinomyces andA. propionica. The catalase test should be doneon aerobically grown cultures, or, if anaerobiccultures are used, they should be allowed tostand in air for 30 min before testing, otherwisesome of the cultures may be negative or weaklypositive. In this laboratory, the four oxygen re-quirement test slants are used for the catalasetest, as this provides a direct comparison of thereaction between aerobically and anaerobicallygrown cells.

Aside from the difference in the catalase re-action, the similarities between A. viscosus andA. naeslundii are so great that A. viscosus maybe considered a catalase-positive variant of A.naeslundii. The cell wall of A. naeslundii containsthe sugars glucose, fucose, rhamnose, mannose,and 2-deoxytalose (10). Therefore, A. viscosusdiffers from A. naeslundii in the carbohydratecomposition of the cell wall, is more aerobic, andis different antigenically. These are consideredadequate differences to maintain these organismsas two separate species, at least for the present.

Since A. viscosus is an aerobic, gram-positivefilamentous organism, a fresh isolate might beconfused with Nocardia. A. viscosus differs inthat it is not acid-fast, does not produce pig-mented colonies, does not produce aerial hyphae,does not grow in a medium containing only aninorganic source of nitrogen, and produces littleor no growth on Sabouraud medium. Also, A.viscosus grows well anaerobically with CO2 andferments a variety of carbohydrates.The similarities and differences between A.

viscosus and R. dentocariosa have been discussedby Georg and Brown (2) and Brown et al. (1).We confirmed the fact that A. viscosus does notproduce coccoidal forms and that it produces acidfrom raffinose, inositol, and starch which are notfermented by R. dentocariosa. We did not findthe nitrite reduction test to differentiate com-pletely between the two genera. The A. viscosus

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HUMAN SiRAINS OF ACTINOMYCES VISCOSUS

cultures all reduced nitrate to nitrite but did notfurther reduce the nitrite in Indole-Nitrite me-dium, whereas about 50% of the Rothia cultures(1) reduced both nitrate and nitrite in this me-dium. However, seven of the human A. viscosusstrains did reduce nitrite when Trypticase SoyBroth with 0.01% nitrite was used for testingnitrite reduction. Serologically there were nocross-reactions between the organisms.

In many laboratories, there is a tendency tocategorically refer to all gram-positive pleomor-phic bacilli as "diphtheroids" without furtherseparation because of the many difficulties in-volved in spiating these bacteria. Morphologi-cally A. viscsus may be in this category, althoughit can be separated from the aerobic corynebac-teria by the filamentous microcolony and theoverall pattern of biochemical reactions. It differsfrom the anaerobic species C. acnes by growingequally well aerobically and anaerobically, hy-drolyzing esculin, fernmeting mebiose, raffinose,sucrose, and salicin, not producing indole, notbeing proteolytic, and not producing a pinksediment in thioglycollate broth. In addition, theanaerobic diphtheroids do not cross-react sero-logically with A. viscosus.

ACKNOWLEDGMENr

This investigton wa supported by Public Health Servicegrants AI-1801 fro the National hItit of Alerg andInfectious D _ses and DE-02675 from the National Institute ofDental Research.

The tichnical. assin of Patricia Coins and Jean A.Setterstrom is gratdully acknowledgd.

LERATURE CrD

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2. Georg, L K, and J. K. Brown. 1967. Rod*a, vz. nov. An

aerobic genus of the family Actihomycetaceae. Int. J.Syst. BacterioL 17:79-88.

2a. Georg, L K., L Pine, and M. A. Gerenser. 1969. Actino-myces viscosus, comb. nov., a catalase-positive, facultativemember of the genus Actinomyces. Int. J. Syst. Bacteriol.19:291-293.

3. Gerencser, M. A., and J. M. Slack. 1967. Isolation andcharacterizaion of Actinomyces propioncs. J. BacterioL94:109-115.

4. Gilmour, M. N., A. Howell, Jr., and B. G. Bibby. 1961. Theclassifation of organisms termed Leptotrichia (Leptodrix)buccasi. L Review of the literature and proposed separationinto Leptotichia buccais Trevisan, 1879 and Bacteionemagem. nov., B. mamruchodi (Mendel, 1919) comb. nov.BacterioL Rev. 25:131-141.

5. Howel, A., Jr. 1963. A filamentous m-croorganinn isolatedfrom periodontal plaque in hamsters. L Isolation, mor-phology and general cultural characteristics. Sabouraudia3:81-92.

6 Howell, A., Jr., and H. V. Jordan. 1963. A filamentous micro-organism isolated from periodontal plaque in hamsterIL Physiological and bi l acteristics. Sabou-rauxlia 393-105.

7. Howell, A., Jr., H. V. Jordon, L K. Georg, and L Pine. 1965.Odonromyces viscosus, gem nov., spec. nov., a filamentous

isolated from periodontal plaque in ham-ses Sabourau 4:56..

8. Howell, A, Jr., W. C. Murphy M, F. Paul, and R. M.Stephan. 1959. Oral strains of Aca omyces. J. Bacteriol.78-:82-95.

9. Jordan, H. V., and P. H. Keyes. L964. Aerobic, gram-positive,filamentous bacteria as etiologic agents of experimentalperiodontal disea in hamsters. Arch. Oral BioL 9:401-414.

10. Pine L, and C. J. Booue. 1967. Comparative cell wall analysesof morphological forms within the genus Acinomycej. J.Bacteriol- 94:875-883.

11. Slack, J. M. 1968. Subgroup on taxonomy of microaerophilicactinomycetes. Report on ation, aims and pro-codures. hnt. J. Syst. Bacteriol. 18:253-262.

12. Slack, J. M., S. Landfried, and M. A. Gerecsr. 1969. Mor-phological, biochemical and serological studies of 64strains of Actinmyces inli J. Bacteriol. 97:873-884.

13. Snyder, M. L, W. W. Bullock, and R. B. Parker. 1967.Morphology ofgram-positive filanemnous bacteria identifiedin dental plaque by flwUrIt antibody technique. Arch.Oral Biol. 12:1269-1273.

14. Wels, A. F., C. E. Miller, and M. K. Nadel. 1966. Rapidfluoresceln and protein assay method for fluorescent-antibody conjugates. AppL MicrobioL 14:271-275.

87Vow. 18, 1969

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