JOURNAL OF BACTERIOLOGY, July 1967, p. 109-115Copyright © 1967 American Society for Microbiology

Vol. 94, No. 1Printed in U.S.A.

Isolation and Characterization of Actinomycespropionicus

MARY ANN GERENCSER AND JOHN M. SLACK

Department of Microbiology, Medical Center, West Virginia University, Morgantown, West Virginia 26506

Received for publication 3 April 1967

Three cultures of Actinomyces have been identified as Actinomyces propionicus.Two of these strains are recent isolates, one, 427, from a case of cervico-facialactinomycosis, and one, 439, from a case of lacrimal canaliculitis. The third strain,346, was described by F. Lentze as A. israelii serological type II. These three strainswere compared with the type strain of A. propionicus ATTC 14157 and with knownstrains of five other Actinomyces species. Morphologically and biochemically thethree new cultures of A. propionicus were identical with the type strain but closelyresembled A. israelii. In serological tests making use of fluorescent antibody, allfour A. propionicus strains gave negative results with antisera for A. israelii, A. bovis,A. naeslundii, and A. eriksonii, but gave positive results with antisera for A. pro-

pionicus 14157 and strain 346. The A. propionicus antisera did not stain other Actino-myces species. A. propionicus contains diaminopimelic acid (DAP) in its cell walland produces propionic acid from glucose. All three new isolates were shown tocontain DAP and to produce propionic acid. By use of the presence of DAP in thecell wall and serological tests as the differential criteria, the three cultures describedin the report were specifically identified as A. propionicus.

Buchanan and Pine (3) described a strain ofActinomyces isolated from human lacrimal canal-iculitis as a new species, Actinomyces propionicus(ATCC 14157, type strain). A. propionicus pro-duced "spider-type" microcolonies and gavebiochemical reactions essentially the same as thoseof A. israelii and A. naeslundii. It differed fromthese and other species of Actinomyces in that itproduced propionic acid from the fermentationof glucose, it did not require carbon dioxide foreither aerobic or anaerobic growth, and diamino-pimelic acid (DAP) was a major component ofthe cell wall.

Georg, Robertstad, and Brinkman (5) includedthis culture in comparative studies of the oxygenrequirements, morphology, biochemical reactions,and serological reactions of Actinomyces species.The morphology, oxygen requirements, and bio-chemical reactions of A. propionicus agreed ingeneral with those reported by Buchanan andPine (3). In serological studies by use of thegel-diffusion technique, A. propionicus antigenreacted only with A. propionicus antiserum. Therewere no cross-reactions between A. propionicusand either A. israelii or A. bovis. However, anti-gens from two strains of A. naeslundii reactedwith the A. propionicus antiserum even though thereciprocal reactions were negative.

Three additional isolates of A. propionicus willbe described. Two were recently isolated in thislaboratory from human infections, and one wasreceived from F. Lentz of Germany.

MATERIALS AND METHODS

Cultures. The following cultures were used in thecomparative studies described in this report: A.israelii (ATCC 10048), A. bovis (ATCC 13683), A.eriksonii (ATCC 15423), A. naeslundii (ATCC 12104),A. propionicus (ATCC 14157), and A. odontolyticus(ATCC 17929).WVU strain no. 346. This culture was received from

F. Lentz as A. israelii serological type II, strainFleischman. It was isolated from a typical case ofhuman actinomycosis (Lentz, personal communica-tion).WVU strain no. 427. This culture was isolated from

a case of cervico-facial actinomycosis occurring in a44-year-old white farmer with a history of a "barley-tuft" caught in the back of his throat. A lump de-veloped on the right side of the neck which graduallyenlarged over a period of 4 months at which time itstarted to drain. No granules were observed in thedrainage material. Drainage material was cultured onBBL Brain Heart Infusion Agar (BHIA) and blood-agar plates and was incubated at 37 C in an atmos-phere of 95% N2-5% CO2. Within 72 hr, small "spider-type" colonies were observed on the BHIA plates andsubcultured.

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WVU strain no. 439. This culture was isolated froma case of lacrimal canaliculitis occurring in a 61-year-old white woman with a 3-year history of exudationat the inner canthus of the left eye with repeated swell-ings and "infections." The lacrimal system had beenprobed and irrigated repeatedly with no relief. Exu-date containing concretions was cultured as describedabove. After 24 hr of incubation, two types of coloniesappeared on the BHIA plates. One was a small fila-mentous colony with dense centers (WVU no. 438),and the other was a "spider" colony (WVU no. 439).Both colony types were subcultured. After 4 to 5days, "molar-tooth" colonies were seen on the blood-agar plates, some of which became dark red after7 to 9 days of incubation. The deep-red colonies whichinitially were filamentous with dense centers wereidentified as A. odontolyticus (1). This is the firstreport of this species being isolated outside of the oralcavity. The nonpigmented colonies which initiallywere "spider" colonies were identified as A. pro-pionicus.

Morphology. The cultures were streaked on BHIAand Heart Infusion Blood Agar and incubated at 37 Cin an atmosphere of 95% N2-5% CO2. Microcolonydevelopment on BHIA was observed microscopicallyat 100 X after 24 to 48 hr of incubation. Macro-colonies were observed on BHIA and blood agar at 7and 14 days. Gram stains and dark-field smears weremade from cultures on various media.

Oxygen requirements. Three-day-old cultures inActinomyces Broth (BBL) were centrifuged, washedonce in 0.85% saline, and resuspended in saline to aturbidity equal to that of a MacFarland no. 3 stand-ard. The suspensions were drawn up into a capillarypipette, and eight slants of BHIA were inoculated byplacing the pipette at the bottom of the slant andstreaking one drop of inoculum from the base to thetop of the slant. For incubation, the cotton plug wasclipped off and pushed into the tube, and a smallpledget of absorbent cotton was added. Equal quanti-ties of various reagents were then added, and thetubes were sealed with a rubber stopper to give fouratmospheric conditions as follows: aerobic, tubes withcotton plugs and no seal; aerobic + C02, 5 drops of10% Na2CO3 + 5 drops of 1 M KH2PO4; anaerobic,5 drops of pyrogallol solution + 5 drops of 10%KOH (pyrogallol solution = 100 g of pyrogallic acid+ 150 ml of distilled water); and anaerobic + CO2,5 drops of pyrogallol solution + 5 drops of 10%Na2CO3. Duplicate tubes were incubated under eachatmospheric condition for 7 days when the amount ofgrowth was estimated visually and recorded 0 to 3+.The best growth for each individual culture was re-corded as 3+, and growth under the various oxygenconditions was compared with this standard.

Biochemical tests. The following tests were done:catalase, indole, nitrate reduction, methyl red, Voges-Proskauer, starch hydrolysis, gelatin liquefaction,H2S production, and acid from carbohydrates. Theinoculum for all tests was taken from well-mixed3-day-old Actinomyces Broth cultures. All cultureswere incubated at 37 C in 95% N2-5% CO2 jars withthe exception of the carbohydrates as noted below.The catalase test was done by adding a drop of 3%

H202 to colonies on BHIA plates or by adding a dropof 3% H202 to a heavy suspension of organisms on amicroscope slide. Catalase tests were also done ongrowth from each of the four oxygen concentrationstested.

Indole-Nitrite Broth (BBL) and M R-V P Medium(BBL) were inoculated in duplicate, and the tests weredone after 3 and 7 days of incubation, respectively.

Starch plates (Nutrient Agar + 0.5% starch) wereinoculated in triplicate with a single streak of theculture across the width of the plate. Tests for hy-drolysis were done by flooding the plate with Gram'siodine after 3, 7, and 14 days of incubation.

Duplicate gelatin agar plates (Actinomyces Broth+ 1.5% agar and 0.4% gelatin) were streaked asabove and tested for liquefaction after 7 and 14 daysby flooding the plate with a mercuric chloride solution(15 g of HgCl2 + 20 ml of concentrated HCl +100 ml of distilled water).

For hydrogen sulfide production, BHIA slants wereinoculated, and a lead acetate paper strip was sus-pended over the slant. Observations for blackening ofthe paper were made at 7 and 14 days.

Carbohydrate fermentation tests were done usingActinomyces Fermentation Broth (BBL) to whichfilter-sterilized carbohydrates were added to a finalconcentration of 1.0% (except starch used at 0.5%).The fermentation tests were incubated under pyro-gallol-carbonate seals and observed for 14 days.DAP. The presence of DAP in the cell wall was

determined by the whole-cell hydrolysate method ofBecker et al. (2).

Serological studies. Antigens for immunization weregrown in nonantigenic peptone-dialysate medium (8).After incubation for 7 to 10 days, the cultures werecentrifuged, washed three times in Merthiolated saline,and suspended in Merthiolated saline to a turbidityequal to a MacFarland no. 8 standard. Of the antigen,1 ml was mixed with 1.0 ml of adjuvant (Algivant;Colab Laboratories, Chicago Heights, Ill.), and this2 ml was injected subcutaneously between the shoulderblades of New Zealand White rabbits. After 2 days ofrest, each rabbit received daily injections in themarginal ear vein of 2, 2, 3, 3, and 4 ml of antigen.One day after the final intravenous injection anothersubcutaneous injection of adjuvant-antigen mixturewas administered. The intravenous course of injectionswas repeated twice with a 1-week rest interval betweencourses. The rabbits were bled 1 week after the lastinjection.

Globulin fractions of the antisera were obtained byammonium sulfate precipitation and conjugated withfluorescein isothiocyanate (4). The excess fluoresceinwas removed by filtration through a Sephadex G-25column. Conjugated antiserum was prepared againstA. propionicus (14157 and 346) and each of theother Actinomyces species.

Gel-diffusion antigens were prepared from culturemedia supernatant fluid by the method of Georg et al.(5) after growing the organisms in Actinomyces Brothfor 7 to 10 days. Gel-diffusion tests were carried outon 2 by 3 inch (5.1 by 7.7 cm) microscope slides byuse of a medium consisting of FTA HemagglutinationBuffer (BBL) with 0.8% Special Agar (Nobel) from

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VOL. 94, 1967 ISOLATION AND CHARACTERIZATION OF A. PROPIONICUS

Difco and 0.001% merthiolate. The tests were incu-bated in a moist chamber at room temperature andread at 1-, 2-, 3-, and 7-day intervals.

RESULTS

Morphology. A. propionicus (ATCC 14157) andWVU cultures 346, 427, and 439 all producedfilamentous "spider" colonies within 24 hr onBHIA plates which were indistinguishable fromthe microcolonies of A. israelii. Within 7 to 14days of incubation, the white heaped-up colonieshad a "molar-tooth" appearance identical to thatproduced by either A. israelii or A. naeslundii. Thedull-orange color previously described (3) wasnot observed within 14 days on BHIA. All fourcultures produced a granular or "bread-crumb"type of growth when grown in broth.

In both Fluid Thioglycollate Broth (BBL) andActinomyces Broth, these cultures produced a verypungent distinctive odor not noted in any othercultures of Actinomyces. However, a similar odorwas noted with Propionibacterium freudenreichii;thus, the pungent odor may be associated withthe production of propionic acid.Gram stains from broth cultures showed gram-

positive filaments with varying degrees of branch-ing. Diphtheroid-like rods were common, andthe spherical cells described by Buchanan andPine (3) were frequently seen. The colonial andcellular morphology of A. propionicus is shown inFig. 1.Oxygen requirements. All four cultures of A.

propionicus gave essentially the same results(Table 1) and grew quite well both aerobically andanaerobically with and without CO2 . It has beenreported (3) that this organism does not requireC02 .

Biochemical tests. The results given by the A.propionicus cultures (Table 2) were quite uniform,except that strain 346 gave only a weakly positivemethyl-red test in 7 days. The four cultures gavepartial hydrolysis of starch as did A. israelii.The slow gelatin liquefaction exhibited by A.

propionicus was unexpected, as Actinomycesspecies are generally considered to be non-proteolytic. None of these cultures showed gelatinliquefaction in a tube test even after 4 weeks ofincubation. The plate test for gelatin liquefactionis not only more sensitive than tube tests but alsois to some extent a rapid test; for example, cul-tures of enterobacteria requiring 2 to 4 weeks toexhibit liquefaction by the tube test were positivein 24 to 72 hr by the plate technique (7). Un-fortunately, the slow liquefaction of gelatin can-not be considered as diagnostic for A. propionicus,as approximately half of A. israelii cultures testedin this laboratory showed some liquefaction ofgelatin within 2 weeks by the plate method.

Fermentation tests. The four A. propionicuscultures also gave similar results in fermentationtests (Table 3), but there was strain variation inthe fermentation of starch and in the speed offermentation of galactose and mannose. The mostobvious strain differences were seen with isolate346. In general, this culture grew more slowly andless abundantly than the other three strains, andbiochemical reactions were slower. Growth wasgreatly stimulated by the addition of a smallamount of rabbit serum (0.1 ml/10.0 ml ofmedium). In the case of the fermentation reac-tions, strain 346 fermented galactose and mannosewhen the tests were held for 21 instead of 14 days.The overall fermentation pattern of A. pro-

pionicus cultures showed some of the charac-teristics of A. israeli and some of the character-istics of A. naeslundii. Failure to fermentarabinose, xylose, ceUobiose, and salicin woulddifferentiate A. propionicus from A. israelii 10048,whereas failure to ferment salicin and fermenta-tion of mannitol would distinguish it from A.naeslundii 12104. However, the degree of variationin fermentation reactions among strains of thesespecies, especially A. israelii, is quite large (6) sothat these differences might not hold true for allisolates.

Fermentation end products. The production ofvolatile organic acids from glucose was deter-mined for us by Yu-Ying F. Li, Mycology Section,National Communicable Disease Center. All threenew isolates and the type strain of A. propionicusproduced both acetic acid and propionic acid asmetabolic end products in large amounts. Acontrol strain of A. israelii produced only aceticacid.DAP. Chromatography of whole-cell hydroly-

sates showed DAP to be present in each of thefour A. propionicus cultures. The results withcontrol whole-cell hydrolysates were negative forA. israelii and positive for Corynebacterium acnes.The results with whole-ceU hydrolysates agreewith analyses of purified cell-wall preparations ofthe control strains.

Serology. By use of fluorescent antibody tech-niques, all four cultures of A. propionicus stainedbrilliantly with undiluted conjugated antiserumagainst the type strain 14157 and isolate 346. Thespherical form of the A. propionicus culturesstained as brightly with conjugated antisera as didthe rod forms. All four strains stained to titerwith antiserum 346, but strain 346 did not stainto titer with serum 14157.

Sorption studies showed that strain 346 is notserologically identical with the other three or-gansims. The 346 antiserum sorbed with cells ofeither 427 or 14157 continued to stain 346, but didnot stain 427, 439, or 14157. On the other hand,

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FIG. 1. Actinomyces propionicus 439 (a) gram stain, BHI agar, 24 hr, 1,200 X; (b) dark-field, BHI agar, 24hr, 1,200 X; (c) Gram stain, thioglycollate broth, 4 days, coccoid forms, 1,200 X; (d) dark-field, thioglycollatebroth, 4 days, coccoid forms, 1,200 X; (e) "spider" microcolony, BHI agar, 48 hr, 300 X; (f) "molar-tooth"mature colony BHI agar, 10 days, 40 X; (g) thioglycollate broth, 7 days.

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VOL. 94, 1967 ISOLATION AND CHARACTERIZATION OF A. PROPIONICUS 113

strain 427 completely sorbed antibodies from stained with conjugated antiserum for A. israelii,14157 antiserum, whereas 346 failed to do so. The A. bovis, A. naeslundii, A. eriksonii, or A. odon-

results of these sorption experiments are shown in tolyticus. These results are listed in Table 5.

Table 4. The gel-diffusion test was used primarily to

None of the four cultures of A. propionicus confirm the serological relationships between A.

TABLE 1. Comparison of oxygen requirements of Actinomyces propionicus and other Actinomyces specieSa

ATITC ATTC ATTC ATTC ATTC ATTCGrowtcondtion WVU WVU WVI 14157 A. 04.104A 17929 A. 13683 A. 15423 A.Growthcndition 346 427 439 propi,,nicus israelii naeslundii odyticu bovis eriksonii

Aerobic.. 2+ 3+ 2+ 2+ 0 2+ + + 0Aerobic+C02. 3+ 3+ 3+ 2+ + 3+ 2+ + 0Anaerobic.......3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+Anaerobic + CO2....3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+

a Best growth was at 3+; growth under the various conditions was compared to this standard.

TABLE 2. Comparison of biochemical tests of Actinomyces propionicus and other Actinomyces species

TestwvU

346

wvu

427

ATTC

49 14157 A.

propionicus

ATTC

10048 A.

israciji

ATTC

12104 A.

naeslundii

ATTC

17929 A.

odonto-

lyticus

Catalase- - - - - - -Nitrate + + + + + + +Indole- - - - - - -Methylired 4d + + + + + -Voges-Proskauer- - - - - - -H2SStarch hydrolysis :41 4 1= -Gelatin liquefaction- + + + + - -

+ after 14 days of incubation.

ATTC

13683 A.

bovis

ATTC

15423 A.

eriksonii

TABLE 3. Fermenztation of carbohydrates by Actinomyces pronionicus and other Actinomvces SDeciesa

ATTC ATTCC ATTC ATTC ATTC ATTCCarbohydrates WVU 346 WVU 427 WVU 439 14157 A. 10048 A. 12104 A. 17929 A. 13683 A. 15423 A.

propionicus israelii tnaeslundii odontolylicus bovis eriksonii

Arabinose - - - - A - - - AXylose - - - - A - - - ARhamnose------ - -

Fructose A A A A A A A A AGalactose - A,s A, s A,s A,ps A - A),s AGlucose A A A A A A A A AMannose - A, s A,s A A A - A,s ACellobiose - - - - A - - ALactose A,s A A A A A,s A A AMaltose A A A A A A - A ASucrose 'A A A A A A A A ATrehalose A A A A A A - - ARaffinose A A A A A A - - AMelezitose - - - - - - - - AGlycerol - - - - - - - -Inositol A A - Al,s -Mannitol A A A A A - - - ASalicin - - - - A A - - AStarch A, s - A,Is - - - A A

- A = acid in 1 to 7 days; A, s =acid in 8 to 14 days; minus sign =no acid.

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GERENCSER AND SLACK

propionicus 14157 and isolates 346 and 427. By useof 14157 antiserum with 14157 and 427 antigens,all the lines produced were lines of identitybetween these two antigens. However, strain 346produced only one weak line with 14157 anti-serum.

DISCUSSION

Although A. propionicus was described as aseparate species in 1962, no other isolates havebeen recognized to date. In this paper, threeadditional strains were compared in detail withthe original isolate, and all were found to beessentially identical morphologically, biochem-ically, and serologically. In addition, the threestrains produced propionic acid from the fer-mentation of glucose. Thus, in this discussion, thename A. propionicus is used to include all fourcultures used in this study.

Additional isolates of A. propionicus probablyhave not been recognized because of the closemorphological resemblance of this organism toother species of Actinomyces, especially A. israelii.

TABLE 4. Antigeniic relationiship between four cul-tures of Actinomyces propionicus with sorbed

antiserum anidfluorescent antibody

AntiserumNo. of A. .-

pro- Serum 346 SerumPionicus Serum 14157 Serum 14157 sorbed 346antigen sorbed with sorbed with with sorbed346 427 14157 with 427

14157 2to3+ - - -

346 - not tested 2+ 2+427 2to3+ _ -

-

439 2 to 3+ not tested _

In this instance, the microcolony produced by A.propionicus is indistinguishable from that formedby A. israelii. Thus, this commonly employeddifferential criterion cannot be applied to thesetwo species.

A. propionicus grows best anaerobically, but itwill also grow aerobically, and CO2 is not requiredas with other species of Actinomyces. In broth, itforms "bread-crumb" colonies.

It resembles both A. israelii and A. naeslundiiin its overall biochemical reactions. However,failure to ferment arabinose, xylose, salicin, andcellobiose, along with partial hydrolysis of starchand late liquefaction of gelatin, will aid in theidentification of A. propionicus. Thus, an organ-ism forming a "spider" microcolony, not fer-menting any of these four carbohydrates anddeveloping a strong Propionibacterium-like odor,could be tentatively identified as A. propionicus.At the present time, A. propionicus is the only

described species of Actinomyces containing DAPin its cell wall. The easiest and most rapid methodfor determining the presence or absence of DAPis to chromatograph whole-cell hydrolysates. Allfour of the A. propionicus strains contained DAP,whereas the other species were negative. This thenis a major differential characteristic.

In our hands, the most rapid and specificmethod of differentiation and identification of thisorganism was the fluorescent-antibody technique.Species-specific conjugated antiserum was pre-pared which did not cross-react with other speciesof Actinomyces. This antiserum was used in thedirect fluorescent-antibody technique and wassuccessfully applied to the identification of A.propionicus in exudate from patients, initial cul-tures, and subcultures.

TABLE 5. Antigenzic relationship of Actiniomyces propionicus with other Actinzomyces as demonstratedbv thefluorescent-antibody techniique

Conjugated antiseraa

Culture no. 4A. israelii 10048 A. naesl1ndii A. bovis 13683 A. eriksonii odontolyticus A. propionicus StrainWVUssraezs ~~12104 15423 17929 14157 346

10048 4+ 2+ _- - -

12104 2+ 4+ - - _ _13683 - - 4+ ---15423 - - - 4+ - - -17929 - - - - 4+ -

14157 - - - -- 4+ 4+346 - - - --4+ 4+427 - - - _ _ 4+ 4+

43ndiluted- - 4+ 4+Undiluted antisera.

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VOL. 94, 1967 ISOLATION AND CHARACTERIZATION OF A. PROPIONICUS

ACKNOWLEDGMENTSWe wish to thank J. L. Campbell of Department of

Oral Surgery and R. R. Trotter of Department ofOphthalmology for making case material available forstudy. Leo Pine of Communicable Disease Centerconfirmed the identity of one strain.

This investigation was supported by Public HealthService grant AL-01801-09 from National Institute ofAllergy and Infectious Diseases.

LITERATURE CITED

1. BATrY, I. 1958. Actinomyces odonitolyticus, a newspecies of actinomycete regularly isolated fromdeep carious dentine. J. Pathol. Bacteriol.75:455-459.

2. BECKER, B., M. P. LECHEVALIER, R. E. GORDON,AND H. A. LECHEVALIER. 1964. Rapid differen-tiation between Nocardia and Streptomyces bypaper chromatography of whole-cell hydroly-sates. J. Appl. Microbiol. 12:421-423.

3. BUCHANAN, B. B., AND L. PINE. 1962. Characteri-zation of a propionic acid producing actinomy-cete, Actinomyces propionicus, Sp. nov. J. Gen.Microbiol. 28:305-323.

4. CHERRY, W. B., M. GOLDMAN, T. R. CARSKI, ANDM. D. MOODY. 1960. Fluorescent antibodytechniques in the diagnosis of communicablediseases. U. S. Public Health Serv. Publ. 729.

5. GEORG, L. K., G. W. ROBERTSTAD, AND S. A.BRINKMAN. 1964. Identification of species ofActinomyces. J. Bacteriol. 88:477-490.

6. HOWELL, A. JR., W. C. MURPHY III, F. PAUL,AND R. M. STEPHAN. 1959. Oral strains ofActinomyces. J. Bacteriol. 78:82-95.

7. McDADE, J. J., AND R. H. WEAVER. 1959. Rapidmethods for the detection of gelatin hydrolysis.J. Bacteriol. 77:60-69.

8. SLACK, J. M., E. H. LUDWIG, H. H. BIRD, ANDC. M. CANBY. 1951. Studies with microaero-philic actinomycetes. I. The agglutination re-action. J. Bacteriol. 61:721-735.

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