Isolation and Characterization of Actinomyces propionicus

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1 JOURNAL OF BACTERIOLOGY, July 1967, p Copyright 1967 American Society for Microbiology Vol. 94, No. 1 Printed in U.S.A. Isolation and Characterization of Actinomyces propionicus MARY ANN GERENCSER AND JOHN M. SLACK Department of Microbiology, Medical Center, West Virginia University, Morgantown, West Virginia 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-facial actinomycosis, 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 strains were compared with the type strain of A. propionicus and with known strains of five other Actinomyces species. Morphologically and biochemically the three new cultures of A. propionicus were identical with the type strain but closely resembled A. israelii. In serological tests making use of fluorescent antibody, all four 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. propionicus and strain 346. The A. propionicus antisera did not stain other Actinomyces species. A. propionicus contains diaminopimelic acid (DAP) in its cell wall and produces propionic acid from glucose. All three new isolates were shown to contain DAP and to produce propionic acid. By use of the presence of DAP in the cell wall and serological tests as the differential criteria, the three cultures described in the report were specifically identified as A. propionicus. Buchanan and Pine (3) described a strain of Actinomyces isolated from human lacrimal canaliculitis as a new species, Actinomyces propionicus (ATCC 14157, type strain). A. propionicus produced "spider-type" microcolonies and gave biochemical reactions essentially the same as those of A. israelii and A. naeslundii. It differed from these and other species of Actinomyces in that it produced propionic acid from the fermentation of glucose, it did not require carbon dioxide for either aerobic or anaerobic growth, and diaminopimelic acid (DAP) was a major component of the cell wall. Georg, Robertstad, and Brinkman (5) included this culture in comparative studies of the oxygen requirements, morphology, biochemical reactions, and serological reactions of Actinomyces species. The morphology, oxygen requirements, and biochemical reactions of A. propionicus agreed in general with those reported by Buchanan and Pine (3). In serological studies by use of the gel-diffusion technique, A. propionicus antigen reacted only with A. propionicus antiserum. There were no cross-reactions between A. propionicus and either A. israelii or A. bovis. However, antigens from two strains of A. naeslundii reacted with the A. propionicus antiserum even though the reciprocal reactions were negative. Three additional isolates of A. propionicus will be described. Two were recently isolated in this laboratory from human infections, and one was received from F. Lentz of Germany. MATERIALS AND METHODS Cultures. The following cultures were used in the comparative 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 This culture was received from F. Lentz as A. israelii serological type II, strain Fleischman. It was isolated from a typical case of human actinomycosis (Lentz, personal communication). WVU strain no This culture was isolated from a case of cervico-facial actinomycosis occurring in a 44-year-old white farmer with a history of a "barleytuft" caught in the back of his throat. A lump developed on the right side of the neck which gradually enlarged over a period of 4 months at which time it started to drain. No granules were observed in the drainage material. Drainage material was cultured on BBL Brain Heart Infusion Agar (BHIA) and bloodagar plates and was incubated at 37 C in an atmosphere of 95% N2-5% CO2. Within 72 hr, small "spidertype" colonies were observed on the BHIA plates and subcultured. 109

2 110 GERENCSER AND SLACK J. BACTERIOL. WVU strain no This culture was isolated from a case of lacrimal canaliculitis occurring in a 61-yearold white woman with a 3-year history of exudation at the inner canthus of the left eye with repeated swellings and "infections." The lacrimal system had been probed and irrigated repeatedly with no relief. Exudate containing concretions was cultured as described above. After 24 hr of incubation, two types of colonies appeared on the BHIA plates. One was a small filamentous 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 5 days, "molar-tooth" colonies were seen on the bloodagar plates, some of which became dark red after 7 to 9 days of incubation. The deep-red colonies which initially were filamentous with dense centers were identified as A. odontolyticus (1). This is the first report of this species being isolated outside of the oral cavity. The nonpigmented colonies which initially were "spider" colonies were identified as A. propionicus. Morphology. The cultures were streaked on BHIA and Heart Infusion Blood Agar and incubated at 37 C in an atmosphere of 95% N2-5% CO2. Microcolony development on BHIA was observed microscopically at 100 X after 24 to 48 hr of incubation. Macrocolonies were observed on BHIA and blood agar at 7 and 14 days. Gram stains and dark-field smears were made from cultures on various media. Oxygen requirements. Three-day-old cultures in Actinomyces Broth (BBL) were centrifuged, washed once in 0.85% saline, and resuspended in saline to a turbidity equal to that of a MacFarland no. 3 standard. The suspensions were drawn up into a capillary pipette, and eight slants of BHIA were inoculated by placing the pipette at the bottom of the slant and streaking one drop of inoculum from the base to the top of the slant. For incubation, the cotton plug was clipped off and pushed into the tube, and a small pledget of absorbent cotton was added. Equal quantities of various reagents were then added, and the tubes were sealed with a rubber stopper to give four atmospheric conditions as follows: aerobic, tubes with cotton plugs and no seal; aerobic + C02, 5 drops of 10% 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 ml of distilled water); and anaerobic + CO2, 5 drops of pyrogallol solution + 5 drops of 10% Na2CO3. Duplicate tubes were incubated under each atmospheric condition for 7 days when the amount of growth was estimated visually and recorded 0 to 3+. The best growth for each individual culture was recorded as 3+, and growth under the various oxygen conditions 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. The inoculum for all tests was taken from well-mixed 3-day-old Actinomyces Broth cultures. All cultures were incubated at 37 C in 95% N2-5% CO2 jars with the 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 drop of 3% H202 to a heavy suspension of organisms on a microscope slide. Catalase tests were also done on growth from each of the four oxygen concentrations tested. Indole-Nitrite Broth (BBL) and M R-V P Medium (BBL) were inoculated in duplicate, and the tests were done after 3 and 7 days of incubation, respectively. Starch plates (Nutrient Agar + 0.5% starch) were inoculated in triplicate with a single streak of the culture across the width of the plate. Tests for hydrolysis were done by flooding the plate with Gram's iodine after 3, 7, and 14 days of incubation. Duplicate gelatin agar plates (Actinomyces Broth + 1.5% agar and 0.4% gelatin) were streaked as above and tested for liquefaction after 7 and 14 days by flooding the plate with a mercuric chloride solution (15 g of HgCl ml of concentrated HCl ml of distilled water). For hydrogen sulfide production, BHIA slants were inoculated, and a lead acetate paper strip was suspended over the slant. Observations for blackening of the paper were made at 7 and 14 days. Carbohydrate fermentation tests were done using Actinomyces Fermentation Broth (BBL) to which filter-sterilized carbohydrates were added to a final concentration of 1.0% (except starch used at 0.5%). The fermentation tests were incubated under pyrogallol-carbonate seals and observed for 14 days. DAP. The presence of DAP in the cell wall was determined by the whole-cell hydrolysate method of Becker et al. (2). Serological studies. Antigens for immunization were grown in nonantigenic peptone-dialysate medium (8). After incubation for 7 to 10 days, the cultures were centrifuged, washed three times in Merthiolated saline, and suspended in Merthiolated saline to a turbidity equal 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 this 2 ml was injected subcutaneously between the shoulder blades of New Zealand White rabbits. After 2 days of rest, each rabbit received daily injections in the marginal ear vein of 2, 2, 3, 3, and 4 ml of antigen. One day after the final intravenous injection another subcutaneous injection of adjuvant-antigen mixture was administered. The intravenous course of injections was repeated twice with a 1-week rest interval between courses. The rabbits were bled 1 week after the last injection. Globulin fractions of the antisera were obtained by ammonium sulfate precipitation and conjugated with fluorescein isothiocyanate (4). The excess fluorescein was removed by filtration through a Sephadex G-25 column. Conjugated antiserum was prepared against A. propionicus (14157 and 346) and each of the other Actinomyces species. Gel-diffusion antigens were prepared from culture media supernatant fluid by the method of Georg et al. (5) after growing the organisms in Actinomyces Broth for 7 to 10 days. Gel-diffusion tests were carried out on 2 by 3 inch (5.1 by 7.7 cm) microscope slides by use of a medium consisting of FTA Hemagglutination Buffer (BBL) with 0.8% Special Agar (Nobel) from

3 VOL. 94, 1967 ISOLATION AND CHARACTERIZATION OF A. PROPIONICUS Difco and 0.001% merthiolate. The tests were incubated in a moist chamber at room temperature and read at 1-, 2-, 3-, and 7-day intervals. RESULTS Morphology. A. propionicus (ATCC 14157) and WVU cultures 346, 427, and 439 all produced filamentous "spider" colonies within 24 hr on BHIA plates which were indistinguishable from the microcolonies of A. israelii. Within 7 to 14 days of incubation, the white heaped-up colonies had a "molar-tooth" appearance identical to that produced by either A. israelii or A. naeslundii. The dull-orange color previously described (3) was not observed within 14 days on BHIA. All four cultures produced a granular or "bread-crumb" type of growth when grown in broth. In both Fluid Thioglycollate Broth (BBL) and Actinomyces Broth, these cultures produced a very pungent distinctive odor not noted in any other cultures of Actinomyces. However, a similar odor was noted with Propionibacterium freudenreichii; thus, the pungent odor may be associated with the production of propionic acid. Gram stains from broth cultures showed grampositive filaments with varying degrees of branching. Diphtheroid-like rods were common, and the spherical cells described by Buchanan and Pine (3) were frequently seen. The colonial and cellular morphology of A. propionicus is shown in Fig. 1. Oxygen requirements. All four cultures of A. propionicus gave essentially the same results (Table 1) and grew quite well both aerobically and anaerobically with and without CO2. It has been reported (3) that this organism does not require C02. Biochemical tests. The results given by the A. propionicus cultures (Table 2) were quite uniform, except that strain 346 gave only a weakly positive methyl-red test in 7 days. The four cultures gave partial hydrolysis of starch as did A. israelii. The slow gelatin liquefaction exhibited by A. propionicus was unexpected, as Actinomyces species are generally considered to be nonproteolytic. None of these cultures showed gelatin liquefaction in a tube test even after 4 weeks of incubation. The plate test for gelatin liquefaction is not only more sensitive than tube tests but also is to some extent a rapid test; for example, cultures of enterobacteria requiring 2 to 4 weeks to exhibit liquefaction by the tube test were positive in 24 to 72 hr by the plate technique (7). Unfortunately, the slow liquefaction of gelatin cannot be considered as diagnostic for A. propionicus, as approximately half of A. israelii cultures tested in this laboratory showed some liquefaction of gelatin within 2 weeks by the plate method. III Fermentation tests. The four A. propionicus cultures also gave similar results in fermentation tests (Table 3), but there was strain variation in the fermentation of starch and in the speed of fermentation of galactose and mannose. The most obvious strain differences were seen with isolate 346. In general, this culture grew more slowly and less abundantly than the other three strains, and biochemical reactions were slower. Growth was greatly stimulated by the addition of a small amount of rabbit serum (0.1 ml/10.0 ml of medium). In the case of the fermentation reactions, strain 346 fermented galactose and mannose when the tests were held for 21 instead of 14 days. The overall fermentation pattern of A. propionicus cultures showed some of the characteristics of A. israeli and some of the characteristics of A. naeslundii. Failure to ferment arabinose, xylose, ceuobiose, and salicin would differentiate A. propionicus from A. israelii 10048, whereas failure to ferment salicin and fermentation of mannitol would distinguish it from A. naeslundii However, the degree of variation in fermentation reactions among strains of these species, especially A. israelii, is quite large (6) so that these differences might not hold true for all isolates. Fermentation end products. The production of volatile organic acids from glucose was determined for us by Yu-Ying F. Li, Mycology Section, National Communicable Disease Center. All three new isolates and the type strain of A. propionicus produced both acetic acid and propionic acid as metabolic end products in large amounts. A control strain of A. israelii produced only acetic acid. DAP. Chromatography of whole-cell hydrolysates showed DAP to be present in each of the four A. propionicus cultures. The results with control whole-cell hydrolysates were negative for A. israelii and positive for Corynebacterium acnes. The results with whole-ceu hydrolysates agree with analyses of purified cell-wall preparations of the control strains. Serology. By use of fluorescent antibody techniques, all four cultures of A. propionicus stained brilliantly with undiluted conjugated antiserum against the type strain and isolate 346. The spherical form of the A. propionicus cultures stained as brightly with conjugated antisera as did the rod forms. All four strains stained to titer with antiserum 346, but strain 346 did not stain to titer with serum Sorption studies showed that strain 346 is not serologically identical with the other three organsims. The 346 antiserum sorbed with cells of either 427 or continued to stain 346, but did not stain 427, 439, or On the other hand,

4 -or -N,.p a A. p.* or.a I4,.O 00. t... 0 Y FIG. 1. Actinomyces propionicus 439 (a) gram stain, BHI agar, 24 hr, 1,200 X; (b) dark-field, BHI agar, 24 hr, 1,200 X; (c) Gram stain, thioglycollate broth, 4 days, coccoid forms, 1,200 X; (d) dark-field, thioglycollate broth, 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. 112

5 VOL. 94, 1967 ISOLATION AND CHARACTERIZATION OF A. PROPIONICUS 113 strain 427 completely sorbed antibodies from stained with conjugated antiserum for A. israelii, antiserum, whereas 346 failed to do so. The A. bovis, A. naeslundii, A. eriksonii, or A. odonresults 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 condtion WVU WVU WVI A A A Growth A A. cndition propi,,nicus israelii naeslundii odyticu bovis eriksonii Aerobic Aerobic +C Anaerobic Anaerobic + CO 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 Test wvu 346 wvu A. propionicus A. israciji A. naeslundii A. Catalase Nitrate Indole Methylired 4d Voges-Proskauer H2S Starch hydrolysis :41 4 1= - Gelatin liquefaction after 14 days of incubation. odontolyticus A. bovis A. eriksonii TABLE 3. Fermenztation of carbohydrates by Actinomyces pronionicus and other Actinomvces SDeciesa C Carbohydrates WVU 346 WVU 427 WVU A A A A A A. propionicus israelii tnaeslundii odontolylicus bovis eriksonii Arabinose A A Xylose A A Rhamnose Fructose A A A A A A A A A Galactose - A, s A, s A, s A,ps A - A),s A Glucose A A A A A A A A A Mannose - A, s A, s A A A - A, s A Cellobiose A - - A Lactose A, s A A A A A, s A A A Maltose A A A A A A - A A Sucrose 'A A A A A A A A A Trehalose A A A A A A - - A Raffinose A A A A A A - - A Melezitose A Glycerol Inositol A A - Al,s - Mannitol A A A A A A Salicin A A - - A Starch 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.

6 114 GERENCSER AND SLACK J. BAcrERIOL. propionicus and isolates 346 and 427. By use of antiserum with and 427 antigens, all the lines produced were lines of identity between these two antigens. However, strain 346 produced only one weak line with antiserum. DISCUSSION Although A. propionicus was described as a separate species in 1962, no other isolates have been recognized to date. In this paper, three additional strains were compared in detail with the original isolate, and all were found to be essentially identical morphologically, biochemically, and serologically. In addition, the three strains produced propionic acid from the fermentation of glucose. Thus, in this discussion, the name A. propionicus is used to include all four cultures used in this study. Additional isolates of A. propionicus probably have not been recognized because of the close morphological resemblance of this organism to other species of Actinomyces, especially A. israelii. TABLE 4. Antigeniic relationiship between four cultures of Actinomyces propionicus with sorbed antiserum anidfluorescent antibody Antiserum No. of A..- pro- Serum 346 Serum Pionicus Serum Serum sorbed 346 antigen sorbed with sorbed with with sorbed with to not tested to3+ _ to 3+ not tested _ In this instance, the microcolony produced by A. propionicus is indistinguishable from that formed by A. israelii. Thus, this commonly employed differential criterion cannot be applied to these two species. A. propionicus grows best anaerobically, but it will also grow aerobically, and CO2 is not required as with other species of Actinomyces. In broth, it forms "bread-crumb" colonies. It resembles both A. israelii and A. naeslundii in its overall biochemical reactions. However, failure to ferment arabinose, xylose, salicin, and cellobiose, along with partial hydrolysis of starch and late liquefaction of gelatin, will aid in the identification of A. propionicus. Thus, an organism forming a "spider" microcolony, not fermenting any of these four carbohydrates and developing 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 DAP in its cell wall. The easiest and most rapid method for determining the presence or absence of DAP is to chromatograph whole-cell hydrolysates. All four of the A. propionicus strains contained DAP, whereas the other species were negative. This then is a major differential characteristic. In our hands, the most rapid and specific method of differentiation and identification of this organism was the fluorescent-antibody technique. Species-specific conjugated antiserum was prepared which did not cross-react with other species of Actinomyces. This antiserum was used in the direct fluorescent-antibody technique and was successfully applied to the identification of A. propionicus in exudate from patients, initial cultures, and subcultures. TABLE 5. Antigenzic relationship of Actiniomyces propionicus with other Actinzomyces as demonstrated bv thefluorescent-antibody techniique Conjugated antiseraa Culture no. 4 A. israelii A. naesl1ndii A. bovis A. eriksonii odontolyticus A. propionicus StrainWVU ssraezs ~~ _ ndiluted Undiluted antisera.

7 VOL. 94, 1967 ISOLATION AND CHARACTERIZATION OF A. PROPIONICUS 115 ACKNOWLEDGMENTS We wish to thank J. L. Campbell of Department of Oral Surgery and R. R. Trotter of Department of Ophthalmology for making case material available for study. Leo Pine of Communicable Disease Center confirmed the identity of one strain. This investigation was supported by Public Health Service grant AL from National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. BATrY, I Actinomyces odonitolyticus, a new species of actinomycete regularly isolated from deep carious dentine. J. Pathol. Bacteriol. 75: BECKER, B., M. P. LECHEVALIER, R. E. GORDON, AND H. A. LECHEVALIER Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole-cell hydrolysates. J. Appl. Microbiol. 12: BUCHANAN, B. B., AND L. PINE Characterization of a propionic acid producing actinomycete, Actinomyces propionicus, Sp. nov. J. Gen. Microbiol. 28: CHERRY, W. B., M. GOLDMAN, T. R. CARSKI, AND M. D. MOODY Fluorescent antibody techniques in the diagnosis of communicable diseases. U. S. Public Health Serv. Publ GEORG, L. K., G. W. ROBERTSTAD, AND S. A. BRINKMAN Identification of species of Actinomyces. J. Bacteriol. 88: HOWELL, A. JR., W. C. MURPHY III, F. PAUL, AND R. M. STEPHAN Oral strains of Actinomyces. J. Bacteriol. 78: McDADE, J. J., AND R. H. WEAVER Rapid methods for the detection of gelatin hydrolysis. J. Bacteriol. 77: SLACK, J. M., E. H. LUDWIG, H. H. BIRD, AND C. M. CANBY Studies with microaerophilic actinomycetes. I. The agglutination reaction. J. Bacteriol. 61: Downloaded from on November 7, 2018 by guest