APPLIED MICROBIOLOGY, May 1973, p. 834-843 Vol. 25, No. 5 Copyright 0 1973 American Society for Microbiology Printed in U.S.A. Identification of Actinomyces, Arachnia, Bacterionema, Rothia, and Propionibacterium Species by Defined Immunofluorescence KENNETH HOLMBERG AND URBAN FORSUM Department of Clinical Bacteriology, Central Hospital, Visterds, Sweden, and Institute of Medical Microbiology, University of Uppsala, Uppsala, Sweden Received for publication 13 February 1973 Fractionated fluorescein-isothiocyanate (FITC)-conjugated immunoglobulin G (dye-to-protein ratio < 10), produced against whole cells of Actinomyces spp., Arachnia, Bacterionena, Rothia, and Propionibacterium spp., give species- specific conjugates with controlled nonspecific staining reactions when appropri- ately diluted on the basis of their antibody content (10 mg/ml). Using this standardization in immunofluorescence, serotype-specific conjugates are also available after dilution for all serotypes of these organisms except for Actino- myces viscosus type 2, and Propionibacterium acnes type 1. Adequately ad- sorbed conjugates could be used to differentiate these serotypes from A. viscosus type 1 and P. acnes type 2, respectively. A serological classification in defined immunofluorescence corresponded to species and serotype designation proposed on the basis of other serological analysis and biochemical characteristics. This includes a separation in immunofluorescence of two serotypes of Propionibacte- rium acnes. The detection of certain actinomycetes of the family Actinomy- cetaceae and Propionibacterium species by the defined immunofluorescence in direct smears prepared from clinical specimens agreed to 88% with parallel cul- turing when including a prereduced (PRAS) medium technique for isolation. Qualitative studies revealed that single cells of these organisms could be spe- cifically identified by immunofluorescence when admixed with morphologically similar bacteria and a large number of other contaminants. A rather substantial literature is developing (46). However, new applications of immuno- which suggests the importance of strains of fluorescent techniques pose special problems gram-positive filamentous or diphtheroidal bac- and require their evaluation and characteriza- teria, or both, in the pathogenesis of periodontal tion with respect to specific staining titer, disease. The pathogenicity of Actinomyces spp. fluorescein concentration, protein concentra- has recently been demonstrated in experimen- tion, fluorescein-to-protein ratio (F/P ratio), tal animals (11, 12, 20, 31, 50). However, the and usable diagnostic titer (cf. 3, 10, 24). etiological role of these organisms in human The aim of this investigation was to deter- periodontal disease remains to be determined. mine the optimal criteria for fluorescein-isothi- This is due in part to the fact that cultivation ocyanate (FITC) conjugated antisera against and isolation of these fastidious organisms re- certain genera of the family Actinomycetaceae quire specific cultural techniques and are time- and certain species of Propionibacterium in consuming procedures (e.g., 19, 27, 29). Thus, direct immunofluorescence. The evaluations the need for specific and rapid identification were made using type species and oral isolates. procedures in ecological and epidemiological The accuracy of identifying these organisms by work has become important. defined immunofluorescence in direct smears Immunofluorescent techniques have been was determined by parallel cultures. found to be of value for detection of Actinomyces spp. in tissue impression smears MATERIALS AND METHODS from experimentally infected mice (34), in tis- Strains. Reference strains were obtained from sue or exudate smears from human tonsillar American Type Culture Collection (ATCC), Rock- materials (4), and in human dental calculus ville, Md., National Collection of Type Cultures 834 VOL. 25, 1973 SPECIES IDENTIFICATION BY IMMUNOFLUORESCENCE 835 (NCTC), Colindale, London, and National Center for (pH 6.3), further purified by chromatography on a Disease Control (CDC), Atlanta, Georgia. Two strains, diethylaminoethyl (DEAE)-cellulose column equili- Actinomyces israelii serotype 2 (WVU 307) and brated and eluted with the dialysis buffer, and finally Arachnia propionica serotype 2 (WVU 346), were concentrated with an ultrafiltration cell (Diaflo model kindly supplied by M. Gerencser, Department of 51, Amicon Corp.) to a protein concentration of 18 to Microbiology, West Virginia University Medical 20 mg/ml. The protein concentration was determined School, Morgantown, W. Va. One strain of Coryne- by measuring the optical density at 280 nm. The IgG bacterium acnes group 2 (D 34) (Propionibacterium preparations gave a single IgG line in immunoelectro- granulosum) was supplied by J. G. Voss, The Proctor phoresis against anti-rabbit plasma protein serum & Gamble Co., Miami Valley Lab., Cincinnati, Ohio. (Behringwerke AG, Germany) (42). Immunization. Type strains (Table 1) were grown Conjugation. Rabbit IgG was conjugated with in Trypticase soy broth (Difco) for 48 to 72 h. After FITC (BDH Biochemicals, Poole, England). FITC centrifugation the cultures were suspended in 0.15 M was added to IgG in the proportion of 30 leg of dye per NaCl with 5% Formalin (vol/vol) for 1 h and washed mg of protein under vigorous stirring at room temper- twice in 0.15 M NaCl. One-milliliter cell suspensions ature for 1 h (pH 9.5). Nonreactive FITC was removed containing roughly 106 organisms per ml were injected by passing the solution through a Sephadex (Phar- intravenously in rabbits every third to fourth day for 4 macia AB, Sweden) G-25 column equilibrated with weeks. The rabbits were bled 7 days after the last phosphate-buffered saline (PBS) (35). injection. Slide agglutination titers of the sera ranged Fractionation of conjugates. To obtain fractions from 1/64 to 1/128 with living cells as antigens. For giving minimal nonspecific staining (24, 51), the each strain, two rabbits were immunized. conjugates were chromatographed on a DEAE-cell- Preparation of IgG. Rabbit immunoglobulin G ulose column equilibrated with 0.0175 M NaHPO4 (IgG) was prepared from sera by precipitation with (pH 6.3). After elution of a small amount of nonconju- ammonium sulfate at 37% saturation. The precipitate gated material with the starting buffer, a stepwise was dissolved in distilled water, and the procedure gradient was applied using 0.125 M NaCl, 0.250 M was repeated twice. The precipitated and redissolved NaCl, and 0.500 M NaCl in 0.0175 M NaHPO4 (pH material was dialyzed against 0.0175 M NaHPO4 6.3), respectively. The fractions were concentrated by TABLE 1. Specific staining titers for homologous strains and nonspecific staining of gingival tissue cells by fractions of FITC conjugates with different F/P ratios Spe- No-Spe- Non- FrIC-labeled antisera F/P cific FITC-labeled antisera | cf ratio specific ratioaP specific (10 mg/ml) (,Ug/mg)g tan- Stain- (10 mg/ml) nstag- stain- Ing Cgng ~ n titer' titer ig Rothia dentocariosa (ATCC 9.0 128 Arachnia proprionica type 1 10.0 16 17931) 13.0 32 (ATCC 14157) 12.0 8 20.0 4 ++ 20.0 4 + Bacterionema matruchotii 7.5 64 Arachnia propionica type 2 5.5 64 (ATCC 14265) 16.0 2 (+) (WVU 346) 12.5 8 20.0 +++ 18.0 1 +++ Actinomyces viscosus type 1 4.0 64 Propionibacterium acnes 7.5 64 (ATCC 15987) 13.0 8 type 1 (NCTC 737) 15.0 4 + 21.0 +++ 21.0 +++ Actinomyces viscosus type 2 4.0 64 Propionibacterium acnes 7.0 64 (ATCC 19246) 12.0 4 type 2 (ATCC 11828) 12.0 4 + 20.0 +++ 17.0 ++ Actinomyces naeslundii 8.0 64 Propionibacterium avidum 9;0 32 (ATCC 12104) 15.0 16 (ATCC 25577) 15.0 4 + 19.0 +++ 20.0 +++ Actinomyces israelii type 1 6.0 64 Propionibacterium 10.0 16 (ATCC 12103) 13.5 2 + granulosum (ATCC 25564) 13.0 8 20.0 +++ 19.0 +++ Actinomyces israelii type 2 5.0 128 Propionibacterium jensenii 10.0 32 (WVU 307) 12.0 8 (ATCC 4867) 16.0 4 + 20.0 2 ++ 18.0 ++ Actinomyces odontolyticus 9.0 32 (ATCC 17982) 15.0 4 + 20.0 1 ++ aExpressed as reciprocal of titer. 836 HOLMBERGjAND FORSUM APPL. MICROBIOL. ultrafiltration (Diaflo model 50, Amicon Corp.) to a acetone for 10 min. For estimation of specific staining final concentration of 10 mg/ml and dialyzed against reactions of type organisms and for identification of PBS (pH 7.2). The protein content and F/P ratio isolates, smears were prepared from 24- to 48-h pure (gg/mg) were estimated as described by Wells et al. cultures. Nonspecific staining of leukocytes were eval- (52). All conjugates were stored at 0 to 5 C or frozen in uated in smears prepared from freshly drawn human several small portions for storage. venous blood. After coagulation for 10 min at room Preparation of FITC-labeled F(ab'), fragments. temperature and then for 20 min at 37 C, the clot was To obtain F(ab')2 fragment of IgG from antisera to removed by washing with 0.15 M NaCl. Rothia dentocariosa, pepsin digestion was performed Staining of smears and recording procedures. (17, 36) using 1 mg of enzyme per 50 mg of IgG in 0.1 One drop of conjugate was placed on a smear and M sodium acetate (pH 3.0) for 7 h. The F(ab'), incubated for 20 min at 37 C. The smear was washed fragments were conjugated with FITC as described with PBS for 15 min and mounted under a cover glass above and fractionated on Sephadex G-25 equili- with phosphate-buffered glycerin (pH 7.2). brated with 0.0175 M Na,PHO4 (pH 4.7) into frac- The preparations were read under a Zeiss fluores- tions with different F/P ratios (17). The first peak to cence microscope equipped with an Osram HBO 200 emerge from the column was collected, and its pH was mercury lamp, oil immersion dark-field condensor, adjusted to 7.2. Conjugated F(ab')2 fragments with and a 4-mm BG 12 filter as primary and a Zeiss 50 high F/P ratios, which showed firmer adsorption to filter as secondary filter. Tissue specimens were read the gel and which therefore eluted in a second peak, with a Zeiss 44 filter as secondary filter.