INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1990, p. 273-286 Vol. 40, No. 3 0020-7713/90/070273-14$02.oo/o Copyright 0 1990, International Union of Microbiological Societies

Actinomyces georgiae sp. nov. , gerencseriae sp. nov. , Designation of Two Genospecies of Actinomyces naeslundii, and Inclusion of A. naeslundii serotypes I1 and I11 and Actinomyces viscosus serotype I1 in A. naeslundii Genospecies 2

J. L. JOHNSON,l LILLIAN V. H. MOORE,l BEVERLY KANEK0,2 AND W. E. C. MOORE1* Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, and Microbial Diseases Laboratory, Department of Health Services, State of California, Berkeley, California 947M2

DNAs of type strains aod representative members of Actinomyces groups from the human periodontal flora and from other habitats were compared by using the S1 nuclease procedure to determine their genetic relatedness. One rather common group from the human periodontal flora, previously called “Actinomyces DOS,” is phenotypically distinct from, and genetically unrelated to, previously described species. We propose the name Actinomyces georgiae for this organism; the type strain is strain ATCC 49285. Another common group from the human periodontal flora is serotype 11, which was found to be genetically distinct from the type strain of A. israelii (serotype I) and from other previously described species of Actinomyces. We propose the name Actinomyces gerencseriae for this organism; the type strain is strain ATCC 23860. A. naeslundii serotype I strains were distinct from the other strains studied. A separate genospecies which included strains of A. naeslundii serotypes I1 and I11 and A. viscosus serotype I1 was delineated. Strains of Actinomyces serotype WVA 963 constitute an additional distinct genospecies. Because there are no reliable phenotypic tests, other than serological analyses, to differentiate Actinomyces serotype WVA 963 and the two genospecies of A. naeslundii, no taxonomic changes are proposed for these three genospecies.

Actinomyces species and serotypes form a major portion scribed as A. naeslundii serotype IV (lo), were not included of the periodontal flora of humans and animals. Although in the study of McCormick et al. there is no direct evidence that these organisms cause Previous reports of DNA hybridization studies have been gingivitis or periodontitis (26), they appear to play a key role limited. Coykendall and Munzenmaier (6), who used a in the colonization of gingival crevices that may relate to membrane filter hybridization analysis method, reported 71 subsequent periodontal disease (18-22; P. K. Kolenbrander, to 107% DNA relatedness between A. naeslundii and human Crit. Rev. Microbiol., in press), Precise identification of strains of A. viscosus. Despite these high levels of DNA these taxa often is difficult because there is considerable relatedness, Coykendall and Munzenmaier did not propose phenotypic variation among strains within species and be- combining the human strains of A. viscosus with A. naeslun- cause specific antisera that are exceedingly helpful in species dii, based on the report by Fillery et al. (8) that the two identification (9) are not available commercially. groups could be separated phenotypically (with 12% dissim- Among the species isolated from humans, it generally has ilarity). However, in discussing their own data, Fillery et al. been accepted that Actinomyces israelii, Actinomyces odon- (8) stated that “these data suggest that the division of human tolyticus, and Actinomyces meyeri are distinct species (31, A. viscosus from A. naeslundii is closer to a serotypic 37). However, high degrees of phenotypic (8, 16, 33-35) and separation than separation at a species level.” Relationships serological (5, 10, 14) relatedness between Actinomyces among the other oral Actinomyces species were not reported naeslundii and human isolates of Actinomyces viscosus have by Coykendall and Munzenmaier. been reported previously. Gerencser (9) noted that “all Part of the problem of species delineation in the genus recent evidence seems to support the earlier suggestions that Actinomyces has been a dearth of information concerning A. naeslundii and A. viscosus are varieties of a single the genetic relatedness among the many types found in the species,” but she retained the current classification of con- human flora. Such evidence is essential for reliable nomen- sidering catalase-negative strains A. naeslundii and catalase- clature and diagnosis. In this study we attempted to provide positive strains A. viscosus, as did Schaal (31). some of the needed information concerning the relationships In a study of polyacrylamide gel electrophoresis patterns of these taxa and in particular to determine the taxonomic of whole-cell preparations from 22 strains of Actinomyces position of serotype WVA 963 (= WVU 963) and strains that species, McCormick et al. (23) found that cluster analysis of reacted with both A. naeslundii and A. viscosus serotype I1 polyacrylamide gel electrophoresis banding patterns permit- antisera. ted clustering first by serotype and then according to spe- cies. Two major divisions were reported. One contained MATERIALS AND METHODS strains of Actinomyces bovis serotype I, A. odontolyticus Bacterial strains and characterization. serotypes I and 11, and A. israelii serotypes I and 11. The The strains which we examined by using DNA hybridization and which were other contained strains of A. naeslundii serotypes I, 11, and I11 and A. viscosus serotypes I and 11. Strains of Actinomy- used in the preparation of fluorescent antibody conjugates ces serotype WVA 963, which previously had been de- are shown in Table 1. Additional strains for which pheno- typic data were summarized were obtained from the Virginia Polytechnic Institute and State University Culture Collec- * Corresponding author. tion and were isolated from dental plaque. These strains

273 274 JOHNSON ET AL. INT. J. SYST.BACTERIOL.

TABLE 1. Strains used in this study"

Species or ATCC Source and commentsb serotype no.' A. georgiae D145A-07T' 492MTd Gingival crevice of healthy adult D028G-6A' Gingival crevice of young adult with rapidly progressive periodontitis D12 1H-02 Gingival crevice of healthy 14-yr-old D145A-07 Gingival crevice of 11-yr-old D146A-10 Gingival crevice of adult with progressive periodontitis D148E-28 Gingival crevice of healthy 12-yr-old E07T-20B Gingival crevice of healthy 4-yr-old

A. naeslundii serotype I 1970T' 12mT CDC W826T, human sinus 12592 W454, antigen strain for conjugate

A. naesfundii serotype I1 12593' 49339d CDC 'W1544, antigen strain for conjugate

A. naesfundii serotype I11 D163E-3 ' 49340d Gingival crevice of adult with progressive periodontitis None CDC WVU 820, antigen strain for conjugate

Actinomyces serotype WVA 963 DO97 W-3' Gingival crevice of adult with progressive periodontitis D135D-26 4933gd Gingival crevice of healthy 12-yr-old D133B-06 Gingival crevice of healthy 11-yr-old D134B-22 Gingival crevice of healthy 11-yr-old D143E-06 Gingival crevice of healthy 12-yr-old D153A-18B Gingival crevice of healthy 11-yr-old None WVU 963, antigen strain for conjugate

A. viscosus serotype I 12571T' 15987T CDC A82gT, naturally occurring periodontal disease in hamsters, antigen strain for A. viscosus serotype I conjugate

A. viscosus serotype I1 12591' 27044 CDC W1053, human sputum, antigen strain for A. viscosus serotype I1 conjugate

Actinomyces serotype NV D163M-19' Gingival crevice of adult with progressive periodontitis D039C-20 Gingival crevice of juvenile with localized rapidly progressive periodontitis D098C-06 Gingival crevice of adult with naturally occurring periodontitis E05K- 11 Gingival crevice of 4-yr-old with gingivitis

A. israelii 1966T' 12102T CDC WWT, human brain abscess, antigen strain for A. israelii serotype I conjugate

A. gerencseriae 12594T 23860T CDC W83gT, human parotid abscess, antigen strain for A. israelii serotype I1 conjugate D 140D-18 Gingival crevice of healthy 11-yr-old

A. meyeri 8617T' 3556gT A. R. Prevot (PIP 2477BT), human with purulent pleurisy 10648 33972 CDC W1148, antigen strain for conjugate

A. bovis serotype I 1965T 13683T Lumpy jaw in cows, antigen strain for conjugate

A. bovis serotype I1 12595 CDC WVU 292, antigen strain for conjugate

A. odontofyticus serotype I 1991-2T' 17929T Deep carious lesions around teeth 12589 CDC W830, antigen strain for conjugate

A. odontofyticus serotype I1 D136E-19B Gingival crevice of healthy 12-yr-old 12590 CDC W1514 (= WVU 482), antigen strain for conjugate

A. hordeovulneris 14010T 35275T Canine ascites fluid

a All strains except the A. georgiae, A. hordeovulneris, and Actinomyces serotype NV strains reacted 4+ with homologous conjugate and did not react with any of the other conjugates; strains of Actinomyces serotype NV reacted 3+ to 4+ with conjugates to A. naeslundii serotype I or I1 and A. viscosus serotype I1 and did not react with any of the other conjugates. Strains of A. georgiae and A. hordeovufnerisdid not react with any of the conjugates. All strains except CDC WVU 820, WVU 963, VPI 10648, VPI 12595, and VPI 12589 were used in the DNA homology studies. VPI, Virginia Polytechnic Institute and State University, Blacksburg; ATCC, American Type Culture Collection, Rockville, Md. ; CDC, Centers for Disease Control, Atlanta, Ga. ; PIP, Pasteur Institute, Paris, France; WVU, West Virginia University, Morgantown. Strain used to prepare reference DNA. Strain that has been deposited with the American Type Culture Collection but has not been returned for depositor approval. VOL. 40, 1990 OF ACTZNOMYCES 275 were limited to those for which cellular fatty acid data were affinity column (7). The following antisera were used: A. available. For those species or serogroups for which multiple israelii serotypes I and 11; A. naeslundii serotypes I, 11, and strains were available, each isolate was obtained from a 111; Actinomyces serotype WVA 963 ; A. viscosus serotypes different person and was characterized during the past 11 I and 11; A. meyeri; and A. odontolyticus serotypes I and 11. years. Thus, the phenotypic variation given below repre- DNA isolation and hybridization. The organisms were sents the variation obtained with multiple lots of media and grown in 300-ml volumes of a medium consisting of peptone- reagents and should express the variation that would be seen yeast extract-glucose (13) supplemented with 1% brain-heart within the species or serogroup. infusion broth (Difco Laboratories, Detroit, Mich.) and Cultures were grown in prereduced anaerobically steril- 0.01% (voVvol) Tween 80. Each flask was inoculated with 8 ized media and were characterized by using the procedures ml of culture grown in chopped meat broth-carbohydrate of Holdeman et al. (13) and Moore et al. (27). Unless medium (13). The flasks were incubated at 37°C with stirring otherwise specified, Tween 80 (0.02%, vol/vol) was added to until there was heavy growth, usually about 40 h. all broth media. After isolation and purification, pure cul- DNA was isolated by using a cetyltrimethylammonium tures were tested for maximum growth and fermentation in bromide (CTAB) procedure which is a variation of a proce- peptone-yeast extract-glucose broth medium with and with- dure that has been used to isolate fungal DNA (T. Flynn, out Tween 80 and were inoculated and incubated (i) anaer- personal communication). A harvested cell pellet (final abically under Co,, (ii) aerobically and restoppered (to weight, 10 g) was transferred to a tared Braun Disintegrator introduce a small amount of oxygen), and (iii) aerobically (Bronwill Scientific, Inc., Rochester, N.Y .) shaking bottle (tubes covered with sterile aluminum foil). Additional phe- along with Tris-EDTA buffer (10 mM Tris hydrochloride, 1 notypic tests were performed by using the inoculation and mM EDTA, pH 8.0). Then 10 ml of 2~ CTAB lysing buffer incubation conditions that produced the best growth and (1 x CTAB lysing buffer is 50 mM Tris hydrochloride, 0.7 M lowest pH. If all conditions produced the same results, the NaCl, 10 mM EDTA, 1% [wt/vol] CTAB, and 1% [voVvol] media used subsequently for characterization included 2-mercaptoethanol, pH 8.0) and 20 ml of glass beads (diam- Tween 80 and were inoculated aerobically and restoppered. eter, 110 pm) were added to the bottle, and the bottle was Cellular fatty acids were determined as previously de- shaken for 5 min with CO, cooling. The lysate was separated scribed (25). Briefly, cells from 10-ml portions of peptone- from the glass beads by filtration through a coarse sintered yeast extract-glucose-Tween 80 cultures were harvested by glass filter. An additional 30 ml of IXCTAB lysing buffer centrifugation and washed once by suspension in 3 ml of was used to wash the lysate from the beads quantitatively. 0.7% (wt/vol) aqueous MgSO, and centrifugation. The cells The pooled lysate was digested with proteinase K (40 pg/ml) were lysed and saponified with 1 ml of basic methanol (45 g for 4 h at 60°C and then extracted with CHCl,. The cetylt- of NaOH, 150 ml of methanol, 150 ml of distilled water), rimethylammonium-nucleic acid salts were then precipitated heated in a boiling water bath for 5 min, mixed with a vortex by diluting the lysate with an equal volume of dilution- mixer, and heated in the boiling water bath for an additional precipitation buffer (50 mM Tris hydrochloride, 10 mM 25 min. To methylate cell constituents, 1ml of HC1-methanol EDTA, 1% [wt/vol] CTAB, pH 8.0). The precipitate was (325 ml of 6.0 N HCl, 164 ml of methanol [certified grade]) washed two or three times with 40-ml volumes of 0.4 M NaCl and 1 ml of sulfuric acid-methanol (162.5 ml of H,SO, to remove the free CTAB. The pelleted precipitate was then [American Chemical Society reagent grade] added to 162.5 dissolved in 8 ml of 2.5 M ammonium acetate, which was ml of distilled water, 275 ml of methanol [certified grade]) facilitated by warming in a 50°C water bath. The tube was were added, and the solution was heated at 80°C for 10 min. then placed on ice, where much of the high-molecular-weight After cooling, methylated components were extracted by RNAs precipitated. After the precipitated RNAs were re- adding 1.25 ml of hexane-ether (200 ml of hexane [high- moved by centrifugation, the DNA and the remaining RNA performance liquid chromatography grade], 200 ml of were collected by ethanol precipitation. The pellet was methyl-tert butyl ether [high-performance liquid chromatog- dissolved in a solution containing 10 ml of 0.1~SSC (Ix raphy grade]) and turning the tubes end over end for 10 min. SSC is 0.15 M NaCl plus 0.015 M trisodium citrate [pH 7.0]), The extract was washed once with 3 ml of a solution 0.5 ml of RNase A (0.5 mg/ml), and 5 pl of RNase T, (200 containing 5.4 g of NaOH in 450 ml of deionized distilled U/pl), and the mixture was incubated at 37°C for 1 h. The water saturated with NaCl. A 2-pl portion of the washed digest was then extracted once with phenol-CHCl,, adjusted extract was chromatographed on a fused-silica capillary to 0.3 M sodium acetate, and ethanol precipitated. The DNA column in a model HP-5890A chromatograph (Hewlett- preparations were dissolved in 0.1X SSC, and the purity of Packard Co., Palo Alto, Calif.) equipped with a flame each preparation was estimated from the amount of hyper- ionization detector and a model HP-3392A integrator chromicity that occurred during its melting profile (17). (Hewlett-Packard). We used the MIS software package For the hybridization experiments, the DNA preparations (Hewlett-Packard) to identify the peaks (by retention time) were uniformly fragmented by three passages through a and to determine the area, the ratio of area to height, the French pressure cell at 16,000 lb/in2. The preparations were equivalent chain length, the total area, and the total area of then denatured by being heated in a boiling water bath for 5 named compounds; the MIS software package was also used min, and, after cooling in ice, the preparations were centri- to calculate the percentage of area for each named com- fuged at 12,000 x g for 15 min to remove any particulate pound compared with the total area of named compounds. material. The DNA concentration of each preparation was Identification was accomplished by using the Virginia Poly- adjusted to 0.4 mg/ml. The preparations were stored at technic Institute and State University Anaerobe Laboratory -20°C. Portions (2 to 3 pg) of the fragmented and denatured Broth Library and methods (Microbial ID, Inc., Newark, preparations from selected reference strains were labeled by Del.). iodination (36). The specific activities of the labeled prepa- Fluorescent antibody. Cells were serotyped with florescent rations ranged from 1 x lo6 to 2 x lo6 cpm/pg of DNA. antibody prepared by one of us (B.K.) by using previously DNA sequence similarity values were determined by using described procedures (7, 11, 12), except that Sepharose 4B the S1 nuclease procedure (17). Each of the reassociation beads rather than Sepharose 6MB beads were used in the vials contained 10 pl of labeled DNA (10 to 20 ng; 30,000 276 JOHNSON ET AL. INT. J. SYST. BACTERIOL. cpm), 50 p,l of unlabeled DNA, 25 p,1 of 5.28 M NaCl in 1mM Growth in prereduced anaerobically sterilized peptone- HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic yeast extract-glucose-Tween 80 broth is turbid (85%) with acid) (pH 7.0), and 25 pl of deionized formamide. The vials smooth (38%), flaky (26%), crumby or granular (24%), or were incubated for 24 h at 65°C (25°C below the thermal stringy to ropy (12%) sediment that occasionally adheres to melting point of native DNA in this buffer system) to a Cot the bottom of the tube. Broth cultures without turbidity value of about 47 mol/liter per s. To measure background often have crumby to coarse granular sediment. Good reassociation values and to determine the amounts of S, growth is produced in peptone-yeast extract (basal medium) nuclease-resistant material in the labeled preparations, the broth cultures, and excellent growth is produced in peptone- denatured unlabeled DNA was replaced with 50 pl(20 pg) of yeast extract medium that contains a fermentable carbohy- sheared native salmon sperm DNA. drate. The pH values of peptone-yeast extract cultures that are inoculated aerobically and restoppered are 6.6 to 7.8; the RESULTS AND DISCUSSION pH values of peptone-yeast extract cultures that are inocu- The results of the DNA hybridization experiments and the lated under CO, are 5.7 to 6.3. The pH of peptone-yeast guanine-plus-cytosine (G+C) contents of the strains which extract-glucose cultures is 4.7 (range, 4.5 to 5.2) and is the we examined are shown in Table 2. Strains of A. israelii same under all conditions of inoculation if growth is equiv- serotype I, A. meyeri, A. odontolyticus serotypes I and 11, A. alent. The acid products (in milliequivalents per 100 ml of bovis, and Actinomyces hordeovulneris were clearly distinct culture) from 4- to 5-day-old peptone-yeast extract-glucose- from the other. species or serotypes. Tween 80 broth cultures are as follows: succinic acid, 1.4 The DNA hybridization data indicated that the organisms (range, 0.3 to 4.0); formic acid, 1.0 (range, 0.4 to 1.8); and which we previously designated “Actinomyces D08” (28, acetic acid, 1.0 (range, 0.2 to 2.7). Small amounts of lactic 29) on the basis of the results of phenotypic tests, polyacryl- acid (0.4 meq/100 ml; range, 0.1 to 0.8 meqllOO ml) are amide gel electrophoresis patterns, and cellular fatty acid detected in 70% of the cultures, and pyruvate (0.6 meq/100 profiles and which were represented by reference DNAs ml; range, 0.2 to 1.5 meq/lOO ml) accumulates in 54% of the from strains VPI D28G-6A and VPI D145A-7 (Table 2) have cultures. Lactate and pyruvate accumulate only in cultures negligible DNA relatedness with the other reference DNAs that are inoculated aerobically and restoppered and are not tested and clearly represent a new species. Because these detected in cultures that are inoculated under CO,. Accu- are facultatively anaerobic, nonsporing, nonmotile, mulation of lactate and pyruvate probably results from an gram-positive, filamentous to diphtheroid bacilli that pro- insufficiency of bicarbonate in the medium or CO, in the duce combinations of lactic and succinic acids, usually along headspace gas. No hydrogen is detected in the headspace with acetic and formic acids, and have G+C contents of 65 gas of peptone-yeast extract-glucose-Tween 80 cultures in- to 69 mol%, they are members of the genus Actinomyces (30, cubated for 4 to 5 days. 31). These organisms are described below as Actinomyces Strains grow well at 37°C. No strain produces urease georgiae (previously “Actinomyces D08”). (urease broth) (13). Fermentation of glycerol is variable. Description of Actinomyces georgiue sp. nov. Actinomyces The major cellular fatty acids are 16:0,18:1 cis-9, and 14:O. georgiae (geor’gi.ae. N. L. gen. n. georgiae, of Georg, There is no reaction with fluorescent antibody conjugates named in honor of Lucile Georg, a pioneer in Actinomyces to A. israelii serotype I, A. israelii serotype I1 (Actinomyces taxonomy). The description below is based on a study of 36 gerencseriae [see below]), A. naeslundii serotypes I, 11, and strains. Strains are facultatively anaerobic, gram-positive, 111, Actinomyces serotype WVA 963, A. viscosus serotype nonmotile, nonsporing rods that occur in pairs, short chains, 11, A. meyeri, and A. odontolyticus serotypes I and 11. and forms and may have swellings; branching is seldom Additional characteristics of the species are shown in seen. Cells of the type strain grown for 24 h in peptone-yeast Tables 3 and 4. Differential characteristics are shown in extract-glucose broth are 0.9 to 1.0 pm wide and 3.3 to 6.1 Table 5. pm long. In broth medium without a fermentable carbohy- The type strain is strain VPI D145A-7 (= ATCC 49285). drate, cells are shorter and may even be coccoid. The G+C content of the DNA ranges from 65 to 69 mol% Although strains grow reasonably well on the surfaces of (Table 1). blood agar plates incubated in air enriched to 10% with CO,, Isolated from human gingival crevices. 55% of the strains produce better surface growth when they A. georgiae accounts for 1.4% of the human healthy are incubated anaerobically in anaerobe jars or streak tubes. periodontal flora (Table 6). Colonies of the type strain incubated anaerobically are 1mm A. israelii serotype 11 strains. Initially, we were surprised at in diameter, circular, entire, pulvinate, translucent, white, the low level of DNA relatedness between A. israelii sero- shiny, and smooth. Colonies of other strains range from 0.5 type I reference DNA and unlabeled DNAs from A. israelii to 2 rnm in diameter and usually are circular, entire, low serotype I1 (strains VPI 12594 and VPI D140D-18). How- convex, opaque (smaller colonies are transparent to translu- ever, Stackebrandt and Charfreitag (39) recently reported cent), white (27% are tan or beige), shiny, and smooth. that an A. israelii serotype I-specific DNA probe failed to Addition of 0.02% Tween 80 to broth medium enhances give a hybridization signal with rRNA from a strain of A. the growth or fermentation of 62% of the strains; it does not israelii serotype I1 and suggested that the A. israelii serotype inhibit the growth or fermentation of any strain tested. In I1 strains may constitute a new species. prereduced anaerobically sterilized broth medium, 53% of According to some numerical taxonomic analyses (16,35), the strains grow better or have a lower pH in peptone-yeast strains of A. israelii serotypes I and I1 cluster together. extract-glucose media when the media are inoculated aero- Other workers (32, 33) have recognized four subgroups bically and the tubes are closed with rubber stoppers than (subspecies) within A. israelii, with two of the subgroups when the media are inoculated anaerobically under CO, or corresponding to the two recognized serotypes. On the basis aerobically and the tubes are covered with sterile aluminum of the low levels of DNA-DNA relatedness with the other foil. Twenty percent of the strains grow equally well under species tested (Table 2), we propose that A. israelii serotype all conditions of inoculation, and 27% grow equally well I1 strains should be recognized as a distinct species, for either under CO, or restoppered after aerobic inoculation. which we propose the name Actinomyces gerencseriae. The TABLE 2. DNA relatednessof strains of Acinomyces species as determinedby S, nuclease experiments % Homology with reference DNA from:

~~~ ~ G+C A. odon- Species or content A. georgiae A. georgiae A. naeslun- A. naeslun- A. naeslun- Serotype VPI no. dii serotype dii serotype dii serotype wA 963 A. viscosus A. viscosus Serotype A. israelii A. mtyeri totyticus serotype” (mol%) VPI VPI serotype I serotype I1 NV VPI serotype I VPI serotype I I1 VPI 111 VPI D28G-6A D145A-07T :z!: 12593 D163E-03 D09z-30VPI 12571 VPI 12591T D163M-19 VPI 1966= 8617T VPI 1991- 2T

A. georgiae D028G-6A 65 100 72 8 7 6 4 3 7 3 D112D-03 65 63 89 8 6 2 4 2 5 6 D121H-02 68 65 90 8 3 4 4 2 4 2 D145A-07T 68 65 100 3 2 2 2 1 2 2 D146A-10 69 68 95 5 2 2 3 2 3 1 D148E-28 68 55 88 5 1 2 3 1 2 1 EO7T-20B 69 60 93 7 1 2 3 3 2 2 A. naeslundii Serotype I 1970T 66 5 3 100 42 36 38 39 35 34 Serotype I1 12593 66 3 1 33 100 51 30 24 62 63 Serotype I11 D163E-03 63 2 2 22 62 100 29 20 55 51

Actinomyces sero- D097W-30 64 4 5 27 36 33 100 24 37 27 type WVA 963 D135D-26 67 3 2 39 32 29 83 32 32 27 D133B-06 70 4 3 45 33 29 74 33 32 27 D134B-22 65 3 2 49 35 31 88 36 32 27 D143E-06 64 5 7 50 34 31 90 36 33 18 D153A-18B 63 6 5 50 36 34 87 38 32 28 A. viscosus Serotype I 12571T 61 3 2 55 44 37 36 100 36 35 Serotype I1 12591 66 5 3 45 79 59 29 36 100 75

Actinomyces sero- D163M-19 63 4 4 40 82 57 40 33 81 100 typeNV D039C-20 65 6 7 43 77 50 33 33 68 72 D098C-06 62 4 4 41 74 49 32 29 66 70 E05K-11 63 3 4 40 69 51 36 35 64 57 A. israelii serotype I 1966T 70 4 1 15 11 11 8 11 10 10 100 0 3 A. gerencseriae 12594= 70 3 3 15 25 16 10 11 21 21 14 0 1 D140D-18 71 7 8 15 9 10 8 11 12 8 23 1 3 A. meyeri 8617T 63 4 3 5 1 2 2 2 2 2 3 100 5 A. bovis serotype I 1965= 63 3 2 12 8 6 6 6 7 6 3 0 2 A. odontotyticus Serotype I 1991-2T 6 3 8 7 6 4 5 8 7 2 0 100 Serotype I1 D136E-19B 62 7 7 5 3 2 3 3 3 1 3 1 41 Serotype I1 12590 1 7 4 2 1 2 0 2 2 2 1 51 A. hordeovulneris 14OlOT 68 4 2 5 2 2 4 2 3 2 2 0 1 * A. geor@ae formerly was referred to as “Actinomyces DO8” (21,22). Actimmyces serotype NV strains cross-react serologically with A. naeslundii and A. viscosus serotype I1 sera. A. gerencseriae formerly was referred to as A. israelii serotype 11. 278 JOHNSON ET AL. INT. J. SYST.BACTERIOL.

TABLE 3. Characteristics of Actinomyces species and serotypes A. naeslundii genospecies A. naeslundii Actinomyces serotype A. georgiue 1 (serotype I) genospecies 2" WVA 963 A. viscosus serotype I: Characteristic Reaction($ % of Reaction($ % of Reaction(s) % of Reaction(s) % of reaction of of 35 strains of 42 strains of 122 strains of 35 strains 1 strain strains positive strains positive strains positive strains positive Acid production from: Am ygdalin -WC 6 - 0 - 3 - 0 Arabinose -a 14 - 0 - 2 - 0 Cellobiose - 3 a- 66d -a 27 V 40 Esculin -a 8 -a 27d - 3 -a 32 Esculin hydrolysis +- 89 + 95 + 95 + 100 Acid production from: Fructose a- 94 a 95 a 99 a 97 Glucose a 100 a 95 a 100 a- 94 Glycogen aw 91 - 4 V 45 -a 26 Inositol -a 23 a- 90 a- 96 a 97 Lactose a- 88 a- 64 a- 73 a- 77 Maltose a 97 a 95 a 98 a- 94 Mannitol -a 31 - 2 -a 6 - 0 Mannose -a 20 a- 88 a- 92 a- 77 Melezitose -a 6 -a 7 -a 34 - 0 Melibiose - 0 a- 88 a- 74 a- 94 Rdnose -a 8 a- 90 a 99 a 97 Rhamnose aw 94 - 2 -a 8 - 3 Ribose a 97 V 4gd a- 74 -a 12 Salicin -a 14 a- 78 -a 29 a- 77 Sorbitol -a 11 -a 7 -a 24 -a 31 Starch a 97 -a 28 a.- 78 a- 72 Starch hydrolysis -+ 14 - 0 - 0 - 3 Acid production from: Sucrose a 100 a 95 a 99 a- 94 Trehalose a 97 a- 71 a- 74 V 52 Xylose a- 94 -a 9 -a 15 - 0 Gelatin hydrolysis (+w) V 51 - 2 - 0 - 0 Milk curd + 97 +- 86 +- 80 + 100 Nitrate reduction -+ 17d + 100 +- 73 +- 94 - - - Catalase production 0 2 V 51 0 Hemolysis Alpha 30 78 48 76 Beta 0 0 0 0 Gamma 70 22 52 24 " A. naeslundii genospecies 2 contains A. naeslundii serotype I1 (8 strains tested), A. naeslundii serotype I11 (45 strains tested), A. viscosus serotype I1 (25 strains tested), and strains that react with both A. naeslundii and A. viscosus serotype I1 conjugates (serotype NV, 44 strains tested). Two analyses of one strain were performed. +, Positive reaction for 95 to 100% of the strains; -, negative reaction or pH greater than 5.7 for 90 to 95% of the strains; a, pH 5.5 or less for 95 to 100% of the strains; v, positive reaction for 40 to 60% of the strains; w, pH 5.5 to 5.7 for 95 to 100% of the strains. Where two reactions are given, the first was the more common. The reactions for the type strains were the more common reactions except where indicated otherwise. The two reactions given account for all of the strains tested; for example, where the reactions were a,w and the percentage of positive strains was 92%, 92% of the strains produced strong acid (pH 4.3 to 5.4) and the remaining 8% produced weak acid (pH 5.5 to 5.7). No strains digested meat, produced indole, or fermented erythritol. The reaction for the type strain was the less common reaction. other two subspecies of A. israelii recognized by Schaal and obically on blood agar plates are 0.2 mm in diameter, colleagues were not represented in our study because we circular, peaked to pulvinate, lumpy, opaque, and white. examined only strains that reacted with the antisera avail- Colonies of other strains are 1.0 to 2.0 mm in diameter able to us. (range, 0.2 to 2.0 mm), slightly irregular to circular, usually Description of Actinomyces gerencseriae sp. nov. Actinomy- opaque (smaller colonies are transparent to translucent), ces gerencseriae (ge.ren.cse'ri.ae. N. L. gen. n. gerencse- lumpy (90%), and white (87%) or buff (13%). riae, of Gerencser, named in honor of Mary Ann Gerencser, Cells from 24-h peptone-yeast extract-glucose broth cul- an authority on Actinomyces species). Formerly, this taxon tures are gram-positive, nonmotile, nonsporing, and usually was called A. israelii serotype 11, but genetically it is filamentous with swellings in the cells. Branching cells are unrelated to the type strain of A. israelii. The DNAs of two seen in some cultures. strains ofA. gerencseriae that were tested, strains VPI 12594 Growth in prereduced anaerobically sterilized broth is (= ATCC 23860T) and VPI D140D-18, have reassociation crumby (44%), stringy (22%), flaky (22%), or grainy (11%) values of 82%. and usually without turbidity (89%). Growth in peptone- The description below is based on a study of 22 strains that yeast extract medium is moderate to good; excellent growth reacted with the A. israelii serotype I1 antisera and not with occurs in medium containing a fermentable carbohydrate. the antiserum of any of the other serotypes tested. The pH of cultures in peptone-yeast extract basal medium The type strain and 12% of the other strains tested are inoculated under CO, is 6.13 (range, 5.7 to 6.6), and the pH obligately anaerobic. The strains that grow on the surfaces of of cultures in peptone-yeast extract medium inoculated blood agar plates incubated in air enriched to 10% with CO, aerobically is 7.0 (range, 6.6to 7.5). The pH in peptone-yeast produce scant to moderately good growth. After incubation extract-glucose-Tween 80 medium incubated for 3 to 5 days for 48 h, surface colonies of the type strain cultured anaer- is 4.7 (range, 4.3 to 5.3). The acid products (in milliequiva- VOL. 40, 1990 TAXONOMY OF ACTINOMYCES 279

TABLE 3-Continued

A. israelii A. gerencseriae A. bovis A. bovis A* odonto'yticus A. odentolyticus A. meyeri serotype I serotype serotype serotype I serotype I1 A. hordeo- vulneris: Reaction(s) % of Reaction(s) % of ':reac- 'I: reac- Reaction(s) % of Reaction($ % of Reaction($ % of reaction of strains tion of tion of of 23 strains of 7 strains of 12 strains 1 strain of9 strains ;zize ::i:spositive 1 strain' 1 strainb strains positive strains positive strains positive

V 56 a- 73d 0 -W 14 -a 17d a- 89 - 0 13 -a 14 -a 17 a 100 a- 69" 0 - 0 - 0 -a 22 V 42d 0 - 0 - 0 + 100 + 100 - +- 61d +- 86 -+ 17

a 100 a 100 100 a 100 a 100 a 100 a 96 100 a- 86 a 100 -a 33 -a 15 22 -a 14 V 58 a- 89 a- 73" 0 - 0 - 0 a 100 a 96 30" V 43 a- 67 a 100 a 100 4 -a 29 a- 67d aw 89" a- 88 0 - 0 -W 8 a 100 a 100 0 - 0 - 0 -a 44 a- 77d 0 - 0 - 0 a- 89 a- 77" 4 - 0 - 0 a 100 a- 92 4 - 0 - 0 -W 11 -a 15 22 -a 14 -a 8 a- 89 a- 92d 4 -W 29 aw 92 a- 89" a- 88d 4 -a 29 - 0 -a 22 -a 8 0 - 0 - 0 a- 66d V 46 30 V 57 a- 92 - 0 - 0 26 - 0 - 0

a 100 a 96 96" a- 71 a 100 a- 66 a 96 0 - 0 -a 8 a 100 a- 92" 17 -W 14 aw 92 -W 11 - 4 74* -+ 29 -W 8 +- 89" + 100 48 V 57 V 50 +- 78 -+ 38d 100 +- 86 - 0 - 0 - 4 0 - 0 - 0

33 4 55 29 45 0 0 - 0 14 0 67 96 + 45 57 55 lents per 100 ml of culture) of peptone-yeast extract-glucose- scesses, cervicofacial patients, retroperito- Tween 80 cultures are as follows: lactic acid, 2.1 (range, 0.3 neal abscesses, hand abscesses, and various other clinical to 4.4); succinic acid, 1.0 (range, 0.2 to 3.0); formic acid, 0.7 specimens. A. gerencseriae accounts for 1.2% of the human (range, 0.2 to 2.0); and acetic acid, 0.6 (range, 0.1 to 1.8). healthy periodontal flora and occurs in 14% of the samples Pyruvate accumulates in about 20% of the cultures (average, examined (Table 6). 0.1 meq/100 ml of culture; range, 0.1 to 0.8 meq/100 ml in A. naeslundii, A. viscosus, and Actinomyces serotype WVA those cultures that are positive). Similar ratios of products 963. A. naeslundii, A. viscosus, and Actinomyces serotype are obtained under anaerobic or restoppered inoculation WVA 963 clearly are more closely related than the other conditions. No hydrogen is detected in the headspace gas. species tested, both as determined by genetic analyses (6) Addition of Tween 80 to broth medium increases the (Tables 2 and 7) and on the basis of phenotypic characteris- growth of 25% of the cultures and does not affect the growth tics (8, 15, 16, 24, 31, 35, 37) (Table 3). They also are the of 75%. No strain produces lecithinase or lipase on egg yolk most common taxa belonging to the genus Actinornyces in agar or urease (urease broth method) (13). Glycerol is not the human gingival flora (Table 6). Classically, A. viscosus fermented by any of the 16 strains tested. The major cellular and A. naeslundii have been separated on the basis of their fatty acids are 16:O and 18:l cis-9. Additional characteristics catalase reactions, with A. viscosus being catalase positive of the species are shown in Tables 3 and 4. DBerential and A. naeslundii being catalase negative, even though there characteristics are shown in Table 5. are some strains of each species that are variant in this This species is distinct from A. israelii serotype I as reaction (37) (Table 8). determined by polyacrylamide gel electrophoresis banding In a numerical taxonomic study of oral gram-positive rods, patterns (23), serological reactions, and the inability of which included the type strain and one other animal strain of strains of A. gerencseriae to ferment L-arabinose. Most A. viscosus, one human strain of A. viscosus, and the type (89%) of the strains of A. israelii serotype I ferment arabi- strain of A. naeslundii, Holmberg and Hallander (15) con- nose. cluded that these reference strains were sufficiently similar The type strain is strain CDC W838 (= VPI 12594 = to form a single species. Similar results were found by ATCC 23860), which was isolated from a human parotid Holmberg and Nord (16), but again only four strains of the A. abscess. naeslundii-A. viscosus complex were tested. A subsequent The G+C content of the DNA is 70 to 71 mol%. study by Fillery et al. (8) of human isolates designated A. Isolated from human gingival crevices, mandibular ab- naeslundii (the type strain and 16 other strains) or A. 280 JOHNSON ET AL. INT. J. SYST. BACTERIOL.

TABLE 4. Methylated cellular components of washed cells of Actinomyces species grown in peptone-yeast extract-glucose-Tween 80 broth % of total chromatographic area in:

A. naeslundii A. naeslundii genospecies 2 Componenta A. georgiae genospecies 1 (n = 128; (serotype I) Serotype I1 Serotype I11 Serotype NV Serotype VII All strains 93/48)’ (n = 197; (n = 15; (n = 122; (n = 102; (n = 40; (n = 279; 182/51) 10/10) 75/39) 81/44)‘ 35/22)d 232165)

10:0 FAME 1.4 f 0.5‘ 2.2 f 1.4 1.2 f 1.7 1.7 f 0.8 1.0 f 0.9 1.8 f 0.8 1.4 f 1.0 12:O FAME 1.6 f 0.4 1.9 rt 0.6 2.4 f 0.9 1.6 f 0.4 2.8 f 1.0 2.9 ’. 0.6 2.2 f 1.0 14:l cis-9 FAME - - 0.5 f 0.4 - 0.4 2 0.4 - - 14:O FAME 12.5 f 2.1 3.0 f 0.8 4.8 f 1.0 2.3 f 0.5 5.2 f 0.9 4.2 ’. 0.7 3.7 f 1.5 15:O is0 FAME ------15:O anteiso FAME ------16:O Aldehyde ------16:O is0 FAME ------16:l cis-7 FAME 1.1 t 0.2 1.0 f 0.5 1.7 f 0.4 1.0 f 0.3 1.7 rt 0.5 1.8 f 0.3 1.4 f 0.5 16:l cis-9 FAME 2.2 f 0.4 2.4 f 0.9 3.8 f 1.4 1.8 f 0.7 3.8 f 1.3 2.1 f 0.8 2.7 f 1.4 16:O FAME 37.5 f 3.0 37.4 f 3.2 35.6 f 4.6 44.5 rt 3.8 35.5 f 5.0 43.7 k 2.4 40.6 2 6.0 16:O DMA 1.8 f 1.3 ------17:O anteiso FAME ------17:O FAME ------16:O 2-OH FAME ------18:2 ~i~-9,12FAME ------18:l cis-9 FAME 34.6 ’. 2.7 44.1 f 3.7 43.2 f 2.5 40.7 rt 2.7 42.5 f 4.0 39.3 f 2.9 41.3 f 3.4 18:O FAME 3.9 2 0.9 5.9 f 2.3 2.4 f 0.9 4.7 f 1.2 2.4 f 0.6 3.1 f 0.9 3.5 f 1.5 18:l cis-9 DMA ------19 cys-9,10/:1 FAME ------20:l cis-11 FAME ------22:O NHC ------17:2 FAME (16.76 ECL) or 17:l cis-8 FAME ------17:l cis-9 FAME or 17:2 FAME (16.80 ECL) ------18:l Cll/T9/T6 FAME or un-17.83 ECL 2.9 ’. 0.7 1.4 f 1.3 3.7 f 2.5 1.4 f 1.3 4.4 f 2.2 1.1 f 1.1 2.6 t 2.3

DMA, Dimethylacetyl; NHC, normal hydrocarbon; ECL, equivalent chain length. The numbers in parentheses are the numbers of analyses (n) and the numbers of isolates/numbers of subjects from which isolates were obtained. ‘. Serotype NV strains react with both A. naeslundii and A. viscosus serotype I1 conjugates. Serotype VII is A. viscosus serotype 11. Mean ’. standard deviation. f-, Minor peak that was highly variable or undetected and was not used in the MIS differential analysis. viscosus (18 strains) led these authors to conclude that “the reference DNAs from A. viscosus serotype I1 and A. division of human A. viscosus from A. naeslundii is closer to naeslundii strains. We found a level of reciprocal relatedness a serotypic separation than separation at a species level.” In of 36% between the type strains of A. viscosus and A. another numerical taxonomic analysis of members of the naeslundii by using the S, nuclease method in this investi- family , Schaal and Schofield (33) exam- gation. The lower value determined by the 8, nuclease ined 40 to 50 strains of A. naeslundii, A. viscosus, and method was expected and fits well with the comparative related organisms and reported that a number of discrete values previously reported when the same strains were clusters are represented within a common cluster. As deter- examined by using different hybridization methods (2). In mined by using the Jaccard coefficient, with which clusters their comparison of reassociation values obtained when the were defined at a similarity level of 55%, the within-group same strains were examined by both membrane competition similarity ranged from about 65 to 75%, and the between- and S, nuclease DNA-DNA relatedness methods, Bouvet group similarity ranged from about 52 to 56%. The type and Grimont (2) showed that strains with 70% or greater strain of A. viscosus and six other animal strains clustered reassociation values as determined by the membrane com- between the type strain and three other strains of A. naeslun- petition method gave S, nuclease reassociation values of 35 dii and strains representing A. naeslundii serotypes I1 and I11 to 50% (6 strains), 50 to 70% (13 strains), and >70% (8 and some of the A. viscosus serotype I1 strains at a similarity strains). Indeed, it appears that some strains that would fit level of about 60%. No taxonomic recommendations were into a species cluster on the basis of membrane competition made. Similar results, with higher similarity values as ana- reassociation results have reassociation values as low as 33 lyzed by using the simple matching coefficient, were re- to 34% when they are examined by using the S, nuclease ported by Schofield and Schaal (33, who observed that method. Therefore, on the basis of DNA reassociation although the two species “are closely related and possibly values, there is reason to combine A. viscosus and A. should be combined,” some phenotypic differences and the naeslundii into a single species, albeit these taxa are at the genetic evidence of Coykendall and Munzenmaier (6) sug- low end of the acceptable range of relatedness for a geno- gest that “they are sufficiently distinct to merit separation, species. It also can be argued that the two taxa can be perhaps at the subspecies level.” A similar position was maintained as separate species, given the lower values maintained by Schaal in Bergey’s Manual of Systematic reported by Coykendall and Munzenmaier (6) when a mem- Bacteriology (31). brane filter method of hybridization was used. In a study of Using the membrane filter method, Coykendall and the intra- and intergeneric relationships of Actinomyces Munzenmaier (6) found that the type strain of A. viscosus species as determined by comparisons of 16s rRNA se- (serotype I) exhibited only 53 and 54% relatedness with quences (39), the highest homology value reported was VOL. 40, 1990 TAXONOMY OF ACTINOMYCES 281

TABLE &Continued

% of total chromatographic area in:

Actinomycesserotype A. viscosus A. israelii A. geren- A. bovis A. odontolyticus WVA 963 serotype I serotype I cseriae A. meyeri A. hordeo- (n = 13; (n = 50; (n = 110; Serotype Serotype II Serotype I Serotype I1 (n = 35; vulneris (n = 107; I 1/1) 27/15) 94/34) (n = 3; 1/11 (n = 3; 1/11 (n = 79; (n = 20; 24/21) (n = 2; 1/1) 74/30) 66/43) 11/11)

2.0 f 1.2 3.9 f 1.5 0.7 f 0.4 1.9 f 0.9 f - 3.1 f 1.1 3.0 f 1.1 3.3 f 1.1 - 1.7 f 0.4 2.7 f 0.7 2.7 f 1.0 1.9 f 0.4 1.0 f 0.2 17.3 f 2.9 2.0 f 0.7 1.7 f 0.5 2.5 f 0.7 1.1 f 0.1 0.6 f 0.4 0.5 f 0.4 0.6 f 0.4 - - - 0.6 f 0.4 1.0 f 0.4 0.6 f 0.4 - 3.6 f 0.8 4.7 f 0.8 4.9 f 1.5 2.8 f 0.9 1.4 f 0.7 11.6 f 1.6 10.2 k 2.9 6.9 f 2.1 6.8 f 1.4 12.9 2 1.6 ------1.0 f 0.4 ------4.4 f 1.7 ------0.6 f 0.5 ------0.8 f 0.2 1.0 f 0.3 1.3 f 0.5 1.2 f 0.6 1.1 f 0.5 - - 1.0 f 0.5 0.7 f 0.5 0.5 f 0.4 - 3.8 f 1.1 5.0 f 0.8 2.6 f 0.5 1.5 f 0.6 1.8 f 0.6 3.0 f 1.5 4.7 f 1.3 6.7 f 1.6 5.6 f 2.8 4.6 f 1.8 33.5 f 3.1 24.2 f 0.8 38.9 f 3.5 43.2 f 3.0 23.7 f 5.2 17.6 f 0.9 23.5 f 5.2 15.6 f 4.0 18.0 f 5.5 24.2 f 2.2 - - - - - 3.6 f 5.7 - - 3.2 f 1.7 ------0.6 f 0.5 - 2.0 f 0.5 - - - - - 0.5 ? 0.4 ------0.8 f 1.2 - - - - - 0.6 f 0.5 ------45.1 f 3.8 47.6 f 2.8 33.4 f 7.6 40.4 f 4.6 56.2 f 7.2 27.8 f 4.9 45.5 f 3.9 49.3 * 7.0 47.0 f 4.7 30.4 f 3.5 3.8 f 1.6 5.0 f 1.6 2.5 f 1.1 3.6 k 1.1 10.1 f 4.0 10.1 f 1.9 2.1 f 0.6 2.1 f 0.6 3.1 f 0.8 5.2 f 2.1 1.3 f 1.8 - 4.3 f 5.6 ------3.0 f 2.6 2.6 f 1.9 ------1.7 f 0.5 ------1.3 f 2.1 ------1.0 f 1.4 ------0.6 f 0.4 1.1 f 0.4 0.8 f 0.6 - 3.1 f 1.0 3.2 f 1.3 3.3 f 0.9 0.7 f 1.0 1.4 f 1.6 4.6 k 0.4 6.4 f 2.2 9.3 f 2.3 6.4 f 2.3 11.7 f 0.5

95.4% between the type strains of A. naeslundii and A. (6) previously reported that strain WVU 820 (our antigen viscosus. However, in the phylogenetic tree constructed by strain for A. naeslundii serotype I1 antisera) exhibited 85% using a distance matrix method of Felsenstein that optimizes DNA relatedness with DNA from the human A. viscosus branch length, Stackebrandt and Charfreitag (39) found that strain which they tested. There also is a close relationship A. viscosus branches between A. odontolyticus and A. bovis between these strains serologically. In the original descrip- rather than with A. naeslundii and A. israelii. These authors tion of A. naeslundii serotype 11, Bragg et al. observed that explain this apparent anomaly by stating that “in the pres- there was extensive cross-reactivity between A, naeslundii ence of varying evolutionary rates, species with the highest serotype I1 and A. viscosus serotype I1 (S. L. Bragg, W. nucleic acid sequence similarity are not necessarily the most Kaplan, and G. Hageage, Abstr. Annu. Meet. Am. SOC. closely related ones” (phylogenetically). In light of this Microbiol. 1975, F6, p. 86). Obviously, not all strains share somewhat confusing situation, we do not propose that A. common antigenic determinants, but many do (e.g., the viscosus I and A. naeslundii be combined at this time. strains that we have referred to as Actinomyces serotype However, our results (Table 7) clearly show that strains of NV). The strains of Actinomyces serotype NV have average A. naeslundii serotype I1 and strains of A. viscosus serotype DNA relatedness values of 70 to 76% with reference DNAs 11 belong in the same species. Coykendall and Munzenmaier from A. naeslundii serotype I1 and A. viscosus serotype I1

TABLE 5. Some differential characteristics of the Actinornyces species tested

~ ~~ A, naeslundii, A. bovis A. odontolyticus A. viscosus, A. meyeri A. hordeo- Characteristic A. israelii Actinomyces A‘ gear- et;~:~eserotype giae Serotype Serotype Serotype Serotype vulneris WVA %3“ I I1 I I1

~~~~ ~~ ~~~ ~~ Acid production from: Fructose +b + + + + + + + + - Raf€inose + + + ------Mannitol + + - Arabinose + - - Trehalose + - - - - - Inosito1 + - - - - Mannose + - - - Nitrate reduced + V -

a Includes A. naeslundii genospecies 1 (serotype I), A. naeslundii genospecies 2 (A. naeslundii serotypes I and 11, A. viscosus serotype 11, and Actinomyces serotype NV), Actinomyces serotype WVA 963, and A. viscosus serotype I. +, Positive reaction for 87 to 100% of the strains tested; -, negative reaction for 88 to 100% of the strains tested. Reactions for which no data are given are not differential. 282 JOHNSON ET AL. INT. J. SYST.BACTERIOL.

TABLE 6. Relative incidence of Actinomyces species and serotypes in human periodontal floras Patients with healthy gingivae Patients with gingivitis and periodontitis Species or serotype % Occurrence % Of flora % Occurrence % Of flora (139 samples)" (4,403 isolates) (1,089 samples)" (34,675 isolates) A, georgiae 2.0 1.4 6.5 0.4 A. gerencseriae 13.7 1.2 16.9 1.5 A. israelii 6.5 0.4 9.4 0.7 A. naeslundii genospecies 1 (serotype I) 54.7 7.6 30.7 2.7 A. naeslundii genospecies 2 A. naeslundii serotype I1 1.4 0.1 1.9 0.1 A. naeslundii serotype I11 25.9 2.8 27.6 3.6 Actinomyces serotype NV 33.1 4.0 30.7 3.1 A. viscosus serotype I1 19.4 1.4 17.2 1.4 A. meyeri 2.9 0.2 6.3 0.3 A. odontolyticus Serotype I 13.0 0.8 19.2 1.2 Serotype I1 3.6 0.1 1.6 0.1 Actinomyces serotype WVA 963 34.5 3.4 26.2 2.4 Total 23.4 17.5

______~~~~ ~ Samples comprised 30 isolated colonies selected in a randomized manner. Any resulting mixed cultures were purified, and all distinct taxa were characterized, giving an average of 31.7 to 31.8 isolates per sample. strains (Table 7) and should be grouped with them. The types I1 and I11 as determined by S, nuclease hybridization strains of A. naeslundii serotype 11, A. viscosus serotype 11, (Table 7) correspond to a value of about 70% as determined and Actinomyces serotype NV exhibit average DNA relat- by the membrane competition method (2), which indicates edness values of 24 to 45% with the type strains of A. that A. naeslundii serotype I11 strains should be associated naeslundii and A. viscosus (Table 7)and should be consid- with the A. naeslundii serotype 11-A. viscosus serotype I1 ered a distinct species. However, other than by serological genospecies. Therefore, we propose that A. naeslundii geno- testing, there is no reliable way to differentiate strains of A. species 2 should contain strains currently belonging to A. naeslundii serotype I1 and A. viscosus serotype I1 from naeslundii serotype 11, A. viscosus serotype 11, and A. strains of A. naeslundii serotype I (Tables 3 and 8). There- naeslundii serotype I11 and the Actinomyces serotype NV fore, we propose that these organisms should be retained in strains. This grouping of A. naeslundii serotypes I1 and I11 in A. naeslundii, in keeping with the recommendation of the same genospecies is consistent with the finding of Schaal Wayne et al. (41) that distinct genospecies "that cannot be and Schofield (33) that A. naeslundii serotypes I1 and I11 and differentiated from another genospecies on the basis of any some human isolates labeled A. viscosus form one of the known phenotypic property not be named until they can be phenotypic subclusters within the larger A. naeslundii-A. differentiated by some phenotypic property." viscosus cluster. We found (Table 7) that strains of A. naeslundii serotype Actinomyces serotype WVA 963 is more distantly related I11 are most closely related to strains of A. naeslundii to the other strains tested (Tables 2 and 7). Gerencser and serotype 11, A. viscosus serotype 11, and Actinomyces sero- Slack (10) previously designated serotype WVA 963 as type NV (51 to 62%) and more distantly related to strains of serotype IV of A. naeslundii, but that designation was not A. naeslundii serotype I, A. viscosus serotype I, and Acti- continued (9), possibly because workers were uncertain nornyces serotype WVA 963 (20 to 37%). Reciprocal relat- whether this serogroup represents a distinct species or is edness values of 51 and 62% between A. naeslundii seio- indeed a serotype of A. naeslundii. On the basis of numerical

TABLE 7. Summary of DNA relatedness data for serotypes of A. naeslundii and A. viscosus"

% Relatedness with reference DNA from:

m m\o

Unlabeled DNA from:

A. naeshndii serotype I1 100 62 63 51 33 24 30 A. viscosus serotype I1 79 100 75 59 45 36 29 Actinomyces serotype NV 76' 7w 100 52' 41' 32" 35' A. naeslundii serotype I11 62 55 51 100 22 20 29 A. naeslundii serotype I 42 35 34 36 100 39 38 A. viscosus serotype I 44 36 35 37 55 100 36 Actinomyces serotype WVA 963 34' 33' 26" 3 1" 43' 33" 100 Data were Summarized from the data in Table 2. Values were determined by using the S1 nuclease method. 'Actinomyces strains that react serologically with A. vkcosus serotype I1 and A. naeslundii serotype I or I1 antisera. Values are the averages of the values shown in Table 2. VOL. 40, 1990 TAXONOMY OF ACTZNOMYCES 283

TABLE 8. Characteristics of serotypes of A. naeslundii genospecies 2 A. naeslundii A. naeslundii A. viscosus Actinomyces serotype I1 serotype I11 serotype I1 serotype NV"

Characteristic -c ,.a- Reaction( s) of s@$))s Reaction( s) of sE::s Reaction( s) of sF:)s Reaction(s) of sc:Ls 8 strains positive 45 strains positive 25 strains 44 strains vositive Acid production from: Am ygdalin - b 0 -a 6 - 0 - 2 Arabinose - 0 - 4 - 0 - 2 Cellobiose - 0 V 53 -a 16 -a 11 Esculin - 0 - 0 - 4 -a 7 Esculin hydrolysis +- 62 + 100 + 100 +- 93 Acid production from: Fructose a 100 a 100 a 100 a 98 Glucose a 100 a 100 a 100 a 100 Glycogen a- 62 V 47 V 44 V 41 Inositol a- 88 a 100 a- 92 a 95 Lactose a- 62 a 96 a- 68 V 54 Maltose a 100 a- 93 a 100 a 100 Mannitol - 0 -a 6 - 0 -a 11 Mannose a 100 a 96 a- 88 a- 89 Melezitose -a 25 V 45 -a 28 -a 30 Melibiose a- 75 a- 87 V 60 a- 70 Raffinose a 100 a 98 a 100 a 100 Rhamnose -a 12 -a 11 - 4 -a 7 Ribose a- 62 a 100 V 48 a- 64 Salicin -a 12 -a 18 -a 32 V 41 Sorbitol - 0 V 60 - 4 - 4 Starch a 100 a- 91 a- 68 a- 66 Sucrose a 100 a 100 a 100 a 98 Trehalose V 50 a 96 a- 68 V 59 Xylose - 0 -a 27 -a 8 -a 9 Milk curd + 100 + 98 +- 72 +- 64 Nitrate reduction + 100 -+ 27 + 100 + 100 Catalase production -+ 38 - 0 +- 84 +- 86 Alpha-hemoly sis 50 56 52 36 Gamma-hemolysis 50 44 48 64 " Actinomyces serotype NV strains react with both A. naeslundii and A. viscosus serotype I1 conjugates. +, Positive reaction for 95 to 100% of the strains; -, negative reaction or pH greater than 5.7 for 95 to 100% of the strains; a, pH less than 5.7 for 95 to 100% of the strains; v, positive reaction for 40 to 60% of the strains. Where two reactions are given, the first is the more common. No strain hydrolyzed starch, digested gelatin or meat, produced indole or beta-hemolysis, or fermented erythritol. analyses, Schaal and Schofield (34) reported that Actinomy- serotype I which we tested is more similar to A. bovis, A. ces strain WVA 963 clustered on the periphery of the A. odontolyticus, A. meyeri, and A. hordeovulneris than to the israelii cluster. In later reports, Schaal and Schofield (33) genospecies of A. naeslundii and strains of Actinornyces and Fillery et al. (personal communication cited by Schaal serotype WVA 963. However, limited weight should be and Schofield) found that strain WVA 963 was more closely given to this observation inasmuch as only one strain of A. related to the A. naeslundii-A. viscosus cluster. Our data viscosus has been tested. indicate that strains of Actinomyces serotype WVA 963 It is possible that the low levels of 14:l cis-9 FAME, 18:2 represent a distinct genospecies that is closely related to A. cis-9,12 FAME, and 18:l cis-9 dimethylacetyl may prove to naeslundii and A. viscosus. However, because these organ- be useful in differentiating A. viscosus serotype I and Acti- isms cannot be differentiated phenotypically (Table 3), we nomyces serotype WVA 963 from A. naeslundii. However, are not proposing a change in their nomenclatural status. because these fatty acids constitute 1% or less of the fatty Genospecies Actinomyces serotype WVA 963 constitutes acids detected, species or serotype differentiation on the 3.4% of the bacterial flora of healthy gingival crevices (Table basis of their presence or absence would be tenuous. The 6). cellular fatty acid profiles which we found for A. naeslundii, Phenotypic reactions of the species and serotypes which A. viscosus, and Actinomyces serotype WVA 963 are similar we studied are shown in Tables 3 and 8, and differential to those reported by Wada et al. (40) for A. viscosus NY1. characteristics (at the 87% level of sensitivity) are shown in However, we did not observe the branched-chain cellular Table 5. Because A. viscosus, strains of Actinomyces sero- fatty acids in cultures of A. naeslundii ATCC 12104T (sero- type WVA 963, and the A. naeslundii genospecies cannot be type I) and A. viscosus ATCC 15987T reported by Wada et differentiated from each other phenotypically, they are al. Even though our cells were grown in medium containing grouped together in Table 5. Tween 80, it is doubtful that the presence of Tween 80 in the The cellular fatty acids of A. viscosus, the genospecies of medium accounted for this much difference in cellular fatty A. naeslundii, and Actinomyces serotype WVA 963 are acid composition, which we cannot explain. shown in Table 4. All of these organisms have major Other species of the genus Actinomyces. Although there amounts of 18:l cis-9 and 16:O fatty acids. In the percentage have been reports, no verified strain of A. bovis (normally of 16:O FAME detected, the one strain of A. viscosus from lumpy jaw of cattle) has been found in human infec- 284 JOHNSON ET AL. INT. J. SYST.BACTERIOL. tions. However, we recently received a strain of A. bovis were originally tested, the reactions with the A. naeslundii isolated from a llama. The characteristics of the strains of A. antisera were equal to those of the control. However, in bovis which we examined are shown in Tables 3 and 4. recent tests with different lots of antisera, the fluorescence The DNA reassociation values for A. odontolyticus sero- with the A. viscosus serotype I1 conjugate was equal to that type II strains with A. odontolyticus serotype I reference of the control antigen, but the fluorescence with A. naeslun- DNA are 41 to 51% as determined by the S, nuclease dii conjugates was much weaker than that originally ob- method. This range is within the lower limit of acceptable served. reassociation values at the species level, so no taxonomic It continues to be exceedingly difficult to differentiate changes are recommended. In addition to succinic, lactic, among the described species of Actinomyces by usual phe- acetic, and formic acids, pyruvic acid accumulates in many notypic tests, although a few tests are helpful. We confirmed A. odontolyticus peptone-yeast extract-glucose-Tween 80 the report of other authors that L-arabinose fermentation cultures (pH 4.8) that are inoculated aerobically and restop- best distinguishes A. gerencseriae (previously A. israelii pered, probably because of the decreased amount of C02in serotype 11) from A. israelii serotype I. Our finding that 89% the headspace gas. Tween 80 enhanced the growth and of A. israelii serotype I strains ferment L-arabinose is within fermentation of most strains and inhibited none. Broth the range of 71 to 100% reported previously (3, 38), and we growth is turbid (97%) with smooth (53%), grainy (30%), or confirmed the previous reports that A. gerencseriae strains stringy to ropy (17%) sediment. Surface colonies are circu- are negative for L-arabinose fermentation. lar, entire, convex (77%), pulvinate (19%), or flat (4%), The cellular fatty acid constituents of the Actinomyces smooth, and shiny. We found that the typical red colonies species and serotypes of Actinomyces species grown at 37°C described for A. odontolyticus could be demonstrated in in peptone-yeast extract-glucose-Tween 80 broth shown in only 58% of the serotype I strains and 43% of the serotype I1 Table 4 are in general agreement with those cited previously strains. The major cellular fatty acids (Table 4) are 18:l cis-9, (l), except that we did not observe the large amounts of 18:O 16:0, and 14:O. Other characteristics of the species and FAME reported by Amdur et al. (1). serotypes are shown in Table 3. The Microbial Identification System (Microbial Indentifi- A. hordeovulneris, which was isolated from dogs (4), was cation System, Dover, Del.), which is based on a principal- included in this study because it is phenotypically similar to component analysis of the relative amounts of these com- A. meyeri (from human periodontal flora and brain ab- pounds, currently correctly identifies new isolates of these scesses) and has similar electrophoretic patterns of soluble genospecies (as first choice) with 66 to 100% accuracy (Table cellular proteins. However, we found that these two species 9). The accuracy of identification of serotypes within A. do not exhibit genetic relatedness (Table 2). The major naeslundii genospecies 2 is only 37 to 65% (first choice) or 55 cellular fatty acids of A. hordeovulneris are 18:l cis-9, 16:0, to 81% (first and second choice). The accuracy is improved and 14:O. Other characteristics of this species are shown in when all serotypes of the genospecies are considered to- Tables 3 and 4. gether because the first or second choice usually is a There is considerable variation in phenotypic reactions serotype included in A. naeslundii genospecies 2. among strains of different Actinomyces species. As pointed The type strain of A. odontolyticus (strain ATCC 17929), out by Slack and Gerencser (37), cultural conditions may which is a serotype I strain, consistently comes out A. affect the percentage of cultures that ferment certain carbo- odontolyticus serotype I1 as the first choice and A. odon- hydrates. We routinely add Tween 80 to media for Actino- tolyticus serotype I as the second choice. Similarly, the type myces species, and for many we purposely admit some air strain (and antigen strain) of A. gerencseriae is identified as into the stoppered tubes of prereduced media to improve A. israelii serotype I (first choice) or A. gerencseriae (sec- growth. We suspect that one problem in obtaining uniform ond, third, or fourth choice). As more verified strains of each results is that the inoculum often is a lumpy or crumby taxon are used to calculate the mean reference pattern, the suspension of cells. Test media may not receive uniform accuracy of the identification method can be expected to amounts of actual cellular inoculum as it is dispensed from improve. The numbers of strains of A. bovis and A. viscosus Pasteur pipettes. However, there obviously are other prob- serotype I available to us are too small to make an estimate lems that are not understood. We have encountered a few of the accuracy of identification based on the patterns of the fresh isolates, usually of A. israelii or A. gerencseriae, that constituents. do not ferment carbohydrates until after they have been Although it appears to be a minor problem with absorbed transferred in the laboratory 10 or more times, even though antisera, as pointed out by Slack and Gerencser (37), there good growth is obtained in all broth media. Sometimes, are some cross-reactions among A. naeslundii, A. israelii, A. passage through thioglycolate supplemented with serum gerencseriae, and A. odontolyticus serotype 11. Therefore, enables us to detect fermentation; other times this does not unless the reference patterns are based entirely upon many appear to help. more DNA homology strains, the accuracy may not increase The reactions for the various taxa shown in Table 3 are in greatly. As a practical matter, we currently must rely on the reasonable agreement with those reported by Slack and strongest fluorescent reactions compared with the reactions Gerencser (37) and the results compiled by Schaal(31), even of the known reference strains. The multiple strains of A. though the specific serological groups often were not deter- georgiae, A. gerencseriae, Actinomyces serotype WVA 963, mined or reported in the original data or were determined and Actinomyces serotype NV were selected for DNA with antisera of different specificities. With our absorbed analysis on the basis of both serological and cellular fatty antisera, we observed very few cross-reactions among the acid results, and they grouped together in DNA-related distinct species revealed by DNA homology data, except for groups as predicted by those analyses (Table 2). the reaction of the Actinomyces serotype NV strains with Among the isolates of Actinomyces species from gingival both A. viscosus serotype I1 and A. naeslundii antisera. crevices (Table 6), there are numerous additional phenotyp- These strains usually reacted with A. naeslundii serotype I1 ically distinct isolates, as well as isolates that have the or A. naeslundii serotype I antisera and rarely reacted with phenotypic characteristics of one of the taxa described A. naeslundii serotype I11 antisera. When the organisms above but do not react with any of the available antisera. VOL. 40, 1990 TAXONOMY OF ACTZNOMYCES 285

TABLE 9. Agreement between cellular fatty acid identifications and identifications made by using fluorescent antibody or phenotypic tests

% of correct cellular fatty acid Species or Identified ~~~~l no. of identifications as‘: Most common other serotype by“: analysesb - First and second choice(s) First choice choicessecond

A. bovis serotype I FA 3 100 100 A. naeslundii serotype 1 A. bovis serotype I1 FA 3 100 100 “Actinomyces D20” A. georgiae PT 143 89 90 “Bifidobacterium D02A” A. gerencseriae FA 119 79 93 A. israelii serotype I, A. naeslundii serotypes I and I11 A. israelii serotype I FA 64 77 78 A. gerencseriae and Bifidobacterium infantis A. meyeri FA 36 92 97 A. georgiae, A. odontolyticus serotypes I and I1 A. naeslundii genospecies 1 FA 246 66 78 Serotype WVA 963, A. gerencseriae, A. naeslundii (serotype I) serotype I11 A. naeslundii genospecies 2 Serotype I1 FA 27 37 55 Actinomyces serotype NV Serotype I11 FA 157 65 78 A. gerencseriae, A. naeslundii serotype I A. viscosus serotype I1 FA 69 48 58 Actinomyces serotype NV, A. naeslundii serotype I1 Actinomyces serotype NV FA 126 62 81 A. naeslundii serotype 11, A. israelii serotype I All genospecies 2 strains FA 379 79 88 A. naeslundii serotype I, A. israelii serotype I

A. odontolyticus serotype I FA 88 73 91 A. odontolyticus serotype I1 A. odontolyticus serotype I1 FA 20 90 100 A. odontolyticus serotype I, A. meyeri A. viscosus serotype I FA 13 100 100 “Actinomyces D01’ ’ Actinomyces serotype WVA 963 FA 129 74 83 A. naeslundii serotype I, A. israelii serotype I

a FA, Fluorescent antibody method; FT,phenotypic test results. Includes additional strains not used for reference patterns (Table 5). Percentage of correct identifications made by cellular fatty acid analysis compared with other serotypes or species of the genera Actinomyces (15 taxa), Bifidobacterium (15 taxa), and Lactobaciffus (65 taxa).

Together, these isolates accounted for 7.6% of the total tibody procedures. J. Bacteriol. 97581-588. healthy flora and 5.4% of the floras of people with gingivitis 4. Buchanan, A. M., J. L. Scott, M. A. Gerencser, B. L. Beaman, or periodontitis (26, 28). Thus, Actinomyces species ac- S. Jang, and E. L. Biberstein. 1984. Actinomyces hordeovulneris counted for 25 to 30% of the bacterial isolates from human sp. nov., an agent of canine actinomycosis. Int. J. Syst. Bacte- (26, 28). riol. 34:439-443. gingival crevices They are associated with early 5. Collins, P. A., M. A. Gerencser, and J. M. Slack. 1973. Enumer- colonization of the teeth and gingival crevices, are known to ation and identification of Actinomycetaceae in human dental attach to other bacteria (19,21), and therefore may influence calculus using the fluorescent antibody technique. Arch. Oral or determine the subsequent composition of the periodontal Biol. 18:145-153. flora. 6. Coykendall, A. L., and A. J. Munzenmaier. 1979. Deoxyribonu- cleic acid hybridization among strains of Actinomyces viscosus ACKNOWLEDGMENTS and Actinomyces naeslundii. Int. J. Syst. Bacteriol. 29:234-240. 7. Duffy, P. S., D. Drouillard, and C. Barbe. 1981. Lymphocyte We gratefully acknowledge the microbiological assistance of Sue sorting on abluminated ciba blue dextran-staphylococcal protein C. Smith, Dianne M. Wall, and Kathy H. Pennington. We are A-conjugated sepharose 6MB affinity column. J. Immunol. indebted to Thomas 0. MacAdoo, Virginia Polytechnic Institute and Methods 45137-151. State University, for confirmation of specific epithets and their 8. Fillery, E. D., G. H. Bowden, and J. M. Hardie. 1978. A derivations and to Mary Ann Gerencser, J.-S. Chen, and D. R. Dean comparison of strains of bacteria designated Actinomyces vis- for critical reviews of the manuscript. cosus and Actinomyces naeslundii. Caries Res. 12:299-312. This work was supported by Public Health Service grants DE- 9. Gerencser, M. A. 1979. The application of fluorescent antibody 05139 and DE-05054 from the National Institute of Dental Research, techniques to the identification of Actinomyces and Arachnia, p. by grant 88-34116-3790 from the U. S. Department of Agriculture, 287-321. In T. Bergen and J. R. Norris (ed.), Methods in and by project 131052 from the Commonwealth of Virginia. microbiology, vol. 13. Academic Press, Inc. (London), Ltd., London. LITERATURE CITED 10. Gerencser, M. A., and J. M. Slack. 1976. Serological identifica- Amdur, B. H., I. E. Szabo, and S. S. Socransky. 1978. Fatty tion of Actinomyces using fluorescent antibody techniques. J. acids of gram-positive bacterial rods from human dental plaque. Dent. Res. 55(Special Issue):A184-A191. Arch. Oral Biol. 23:23-29. 11. Harrell, W. K., H. Ashworth, L. E. Britt, J. R. George, S. B. Bouvet, P. J. M., and P. A. D. Grimont. 1986. Taxonomy of the Gray, Jr., J. H. Green, H. Gross, and J. E. Johnson. 1976. genus Acinetobacter with the recognition of Acinetobacter Procedural manual for production of bacterial, fungal, parasitic baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Ac- reagents, 3rd ed., p. 87-90. Centers for Disease Control, At- inetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. lanta, Ga. and emended descriptions of Acinetobacter calcoaceticus and 12. Herbert, G. A., B. Pittman, R. M. McKinney, and W. B. Acinetobacter lwofi. Int. J. Syst. Bacteriol. 36:228-240. Cherry. 1972. The preparation and physiochemical characteri- Brock, D. W., and L. K. Georg. 1969. Determination and zation of fluorescent antibody reagents. Centers for Disease analysis of Actinomyces israelii serotypes by fluorescent-an- Control, Atlanta, Ga. 286 JOHNSON ET AL. INT. J. SYST.BACTERIOL.

13. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). 1977. 27. Moore, W. E. C., D. E. Hash, L. V. Holdeman, and E. P. Cato. Anaerobe laboratory manual, 4th ed. Department of Anaerobic 1980. Polyacrylamide slab gel electrophoresis of soluble pro- Microbiology, Virginia Polytechnic Institute and State Univer- teins for studies of bacterial floras. Appl. Environ. Microbiol. sity, Blacksburg. 39:900-907. 14. Holmberg, K., and U. Forsum. 1973. Identification of Actino- 28. Moore, W. E. C., L. V. Holdeman, E. P. Cato, R. M. Smibert, myces, Arachnia, Bacterionema, Rothia, and Propionibacte- J. A. Burmeister, K. G. Palcanis, and R. R. Ranney. 1985. rium species by defined immunofluorescence. Appl. Microbiol. Comparative bacteriology of juvenile periodontitis. Infect. Im- 25834-843. mun. 48507-519. 15. Holmberg, K., and H. 0. Hallander. 1973. Numerical taxonomy 29. Moore, W. E. C., L. V. Holdeman, R. M. Smibert, D. E. Hash, and laboratory identification of Bacterionema rnatruchotii, J. A. Burmeister, and R. R. Ranney. 1982. Bacteriology of Rothia dentocariosa, Actinomyces naeslundii, Actinomyces vis- severe periodontitis in young adult humans. Infect. Immun. cosus, and some related bacteria. J. Gen. Microbiol. 76:43-46. 38:1137-1148. 16. Holmberg, K., and C.-E. Nord. 1975. Numerical taxonomy and 30. Schaal, K. P. 1986. Genus Arachnia Pine and Georg 1969, laboratory identification of Actinomyces and Arachnia and 269AL, p. 1332-1342. In P. H. A. Sneath, N. S. Mair, M. E. some related bacteria. J. Gen. Microbiol. 91:17-44. Sharpe, and J. G. Holt (ed.), Bergey’s manual of systematic 17. Johnson, J. L. 1981. Genetic characterization, p. 450-472. In P. bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, 31. Schaal, K. P. 1986. Genus Actinomyces Harz 1977,133AL,p. W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual of 1383-1418. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and methods for general bacteriology. American Society for Micro- J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, biology, Washington, D. C. vol. 2. The Williams & Wilkins Co., Baltimore. 18. Jordan, H. V. 1982. Pathogenicity of Actinomyces species, p. 32. Schaal, K. P., and R. Gatzer. 1985. Serological and numerical 169-178. In R. J. Genco and S. E. Mergenhagen (ed.), Host- phenetic classification of clinically significant fermentative acti- parasite interactions in periodontal diseases. American Society nomycetes. In T. Arai (ed.), Filamentous microorganisms. for Microbiology, Washington, D.C. Biomedical aspects. Japanese Scientific Society Press, Tokyo. 19. Kolenbrander, P. E. 1988. Intergeneric coaggregation among 33. Schaal, K. P., and G. M. Schofield. 1981. Current ideas on the human oral bacteria and ecology of dental plaque. Annu. Rev. taxonomic status of the Actinomycetaceae. Zentralbl. Bakte- Microbiol. 42: 627-656. riol. Mikrobiol. Hyg. Abt. 1 Suppl. 11:67-78. 34. Schaal, K. P., and G. M. Schofield. 1981. Taxonomy of Actino- 20. Kolenbrander, P. E., R. Andersen, and L. V. Holdeman. N. mycetaceae. Rev. Inst. Pasteur Lyon 14:27-39. 1985. Coaggregation of oral Bacteroides species with other bacteria: central role in coaggregation bridges and competitions. 35. Schofield, G. M., and K. P. Schaal. 1981. A numerical taxonomic Infect. Immun. 48:741-746. study of members of the Actinomycetaceae and related taxa. J. Gen. Microbiol. 127:237-259. 21. Kolenbrander, P. E., R. N. Andersen, and L. V. H. Moore. 36. Selin, Y. M., B. Harich, and J. L. Johnson. 1983. Preparation of 1989. Coaggregation of Fusobacterium nucleatum, Selenomo- labeled nucleic acids (nick translation and iodination) for DNA nus flueggei, Selenomonas infelix, Selenomonas noxia, and homology and rRNA hybridization experiments. Cum. Micro- Selenomonas sputigena with strains from eleven genera of oral biol. 8:127-132. bacteria. Infect. Immun. 57:3194-3203. 37. Slack, J. M., and M. A. Gerencser. 1975. Actinomyces, filamen- 22. Kolenbrander, P. E., Y. Inouye, and L. V. Holdeman. 1983. tous bacteria. Biology and pathology. Burgess Publishing Co., New Actinomyces and Streptococcus coaggregation groups Minneapolis. among human oral isolates from the same site. Infect. Immun. 38. Slack, J. M., S. Landfried, and M. A. Gerencser. 1969. Morpho- 41501-506. logical, biochemical, and serological studies on 6 strains of 23. McCormick, S. S., H. F. Mengoli, and M. A. Gerencser. 1985. Actinomyces israelii. J. Bacteriol. 97:873-884. Polyacrylamide gel electrophoresis of whole-cell preparations of 39. Stackebrandt, E., and 0. Charfreitag. 1990. Partial 16s rRNA Actinomyces spp. Int. J. Syst. Bacteriol. 35:429-433. primary structure of five Actinomyces species: phylogenetic 24. Melville, T. H. 1965. A study of the overall similarity of certain implications and development of an Actinomyces israelii- actinomycetes mainly of oral origin. J. Gen. Microbiol. 40: specific oligonucleotide probe. J. Gen. Microbiol. 136:37-43. 309-315. 40. Wada, H., H. Kurihara, and H. Okada. 1982. Fatty acid 25. Moore, L. V. H., J. L. Johnson, and W. E. C. Moore. 1987. composition in phenol extracts from antinomyces species. J. Selenomonas noxia sp. nov., Selenomonas flueggei sp nov., Dent. Res. 61:1282-1286. Selenomonas infelix sp. nov., Selenomonas dianae sp. nov., 41. Wayne, L. G., R. R. Colwell, P. A. D. Grimont, 0. Kandler, and Selenomonas artemidis sp. nov., from the human gingival M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Mur- crevice. Int. J. Syst. Bacteriol. 36:271-280. ray, E. Stackebrandt, M. P. Starr, and H. G. Truper. 1987. 26. Moore, L. V. H., W. E. C. Moore, E. P. Cato, R. M. Smibert, Report of the Ad Hoc Committee on Reconciliation of Ap- J. A. Burmeister, A. M. Best, and R. R. Ranney. 1987. Bacteri- proaches to Bacterial Systematics. Int. J. Syst. Bacteriol. ology of human gingivitis. J. Dent. Res. 66:989-995. 37:463-464.