INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol. 24, No. 2 April 1974, p. 154-159 Printed in U.S.A. Copyright 0 1974 International Association of Microbiological Societies Morphological, Biochemical, and Serological Characterization of Rothia dentocariosa R. J. LESHER, M. A. GERENCSER, and V. F. GERENCSER Department of Microbiology, West Virginia University School of Medicine, Morgantown, West Virginia 26506 Fifty strains of Rothia dentocariosa Georg and Brown were characterized morphologically, biochemically, and serologically. All strains had characteristics agreeing with previous morphological descriptions of this organism, although there was greater biochemical and serological strain variation than previously reported. Four biotypes were established on the basis of variability in reduction of nitrite, production of urease, hydrolysis of esculin, and formation of acid from lactose, mannitol, mannose, raffinose, rhamnose, salicin, and trehalose. Three serotypes and a group of fluorescent antibody-negative strains were identified on the basis of the fluorescent antibody technique. A relationship between the four biotypes and the three serotypes was established. The monospecific genus Rothia, with type prepared with the neotype strain of R. species Rothia dentocariosa, was proposed by dentocariosa, ATCC 1793 1. Similar aberrant Georg and Brown (2) to accommodate strains have been submitted by others to the microorganisms previously designated as Acti- Center for Disease Control for identification (L. nomyces dentocariosus, Nocardia dentocario- Georg, personal communication). sus, and Nocardia salivae. Brown, Georg, and By comparing the morphological, biochemi- Waters (1) subsequently studied 50 isolates cal, and serological characteristics of R othia having the morphological and biochemical with Rothia-like organisms, we hope to clarify characteristics of Rothia. Hammond (3) showed the uncertain status of the latter. that all of his strains of R. dentocariosa contained a soluble polysaccharide antigen (RPS), and the detection of this antigen by the MATERIALS AND METHODS fluorescent-antibody (FA) technique is useful Bacterial strains. The sources and designations of in identifying this organism. the fifty strains used in this study are given in Table 1. Rothia is included in the family Actinomy- All cultures were maintained by monthly transfer cetaceae because of its branching, filamentous on trypiic soy agar (TSA) (Difco) slants. Prior to use, morphology and its ability to septate into the cultures were transferred twice in tryptic soy bacillary, diphtheroidal, or coccal cells, or a broth (TSB) (Difco). mixture of these; its colonial morphology, Colonial morphology. TSB cultures were streaked including spider microcolonies and mature onto two TSA plates and incubated aerobically and colonies which may be heaped and rough or anaerobically at 37 C. The anaerobic plate was incubated in a Torbal jar containing N,:H,:CO, entirely smooth; and its cell wall composition. (80:20:10). Microcolonies were observed after 18 and However, Rothia differs from the genera 24 h of incubation at a magnification of XlOO to A ctinomy ces, A rachnia, Bifido bacterium, and X400. Mature colonies were observed at X25 to X40 Bacterionema in its aerobic tendencies, its after 7 days of incubation. production of catalase, its lack of growth Cellular morphology. Gram-stained smears and wet stimulation by COZY and its production of mounts for dark-field microscopy were prepared and lactose as the major end product from glucose examined from 2- and 7day-old cultures in TSB and fermentation. on TSA. Among the organisms isolated from dental Biochemical Tests. Tests for catalase, indole, nitrate reduction, nitrite reduction ,esculin hydrolysis, gelatin calculus in this laboratory were a number of liquefaction, hydrogen sulfide production using BHI filamentous, aerobic, gram-p ositive, catalase- agar, urease production, and the production of acid positive strains resembling Rothia morpho- from carbohydrates were done by the methods of logically but possessing variant biochemical Brown et al. (1). reactions and not reacting with antisera Additional biochemical tests used were growth on 154 VOL. 24, 1974 CHARACTERIZATION OF ROTHIA DENTOCARIOSA 155 Sabouraud dextrose agar (Difco), liquefaction of formation of flat crystals of benzoic acid. Loeffler coagulated serum (BBL) slants, digestion of The production of ammonia from arginine was casein, hydrolysis of hippurate, deoxyribonuclease tested after 7 and 14 days. The basal medium production, production of ammonia from arginine, contained the following: yeast extract, 0.1%; Trypti- and the oxidation-fermentation (0-F) test (5). case, 0.1%; K,PO,, 0.03%; and NaCl, 0.5%. The test DNase test agar (BBL) was inoculated and medium also contained 0.3% arginine. Duplicate tubes incubated for 7 days. After incubation, the plates were of basal and test media were inoculated and tested for flooded with 1 N HCl and observed for the formation ammonia production by mixing 1.0 ml of each of clear zones around the colonies. medium with 1.0 ml of Nessler reagent and observing TSB broth containing 1.0% sodium hippurate was for the formation of a deep orange to brick-red color inoculated and incubated for 7 days. The cultures in the presence of ammonia. were centrifuged, and 1.O ml of supernatant was added The oxidation-fermentation test was performed as to 1.5 ml of 50% H,SO,. After 4 h at room described by Hugh and Leifson (5). The medium used temperature, the tubes. were observed for the to test for fermentation of glucose contained 24 g of TABLE 1. Sources of strains studied WVU' number Previous designation and source Rothia dentocariosa 477 CDC 808b, leg-stump drainage 478 CDC W876, blood 479 CDC W853, throat 1317 WUisolate, dental calculus 1489 ATCC' 1793 1, type strain human dentine 1524 ATCC 14189 1525 ATCC 14190 1526 ATCC 14191 1560 I. R. Rothenberg 25684, sputum 1562 1. R. Rothenberg 28342, sputum Rothia-like 804,841,842 WUisolates, dental calculus 858,874,918 936,945,972 992,997,999 1013,1027,1072 1073,1088,1192,1200 1532 CDC W874a, throat 1533 CDC X599a, throat 1534 CDC W1581 1535 CDC W1591, eye 1536 CDC X566aS 1537 CDC X5486, sputum 1538 CDC W1535, throat 1539 CDC X667, sputum 1540 CDC X666, throat 1541 CDC X690 1549 CDC W1578, sputum 1550 CDC W712, sputum 1551 CDC W874B, throat 1552 CDC X368, CSF 1553 CDC X569g, Roth D108 1554 CDC X358, Davis BC1 1555 CDC X359, Davis NSE210 1556 CDC X483, blood 1557 CDC W781, throat 1558 CDC X303, NCI'C 10207 1559 CDC X567a - ~~ 'WVU, West Virginia University, Morgantown, W. Va. CDC, Center for Disease Control, Atlanta, Ga.; all cultures provided by L. Georg. American Type Culture Collection, Rockville, Md. 156 LESHER, GERENCSER, AND GERENCSER IN". J. SYST. BACTERIOL. fluid thioglycolate medium without glucose or indi- TABLE 3. Biochemical reactions of 50 cator (BBL) per liter in addition to the components Rothia strains suggested by Hugh and Liefson (5). Serological tests. Strains (WVU 477, 936, 999, 1 No. % 1088, and 1489) were used for antiserum production. Test positive Positive Cells grown in TSB at 37 C for 24 h were harvested by centrifugation, washed twice in sterile saline, resus- Catalase production ........... 50 100 pended in saline to a turbidity of a no. 4 MacFarland Indole production ............. 0 0 tube, and stored in 4-ml quantities at -30 C. Nitrate reduction ............. 48 96 For immunization, one vial of antigen was thawed Nitrite reduction (0.001%) ...... 44 88 prior to injection. The immunization schedule is given Nitrite reduction (0.01%) ....... 33 66 in Table 2. Esculin hydrolysis ............. 47 94 The procedures used for determining the working Urease production ............. 10 20 titer of the conjugate and for staining the smears were Deoxyribonuclease productidn ... 45 90 essentially the same as those described by Slack, Gelatin liquefaction ........... 0 0 Landfried, and Gerencser (6). Casein hydrolysis ............. 0 0 Sorption of the antisera was done by incubating 1 Serum liquefaction ............ 0 0 ml of a 1:2 dilution of the conjugate with 0.1 ml of washed, packed cells of the sorbing strain at 56 C for 1 Acid from:' h followed by overnight refrigeration. The conjugate Glucose ................... 50 100 was removed from the cells by centrifugation, and the Glycerol.. ................. 50 100 sorption was repeated. A sorbed antiserum was Lactose ................... 11 22 considered satisfactory if it stained the homologous Maltose ................... 50 100 strain with an intensity of 4+ at a final dilution of Mannose.. ................. 46 92 1 :40 and did not stain the sorbing strain at 1 :2. Mannitol .................. 10 20 Raffinose .................. 10 20 Rhamnose ................. 14 28 RESULTS Ribose .................... 35 70 Salicin .................... 42 84 Cellular morphology. Cells in TSA and TSB Sucrose ................... 50 100 Tr ehalose .................. 44 88 were gram positive and pleomorphic; coccoid, cocco-bacillary, and filamentous forms were a None of the strains fermented adonitol, arabinose, present. Cultures would occasionally be com- cellobiose, glycogen, inositol, sorbitol, starch, or pletely coccoid or diphtheroidal. Aging cultures xylose. tended to become gram negative. Colonial morphology. Young (18- to 24-h- old) colonies on TSA plates averaged 1 mm in Mature colonies (7 to 14 days) were creamy diameter. When examined with a microscope, white in color and varied from 1 to 4 mm in young colonies grown