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APPLIED MICROBIOLOGY, Jan. 1969, p. 150-156Copyright @ 1969 American Society for Microbiology
Laboratory Identification of Rothia dentocariosaand Its Occurrence in Human
Clinical MaterialsJUNE M. BROWN, LUCILLE K. GEORG, AND LINDA C. WATERS
National Communicable Disease Center, Atlanta, Georgia 30333
Received for publication 18 October 1968
Fifty isolates of Rothia dentocariosa from diverse clinical sources were charac-terized by 28 separate tests. An attempt was made to select practical tests that couldbe completed in a minimal length of time. Rothia is also compared with Actinomycesand Nocardia with which it is often confused. Of the isolates 100% were posi-tive in the following reactions: catalase production, nitrate and nitrite reduction,esculin hydrolysis, and acid production from glucose, sucrose, maltose, salicin, andglycerol. The importance of recognizing this organism is based on the fact that it isfrequently isolated from human clinical materials and must be differentiated frommorphologically similar organisms of the genera Actinomyces and Nocardia, whichcontain pathogenic members.
Georg and Brown (2) created the genus Rothia,in the family Actinomycetaceae, to accommodatean organism previously known as Actinomycesdentocariosus (6), Nocardia dentocariosus (8),and N. salivae (1).The organism, now known as R. dentocariosa,
was apparently first isolated from carious dentinein humans by Onisi in 1949 (6). Onisi believedthe organism belonged in the order Actinomy-cetales and proposed the name A. dentocariosusbecause of morphological and physiologicalsimilarities to species of Actinomyces found inthe mouth. He described the organism as beinghighly pleomorphic, facultatively anaerobic, andas showing both coccoid and branched fila-mentous elements. In 1957, Roth (8), whilestudying isolates from carious dentine, found asimilar group of organisms. Although Onisi'soriginal isolates were not available for compari-son, she believed her isolates were the sameorganism as Onisi's A. dentocariosus. However,she placed them in the genus Nocardia, as N.dentocariosus, because of their preference foraerobic conditions of growth.
Studying in England in 1960, Davis and Freer(1), unaware of Onisi's or Roth's studies, alsodescribed a group of similar isolates from thehuman mouth. These investigators placed theisolates in the genus Nocardia on the basis ofmorphological and physiological characteristicsand suggested the name N. salivae. They pre-sented evidence, however, that the cell wall
constituents were not compatible with those ofNocardia species, since diaminopimelic acid(DAP) was not present.On the basis of a comparative study of Roth's,
and Davis and Freer's isolates, and similar iso-lates obtained in the National CommunicableDisease Center, Mycology Section DiagnosticService, Georg and Brown placed all of theseorganisms in synonymy. However, since neitherthe genus Actinomyces nor the genus Nocardiawas appropriate, the new genus Rothia was de-scribed (2).The purpose of this report is to establish prac-
tical methods for the identification of R. dento-cariosa and its differentiation from morphologi-cally and physiologically similar organisms. Thesemethods are necessary because R. dentocariosaoccurs commonly in human clinical materials,and few diagnostic laboratories are aware ofappropriate procedures for its cultivation or thecriteria necessary for its identification. It is par-ticularly important to differentiate this organismfrom pathogenic members of the genera Actino-myces and Nocardia.
MATERIALS AND METHODS
Cultures. Fifty cultures were included in this study.Thirty-nine isolates were derived from the NationalCommunicable Disease Center Mycology Section'sroutine diagnostic service. In addition, through thecourtesy of Genevieve Roth and G.H.G. Davis, sixcultures from Roth's collection and five cultures from
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ROTHIA DENTOCARIOSA IN CLINICAL MATERIALS
Davis and Freer's collection were included. None ofOnisi's original isolates were available. The cultures,with their clinical sources and other descriptive data,are given in Table 1.
Morphology. Gram stains were made and studiedfrom 24- and 48-hr Trypticase Soy Broth (TSB;BBL)and 48-hr Trypticase Soy Agar (TSA;BBL) plates.Young colonies were observed at 40X magnificationunder a dissecting microscope on TSA plates after 24-
hr aerobic incubation. In some instances, young micro-colonies were examined on Brain Heart Infusion Agar(BHIA;Difco) plates, incubated under anaerobic con-ditions to compare them with those produced by themicroaerophilic to anaerobic Actinomyces species.Mature colonies were observed on TSA and BHIAplates incubated aerobically for 7 days. All cultureswere incubated at 37 C.Oxygen requirements. TSB cultures (24 hr) were
Table 1. Origin of Rothia dentocariosa isolates studied
NCDC --NCDC-___stock Original source Clinical source Stock Original source Clinical sourceno. no.
Roth XD. IA (ATCC17931 type strain)
NCTC 10,207Roth 24.2ARoth 24.6ARoth 30.11ADavis NS-DONIDavis NS4Davis NS-BlCalifornia State De-partment of Health
Washington State De-partment of Health
Philip Golding, Colum-bus, Ohio
Doctor's HospitalColumbus, Ohio
Doctor's HospitalColumbus, Ohio
Roth DIOBRoth D6AMissouri State Depart-ment of Public Healthand Welfare
Maine State Departmentof Health and Welfare
Roth XP6AMaine State Department
of Health and WelfareNew York State De-partment of Health
New York State De-partment of Health
V.A. Hospital, Bronx,N.Y.
Pennsylvania State De-partment of Health
Pennsylvania State De-partment of Health
Albert Einstein MedicalCenter, Philadelphia,Pa.
Albert Einstein MedicalCenter, Philadelphia,Pa.
Florida State Board ofHealth
Carious teeth
SalivaCarious teethCarious teethCarious teethSalivaSalivaSalivaCerebral spi-nal fluid
Chest abscess
Bronchialaspirate
Unknown
Unknown
Carious teethCarious teethThroat
Throat
Carious teethThroat
Postoperativewound
Throat
Drainagefrom legstump
Throat
Nose
Gums
Gums
Throat ab-scess
W875
W876
W928
W942
W944
W949
W960
W962
W963
W964
W1008
W1016
W1023
W1052
W1088
W1134
W1135
W1138
W1139
W1141
W1144
W1165
W1169
Ohio State Departmentof Health
Alabama State Depart-ment of Public Health
Michigan Department ofHealth
Pennsylvania State De-partment of Health
Geisinger Medical Cen-ter, Danville, Pa.
Florida State Board ofHealth
Florida State Board ofHealth
Michigan Department ofHealth
Michigan Department ofHealth
Michigan Department ofHealth
Massachusetts StateDepartment of Health
New York State De-partment of Health
Montana State Board ofHealth
New York State De-partment of Health
Indiana State Board ofHealth
Ohio State Departmentof Health
Ohio State Departmentof Health
Florida State Board ofHealth
Florida State Board ofHealth
Pennsylvania State De-partment of Health
Pennsylvania State De-partment of Health
Pennsylvania State De-partment of Health
Indiana State Board ofHealth
Sputum
Blood
Throat
Throat
Throat
Urine
Throat
Sputum
Sputum
Throat
Leg ulcers
Sputum
Throat
Heart's bloodat autopsy
Tissue
Sputum
Sputum
Throat
Throat
Throat
Throat
Sputum
Sputum
W858
X303X346X347X348X355X356X358X368
X482
X545
X566a
X567a
X596X598X614a
X666
X690X703
W753
W781
W808
W815
W816
W841
W842
W853
I
-I
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BROWN, GEORG, AND WATERS
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-: t;. :0ji -. aS; iX-__ ff- ff T-X_ ;$f pASt ASE; tit$;:04 0:D fiA ffAL^ ......... ;.;. .... X Ad j iDA$ lS . i.t f iFEy wq .) 0 itX0 t Sj t .t:F-: t X S-t t 7 : ff tS: 0 _ i f X f ff; 4 y ;; 0 X f i : X X:; , f.itS: -s : 0 X i- 0 f i i 0
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::: :- . i A ; :.FIG. 1. Cellular morphology from gram-stained smears of R. dentocariosa. (a) Coccoid forms from 3-day-old
TSB culture (Wi183); X 2,300. (b) Bacillary forms from 2-day-old TSB culture (X303); X 1,000. (c) Mixtureofcoccoid and filamentous forms from 7-day-old TSB culture (X303); X 1,000. (d) Filamentous forms showingclubs and branches from 7-day-old TSB culture (X528); X 1,000.
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FIG. 2. Colonial morphology of R. dentocariosa. (a) Smooth convex colonies from 48-hr aerobic TSA plate(X529); X 6. (b) "Spider"-type colonyfrom 48-hr anaerobic TSA plate (W963); X 380. (c) Rough colonyfrom7-day-old aerobic TSA plate (X529); X 6. (d) Rough colony from 7-day-old aerobic TSA plate (X368); X 12.(e) Rough, pebbly colonies from 7-day-old aerobic TSA plate (X529); X 13. (f) Smooth and pebbly colonies from7-day-old aerobic TSA plate (X368); X 6.
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BROWN, GEORG, AND WATERS
inoculated onto six BHIA slants and incubated aero-bically, microaerophilically (CO2 seal), and anaero-bically (pyrogallol plus C02), according to the methodof Georg et al. (3).
Biochemical tests. The following tests were per-formed: catalase formation, indole production, nitratereduction, nitrite reduction, esculin hydrolysis, milkreactions, gelatin liquefaction, H2S and urease produc-tion, and acid production from carbohydrates. Theinoculum for all tests was taken from 24-hr TSB cul-tures.The catalase test was carried out by overlaying
growth on a DHIA slant with 3% H202 and watchingfor a stream of bubbles within 10 min. The indole pro-duction was determined on Indole-Nitrite Medium(BBL). After maximal growth was reached, the indolewas extracted by adding xylene and was detected byoverlaying with Ehrlich's reagent. Reduction of nitratewas demonstrated on Indole-Nitrite Medium aftermaximal growth was reached (usually at 2 days).Nitrite reduction was determined in TSB containing0.01% potassium nitrite. Tests were read after 7 daysof incubation. Infusion broth containing 0.1% agarand 0.1% esculin was used to detect esculin hydrolysis.A few drops of 1.0% aqueous ferric citrate was addedto a 7-day-old culture. Presence ofa brown-black colorindicated that esculin was hydrolyzed. Milk reactionswere studied in nonhomogenized milk containing ironfilings. Brom-cresol-purple was added after 7 days ofincubation to determine acidity. Gelatin liquefactionwas determined at 7, 14, and 21 days in ThiogelMedium (BBL). HaS production was determined byusing lead acetate papers suspended over streakedslants of Triple Sugar Iron (TSI) medium andHeart Infusion (HI) agar. In addition, the butt ofthe TSI agar slant was stabbed. Urease productionwas determined by inoculating Urea Broth (Difco) towhich enriched thioglycollate broth without glucoseor indicator (1 ml of urea broth to 8 ml of thio-glycollate broth) had been added. The basal mediumfor the fermentation of carbohydrates was meat ex-tract-peptone broth with Andrade's indicator [meatextract (Difco), 3.0 g; peptone (Difco), 10.0 g;NaCi, 5.0 g; Andrade's indicator, 10.0 ml; anddistilled water, 1,000 ml at pH 7.4]. Seitz-filteredaqueous sugar solutions were used at a final concen-tration of 1.0%, except arabinose, glycerol, inositol,and salicin, which were used at concentrations of0.5%. Final readings were made in 7 days, except forglycerol which was read after 3 weeks.
TABLE 2. Effect of oxygen on growth ofRothiadentocariosa
Amt of growth of 50 isolatesa
Conditions under which organisms ____fgrwthf__________were grown
4+ 3+ 2+ 1+ 0
Aerobic 46 4 0 0 0Microaerophilic + C02...31 18 1 0 0
Anaerobic + CO2........0 0 5 14 31
a Amount of growth was estimated visually andrecorded as 0 to 4+.
TABLE 3. Biochemical reactions of Rothiadentocariosa
Reaction of 50Tests isolates tested
(% positive)
Catalase production ................ 100Nitrate reduction................... 100Nitrite reductions................... 100Indole production .................. ObMilk reaction....................... 0Gelatin liquefaction................. 0TSI (acid from slant)............... 100TSI (acid in butt)................... 100H2S production.Lead acetate paper over TSI 96Lead acetate paper over HIA .... 2
Esculin hydrolysis .................. 100Urease production.................. 0
a Medium used, TSB with 0.01% potassiumnitrite.
bAll negative reactions remained negative to 21days.
RESULTSMorphology. Smears from broth cultures
showed gram-positive, very pleomorphic orga-nisms, varying from coccoid to filamentous forms.Most isolates were completely coccoid, whereasothers ranged from coccoid to bacillary forms.Occasionally, a culture would manifest onlyfilamentous forms with rudimentary branches andclavate ends. The loss ofgram positivity was notedin some filamentous elements. Stains made fromthe growth of 24-hr aerobic TSA plates revealedthe same marked pleomorphism as those frombroth, but they revealed a predominance ofbranched filaments. Young colonies on aerobicTSA plates averaged about 1.0 mm. After 24 hrof incubation, a small percentage of the cultureshad a filamentous border, but most had entireedges and were smooth to slightly rough with amucoid consistency. In 7 days, mature coloniesvaried from 1 to 4 mm and were usually raisedwith strikingly rough and highly cerebriform sur-faces and irregular or scalloped edges. Occasionalisolates developed smooth, convex, entire colo-nies, or mixtures of smooth and extremely rough-surfaced colonies. The cellular morphology of R.dentocariosa is shown in Fig. 1 a-d. Colonialmorphology is illustrated in Fig. 2 a-f. Althoughthe gross and microscopic morphology of thisorganism is highly variable, its general physio-logical characteristics are remarkably uniform, asdemonstrated by its oxygen requirements andreactions in various biochemical tests.Oxygen requirements. All cultures grew better
aerobically except one. The addition of CO2
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TABLE 4. Carbohydrate fermentations ofRothiadentocariosa
Reactions of 50Acid production from isolates tested(%0/ positive at
7 days)
Glucose.......................... 100Mannitol........................... 0Lactose........................... 0Sucrose........................... 100Maltose........................... 100Salicin........................... 100Glycerol........................... 72aStarch........................... 0Arabinose.......................... 0Xylose........................... 0Inositol ........................... o
a Of the isolates 100l% produced acid fromglycerol after 21 days of incubation.
seemed to reduce growth. Growth anaerobicallywas poor or absent (Table 2).
Biochemical tests. Results of all cultures werefairly uniform (Tables 3 and 4). Only the fermen-tation of glycerol varied. In 7 days, 72% of theisolates produced acid, but after 21 days of incu-bation, all isolates produced acid from glycerol.
DISCUSSIONThe morphological data presented in this report
are consistent with those of other investigators. Afew differences in physiological properties werenoted, the main difference being the lack ofdemonstrable proteolytic activity in gelatin andmilk. Roth and Thurn (9) obtained variableresults with gelatin. However, when bufferedneopeptone gelatin was used, they obtainedliquefaction with all isolates. With these authors,litmus milk was regularly peptonized, but onlyafter long incubation. Howell and Jordan (4)also reported gelatin hydrolysis. In our hands,the methods these investigators used were toosensitive, and the results produced were difficultto interpret.
Nitrite reduction varied depending on themedium and incubation time. TSB with 0.01%potassium nitrite was superior to the Indole-Nitrite Medium containing potassium nitrate.With the Indole-Nitrite Medium, 47% of theisolates were positive for nitrite reduction in 15days, whereas with the TSB (containing 0.01%KNO2) 100% of the isolates were positive within7 days. In both methods, the presence of nitritewas demonstrated by standard methods. Thereduction of nitrite by these organisms is par-ticularly helpful in differentiating R. dentocariosafrom Actinomyces (Odontomyces) viscosus. Al-
though other investigators have reported theVoges-Proskauer test as positive with R. dento-cariosa, our results with this organisms wereneither consistent nor reproducible.
Results of carbohydrate fermentation tests weresimilar to those obtained by Roth (8), Onisi andNuckolls (7), Davis and Freer (1), and Howelland Jordan (4) except for tests with salicin. Theseauthors did not report the production of acidfrom salicin, whereas all isolates in this seriesfermented salicin. However, the basal mediumused was different.
Judging from our experience, most diagnosticlaboratories are unable to identify R. dento-cariosa. Isolates are frequently suspected of beingNocardia species because they grow aerobicallyand show branched filamentous forms. In general,however, they present soft moist colonies lackingaerial mycelium. The inability of R. dentocariosato grow on Sabouraud Dextrose Agar (Difco),commonly employed for cultivating Nocardiaspecies, distinguishes R. dentocariosa from No-cardia species quite simply. R. dentocariosa re-quires an enriched medium, such as TSA, forgrowth. In addition, as R. E. Gordon showed (per-sonal communication), all Nocardia species oxidizesugars but do not ferment them, whereas R.dentocariosa readily ferments sugars with theproduction of acid. Hugh and Leifson's method(5) can be used to demonstrate the ability tooxidize sugars without fermenting them.Other investigators have mistaken isolates of R.
dentocariosa for Streptococcus or Corynebacte-rium, especially when a cultureconsisted largely ofcoccoid or diphtheroidal forms. Isolates may bemistaken also for Actinomyces. As indicated inthe preceding paragraph, R. dentocariosa isbasically an aerobe; however, it is frequentlyisolated in thioglycollate broth or on agar platesincubated anaerobically. Anaerobic conditions,which generally are unfavorable to the growth ofR. dentocariosa. apparently stimulate the develop-ment of filamentous forms. The young colony onBHIA plates (incubated anaerobically) is highlyfilamentous and similar to the "spider-type"microcolony of several Actinomyces species. R.dentocariosa can easily be distinguished fromActinomyces on the basis of different oxygenrequirements and the morphology in aerobiccultures. R. dentocariosa has a marked preferencefor aerobic conditions. Furthermore, this orga-nism may be completely coccoid in some cultures,particularly in broth. This characteristic has neverbeen noted with Actinomyces.The occurrence of this organism in the human
mouth is well established. R. dentocariosa hasbeen isolated also from other clinical sourcessuch as blood, spinal fluids, and abscesses. How-
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BROWN, GEORG, AND WATERS
ever, the etiological relationship of this organismto disease is not understood. Attempts to produceinfections in experimental animals have beenunsuccessful.
LITERATURE CITED
1. Davis, G. H. G., and J. H. Freer, 1960. Studies upon an oralaerobic actinomycete. J. Gen. Microbiol. 23:163-178.
2. Georg, L. K., and J. M. Brown. 1967. Rothia, gen. nov., an
aerobic genus of the family Actinomycetaceae. Intern. J.Systematic Bacteriol. 17:79-88.
3. Georg, L. K., G. W. Roberstad, and S. A. Brinkman. 1964.Identification of species of Actinomyces. J. Bacteriol. 88:477-490.
4. Howell, A., and H. V. Jordan. 1963. A filamentous micro-
APPL. MICROBIOL.
organism isolated from peridontal plaque in hamsters. H.Physiological and biochemical characteristics. Sabouraudia3:93-105.
5. Hugh, R., and E. Leifson. 1953. The taxonomic significance offermentative versus oxidative metabolism of carbohydratesby various gram negative bacteria. J. Bacteriol. 66:24-26.
6. Onisi, M. 1949. Study on the Actinomyces isolated from thedeeper layers of carious dentine. Shikagaku Zasshi 6:273-282.
7. Onisi, M., and J. Nuckolls. 1958. Description of actinomycetesand other pleomorphic organisms recovered from pigmentedcarious lesions of the dentine of human teeth. Oral SurgOral Med. Oral Pathol. 11:913-930.
8. Roth, G. D. 1957. Proteolytic organisms of the carious lesion.Oral Surg. Oral Med. Oral Pathol. 10:1105-1117.
9. Roth, G. D., and A. N. Thum. 1962. Continued study of oralNocardia. J. Dental Res. 41:1279-1292.
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