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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


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


Geisinger Medical Cen-ter, Danville, Pa.






Massachusetts StateDepartment of Health


Montana State Board ofHealth


Indiana State Board ofHealth








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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::: :- . 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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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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ROTHIA DENTOCARIOSA IN CLINICAL MATERIALS

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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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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