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Mycoplasma Orale “Types” 2 and 3, Respectively E INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol. 24, No. 2 April 1974, p. 252-255 Printed in U.S.A. Copyright 0 1974 International Association of Microbiological Societies Proposal of Mycoplasma buccale nom. nov. and Mycoplasmafaucium nom. nov. for Mycoplasma orale “Types” 2 and 3, Respectively E. A. FREUNDT, D. TAYLOR-ROBINSON, R. H. PURCELL, R. M. CHANOCK, and F. T. BLACK Institute of Medical Microbiology, University of Aarhus, Aarhus, Denmark; MRC Clinical Research Centre, Harrow, Middlesex, England; and Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20014 Following recommendations made by the Subcommittee on the Taxonomy of Mycoplasrnatales of the International Committee on Systematic Bacteriology, it is proposed that Mycoplasma orale 2 and Mycoplasrna orale 3 be recognized as two separate species, Mycoplasrna buccale nom. nov. (type strain: CH20247; ATCC 23636) and Mycoplasrna fauciurn nom. nov. (type strain: DC-333; ATCC 25293), respectively. The general properties and distinctive characteristics of the newly named species are summarized. At present, three “types” of Mycoplasrna (22) be recognized as a species under the new orale are recognized: M. orale 1 Taylor- name Mycoplasrna buccale (L. adj. buccalis Robinson et al. 1964 (21), M. orale 2 Taylor- buccal), and (ii) Mycoplasrna orale 3 Fox et al. Robinson et al. 1965 (22), and M. orale 3 Fox 1969 (7) be recognized as a species under the et al. 1969 (7). However, the authors who new name Mycoplasma fauciurn (L. noun described the latter two “types” or “serotypes” fauces the throat; L. gen. pl. noun fauciurn of did, in fact, regard them as distinct new species throats). rather than subspecies of M. orale 1. In Consequently, Mycoplasma orale 1 should consequence, the Subcommittee on the Tax- henceforward be referred to simply as Myco- onomy of Mycoplasrnatales of the International plasma orale. Committee on Systematic Bacteriology (1 8), in We designate strain CH20247 (22) as the considering the taxonomic status of the three type strain of M. buccale; this strain has been proposed “types” of M. orale, recommended deposited in the American Type Culture Collec- that M. orale 1 and 2 be recognized as two tion (ATCC) under the number 23636. The separate species and that a binary name be type strain of M,faucium is DC-333 (7); it has proposed for M. orale 2. As to the taxonomic been deposited in the ATCC under the number status of M. orale 3, it was agreed by the 25293. Both strains were isolated from the Subcommittee that a recommendation should human oropharynx. Their characters agree await further comparative studies based on throughout with those described for the species nucleic-acid homology and gel-electrophoresis they represent. techniques. In reconsidering presently available The following descriptions of the two newly data, including those from polyacrylamide named species are based mostly on previously gel-electrophoresis studies recently published published work, supplemented with a few new (241, the Subcommittee later felt that there was observations in order to comply with the sufficient evidence to justify recognizing M. proposed minimal standards for descriptions of orale 3 as a separate species as well (Subcom- species of the order Mycoplasmatales (1 9). mittee on the Taxonomy of Mycoplasmatales: Minutes of Meeting, 8 January 1973, New York, unpublished data). Following the recommendations of the Sub- Mycoplasma buccale nom. nov. committee on the Taxonomy of Mycoplas- matales, we herewith propose that: (i) Myco- Morphology and staining characteristics. plasma orale 2 Taylor-Robinson et al. 1965 Dark-field microscopy of broth cultures of 25 2 VOL. 24. 1974 MYCOPLASMA ORALE “TYPES” 2 AND 3 253 strain CH20247 in the logarithmic growth colony-forming mycoplasmas, being most sen- phase showed minute coccoid, coccobacillary, sitive to tetracyclines (20). and very short filamentous forms. Thin-section Storage. Stock cultures survive for years at electron micrographs of colonies on solid -30 and -70 C and indefinitely in the medium fixed and embedded in situ revealed freeze-dried state (1). pleomorphic cells bounded by a triple-layered Effect on erythrocytes. Zones of alpha-type membrane and devoid of a cell wall. Gram- (incomplete) hemolysis develop around col- negative. (F. T. Black and E. A. Freundt 1973, onies overlaid with guinea-pig or sheep erythro- unpublished data). cytes (2, 22); the hemolysin has been identified Colonial appearance. Colonies on agar me- as hydrogen peroxide (4, 15). The colonies do dium containing 20% horse serum attain a not adsorb human, rat, guinea-pig, or chicken maximum diameter of approximately 200 pm erythrocytes (1 2, 16). in 3 to 4 days. Fully developed colonies show Sensitivity to SPS and digitonin. Growth is the typical “fried egg” appearance of Myco- inhibited by 5% sodium-polyanethol-sulfonate plasma colonies, with a small central nipple (SPS) and by 1.5% digitonin, as demonstrated (22). by disk growth-inhibition tests on agar (8). Absence of reversion. No reversion to a Electrophoretic patterns of cell proteins. The bacterium occurred when an early-passage cul- cell protein pattern is clearly distinct from the ture of strain CH20247 was subcultured five patterns of other Mycoplasma species of human consecutive times on media devoid of penicillin origin, including M. orale and M. faucium (24). and thallium acetate (E. A. Freundt, 1973, Serological characteristics. Serologically dis- unpublished data). tinct by disk growth-inhibition, metabolism Filterability characteristics. A suspension of inhibition, immunofluorescence, indirect he- strain CH20247 used for filtration contained 4 magglutination, and complement-fixation tests X lo8 colony-forming units (CFU)/ml, and from other Mycoplasma species of human after passage through 450-, 200-, and 100-nm source, including M. orale and M. faucium, as membrane filters (Acropor), the filtrates well as from a number of other Mycoplasma yielded 4 X lo6, 4 X lo6, and 0 CFU/ml, and Acholeplasma species tested (7, 9, 22). In respectively (F. T. Black, 1973, unpublished the complement-fixation test, a marked cross- data). reaction between M. buccale and M. orale has Gaseous requirements. The organism is a been demonstrated (22). Also, sharing of facultative anaerobe, although the growth of antigens with other species of human origin is fresh isolates is better under anaerobic condi- demonstrable by means of double immunodif- tions, i.e., in an atmosphere of 5% COZ and fusion in agar (22). 95% nitrogen (2, 21). Properties of DNA. Determinations of the Growth requirements. Growth occurs best at guanine plus cytosine (G+C) contents range from 36 to 37 C in a medium of pH 7.0 to 8.0 25% by buoyant density (18) to 26.4 k 0.2% by containing PPLO broth (Difco), seven parts; T, (9). The relative relatedness to other human uninactivated horse serum, two parts; and 25% Mycoplasma species including M. orale, as yeast extract (Fleischmann type 20-40 or determined by nucleic-acid homology tests, is Distillers Company Ltd. dry yeast), one part 710% (17). (23). Deoxyribonucleic acid (DNA) (0.02%) Habitat. This species is an apparently infre- appears to stimulate growth (6), but the quent parasitic inhabitant of the human oro- evidence for this is not conclusive. Fresh yeast pharynx, comprising approximately 1 to 2% of extract is not required for growth (7). Choles- all mycoplasmas recovered from the orophar- terol is required (14). ynx (7, 22); it is the predominant species of the Biochemical characteristics. Carbohydrates mycoplasmal flora of the oropharynx of non- and urea are not hydrolyzed, but arginine is human primates (3, 6, 10, 11, 13). hydrolyzed. 2, 3, 5-Triphenyl-tetrazolium chlo- ride is reduced anaerobically but not aerobi- cally. The phosphatase test is positive. “Film and spots” are not produced on either horse Mycoplasma faucium nom. nov. serum agar or egg-yolk medium. Gelatin, casein, and coagulated horse serum are not liquefied Morphology and staining characteristics. The (2, 5, 7, 22). Growth is inhibited by 0.001% morphology and staining characteristics are the methylene blue (7). same as for M. buccale (F. T. Black and E. A. Antibiotic sensitivity. This species has the Freundt 1973, unpublished data). same sensitivity to antibiotics as other large- Gaseous requirements and absence of rever- 254 FREUNDT ET AL. INT. J. SYST. BACTERIOL. sion. The gaseous requirements and absence of immunodiffusion in agar (7). reversion are the same as for M. buccale (7). Properties of the DNA. Not determined. Colonial appearance. Colonies on agar me- Habitat. This species apparently is a rare dium containing 20% horse serum resemble member of the normal flora of the human those of M. buccale in size and general oropharynx, comprising less than 2% of the appearance. However, they develop somewhat total number of mycoplasmas recovered (7). more superficially and are more loosely at- Occasionally it is recovered from the oro- tached to the agar surface than the colonies of pharynx of nonhuman primates (6). other mycoplasmas (7). Filterability characteristics. A suspension of REPRINT REQUESTS strain DC-333 -used for filtration contained 1.2 X lo6 CFU per ml, and after passage through Address reprint requests to: Dr. E. A. Freundt, 450-, 220-, and 100-nm filters, the filtrates Institute of Medical Microbiology, University of yielded 4 X lo4, 4 X lo3, and 0 CFU/ml, Aarhus, DK-8000 Aarhus C, Denmark. respectively (F. T. Black, 1973, unpublished data). LITERATURE CITED Growth requirements. Although growth oc- 1. Addey, J. P., D. Taylor-Robinson, and M. Dimic. curs in essentially the same medium used for M. 1970. Viability of mycoplasmas after storage in buccale, this species appears to be more frozen or lyophilized states. J. Med. Microbiol. fastidious. Fresh yeast is required (7), and 3~137-145. L-cystein (0.1%) appears to stimulate growth 2. Aluotto, B. B., R. G. Wittler, C. 0. Williams, and significantly (R.
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