INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1995, p. 605-612 Vol. 45, No. 3 0020-7713/95/$04.00+0 Copyright 0 1995, International Union of Microbiological Societies Revised Minimum Standards for Description of New Species of the Class MoZZicutes (Division Tenericutes) INTERNATIONAL COMMITTEE ON SYSTEMATIC BACTERIOLOGY SUBCOMMITTEE ON THE TAXONOMY OF MOLLICUTEST In this paper the Subcommittee on the Taxonomy of Mollicutes proposes minimum standards for descrip- tions of new cultivable species of the class MoZZicutes (trivial term, mollicutes) to replace the proposals published in 1972 and 1979. The major class characteristics of these organisms are the lack of a cell wall, the tendency to form fried-egg-type colonies on solid media, the passage of cells through 450- and 220-nm-pore-size membrane filters, the presence of small A-T-rich genomes, and the failure of the wall-less strains to revert to walled bacteria under appropriate conditions. Placement in orders, families, and genera is based on morphol- ogy, host origin, optimum growth temperature, and cultural and biochemical properties. Demonstration that an organism differs from previously described species requires a detailed serological analysis and further definition of some cultural and biochemical characteristics. The precautions that need to be taken in the application of these tests are defined. The subcommittee recommends the following basic requirements, most of which are derived from the Internutional Code ofNomencEature @Bacteria, for naming a new species: (i) designation of a type strain; (ii) assignment to an order, a family, and a genus in the class, with selection of an appropriate specific epithet; (iii) demonstration that the type strain and related strains differ significantly from members of all previously named species; and (iv) deposition of the type strain in a recognized culture collection, such as the American Type Culture Collection or the National Collection of Type Cultures. The publication of the description should appear in a journal having wide circulation. If the journal is not the International Journal of Systematic Bacteriology, a reprint must be submitted to that journal so that the name may be considered for inclusion in a validation list as required by the International Code ofNomenclature of Bacteria. The Subcommittee on the Taxonomy of Mollicutes first pub- age of the culture. Some species have specialized terminal lished a “Proposal for Minimum Standards for Descriptions of structures that have been shown to carry adhesins, proteins New Species of the Order Mycuplasmatales” in 1972 (38). This that may mediate attachment to eucaryotic cells and tissues, document, which followed an earlier proposal for general including mammalian cell surfaces (4, 70). The replication of guidelines for nomenclature of these organisms (37), was re- the mollicute genome precedes, but is not necessarily synchro- vised in 1979 (33). In view of the greatly increased number of nized with, cell division. Thus, budding and fragmentation of mollicutes described, the molecular phylogenetic schemes pro- filamentous forms, as well as classical binary fission, may be posed for members of the group, the recently proposed taxo- observed. Cells may be motile or nonmotile. Neither spores nomic revisions of the class Mollicutes (98), and the develop- nor walled stages are known. All species described thus far can ment of new technologies for classification, the subcommittee be grown on artificial cell-free media of diverse complexity. deemed it necessary to again update the recommendations for However, members of a large phylogenetically cohesive group minimum standards. of unnamed mollicutes, the phytoplasmas (43, 83), have not Description of the class MoZlicutes. Members of the class been cultivated and are therefore not within the scope of this Mollicutes (10, 23, 66, 67, 69, 102, 110) are eubacteria that are document. Most species of cultivable mollicutes are faculta- bounded by a single membrane; they lack a cell wall and cannot tively anaerobic, but a few are obligate anaerobes that are synthesize cell wall precursors, such as muramic and diamin- rendered nonviable by exposure to minute concentrations of opimelic acids (53, 62). oxygen. Colonies on solid medium are usually no larger than 1 The organisms can be very small; the diameters of some to 2 mm in diameter; they are usually considerably smaller. viable spherical cells are in the range of 300 nm (8), and viable The colony diameters are often about 0.5 mm but in some helical filaments can be as small as 200 nm in diameter (102, cases may be as small as 0.01 mm. The organisms tend to 110). The cells vary in shape from coccoid to helical or fila- penetrate and grow beneath the surface of solid media. Under mentous. Some species are pleomorphic. In a minority of spe- suitable conditions, most nonmotile species form colonies that cies there is a tendency for filaments to produce truly branched have a characteristic fried-egg appearance (64). Colonies of mycelioid structures. Cell shape in many instances appears to motile species are usually diffuse, with a number of satellite depend on both the nutritional qualities and the osmotic pres- colonies nearby. sure of the medium used to grow the organisms, as well as the Species in the class exhibit absolute resistance to penicillin and its analogs and are also resistant to rifampin (27, 61, 95). Although most species that have been tested are susceptible to many broad-spectrum antibiotics, including the tetracyclines, p R. FrWhitcornb (Chair), J. G. Tully (Secretary), J. M. BovC, J. M. lateral transfer of tetracycline resistance genes from walled Bradbury, G. Christiansen, I. Kahane, B. C. Kirkpatrick, F. Laigret, R. H. Leach, H. C. Neimark, J. D. Pollack, S. Razin, B. B. Sears, and bacteria (streptococci) to mollicutes can occur in the human D. Taylor-Robinson. Corresponding author: J. G. Tully. Mailing ad- genital tract (75). Growth and metabolism are specifically in- dress: Mycoplasma Section, NIAID, Bldg. 550, Frederick Cancer Res. hibited by homologous antibody. Many characteristics of mol- Dev. Center, Frederick, MD 21702. Phone: (301) 846-1192. Fax: (301) licute species, such as their pleomorphism, their tendency to 846-5165. penetrate and grow beneath the surface of solid medium, and 605 606 SUBCOMMITTEE ON THE TAXONOMY OF MOLLICUTES INT.J. SYST. BACTERIOL. TABLE 1. Taxonomy and characteristics of members of the class Mollicutes No. of G+C Genome Cholesterol Other distinctive Classification recognized content size Habitat(s) requirement features species (mol%) (kbp)” Order I: Mycoplasmatales Family I: Mycoplasmataceae Genus I: Mycoplasma 100 23-40 600-1,350 Yes Humans, animals Optimum growth usually occurs at 37°C Genus 11: Ureaplasma 6 27-30 760-1,170 Yes Humans, animals Urea hydrolysis Order 11: Entomoplasmatales Family I: Entomoplasmataceae Genus I: Entornoplasma 5 27-29 79O-1,140 Yes Insects, plants Optimum growth occurs at 30°C Genus 11: Mesoplasma 12 27-30 870-1,100 No Insects, plants Optimum growth occurs at 30°C; sustained growth in serum-free medium occurs only in the presence of 0.04% Tween 80 Family 11: Spiroplasmataceae Genus I: Spiroplasma 17 25-30 940-2,240 Yes Insects, plants Helical filaments; optimum growth occurs at 30 to 37°C Order 111: Acholeplasmatales Family I: Acholeplasmataceae Genus I: Acholeplasma 13 26-36 1,50CL1,650 No Animals, some plants Optimum growth occurs at and insects 30 to 37°C Order IV: Anaeroplasmatales Family I: Anaeroplasmataceae Genus I: Anaeroplasma 4 29-34 1,500-1,600 Yes Bovine and ovine rumina Oxygen-sensitive anaerobes Genus 11: Asteroleplasma 1 40 1,500 No Bovine and ovine rumina Oxygen-sensitive anaerobes a Genome size as reported in literature. their absolute resistance to penicillin, are directly related to the orders, families, and genera (98) and some of the criteria used lack of a cell wall. In most species of the class, the guanine- to distinguish the higher taxa. plus-cytosine (G+C) content of the DNA is between 23 and 35 Proposal. A new mollicute species must be shown to belong mol%, although in a few species the G+C contents are higher to the class, to be assignable to a genus (old or new), and to be (up to 40 mol%) (31,41,42). The genome size varies from 600 significantly different from all previously named species. A to 2,200 kbp (13, 14, 40, 59, 60, 63, 76). pure culture is essential for determinative studies, and the Mollicutes may be commensal organisms or frank or oppor- following data should, therefore, be provided in the published tunistic pathogens. Certain species are etiologic agents of dis- description. eases of vertebrates, invertebrates, or plants. Mollicutes patho- (i) Strain purification and preliminary cloning. The pure genic for plants multiply in and are transmitted by insect culture that is required for a type strain must be derived from vectors. the inevitably mixed population of mollicute cells that is The vast majority of plant-pathogenic mollicutes belong to a present in cultures of an uncloned isolate. Purification is group previously called the mycoplasma-like organisms but achieved by cloning at least three times, which at each stage now properly called phytoplasmas (43,49,54,83,84,90). A few involves gentle filtration of a broth culture through membrane Spiroplasma species are also plant pathogens (102). Both of filters with the smallest possible pore diameter (usually 200 to these types of mollicutes are maintained in a biological cycle 300 nm) (94). The filtrate and dilutions of the filtrate are involving the phloem tissues of their host plants and their cultured on solid medium, and an isolated colony is subse- homopterous insect vectors. The phytoplasmas are true molli- quently picked from a plate on which few colonies have devel- cutes, as judged by the sequences of their 16s rRNA and oped. The filtration and cloning sequence is repeated three ribosomal protein genes (49, 50, 57, 84, 90).