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). These mollicutes times sequentially to optimize the probability that the clone have not been cultivated despite extensive attempts in many eventually selected as the type strain is derived from a single laboratories. Murray and Schleifer (56) recently proposed a cell. Triple cloning by limiting dilution in liquid media may be provisional taxonomic status for noncultivable organisms that an acceptable alternative method (34). All tests upon which the have been partially characterized and for which specific taxonomic description is based should be carried out with the genomic and phenotypic characteristics have been determined. cloned strain. Under these circumstances, the subcommittee recommends (ii) Determination at the level of the class Mollicutes. (a) that formal naming of phytoplasma taxa at the species level Absence of reversion. If it is possible that the organism is a should be deferred. A classification scheme for these organ- bacterial L-phase variant, it is necessary to test the isolate for isms is being discussed by the Subcommittee on the Taxonomy reversion to eubacterial morphology soon after the cloning of Mollicutes (35, 36) and by an international collaborative procedure is completed. In these tests (l), the cloned organism working team on phytoplasmas sponsored by the International should fail to revert to a walled bacterium during early pas- Organization for Mycoplasmology. sages in a medium that does not contain antibiotics or other Table 1 shows the current classification of the Mollicutes into substances (e.g., high salt concentrations) known to induce VOL. 45, 1995 MINIMUM STANDARDS FOR DESCRIPTION OF NEW SPECIES 607 bacterial L-phase variants. Appropriate media for attempting terol are usually determined by direct quantitative comparison to demonstrate reversion might include a standard bacterio- of the growth responses of an organism in the presence and logical broth, a semisolid version of this medium, and conven- absence of cholesterol or other sterols (71, 96). These tests tional blood agar. require serum-free media containing various supplements of It must be remembered that the capability of L-phase vari- fatty acids and albumin, as well as increasing concentrations (1 ants to revert to typical bacterial forms varies considerably to 20 pg/ml) of solubilized cholesterol. The growth responses between species and between strains of the same species; it are measured by determining the increases in the amount of also varies with cultural conditions. Reversion usually occurs total cellular protein or the number of CFU on solid medium. more readily in liquid or semisolid media than on solid me- Members of the family Mycuplasmataceae and many organisms dium. Aerobic growth conditions may favor reversion of some assigned to the families Entomoplasmataceae, Spiroplasmata- organisms. DNA base compositions (G+C contents) and fil- ceae, and AnaerupZasmataceae exhibit minimal or negligible terability test results (see below) may also suggest that organ- growth in serum-free media but enhanced growth in the pres- isms are L-phase variants. ence of increasing concentrations (up to 20 pg/ml) of choles- (b) Ultrastructure of the limiting cell membrane. Placement terol. Acholeplasmas, mesoplasmas, asteroleplasmas, and of the organism in the class Mollicutes requires that an appro- some spiroplasmas can grow well in either serum-free medium priate thin-sectioning electron microscopic procedure be per- alone or serum-free medium supplemented with polyoxyethyl- formed to show that the organism is bounded by a single ene sorbitan monooleate (Tween 80). membrane and lacks a cell wall (19). This information, in An additional modification of the cholesterol tests (82) in- conjunction with the failure of the organism to revert to a volves inoculation of serum-free media (with or without 0.04% bacterial form and evidence that the organism is not an archae- Tween 80 or 15% fetal bovine serum) with test organisms. An bacterium (32), permits initial assignment to the class Mulli- attempt is made to sustain passage through 23 serial 10-fold cutes. In this regard, it should be noted that noncultivable, dilutions. The endpoint selected represents a theoretical dilu- wall-less organisms have been reported from diverse natural tion factor (based on Avogadro’s number) that would exclude ecosystems. It is still possible that a wall-less bacterium (other the possibility of carryover of sterol in the inoculum. Successful than an archaebacterium) that is phylogenetically unrelated to passage of nonhelical mollicutes that fail to grow in serum-free the mollicutes might eventually be discovered. broth in the Tween 80-containing medium in this test suggests (c) Filterability. The minimum pore diameter that permits that the organism should be assigned to the genus Mesoplasma. passage of viable organisms should be determined (94). High- This test has also proved to be useful for determining the sterol pressure filtration, which can force wall-less cells through pores requirements of spiroplasmas. smaller than their diameter, should be avoided. Cells of all Several important factors related to cholesterol or sterol currently described mollicute species pass through membrane tests need emphasis. While tests to determine the digitonin or filters having a pore diameter of 450 nm if cultures in liquid sodium polyanethol sulfonate sensitivity of mollicutes (25) are media are used. Most species also pass through membrane useful for preliminary assessment of the sterol requirement of filters with 300- and 200-nm pores, although the titer is often an organism, they are not definitive. Sterol growth require- significantly reduced after passage through such pores. On the ments of mollicutes cannot be assessed simply by a few pas- other hand, bacterial L-phase variants frequently either fail to sages of the organisms on serum-free agar or in broth media pass through 300-nm-pore-size membrane filters or exhibit because of the carryover of cholesterol and/or other sterols in greatly reduced titers, and most do not pass through 200-nm- the inocula. Control strains of both sterol-requiring and non- pore-size membranes (104). sterol-requiring mollicutes should always be included in tests (d) Nucleic acid procedures. The three procedures de- to assess the sterol requirements of putative new species. The scribed below also are often useful in placing an organism in amounts of growth of spiroplasmas that occur in cholesterol the class MolZicutes. (i) Determination of the G+C base com- tests should not be determined by dark-field examination for position is a requirement for a new species description (see numbers of helices, since some Spiroplasma species can grow below) and can also be useful in a preliminary assay for mol- as nonhelical organisms (82). Some species in the Mycoplas- licutes. An observed G+C value of more than 40 mol% should mataceae or Spiroplasmataceae may have such fastidious raise a strong concern that the organism might not be a mol- growth requirements that the conventional serum-free base licute. (ii) Genome size determination may assist placement of medium supplemented only with cholesterol might not provide the organism (see below). The genome sizes of the mollicutes their minimal nutritional needs. In such instances (101, 103), that have been examined to date have been found to range comparative tests in which different quantities of both choles- from about 600 to 2,200 kbp. A wall-less organism with a terol and animal serum are added can provide an estimate of genome larger than 3,000 kbp might be an L-phase variant. (iii) the growth requirement for sterol. Sequence analysis of the 16s ribosomal DNA of a candidate (b) Cellular morphology. It should be emphasized that mol- organism can provide unambiguous evidence that the organism licute morphology depends to a considerable extent on the is a mollicute. This information may be obtained by sequencing composition of the medium and the age of the culture. Deter- material amplified by the PCR with primers specific for eubac- mination of the taxonomic position of a new mollicute requires terial 16s ribosomal DNA (106). The 16s ribosomal DNA examination of the morphology of a typical population of the sequences of many mollicutes and eubacteria are available for organism in a log-phase culture, usually by dark-field or phase- comparison in gene data banks (107). contrast microscopy (12,93). Members of the family Spiroplas- (iii) Determination at the level of order and family. The mataceae are helical under most circumstances in liquid media, appropriate order and family within the class Mullicutes are in which they exhibit flexional and, in some cases, translational determined by the presence or absence of a sterol requirement, motility (110, 118). Microscopic techniques may reveal that the gross cellular morphology, the colony appearance, the op- nonhelical organisms are pleomorphic; often such organisms timum growth temperature, the genome size, and the atmo- occur as small coccoid bodies, bipolar forms, and/or finely spheric oxygen requirement for growth. The following tests are branched or unbranched filaments of varying lengths. Exami- mandatory. nation of methanol-fixed organisms stained with Giemsa solu- (a) Sterol requirement. Growth requirements for choles- tion (112) may be useful for determining the morphology of 608 SUBCOMMITTEE ON THE TAXONOMY OF MULLICUTES IW. J. SYST. BACTERIOL. certain Mycoplasma species. However, since nonhelical as well UGA as a tryptophan codon, while only UGG encodes tryp- as helical organisms may exhibit motility, examination of live tophan in members of the orders Acholeplasmatales and material is mandatory. Anaeroplasmatales (7, 74, 87, 90, 121). Fixatives used in electron microscopic techniques frequently (iv) Determination at the level of genus. The results of the distort the helical morphology of spiroplasmas. Therefore, it is tests performed to assign a mollicute to the correct order and useful to examine both live and fixed cells by dark-field mi- family are of considerable value for assigning the new species croscopy before embedding and sectioning (20). Although to the correct genus. The genera listed in Table 1 are recog- shape and form must be determined for a description, it is not nized as follows. possible to identify an organism as a member of the Mollicutes (a) Genus Mycoplasmu. The genus Mycoplasma comprises solely on the basis of the results of conventional light micros- those nonhelical mollicutes from vertebrates that (i) are not copy or scanning electron microscopy of cells. obligately anaerobic (oxygen sensitive), (ii) require cholesterol (c) Colony appearance. It is necessary to demonstrate the or sterols for growth, (iii) have an optimum temperature of ability of a candidate strain to grow on solid medium and to 37°C or above, (iv) are not capable of urea hydrolysis, and (v) determine the morphology of the colonies produced. At least have a genome size of 600 to 1,350 kbp (Table 1). some colonies of most nonhelical mollicutes exhibit a fried-egg (b) Genus Ureaplasma. Organisms that have features similar morphology, which is characterized by a central zone in which to the features described above for the genus Mycoplasma but the organisms grow into the medium and a peripheral zone of are capable of urea hydrolysis and have a genome size of 760 surface growth (64). However, many Spiroplasma species and a to 1,170 kbp (Table 1) are assigned to the genus Ureaplasma small minority of nonhelical mollicutes do not form such col- (76, 89). Demonstration of urea hydrolysis, which results in an onies under any currently known cultural conditions. Spiro- increase in the pH of the medium because of ammonia pro- plasmas often form diffuse colonies that tend to generate sat- duction, is a minimum requirement for assigning a new species ellite colonies. This type of growth is highly characteristic of to the genus Ureaplasma. However, although no other molli- motile organisms and therefore is normal for members of the cute genera are known to possess this enzymatic activity, this family Spiroplasmataceae (102, 110). It is significant that non- test is mandatory for characterization of new strains, since this motile spiroplasma variants do form fried-egg colonies (92). property is known to occur in other procaryotes obtained from Colony appearance is strongly dependent on the composi- habitats other than the urinary tract. A standardized technique tion of the medium. Although most mollicutes can grow be- for determining urea hydrolysis in mollicutes has been de- neath the surface of solid medium, this either does not occur or scribed (65). is difficult to demonstrate on some media with certain strains. (c) Genera of the Entomoplasmutales. Nonhelical mollicutes In particular, the motility of spiroplasmas may prevent typical from arthropods or plant surfaces that (i) are not obligately colony formation on conventional “soft” agar (containing 0.8 anaerobic, (ii) have a sterol requirement for growth, (iii) have to 1% purified agar). The use of so-called “hard” agar (con- optimum growth temperatures near 30°C, (iv) have genome taining 2.0 to 2.5% purified agar) may permit demonstration of sizes of about 790 to 1,140 kbp (Table l), and (v) exhibit fried-egg spiroplasma colonies, particularly if the medium is phylogenetic affinity to the spiroplasmas are assigned to the suboptimal (103). Some nonmotile mollicutes produce surface genus Entomoplasma (98). Nonhelical mollicutes isolated from growth with little or no central ingrowth, although use of a arthropods or plant surfaces that have similar features but (i) softer agar may encourage the latter type of growth. Other exhibit sustained growth in serum-free medium containing mollicutes produce central ingrowth without the formation of 0.04% polyoxyethylene sorbitan and (ii) have genome sizes of peripheral surface growth, particularly on suboptimal media. about 870 to 1,100 kbp (Table 1) are assigned to the genus Therefore, a substantial effort to modify the agar content of Mesoplasma. These two genera make up the family Ento- solid media should be made before it is concluded that a moplasmataceae (98). Helical mollicutes isolated from arthro- fried-egg morphology cannot be demonstrated for a nonhelical pods, plant surfaces, or plant phloem that (i) are not obligately mollicute. anaerobic, (ii) do or do not have a sterol requirement, and (iii) (a) Optimum growth temperature. Temperature require- have genome sizes of about 940 to 2,200 kbp (Table 1) are ments for growth are useful for differentiating some families. classified in the genus Spiroplasma and family Spiroplasmatu- Members of the order Entomoplasmatales, including species ceae (102, 109, 110). belonging to the genera Spiroplasma, Mesoplasma, and Ento- Note that since members of the Mycoplasmataceae have moplasma, and some members of the order Acholeplasrnatales on rare occasions been isolated from plant sources, it is theo- usually grow best at 30 to 32°C (44, 45, 116), while other retically possible for members of the Entomoplasmataceae members of the class grow best at 37°C (98). Tests for assessing to be isolated from vertebrates. Therefore, although host as- the optimum temperature and the temperature range that rep- sociation provides an important clue to an organism’s iden- resents observed growth patterns of currently described mol- tity, it is always important to examine the critical proper- licutes (10 to 41°C) have been described elsewhere (44, 45, ties that allow placement of a candidate strain in an existing 116). genus, as well as to assess the reason why it may be found in (e) Aerobiosis or anaerobiosis. Mollicutes that have a strict that site. requirement for anaerobic environments and exhibit sensitivity (a) Genus Acholeplasma. The genus Acholeplasma consists of to oxygen are classified in the family Anaeroplasrnataceae, the nonhelical mollicutes from vertebrates, plants, or arthropods single family in the order Anaeroplasmatales. At the present that (i) are not obligately anaerobic, (ii) are able to grow in time, such organisms have been found only in ovine and bovine media lacking sterol, (iii) exhibit growth temperature optima rumina. The procedures required to describe these organisms of 30 to 37”C, and (iv) have a genome size of about 1,500 to have been presented elsewhere (78-80). 1,650 kbp (Table 1) (97, 98). (f) Codon usage. If facilities for establishing UGA codon (e) Genera of the Anaeroplasrnataceae. Nonhelical mollicutes usage by a candidate organism are available, this information from vertebrates that are obligately anaerobic and require ste- can be useful in assigning the organism to an order and/or rol are placed in the genus Anaeroplasma, while obligately family; however, this information is not essential. Members of anaerobic organisms that do not require sterol are placed in the orders Mycoplasmatales and Entomoplasmutales utilize the genus Asteroleplasma (80). VOL.45, 1995 MINIMUM STANDARDS FOR DESCRIPTION OF NEW SPECIES 609

(v) Determination at the level of species: definition of spe- didate organism with specific and potent antisera or conjugates cies. The species concept for members of Mollicutes is based on of all previously described species belonging to the genus. arbitrary criteria, as is the species concept for all procaryotic Some mollicutes (particularly Spiroplasma species) exhibit taxa. Ideally, mollicute species can be regarded as clusters of nonspecific autofluorescence when they are grown on certain morphologically and biologically similar strains whose ge- agar formulations, which complicates immunofluoresence nomes exhibit relatively high levels (>70%) of relatedness (67, tests. In these instances, the deformation test (109, 115, 119, 109, 110). The value >70% was chosen to conform with cur- 120) has special value for screening candidate species against rently accepted practice in microbial systematics (30, 105). In antisera of previously established spiroplasma species or practice, extensive studies of DNA hybridization among strains groups. A combined deformation-metabolism inhibition test may not be feasible. It is therefore usually necessary to estab- system has been used to provide both screening and refined lish patterns of relationships by examining phenotypic markers. analysis and definition of Spiroplasma species and subgroup The techniques that may be used include serologic or meta- relationships (109, 117, 119). bolic techniques or molecular methods such as Southern hy- Note that the recommendations relating to serological tech- bridization and DNA sequencing with the PCR (72, 99). The niques are based on an extensive background of use within the following information should be provided in the description of field of mollicute taxonomy and on a unique record of the a species. sensitivity and specificity of these techniques for distinguishing (a) General biological information. The type strain and re- mollicutes. However, it is recognized that some existing species lated strains which form the basis of the description of the (e.g., Mycoplasma hominis and Mycoplasma iowae) exhibit sub- proposed new species and the origins or sources of these stantial serologic heterogeneity, so that an occasional isolate strains should be given in detail, along with a description of the may not react in some serologic tests with antiserum to the type culture media used. Any special growth factor(s) or medium strain of the species. For these specific mollicutes, it is advis- constituent(s) required by the organism should be noted, and able to utilize typing antisera to several strains of that species any possible problems encountered in cultivation should be in at least two different serologic techniques. discussed. Media that support multiplication of the organism (c) Fermentation of glucose. The ability of the organism to should be specified, and evidence of penicillin resistance catabolize glucose to acid should be assessed, preferably under should be included. The incubation temperatures used (includ- both aerobic and anaerobic cultivation conditions (1, 24, 68). ing the temperature range and optimum temperature[s] for The composition of the medium, the temperature, and the growth), pH requirements, and atmospheric conditions re- length of incubation used in these tests should be defined. quired for growth are critical for a complete description. (d) Hydrolysis of arginine. The ability of the organism to Data regarding pathogenicity or lack of pathogenicity for the hydrolyze arginine with an increase in pH resulting from the natural host(s) should also be provided, together with habitat production of ammonia should be determined (1, 3). A mod- information and experimental pathogenicity data obtained ified test performed with arginine concentrations that vary with vertebrates, invertebrates, or plant species. from 2 to 10 g/liter could also be used, since some organisms The following tests should be performed to establish the are inhibited by higher concentrations of arginine (47). It properties of the species. should also be emphasized that in certain cases demonstration (b) Serological relatedness. The small number of determi- of arginine hydrolysis in spiroplasmas can be clearly defined native characteristics used for species recognition leads to only when glucose or some other energy source is supplied at great reliance on serology. The candidate organism (the puta- the same time (91). The most useful procedure is to add a low tive type strain) should be compared serologically with type or concentration of glucose (0.1%) to arginine-containing broth; reference strains of all other named species belonging to the although this technique may produce an initial decrease in pH, presumptive genus. If the strain cannot be placed in one of the it may permit subsequent increases in the pH as the spiro- previously described genera, it should be compared with all plasma grows (16). previously named species of Mollicutes. The strain should be Experience has revealed the difficulties and pitfalls that are examined by at least two serological methods. The growth encountered when utilization tests are performed with com- inhibition and agar plate immunofluorescence tests are recom- plex growth media. Substances other than the substrate may be mended for nonhelical organisms, and the growth inhibition, metabolized, with a concomitant change in pH. If possible, it is metabolism inhibition, and spiroplasma deformation tests are advisable to perform biochemical tests in less complex media preferable for helical mollicutes. (e.g., in the absence of yeast extract or serum or with serum The growth inhibition test (5,17,18,22,85,111) is the most replaced by bovine serum fraction [97, lOS]), since such media useful technique for serologic characterization of new molli- generally contain fewer substances that can be metabolized to cute species. Reciprocal testing of antisera and antigens within produce a change in pH. Repeated subculture in the presence the candidate genus is usually not required, provided that of the substrate may be required to detect hydrolysis of certain antigen from the putative new species is tested against specific substances. This is especially important in tests for hydrolysis and potent antisera to all previously described species in the of arginine, especially if the organism also metabolizes glucose. genus. However, if only a few species of the genus have been It is also essential that each test for pH change be carefully described, reciprocal testing of both antigen and antisera is controlled by observations of uninoculated medium with sub- recommended as additional confirmation of serologic distinc- strate, inoculated medium without substrate, and organisms tiveness. If the candidate organism cannot be grown easily on with known requirements. agar, the metabolism inhibition test (88) may be used as an (e) Detection of P-D-glucosidase. The levels of p-D-glucosi- alternative method. This procedure has been used successfully dase in various acholeplasmas can be a useful way to distin- in serologic analyses of Ureaplasma strains (77, 86, 88) and guish species. The test for this enzyme is based on hydrolysis of Spiroplasma species or groups (100, 109, 117). esculin or arbutin (11, 81, 113). The agar plate immunofluorescence test can be performed (f) Genetic characteristics. At a minimum, it is recom- as either a direct or an indirect fluorescent antibody test (6,21, mended that genome size and DNA base composition (G+C 26, 29). As with growth inhibition tests, at a minimum the content) should be determined for a candidate species. DNA comparison should involve testing of agar colonies of the can- base composition should be determined by at least one of the 610 SUBCOMMITTEE ON THE TAXONOMY OF MOLLICUTES INT. J. SYST. BACTERIOL. following recommended techniques: melting temperature, basis for cytadsorption of Mycoplasma pneumoniue. J. Bacteriol. 151:1514- buoyant density, high-pressure liquid chromatography (15), 1522. 5. Black, F. T. 1973. Modification of the growth inhibition test and its appli- and isopycnic centrifugation (39, 42). Recent studies have cation to human T-mycoplasmas. Appl. Microbiol. 25528-533. shown that the genome sizes of mollicutes can be determined 6. Black, F. T., and A. Krogsgaard-Jensen. 1974. Application of indirect more easily and more accurately with pulsed-field gel electro- immunofluorescence, indirect haemagglutination and polyacrylamide-gel phoresis (13, 14, 58-60, 63, 76) than with previously described electrophoresis to human T-mycoplasmas. Acta Pathol. Microbiol. Scand. Sect. B 82:345-353. techniques that involve renaturation kinetics (2). Pulsed-field 7. Blanchard, A. 1990. Ureaplasma urealyticum urease genes: use of a UGA gel electrophoresis genome size measurements are made di- tryptophan codon. Mol. Microbiol. 4669476. rectly by using whole-cell suspensions in agarose; thus, in this 8. Boatman, E. S. 1979. Morphology and ultrastructure of the Mycoplasma- technique the essential integrity of the genome is maintained. tales, p. 63-102. In M. F. Barile and S. Razin (ed.), The mycoplasmas, vol. 1. Academic Press, New York. It is now clear that the genome sizes of mollicutes form a 9. Bonnet, F., J. M. Bov6, R. H. Leach, G. S. Cottew, D. L. Rose, and J. G. continuum. The range of values obtained for certain genera is Tully. 1993. Deoxyribonucleic acid relatedness between field isolates of so broad that this character is no longer as valuable as it once mycoplasma F38 group, the agent of contagious caprine pleuropneumonia, was for differentiating higher taxa (Table 1) (98). However, and strains of Mycoplasma capricolum. Int. J. Syst. Bacteriol. 43597-602. 10. Bod, J. M. 1993. Molecular features of mollicutes. Clin. Infect. Dis. genome size is useful for secondary assignment of candidate l’I(Supp1. 1):S1043 1. organisms to the genus and species levels and therefore should 11. Bradbury, J. M. 1977. Rapid biochemical tests for characterization of the be determined for organisms that are being considered for Mycoplasmatales. J. Clin. Microbiol. 5531-534. species status. 12. Bredt, W. 1983. Phase-contrast microscopy. Methods Mycoplasmol. 1:31- 33. (vi) Determination at the level of subspecies. In general, the 13. Carle, P., F. Laigret, J. G. Tully, and J. M. BovC. 1995. Heterogeneity of tests used at the subspecies level are similar to those used at genome sizes within the genus Spiroplasma. Int. J. Syst. Bacteriol. 45178- the species level. In the Mollicutes the rank of subspecies 181. should be reserved for important strains which differ consis- 14. Carle, P., D. L. Rose, J. G. Tully, and J. M. Bov6.1992. The genome size of spiroplasmas and other mollicutes. IOM Lett. 2263. tently but which nevertheless prove to be too closely related on 15. Carle, P., C. Saillard, and J. M. Bov6. 1983. Determination of guanine plus the basis of serological properties or nucleic acid hybridization cytosine content of DNA. Methods Mycoplasmol. 1:301-308. data to warrant species rank (9, 48). In general, a subspecies 16. Clark, T. B., R. F. Whitcomb, J. G. Tully, C. Mouches, C. Saillard, J. M. should not be proposed when information concerning the se- Bov6, H. Whblewski, P. Carle, D. L. Rose, and D. L. Williamson. 1985. Spiroplasma mellifemm sp. nov., a new species from the honeybee (Apis rological or biological properties of a strain is limited or when mellifera). Int. J. Syst. Bacteriol. 33296-308. data on nucleic acid hybridization between the candidate or- 17. Clyde, W. A, Jr. 1964. Mycoplasma species identification based upon ganism and the established species to which it exhibits a sero- growth inhibition by specific antisera. J. Immunol. 92958-965. logical relationship are lacking. In the absence of such defini- 18. Clyde, W. A., Jr. 1983. Growth inhibition tests. Methods Mycoplasmol. 1:405410. tive information, temporary use of infrasubspecific ranks 19. Cole, R M. 1983. Transmission electron microscopy: basic techniques. (strain, serovar, pathovar, biovar, etc.) is much preferred, since Methods Mycoplasmol. 1:43-50. the use of such designations provides more flexibility than 20. Cole, R. M., J. G. Tully, T. J. Popkin, and J. M. Bov6. 1973. Morphology, Latin tertiary combinations (i.e., subspecies names) and en- ultrastructure, and bacteriophage infection of the helical mycoplasma-like organism (Spiroplasma cim’ gen. nov., sp. nov.) cultured from “stubborn” courages the accumulation of additional data relevant to clas- disease of citrus. J. Bacteriol. 115367-386. sification (109). 21. Del Giudice, R. A., N. F. Robillard, and T. R. Carski. 1967. Immunofluo- (vii) Optional taxonomic tests. Useful supporting informa- rescence identification of Mycoplasma on agar by use of incident illumina- tion at various taxal levels may also be obtained from other tion. J. Bacteriol. 93:1205-1209. 22. Edward, D. G., and W. A. Fitzgerald. 1954. Inhibition of growth of pleuro- tests. Workers are encouraged to develop and employ new pneumonia-like organisms by antibody. J. Pathol. Bacteriol. 6823-30. technologies for classification. Although special facilities may 23. Edward, D. G., and E. A. Freundt. 1967. Proposal for Mollicutes as name of be required for certain procedures, investigators are neverthe- the class established for the order Mycoplasmatales. Int. J. Syst. Bacteriol. less encouraged to carry out as many of the optional tests as 1R267-268. 24. Edward, D. G., and W. B. Moore. 1975. A method for determining utiliza- possible. In particular, the PCR may identify sequences that tion of glucose by mycoplasmas. J. Med. Microbiol. 8:45 1-454. can be used to design probes specific to higher taxa (72, 99). 25. Freundt, E. A,, B. E. Andrews, H. Erne, M. Kunze, and F. T. Black. 1973. The following tests may be useful for some organisms: (i) The sensitivity of Mycoplasmatales to sodium-polyanethol-sulfonateand fermentation of carbohydrates other than glucose and/or re- digitonin. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 229104-112. lated compounds, including mannose, sucrose, and trehalose; 26. Furr, P. M., and D. Taylor-Robinson. 1984. Microimmunofluorescence (ii) adsorption of erythrocytes (preferably from guinea pig or technique for detection of antibody to Mycoplasma genitalium. J. Clin. sheep cells) to colonies on solid medium (1,28,52); (iii) poly- Pathol. 3R1072-1074. acrylamide gel electrophoresis of cellular proteins (55); (iv) 27. Gadeau, A. P., C. Mouches, and J. M. Bod. 1986. Probable insensitivity of mollicutes to rifampin and characterization of spiroplasmal DNA-depen- genomic restriction fragment length polymorphism (73, 122); dent RNA polymerase. J. Bacteriol. 166824-828. and (v) random amplification of polymorphic DNA (114). 28. Gardella, R. S., and R. A. Del Giudice. 1983. Hemagglutination, hemad- Finally, it is important that workers who propose new taxo- sorption, and hemolysis. Methods Mycoplasmol. 1:379-384. nomic descriptions for mollicutes or for other procaryotes have 29. Gardella, R. S., R. A. Del Giudice, and J. G. Tully. 1983. 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