INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1991, p. 229-233 Vol. 41, No. 2 0020-7713/91/020229-05$02.oo/o Copyright 0 1991, International Union of Microbiological Societies Mycoplasma spermatophilum, a New Species Isolated from Human Spermatozoa and Cervix AURIOL C. HILL Medical Research Council Toxicology Unit, Woodmansterne Road, Carshalton, Surrey SM5 4EF, United Kingdom A mycoplasma isolated from human spermatozoa and a human cervix was shown to be serologically distinct from 98 previously recognized MycopEasma and AchoZepZasma spp. Six mycoplasma colonies were cloned and examined in detail for morphology, growth, and biochemical characteristics; five of these were from sperm samples and one was from a cervix. These strains were closely related and had the following properties: guanine-plus-cytosine content of 32 mol%, requirement for sterol, and anaerobic growth. Glucose was not metabolized, and arginine and urea were not hydrolyzed. Strain AH159 (= NCTC 11720) is the type strain of a new species, Mycoplasma spermatophilum. Twelve named Mycoplasma and Acholeplasma species colony isolated from each patient was cloned to produce a have been isolated from the respiratory or genital tracts of pure culture; this was done by initially filtering a broth humans (6). Mycoplasma buccale, Mycoplasma faucium, culture through a 220-nm-pore-size membrane filter, cultur- Mycoplasma lipophilum , Mycoplasma orale, Mycoplasma ing the filtrate on solid medium, transferring a single result- pneumoniae, and Mycoplasma salivarium are found almost ing colony to another agar plate, and inoculating the subse- exclusively in respiratory tracts. Mycoplasma fermentans quent growth into broth. This whole procedure was repeated has been found infrequently in urogenital tracts, while My- an additional four times; thus, the organisms were filter coplasma primatum, a species commonly present in nonhu- cloned five times (29). The clones were designated strains 9, man South American primates (15), has on occasion been 47, 50, 58, AH159T, and Monks. isolated from humans. Acholeplasma laidlawii, which is also Mycoplasma species. Mycoplasma type strains were ob- uncommon in humans, has been isolated from many animal tained from the National Collection of Type Cultures, Colin- species and plants (26). dale, England, and from H. Atobe, M. F. Barile, J. M. The more common human genital mycoplasmas are Urea- Bradbury, R. H. Fallon, E. A. Freundt, J. T. Heywood, plasma urealyticum and Mycoplasma hominis, although F. T. W. Jordan, D. E. Jasper, H. Kirchoff, R. H. Leach, each of these species has also been found in human respira- G. J. McGarrity, D. L. Rose, G. Smith, D. Taylor-Robinson, tory systems. Both of these species have been implicated in and J. G. Tully. certain diseases (e.g., nongonococcal urethritis and pelvic The following Mycoplasma spp. type strains were used: inflammatory disease) (30). The association of ureaplasmas M. agalactiae PG2, M. alkalescens PG51, M. alvi Ilsley, M. with some other diseases has not been proved as controls are anatis 1340, M. anseris 1219, M. arginini G230, M. arthrit- often similarly infected. It is possible that serotyping of idis PG6, M. bovigenitalium PG11, M. bovirhinis PG43, M. ureaplasmas may clarify this position. bovis PG45, M. bovoculi M165/69, M. buccale CH-20247, M. Recently, a fastidious mycoplasma was isolated from californicum ST-6, M. canadense 275C, M. canis PG14, M. human genital tracts by using SP4 medium (37). This myco- capricolum California kid, M. caviae G122, M. caviphaiyn- plasma, Mycoplasma genitalium was recovered from pa- gis 117C, M citelli RG-2C, M. cloacale 383, M. collis 58B, tients with nongonococcal urethritis (36), but the respiratory M. columbinasale 694, M. columbinum MMPl, M. colum- tract is probably the primary host site of colonization (3). borale MMP4, M. conjunctivae HRC581, M. cricetuli CH, Strains 9,47,50,58, AH159T (T = type strain), and Monks M. cynos H831, M. dispar 46212, M. edwardii PG24, M. were isolated from the urogenital tracts of patients with ellychniae ELCN-1, M. entemophilum TAC, M. equigeni- infertility problems and could not be identified as any of the talium T37, M. equirhinis M432f72, M.fastidiosum 4822, M. previously described human species. In this paper I describe faucium DC333, M. felifaucium PU, M. feliminutum Ben, the characteristics of these organisms and propose that they M. felis Co, M. fermentans PG18, M.jocculate MS42, M. are members of a new Mycoplasma species. gallinaceum DO, M. gallinarum PG16, M. gallisepticum PG31, M. gallopavonis WR1, M. gateae CS, M. genitalium MATERIALS AND METHODS G-37, M. glycophilum 486, M. hominis PG21, M. hyophar- yngis H3-6%F, M. hyopneumoniae J, M. hyorhinis BTS7, M. Mycoplasma strains. Mycoplasmas were isolated from the hyosynoviae S16, M. iners PG30, M. iowae 695, M. lactucae spermatozoa of five patients attending an in vitro fertilization 831-C4, M. lipofaciens R171, M. lipophilum MaBy, M. clinic (16) and from the cervix of one woman attending a luminosum PIMN-1, M. lucivorax PIPN-2, M. maculosum clinic for infertility (strain Monks). The cervix isolate was PG15, M. melaleucae MI, M. meleagridis 17529, M. moatsii obtained from a cervical swab rubbed over the surface of an MK405, M. mobile 163K, M. molare H542, M. muris RIII4, agar culture medium. Semen and washed sperm were inoc- M. mustelae MX9, M. mycoides subsp. Capri PG3, M. ulated onto the agar medium and SP4 agar and into glucose, mycoides subsp. mycoides PG1, M. neurolyticum Type A, arginine, and urea broth media and SP4 liquid medium (13, M. opalescens MH5408, M. orale CH-19299, M. ovipneu- 32, 37). Most of the inoculated liquid media were diluted moniae Y98, M. oxoniensis 128, M. pirurn HRC70-159, M. 1/10; the one exception was urea medium, which was diluted pneumoniae FH, M. primatum HRC292, M. pullorum CKK, 10-fold to lo6. The amount of inoculum depended on the M. pulmonis PG34 (ASH), M. putrefaciens KS-1, M. sali- material available from the in vitro fertilization clinic. One varium PG20, M. somnilux PYAN-1, M. spumans PG13, M. 229 230 HILL INT. J. SYST.BACTERIOL. sualvi Mayfield (clone B), M. subdolum TB, M. synoviae colonies were passaged three times on similar plates to WVU1853, M. testudinis 01008, and M. verecundum 107. ensure that growth was not due to a carry-over of serum The following Acholeplasma spp. type strains were used: from the original culture, giving misleading results. A. axanthum S743, A. equifetale N93, A. florum LI, A. The clones were also tested indirectly for sterol depen- granularurn BTS39, A. hippikon C1, A. laidlawii PG8, A. dence by a paper disk inhibition method (ll), using either modicum PG49, A. morum 72-043, A. oculi 19L, and A. dried disks that originally contained 0.02 ml of a 1.5% parvum H23M. (wt/vol) ethanolic solution of digitonin (Sigma Chemical Co. , Medium and growth conditions. The culture medium used St. Louis, Mo.) or wet disks that contained 0.02 ml of a 20% has been described previously (13, 32, 37). The purified agar (wt/vol) aqueous solution of sodium polyanethol sulfonate used in the growth medium was either Ionagar no. 2 (Oxoid (Koch-Light Laboratories Ltd., Colnbrook, England) on Ltd., Basingstoke, England) or Lab M agar (Lab M, Lon- basal growth medium containing Lab M agar. The widths of don, England). Depending on their biochemical activities, zones of growth inhibition were measured in millimeters. the mycoplasmas were grown in liquid medium containing Biochemical activity. The six clones were examined for -1% (wt/vol) glucose (pH 7.8) or in liquid medium containing carbohydrate metabolism; for hydrolysis of esculin, arginine 0.5 to 1% (wt/vol) arginine (pH 7.3). The species grown in (1 and 0.1%), and urea; for reduction of methylene blue, medium containing arginine were not inhibited by 1% argi- resazurin, tetrazolium, and tellurite; and for phosphatase nine. Agar cultures were incubated at 35 to 37°C either in a activity (1, 2, 18, 28, 34, 39). lhumid chamber or under anaerobic conditions in a GasPak Erythrocyte techniques. The clones were examined for :system. Liquid cultures were stored at -70°C in ampoules. hemolytic activity, hemadsorption, and hemagglutination Growth requirements and characteristics. The clones were with guinea pig, human, and sheep erythrocytes (1, 19). subcultured onto solid medium and incubated under both Polyacrylamide gel electrophoresis. Electrophoresis was aerobic and GasPak anaerobic conditions at 35 to 37°C. The carried out as described by Mouches and Bovk (21), using a levels of susceptibility to methylene blue were investigated one-dimensional slab, Centrifuged cell suspensions were by adding 0.002% (wt/vol) methylene blue to the basal solid dissolved in a solution containing 20% glycerol, 5% 2-mer- medium and comparing the growth of inoculated mycoplas- captoethanol, and 3% sodium dodecyl sulfate in 0.0625 M mas with the growth of mycoplasmas cultivated on agar Tris buffer (pH 6.8). To a 12.5% acrylamide gel lo-, 25, or medium without methylene blue. Lipolytic activity was 50-pl portions of a cell sample were added. Electrophoresis tested by inoculating mycoplasmas onto basal medium en- was performed at room temperature for 1 h at a constant riched with 10% egg yolk emulsion (9). The incubated plates current (20 mA). The gels were stained with Coomassie blue were examined after 3, 7, and 14 days for visual evidence of (Sigma). lipolysis (clearing) or film production. DNA base composition. DNAs were extracted from centri- Absence of reversion. The clones were subcultured by five fuged broth culture deposits of strains AH159T, 47, and passages in both solid and liquid media that contained no Monks by using the method of Gross-Bellard et al. (1.2), and microbial inhibitors in order to determine whether the or- the guanine-plus-cytosine contents were determined from ganisms reverted to bacterial forms. Agar culture colonies of the buoyant densities of the DNAs in cesium chloride by each clone were also treated with Dienes stain in order to ultracentrifugation (27).