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 Isolated from Human Spermatozoa and Cervix AURIOL C. HILL Medical Research Council Toxicology Unit, Woodmansterne Road, Carshalton, Surrey SM5 4EF, United

A 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 (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 are Urea- Bradbury, R. H. Fallon, E. A. Freundt, J. T. Heywood, plasma urealyticum and , 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). DNA extracted from Escherichia differentiate mycoplasma colonies from bacterial L forms coli with a known guanine-plus-cytosine content was in- (33). cluded as a control. Morphological studies. Mycoplasma colonies grown on Serological studies. Antisera were prepared as described agar were examined microscopically at a magnification of by Morton and Roberts (20) and Hill (13). The following x 100 after 4,7, and 14 days of incubation. The colonies were three serological methods were used: growth inhibition tests transferred to slides and stained with Giemsa stain (10, 17). with antiserum-impregnated sterile paper disks (5) , metabo- Liquid cultures were observed by dark-field microscopy, lism inhibition tests in microtiter plates (14, 23, 24, 31), and and organisms were stained with Giemsa stain (17). Organ- immunoperoxidase tests with colonies grown on agar (22). isms grown in broth were harvested by centrifugation, and All of the tests were carried out in duplicate. Type strain the cell pellet was fixed in 2% (vol/vol) glutaraldehyde and AH159 and strains 47, 50, and Monks were reacted with postfixed in 1% (wt/vol) osmium tetroxide for 1 h. Thin antisera prepared against the species listed above. Antisera sections of these preparations were stained with uranyl to the four clones were tested with the previously described acetate and lead citrate and examined by electron micros- Mycoplasma and Acholeplasma species. Antisera to the four COPY (38). clones were reacted with each other to demonstrate their Filtration studies. Cultures (after 24 h of incubation) were relationships. The strains (antigens) could not be tested with diluted 1:lO in liquid medium, and the resulting preparations antisera in the metabolism inhibition test because of their were filtered and refiltered through a series of membrane lack of biochemical activity. filters (Millipore Corp., Bedford, Mass.) with pore diameters of 220, 300,450,650, and 800 nm. The number of CFUs per RESULTS AND DISCUSSION ml in each filtrate was compared with the number in the unfiltered culture dilution. All six strains were visible as very small colonies on Sterol dependence. Single colonies of strains AH159T, 47, primary isolation after 2 to 3 weeks of incubation on agar and Monks were seeded onto serum-free solid media sup- medium (13, 32) and (for strains 9, 47, and 58) on SP4 plemented with 0.5% bovine serum albumin, 0.5% glucose, medium under anaerobic conditions. The number of colonies and 10 pg of palmitic acid per ml. Cholesterol, dissolved in was small (5 to 20 colonies). Five strains were isolated from Tween 80, was added to give final concentrations of 20, 10, washed sperm samples, but in only two cases were the 5, and 1 pg/ml. Plates containing no cholesterol were in- strains recovered from the semen as well. After several cluded (7, 8, 25, 35). The mycoplasmas were also subcul- passages the strains grew much more rapidly, but no growth tured onto the basal agar medium without serum. When was visible aerobically even after prolonged incubation or growth occurred on any of the serum-free media, single after many passages on mycoplasma culture medium. The VOL. 41, 1991 MYCOPLASMA SPERMATOPHILUM SP. NOV. 231

served. Liquid cultures stained with Giemsa stain contained pleomorphic forms characteristic of mycoplasmas. The ultrastructure of three isolates (strains AH159T, 47, and Monks) was typical of mycoplasma morphology when thin sections were viewed by electron microscopy (4). The cells were bounded by a single trilaminar unit membrane and lacked any evidence of material (Fig. 2). Filtration of a broth culture of strain AH159T showed that reduced numbers of the 1.4 X lo8 CFU/ml in the original dilution passed through the 300-nm-pore-size membrane (8 x lo7 CFU/ml) and 220-nm-pore-size membrane (5 x 106 CFU/ml). Strains AH159*, 47, and Monks required cholesterol for growth. They could not be cultured on medium without cholesterol, but they grew and were passaged on medium containing 10 p,g of cholesterol per ml. They were suscepti- ble to digitonin and sodium polyanethanol sulfonate, with zones of growth inhibition of 8 and 6 mm, respectively; FIG. 1. Colonies of strain AH159= grown under anaerobic con- ditions for 4 days on an agar medium. Bar = 50 pm. under these conditions growth was semiconfluent. The biochemical activities of the six test strains were identical and are summarized in the species description below. In appropriate tests all six strains were hemolytic for colonies (Fig. 1) initially showed little center, but the typical guinea pig, human, and sheep erythrocytes but did not fried egg appearance became more obvious after several hemadsorb or hemagglutinate these cells. passages. Although initially it was difficult to obtain good The guanine-plus-cytosine content of this species was growth in liquid medium, the titers improved as the organ- determined from its buoyant density in cesium chloride to be isms became adapted to the medium. No growth was ob- 32 mol%. This value is within the range of values for the served on methylene blue agar or medium without serum. previously characterized Mycoplasma species. No film was produced on egg yolk agar. None of the strains The serological techniques used revealed similar levels of reverted to a bacterial form when the organisms were cross-reactions among the four strains tested. No significant subcultured on medium without bacterial inhibitors. The cross-reactions were detected with any of the species listed colonies rapidly stained with Dienes reagent, confirming that in Materials and Methods (Table 1). the isolates were true mycoplasmas (members of the Molli- The six strains had similar electrophoretic protein pat- cutes) rather than bacterial L forms (33). terns, and these patterns were distinct from those of M. No motility was observed when the strains were examined feliminutum, which also lacks the ability to metabolize by dark-field microscopy, and helical forms were not ob- glucose and the ability to hydrolyze arginine.

FIG. 2. Electron micrograph of a section of strain AH159T cells stained with uranyl acetate and lead citrate. Bar = 100 nm. 232 HILL INT.J. SYST.BACTERIOL.

TABLE 1. Serological reactions of mycoplasma species and The main characteristics of M. spermatophilum sp. nov. strain AH159T with antiserum to strain AH159= are described below. Morphological and physical characteristics. Pleomorphic Reaction in the: cells bounded by a single unit membrane. Colonies have a Growth Metabolism Immuno- typical fried egg appearance after several passages on agar. Antigen(s) inhibition inhibition peroxidase test testa testb Organisms are filterable through 220-nm-pore-size mem- (mm) (titer) (titer) brane filters and are resistant to . Growth characteristics. Requires sterol; inhibited by digi- P,H159= 5 NT‘ 4,000 tonin and sodium polyanethol sulfonate. Optimal growth A11 other speciesd 0 <16-32 <10&100 occurs at 35 to 37°C under anaerobic conditions. ~ ~~ a In the metabolism inhibition test the antiserum was diluted 1/16 to Metabolic characteristics. No carbohydrates are metabo- U32,OOO. lized; arginine, urea, and esculin are not hydrolyzed. Resa- In the immunoperoxidase test the antiserum was diluted 1/100 to 1/16,000. zurin is reduced (weakly), but methylene blue, tellurite, and NT, Not tested. Species listed in Materials and Methods. tetrazolium are not reduced. Slight production of phos- phatase. Serological characteristics. Serologically distinct from all Strain Monks was sent to R. H. Leach (Colindale, United other Mycoplasma species. Kingdom) to confirm my findings that it was distinct from M. DNA base composition. The DNA base composition is 32 Jeliminutum (with similar biochemical reactions and anaero- mol% guanine plus cytosine. biasis). Leach found a positive one-way reaction in the Habitat. The habitat is human semen and cervixes. immunoperoxidase test with M. feliminutum antiserum re- Type strain. The type strain is strain AH159 (= NCTC ceived from H. Ern@ (Aarhus, Denmark). Therefore, this 11720). antiserum was absorbed against M.feliminutum BenT. There was no reaction with M. feliminuturn, but the level of the ACKNOWLEDGMENTS reaction with strain Monks remained at 1,000. Therefore, the I thank C. Colhoun and I. Whisson for supplying the photographs positive reaction was due to some factor unconnected with and G. A. Stalley for the electrophoresis tests. .%I.feliminutum. There was no reaction between strain Monks and fluorescein-conjugated M. feliminutum antise- REFERENCES ’rum (in the immunofluorescence test) received from M. F. 1. Aluotto, B. B., R. G. Wittler, C. 0. Williams, and J. E. Faber. Barile (United States). 1970. Standardized bacteriologic techniques for the characteri- It can be difficult to demonstrate glucose fermentation or zation of Mycoplasma species. Int. J. Syst. Bacteriol. 20:35-58. arginine hydrolysis by M.fermentans ; however, in addition 2. Barber, T. L., and J. Fabricant. 1971. Identification of Myco- to these properties, the new strains differ from M. fermen- plasmatales: characterization procedures. 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