Hydrolysis of Aesculin and Phosphatase Production by Members of the Order Mycoplasmatales Which Do Not Require Sterol

Abstract

Two bacteriological methods, one to demonstrate phosphatase production and the other to demonstrate aesculin hydrolysis, were tested on sterol-requiring My coplasma and sterol-nonrequiring Acholeplasma strains to determine their utility in differentiating species. The phosphatase test, which gave weak and variable reactions with most of the sterolnonrequiring strains, was judged ineffectual for differentiating these strains. The aesculin test distinguished all Mycoplasma strains and Acholeplasma granularum, both of which gave negative reactions, from most Acholeplasma laidlawii strains and from Acholeplasma axanthum, which gave positive reactions.T h e bacteriological characterization of Mycoplasma species by standardized methods was recently described by Aluotto et al. (1). Of the 22 species studied by these authors, half displayed distinctive patterns of biochemical activities. The remaining strains fell into groups containing several species with biochemically similar patterns. One such group included Mycoplasma gallisepticum, Mycoplasma histotropicum [now regarded as a strain of Mycoplasma pulmonis (3)] , Mycoplasma granularum, and Mycoplasma laidlawii. Application of the test of Razin and Tully ( 6 ) for determining cholesterol requirement readily permits differentiation of the sterol-requiring species in this group from the two sterol-nonrequiring species, M. granularum and M. laidlawii. [For these and other sterol-nonrequiring species, a new generic name, Acholeplasrna, was recently proposed (4) and will be employed hereafter in this report.] Acholeplasma granularum and Acholeplasma laidlawii can be differentiated from each other by serological methods and gel electrophoretic patterns (7, 8), but a simple biochemical test for distinguishing them has been lacking. These strains react somewhat differently from each other when tested for phosphatase production and hemolysis; however, neither test alone is capable of distinguishing the species. The phosphatase reaction was reported t o be weakly positive for A. laidlawii type A but negative for A . laidlawii type B and A. granularum (1). The type of hemolytic reaction for various A . laidlawii strains was examined (unpublished data) and found t o vary from strain t o strain. Now that a new and distinct species of Acholeplasma has been recognized (9), further biochemical tests for characterizing and differentiating them are all the more desirable.A survey of biochemical reactions applicable t o the problems of Mycoplasma and Acholeplasma characterization and differentiation pointed to aesculin hydrolysis as a possibility. A group of more than 22 types or representative strains of Mycoplasma and 32 Acholeplasma strains were, therefore, examined for ability to hydrolyze aesculin. In addition, the test for phosphatase activity was reexamined on all Acholeplasma strains t o clarify and expand the findings of Aluotto et al. (1). MATERIALS AND METHODS Organisms

Mycoplasma oxoniensis, a New Species Isolated from Chinese Hamster Conjunctivas

“…The two test clones were examined for carbohydrate metabolism for hydrolysis of esculin, arginine (1 and 0.1%), and urea for reduction of methylene blue, resazurin, tetrazolium, and tellurite, and for phosphatase activity by using standard methods (1, 2, 16, 25,30,33).…”

Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes)

“…For example, sensitivities to rifampicin (Gadeau et al, 1986;Pellegrin et al, 1990;Gaurivaud et al, 1996), digitonin (Freundt et al, 1973;Tully, 1983c) or amphotericin B (Rottem, 1972) characterize some taxa. Tests that detect a-D-glucosidase (Williams & Wittler, 1971;, benzyl viologen reduction (Pollack et al, 1996a) or adsorption of erythrocytes to colonies on solid medium (Manchee & Taylor-Robinson, 1968;Aluotto et al, 1970; are of interest in some species. PAGE of cellular proteins (Razin, 1968;Rodwell & Rodwell, 1978;Mouches et al, 1982Mouches et al, , 1983Saillard et al, 1990) may be an effective technique for special interspecies comparisons.…”

Mycoplasma citelli, a New Species from Ground Squirrels

“…The procedures used to determine carbohydrate fermentation, arginine and urea hydrolysis, phosphatase activity, tetrazolium reduction, serum or gelatin digestion, and film-and-spot reaction have been described earlier (1,23). Most of these tests were performed on a broth medium containing 1% bovine serum fraction rather than on a medium with 20% horse serum.…”