In vitro susceptibilities of Mycoplasma penetrans were determined. MICs and MBCs were determined. The. MICs at which 50% of the isolates are inhibited ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1995, p. 1386–1387 0066-4804/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 39, No. 6
In Vitro Antibiotic Susceptibility Testing of Clinical Isolates of Mycoplasma penetrans from Patients with AIDS MICHAEL M. HAYES, HSIN-HSIN FOO, JORGE TIMENETSKY,†
AND
SHYH-CHING LO*
American Registry of Pathology, Department of Infectious and Parasitic Disease Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306-6000 Received 13 February 1995/Returned for modification 30 March 1995/Accepted 10 April 1995
In vitro susceptibilities of Mycoplasma penetrans were determined. MICs and MBCs were determined. The MICs at which 50% of the isolates are inhibited (micrograms per milliliter) for broth dilution testing were as follows: azithromycin, 0.039; chloramphenicol, 0.625; ciprofloxacin, 0.156; clindamycin, 0.078; doxycycline, 0.312; erythromycin, 0.312; gentamicin, >10; levofloxacin, 0.078; lincomycin, 0.625; streptomycin, >10; and tetracycline, 1.25. Bactericidal activity was significant only for ciprofloxacin (MBC at which 50% of the isolates are killed, 0.312 mg/ml) and levofloxacin (MBC at which 50% of the isolates are killed, 0.312 mg/ml). racycline hydrochloride were obtained from Sigma Chemical Co., St. Louis, Mo. Azithromycin dihydrate was provided by Pfizer, Inc. Ciprofloxacin hydrochloride was provided by Miles, Inc. Levofloxacin was provided by Daiichi Pharmaceutical Corporation. Antibiotic stock solutions were prepared as previously described (3). The potency of the stock solutions was tested by recommended methods with Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) as reference strains. All antibiotic stocks were within recommended quality control ranges (11). For antibiotic susceptibility testing of M. penetrans, a broth dilution procedure in 96-well microtiter plates was used (3). We used a broth dilution procedure for susceptibility testing because of the quantitative results and high reproducibility of this type of method (3, 17, 18, 21). The postfreeze titer of mycoplasma stocks was first determined by titration in 96-well microtiter plates (20-ml stock, 10-fold serial dilution in SP4, in quadruplicate). Plates were sealed and incubated at 378C for 2 weeks. The end point for M. penetrans strains was read as the highest dilution showing an acidic color change. Color change unit (CCU) values were calculated as a 50% end point (14). The number of CFU was determined by serial dilution of mycoplasma stocks, streaking 10 ml of each dilution (in duplicate) on SP4 solid medium (1% agarose), and then counting the mycoplasma colonies after incubation for 1 week at 378C. The results of the CCU and CFU determinations were in close agreement, usually showing less than a fourfold variation. For susceptibility tests, SP4 was dispensed in 96-well microtiter plates (100 ml per well). The antibiotic stock solutions were titrated by serial twofold dilution. One well per titration served as an antibiotic-free control. M. penetrans stock culture was diluted in SP4 to give an estimated concentration of 2 3 104 CCU/ml and incubated at 378C for 2 to 3 h. One hundred microliters of the diluted stock culture was dispensed in each well. The diluted stocks were simultaneously titrated in a separate 96-well plate (67 ml, fourfold dilutions, in quadruplicate) so that a precise titer could be determined. The plates were sealed and incubated at 378C. CCU titrations, CFU titrations, and susceptibility tests were performed aerobically. The initial MIC was read as the lowest concentration of antibiotic preventing color change when the antibiotic-free control culture first showed an acidic color change. MBCs were determined for those antibiotics which had significant inhibitory activity in the broth dilution assay. Antibiotic solutions were titrated in 96-well plates, and M. penetrans stock cultures were diluted to give a final concentration of 106 CFU/ml when 100
A previously unknown mycoplasma was recently isolated from urine sediments of human immunodeficiency virus (HIV)infected patients with AIDS (8). Biochemical, serological, and DNA analyses revealed that the mycoplasma was a new species, now named Mycoplasma penetrans (7). M. penetrans has the properties of adhesion, hemadsorption, and cytadsorption which are associated with in vivo virulence of mycoplasmas (1, 2, 10). It is invasive and capable of penetrating the cytoplasm of mammalian cells. Invasion produces cytopathic effects and results in cell death (6). Enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses revealed a high prevalence of antibodies to M. penetrans in AIDS patients infected with HIV (40%). In comparison, seropositivity is rare in HIVnegative low-risk control subjects (0.3%) and in HIV-negative patients attending sexually transmitted disease clinics (0.9%). HIV-negative patients with disease states associated with immune dysfunctions and/or low leukocyte counts were seronegative for M. penetrans (0%) (19). Seropositivity to M. penetrans was found primarily among male homosexuals with AIDS or asymptomatic HIV-infected male homosexuals but not in HIV-infected intravenous drug users or hemophilia patients. Serological evidence of M. penetrans infection was significantly associated with Kaposi’s sarcoma in male homosexuals (20). We have tested nine strains of M. penetrans isolated from HIV-infected patients for susceptibility to antibiotics. M. penetrans strains GTU54(6A1), GTU73(1A1), TX270(1A1), TX343 (B12), TX662(1A3), TX682(5B1), and TX984(1A1) were isolated from urine specimens from AIDS patients. B77(A1A) was isolated from the semen of a patient with AIDS. GTU54 (6A1), GTU73(1A1), and B77(A1A) were isolated from the same patient at different times. HGARA was recently isolated in the laboratory of C. Bebear (Universite´ de Bordeaux, Bordeaux, France) and was provided to our laboratory by J. G. Tully (National Institute of Allergy and Infectious Diseases, Frederick, Md.). All M. penetrans stock cultures were grown in SP4 broth medium (0.1% glucose, 0.01% arginine [without antibiotics, thallium acetate, or amphotericin B]) aerobically (7). Chloramphenicol (crystalline), clindamycin hydrochloride, doxycycline hydrochloride, erythromycin base, gentamicin sulfate, lincomycin hydrochloride, streptomycin sulfate, and tet* Corresponding author. † Present address: Insituto de Ciencias Biomedicas da Universidae de Sa˜o Paulo, Depto. de Microbiologia, Sa ˜o Paulo-SP Brazil CP 05508 900. 1386
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NOTES
TABLE 1. Broth dilution antibiotic susceptibility testing of M. penetrans MIC (mg/ml) Antibiotic
Azithromycin Chloramphenicol Ciprofloxacin Clindamycin Doxycycline Erythromycin Gentamicin Levofloxacin Lincomycin Streptomycin Tetracycline a
Range
50%a
0.019–0.156 0.156–1.25 0.039–0.625 0.039–0.156 0.078–0.625 0.078–0.625 .10 0.078–0.156 0.312–2.5 5–.10 0.625–5
0.039 0.625 0.156 0.078 0.312 0.312 .10 0.078 0.625 .10 1.25
50%, MIC at which 50% of the isolates are inhibited.
ml was added to the 96-well plates. The initial number of CFU per milliliter for each strain was determined by plating dilutions (in duplicate) from the antibiotic-free control wells on SP4 solid medium. After 24 h of incubation, dilutions were made from wells containing antibiotics and plated on SP4 solid medium (in duplicate). All SP4 plates were incubated for 1 week, and the number of CFU per milliliter was determined. The MBC was defined as the concentration of antibiotic that reduced the number of CFU per milliliter from the initial value to 0.1% of the initial value (3, 12). In this study, we tested nine M. penetrans strains. The results of the broth dilution susceptibility tests are shown in Table 1. For each particular antibiotic, the range of MICs is relatively narrow, usually 16-fold or less. These results are consistent with those from a previous report of susceptibility testing of a single strain of M. penetrans (13). Our study differs from that in the previous report in four respects. First, we tested a larger number of M. penetrans strains (nine versus one); second, we tested additional antibiotics (i.e., chloramphenicol, ciprofloxacin, gentamicin, lincomycin, and streptomycin); third, our results are quantitative, not qualitative reports of sensitivity and resistance as in the earlier study; and fourth, we determined the bactericidal effects of the more promising antibiotics. The MBC susceptibility tests (Table 2) demonstrate that only the quinolone compounds ciprofloxacin and levofloxacin had a significant bactericidal effect, which is consistent with reports about other mycoplasma species (4, 18). While both the macrolide azithromycin and the fluoroquinolones ciprofloxacin and levofloxacin may accumulate intracellularly in high con-
TABLE 2. In vitro bactericidal testing of M. penetrans MBC (mg/ml)a Antibiotic
Azithromycin Chloramphenicol Ciprofloxacin Clindamycin Doxycycline Erythromycin Levofloxacin
Range
50%
2.5–.10 5–.10 0.312–1.25 5–.10 2.5–.10 2.5–.10 0.156–0.312
5 .10 0.312 10 .10 .10 0.312
a Concentration of antibiotic which would reduce the number of CFU in the initial inoculum by 99.9%. b 50%, MBC at which 50% of the isolates are killed.
1387
centrations (15), only the fluoroquinolones have a significant bactericidal effect on M. penetrans. The fluoroquinolones are now commonly used to treat bacterial infections in immunocompromised patients, such as those undergoing chemotherapy (9). It is important to note that mycoplasma infections in vivo may be difficult to eradicate, especially in patients who are immunocompromised (5, 16). This study was supported in part by NIH grant AI-31830. REFERENCES 1. Chandler, D. K. F., S. Razin, E. B. Stephens, R. Harasawa, and M. F. Barile. 1982. Genomic and phenotypic analyses of Mycoplasma pneumoniae strains. Infect. Immun. 38:604–609. 2. Gardella, R.-S., and R. A. DelGiudice. 1983. Methods Mycoplasmol. 1:379. 3. Hayes, M. M., H.-H. Foo, H. Kotani, D. J. Wear, and S.-C. Lo. 1993. In vitro antibiotic susceptibility testing of different strains of Mycoplasma fermentans isolated from a variety of sources. Antimicrob. Agents Chemother. 37:2500– 2503. 4. Kenny, G. E., T. M. Hooton, M. C. Roberts, F. D. Cartwright, and J. Hoyt. 1989. Susceptibilities of genital mycoplasmas to the newer quinolones as determined by the agar dilution method. Antimicrob. Agents Chemother. 33:103–107. 5. Lo, S.-C., C. L. Buchholz, D. J. Wear, R. C. Hohm, and A. M. Marty. 1991. Histopathology and doxycycline treatment in a previously healthy non-AIDS patient systemically infected by Mycoplasma fermentans (incognitus strain). Mod. Pathol. 6:750–754. 6. Lo, S.-C., M. M. Hayes, H. Kotani, P. F. Pierce, D. J. Wear, P. B. Newton III, J. G. Tully, and J. W.-K. Shih. 1993. Adhesion onto and invasion into mammalian cells by Mycoplasma penetrans: a newly isolated mycoplasma from patients with AIDS. Mod. Pathol. 6:276–280. 7. Lo, S.-C., M. M. Hayes, J. G. Tully, R. Y.-H. Wang, H. Kotani, P. F. Pierce, D. L. Rose, and J. W.-K. Shih. 1992. Mycoplasma penetrans sp. nov., from the urogenital tract of patients with AIDS. Int. J. Syst. Bacteriol. 42:357–364. 8. Lo, S.-C., M. M. Hayes, R. Y.-H. Wang, P. F. Pierce, H. Kotani, and J. W.-K. Shih. 1991. Newly discovered mycoplasma isolated from patients infected with HIV. Lancet 338:1415–1418. 9. Maiche, A. G. 1991. Use of quinolones in the immunocompromised host. Eur. J. Microbiol. Infect. Dis. 10:361–367. 10. Manchee, R. J., and D. Taylor-Robinson. 1969. Studies on the nature of receptors involved in attachment of tissue culture cells to mycoplasmas. Br. J. Exp. Pathol. 50:66–75. 11. National Committee for Clinical Laboratory Standards. 1990. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. M7-A2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 12. National Committee for Clinical Laboratory Standards. 1992. Methods for determining bactericidal activity of antimicrobial agents. M26-T. National Committee for Clinical Laboratory Standards, Villanova, Pa. 13. Poulin, S. A., R. E. Perkins, and R. B. Kundsin. 1994. Antibiotic susceptibilities of AIDS-associated mycoplasmas. J. Clin. Microbiol. 32:1101–1103. 14. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27:493–497. 15. Rovold, R. A., and S. C. Piscitelli. 1993. New oral macrolide and fluoroquinolone antibiotics: an overview of pharmacokinetics, interactions, and safety. Clin. Infect. Dis. 17(Suppl. 1):S192–S199. 16. Smith, C. B., W. T. Friedewald, and R. M. Chanock. 1967. Shedding of Mycoplasma pneumoniae after tetracycline and erythromycin therapy. N. Engl. J. Med. 276:1172–1175. 17. ter Laak, E. A., A. Pijpers, J. H. Noordergraaf, E. C. Schoevers, and J. H. M. Verheijden. 1991. Comparison of methods for in vitro testing of susceptibility of porcine Mycoplasma species to antimicrobial agents. Antimicrob. Agents Chemother. 35:228–233. 18. Waites, K. B., L. B. Duffy, T. Schmid, D. Crabb, M. S. Pate, and G. H. Cassell. 1991. In vitro susceptibilities of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum to sparfloxacin and PD 127391. Antimicrob. Agents Chemother. 35:1181–1185. 19. Wang, R. Y.-H., J. W.-K. Shih, T. Grandinetti, P. F. Pierce, M. M. Hayes, D. J. Wear, H. J. Alter, and S.-C. Lo. 1992. High frequency of antibodies to Mycoplasma penetrans in HIV-infected patients. Lancet 340:1312–1316. 20. Wang, R. Y.-H., J. W.-K. Shih, S. H. Weiss, T. Grandinetti, P. F. Pierce, M. Lange, H. J. Alter, D. J. Wear, C. L. Davies, R. K. Mayur, and S.-C. Lo. 1993. Mycoplasma penetrans infection in male homosexuals with AIDS: high seroprevalence and association with Kaposi’s sarcoma. Clin. Infect. Dis. 17:724– 729. 21. Whitear, K. G., D. D. Bowtell, E. Ghiocas, and K. L. Hughes. 1983. Evaluation and use of a microbroth dilution procedure for testing sensitivity of fermentative avian mycoplasmas to antibiotics. Avian Dis. 27:937–949.
