Potato dextrose agar antifungal susceptibility testing for yeasts and molds: evaluation of phosphate effect on antifungal activity of CMT-3.
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Journal: 2002/August - Antimicrobial Agents and Chemotherapy
ISSN: 0066-4804
PUBMED: 11959582
Abstract:
The broth macrodilution method (BMM) for antifungal susceptibility testing, approved by the National Committee for Clinical Laboratory Standards (NCCLS), was found to have deficiencies in testing of the antifungal activity of a new type of antifungal agent, a nonantibacterial chemically modified tetracycline (CMT-3). The high content of phosphate in the medium was found to greatly increase the MICs of CMT-3. To avoid the interference of phosphate in the test, a new method using potato dextrose agar (PDA) as a culture medium was developed. Eight strains of fungi, including five American Type Culture Collection strains and three clinical isolates, were used to determine the MICs of amphotericin B and itraconazole with both the BMM and the PDA methods. The MICs of the two antifungal agents determined with the PDA method showed 99% agreement with those determined with the BMM method within 1 log(2) dilution. Similarly, the overall reproducibility of the MICs with the PDA method was above 97%. Three other antifungal agents, fluconazole, ketoconazole, and CMT-3, were also tested in parallel against yeasts and molds with both the BMM and the PDA methods. The MICs of fluconazole and ketoconazole determined with the PDA method showed 100% agreement within 1 log(2) dilution of those obtained with the BMM method. However, the MICs of CMT-3 determined with the BMM method were as high as 128 times those determined with the PDA method. The effect of phosphate on the antifungal activity of CMT-3 was evaluated by adding Na2HPO4 to PDA in the new method. It was found that the MIC of CMT-3 against a Penicillium sp. increased from 0.5 microg/ml (control) to 2.0 microg/ml when the added phosphate was used at a concentration of 0.8 mg/ml, indicating a strong interference of Na2HPO4 with the antifungal activity of CMT-3. Except for fluconazole, all the other antifungal agents demonstrated clear end points among the yeasts and molds tested. Nevertheless, with its high reproducibility, good agreement with NCCLS proposed MIC ranges, and lack of interference of phosphate, the PDA method shows promise as a useful assay for antifungal susceptibility testing and screening for new antifungal agents, especially for drugs that may be affected by high (supraphysiologic) phosphate concentrations.
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Antimicrob Agents Chemother 46(5): 1455-1461
PMID: 11959582

Potato Dextrose Agar Antifungal Susceptibility Testing for Yeasts and Molds: Evaluation of Phosphate Effect on Antifungal Activity of CMT-3

Department of Oral Biology and Pathology, School of Dental Medicine, Department of Laboratories, University Hospital and Medical Center, State University of New York at Stony Brook, Stony Brook, New York2
Corresponding author. Mailing address: Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794. Phone: (631) 632-8912. Fax: (631) 632-9705. E-mail: ude.koorbynots@bulog.enrol.
Received 2001 Aug 21; Revised 2001 Oct 29; Accepted 2001 Nov 20.
Copyright © 2002, American Society for Microbiology

Abstract

The broth macrodilution method (BMM) for antifungal susceptibility testing, approved by the National Committee for Clinical Laboratory Standards (NCCLS), was found to have deficiencies in testing of the antifungal activity of a new type of antifungal agent, a nonantibacterial chemically modified tetracycline (CMT-3). The high content of phosphate in the medium was found to greatly increase the MICs of CMT-3. To avoid the interference of phosphate in the test, a new method using potato dextrose agar (PDA) as a culture medium was developed. Eight strains of fungi, including five American Type Culture Collection strains and three clinical isolates, were used to determine the MICs of amphotericin B and itraconazole with both the BMM and the PDA methods. The MICs of the two antifungal agents determined with the PDA method showed 99% agreement with those determined with the BMM method within 1 log2 dilution. Similarly, the overall reproducibility of the MICs with the PDA method was above 97%. Three other antifungal agents, fluconazole, ketoconazole, and CMT-3, were also tested in parallel against yeasts and molds with both the BMM and the PDA methods. The MICs of fluconazole and ketoconazole determined with the PDA method showed 100% agreement within 1 log2 dilution of those obtained with the BMM method. However, the MICs of CMT-3 determined with the BMM method were as high as 128 times those determined with the PDA method. The effect of phosphate on the antifungal activity of CMT-3 was evaluated by adding Na2HPO4 to PDA in the new method. It was found that the MIC of CMT-3 against a Penicillium sp. increased from 0.5 μg/ml (control) to 2.0 μg/ml when the added phosphate was used at a concentration of 0.8 mg/ml, indicating a strong interference of Na2HPO4 with the antifungal activity of CMT-3. Except for fluconazole, all the other antifungal agents demonstrated clear end points among the yeasts and molds tested. Nevertheless, with its high reproducibility, good agreement with NCCLS proposed MIC ranges, and lack of interference of phosphate, the PDA method shows promise as a useful assay for antifungal susceptibility testing and screening for new antifungal agents, especially for drugs that may be affected by high (supraphysiologic) phosphate concentrations.

Abstract

Fungal infections have been reported to have dramatically increased in the past decade, and these often occur as systemic infections or as coinfections with other diseases, such as AIDS or cancer, or in patients who are immunocompromised (1, 2, 5, 18). In fact, candidiasis has become the most common infection in AIDS and cancer patients. Unfortunately, in addition to the limited number of antifungal drugs currently available, fungal infections tend to rapidly develop resistance to these drugs. For these reasons, fungal infections now show much higher mortality rates than bacterial infections (19).

The rapid increase in fungal infections and the growing number of new antifungal agents indicate an increasing need for rapid and accurate methods for antifungal screening and susceptibility testing. The National Committee for Clinical Laboratory Standards (NCCLS) recently approved a standardized method for antifungal susceptibility testing of yeasts, M27-A (7), and proposed a method for testing of filamentous fungi, M38-P (8), based on a broth macrodilution method (BMM). Subsequently, several modifications of these methods were proposed, including flow cytofluorometric detection (9, 20), colorimetric microdilution (4, 10), E-test (13, 16), modified agar dilution (21), semisolid agar test (14), and capacitance method (3). Most of these methods were developed for the determination of MICs of agents against yeasts.

CMTs are derivatives of tetracycline in which the dimethylamino group at position 4 of the multiple rings has been removed. They show no effective antibacterial activity, unlike the parent drug, tetracycline, but do exhibit other therapeutic properties, such as the abilities to inhibit host-derived tissue destructive enzymes, the matrix metalloproteinases, and to reduce the levels of proinflammatory cytokines, such as interleukin 1β and tumor necrosis factor alpha (6, 15, 17). In the experiments described below, we unexpectedly found that the nonantibacterial CMTs exhibited antifungal activity in vitro and that this activity was severely compromised by the high (nonphysiologic) level of phosphate in RPMI 1640 medium, which significantly increased the MICs of this new group of antifungal agents. As a result, a new and rapid method using potato dextrose agar (PDA) as a culture medium was developed for antifungal susceptibility testing of CMTs. Therefore, the purpose of the current study was to evaluate the reproducibility and accuracy of this new method compared to those of the NCCLS macrodilution methods (M27-A and M38-P), to evaluate the effects of phosphate on the antifungal activity of CMT-3, and to demonstrate the usefulness of the new method as an antifungal susceptibility test for both yeasts and molds.

REFERENCES

REFERENCES

References

  • 1. Beck-Sague, C., and W. R. Jarvis. 1993. Secular trends in the epidemiology of nosocomial fungal infections in the United States. 1980-1990, National Nosocomial Infections Surveillance Syst. J. Infect. Dis.167:1247-1251. [[PubMed]
  • 2. Berrouane, Y. F., L. A. Herwaldt, and M. A. Pfaller. 1999. Trends in antifungal use and epidemiology of nosocomial yeast infections in a university hospital. J. Clin. Microbiol.37:531-537.
  • 3. Chang, H. C., J. J. Chang, A. H. Huang, and T. C. Chang. 2000. Evaluation of a capacitance method for direct antifungal susceptibility testing of yeasts in positive blood cultures. J. Clin. Microbiol.38:971-976.
  • 4. Davey, K. G., A. Szekely, E. M. Johnson, and D. W. Warnock. 1998. Comparison of a new commercial colorimetric microdilution method with a standard method for in vitro susceptibility testing of Candida spp. and Cryptococcus neoformans. Antimicrob. Agents Chemother.42:439-444. [[PubMed]
  • 5. Dupont, B., J. R. Graybill, D. Armstrong, R. Laroche, J. E. Touze, and L. J. Wheat. 1992. Fungal infections in AIDS patients. J. Med. Vet. Mycol.30(Suppl. 1):19-28. [[PubMed]
  • 6. Golub, L. M., N. S. Ramamurthy, A. Llavaneras, M. E. Ryan, H. S. Lee, Y. Liu, S. Bain, and T. Sorsa. 1999. A chemically modified non-antimicrobial tetracycline (CMT-8) inhibits gingival matrix metalloproteinases, periodontal breakdown, and extraoral bone loss in ovariectomized rats. Ann. N. Y. Acad. Sci.878:290-310. [[PubMed]
  • 7. National Committee for Clinical Laboratory Standards. 1997. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
  • 8. National Committee for Clinical Laboratory Standards. 1998. Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard M38-P. National Committee for Clinical Laboratory Standards, Wayne, Pa.
  • 9. Peyron, F., A. Favel, H. Guiraud-Dauriac, M. E. I. Mzibri, C. Chastin, G. Dumenil, and P. Regli. 1997. Evaluation of a flow cytofluorometric method for rapid determination of amphotericin B susceptibility of yeast isolates. Antimicrob. Agents Chemother.41:1537-1540.
  • 10. Pfaller, M. A., and A. L. Barry. 1994. Evaluation of a novel colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates. J. Clin. Microbiol.32:1992-1996.
  • 11. Pfaller, M. A., S. A. Messer, A. Bolmstrom, F. C. Odds, and J. H. Rex. 1996. Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates. J. Clin. Microbiol.34:1691-1693.
  • 12. Pfaller, M. A., S. A. Messer, and S. Coffman. 1995. Comparison of visual and spectrophotometric methods of MIC endpoint determinations by using broth microdilution methods to test five antifungal agents, including the new triazole D0870. J. Clin. Microbiol.33:1094-1097.
  • 13. Pfaller, M. A., S. A. Messer, A. Karlsson, and A. Bolmstrom. 1998. Evaluation of the Etest method for determining fluconazole susceptibilities of 402 clinical yeast isolates by using three different agar media. J. Clin. Microbiol.36:2586-2589.
  • 14. Provine, H., and SHadley. 2000. Preliminary evaluation of a semisolid agar antifungal susceptibility test for yeasts and molds. J. Clin. Microbiol.38:537-541. [Google Scholar]
  • 15. Seftor, R. E., E. A. Seftor, J. Delarco, D. Kleiner, J. Leferson, W. Stetler-Stevenson, T. F. McNamara, L. M. Golub, and M. J. C. Hendrix. 1998. Chemically modified tetracyclines inhibit human melanoma cell invasion and metastasis. Clin. Exp. Metastasis16:217-225. [[PubMed]
  • 16. Simor, A. E., G. Goswell, L. Louie, M. Lee, and M. Louie. 1997. Antifungal susceptibility testing of yeast isolates from blood cultures by microbroth dilution and the E test. Eur. J. Clin. Microbiol. Infect. Dis.16:693-697. [[PubMed]
  • 17. Sorsa, T., N. S. Ramamurthy, T. Vernillo, X. Zhang, Y. Konttinen, B. Rifkin, and L. M. Golub. 1998. Functional sites of chemically modified tetracyclines: inhibition of the oxidative activation of human neutrophil and chicken osteoclast pro-matrix metalloproteinases. J. Rheumatol.25:975-982. [[PubMed]
  • 18. Wanke, B., M. D. Lazera, and M. Nucci. 2000. Fungal infections in the immunocompromised host. Mem. Inst. Oswaldo Cruz95(Suppl. 1):153-158. [[PubMed]
  • 19. Weinstein, M. P., M. L. Towns, S. M. Quartey, S. Mirrett, L. G. Reimer, G. Parmigiani, and L. B. Reller. 1997. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of microbiology, epidemiology, and outcome of bacteria and fungemia in adults. Clin. Infect. Dis.24:584-602. [[PubMed]
  • 20. Wenisch, C., K. F. Linnau, B. Parschalk, K. Zedtwitz-Liebenstein, and A. Georgopoulos. 1997. Rapid susceptibility testing of fungi by flow cytometry using vital staining. J. Clin. Microbiol.35:5-10.
  • 21. Yoshida, T., K. Jono, and K. Okonogi. 1997. Modified agar dilution susceptibility testing method for determination of in vitro activities of antifungal agents, including azole compounds. Antimicrob. Agents Chemother.41:1349-1351.
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