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Antimicrobial Agents and Chemotherapy, April 2003, p. 1200-1206, Vol. 47, No. 4
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.4.1200-1206.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Sublethal Injury and Resuscitation of Candida albicans after Amphotericin B Treatment

Robert S. Liao,1 Robert P. Rennie,1,2* and James A. Talbot1

Department of Medical Microbiology and Immunology, University of Alberta,1 National Centre for Mycology, University of Alberta Hospital, Walter C. Mackenzie Health Sciences Centre, Edmonton, Alberta T6G 2J2, Canada2

Received 25 June 2002/ Returned for modification 18 July 2002/ Accepted 30 December 2002

Amphotericin B treatment was previously shown to inhibit Candida albicans reproduction and reduce the fluorescence of vitality-specific dyes without causing a corresponding increase in the fluorescence of the mortality-specific dyes bis-(1,3-dibutylbarbituric acid)trimethine oxonol and SYBR Green I. In the present study, we have confirmed these results and have shown that the numbers of CFU are reduced by 99.9% by treatment with 0.5 µg of amphotericin B per ml for 10 h at 35°C. This reduction was not due to fungal cell death. First, the level of reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide increased in the presence of concentrations of amphotericin B that caused greater than 90% reductions in the numbers of CFU. Second, fungal cells treated with amphotericin B at a concentration of 0.5 µg/ml were resuscitated by further incubation at 22°C for 15 h in the continued presence of amphotericin B. Third, recovery of the ability to replicate was prevented by sequential treatment with 20 µg of miconazole per ml, which also increased the fluorescence of mortality-specific dyes to near the maximal levels achieved with 0.9 µg of amphotericin B per ml. Sequential treatment with fluconazole and flucytosine did not increase the levels of staining with the mortality-specific dyes. Itraconazole was less effective than ketoconazole, which was less effective than miconazole. The practice of equating the loss of the capacity of C. albicans to form colonies with fungal cell death may give incorrect results in assays with amphotericin B, and the results of assays with caution with other antifungal agents that are lipophilic or that possess significant postantifungal effects may need to be interpreted.

* Corresponding author. Mailing address: Department of Microbiology and Public Health, 2B3.08 Walter Mackenzie Centre, University of Alberta Hospital, 8440-112 St., Edmonton, Alberta T6G 2J2, Canada. Phone: (780) 407-7242. Fax: (780) 407-3864. E-mail: r.rennie{at}provlab.ab.ca.

Antimicrobial Agents and Chemotherapy, April 2003, p. 1200-1206, Vol. 47, No. 4
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.4.1200-1206.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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