Sublethal Injury and Resuscitation of Candida albicans after Amphotericin B Treatment

Abstract

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 ⌱. 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 g 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 g/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 g of miconazole per ml, which also increased the fluorescence of mortality-specific dyes to near the maximal levels achieved with 0.9 g 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.Candida is the fourth most common cause of nosocomial bloodstream infections in the United States (10), and the rate of primary bloodstream infections continues to increase (3). The most frequently isolated Candida species in these infections is Candida albicans. Candidemia is also frequently refractory to therapy, and the rate of mortality attributable to candidemia has been estimated to be 38% (36). Despite recent advances in the development of antifungal agents, amphotericin B (AMB) still remains the drug of choice for the treatment of life-threatening systemic infections (14), and the activity of AMB is the standard to which the activities of other antifungal agents are compared. AMB binds with the sterol ergosterol, resulting in the loss of membrane integrity and osmotic stability (5).The process of cell replication deactivation is believed to involve stepwise changes in the physiological state of a cell that render an intermediate form that is incapable of initiating replicative processes but that is still capable of metabolism (21, 27). Thus, the loss of replication competency is an early event in a phase of decline that eventually leads to cell death (19,27).We have previously examined the effects of AMB on the vitality and mortality of C. albicans cell...

Plasma membrane damage toCandida albicanscaused by chlorine dioxide (ClO2)

“…The low level of membrane disruption can be further demonstrated by the poor influx of PI. PI is most commonly used as an indicator for cell death or cells with compromised membrane because it is not taken up by cells with an intact plasma membrane, and thus, can only enter cells that have lost their membrane integrity (Jepras et al 1995;Comas and Vives-Rego 1997;Novo et al 2000;Amor et al 2002;Liao et al 2003). In our study, although the PI staining rates showed dose-and timedependent increases, the PI did not significantly enter cells until the time cultures were exposed to 20 mmol l )1 of ClO 2 for 5 min, during which 99AE99% of cells were inactivated (Figs 5 and 6).…”

New Insights Regarding Yeast Survival following Exposure to Liposomal Amphotericin B

“…The exact reasons for this lower-than-expected response still remain unclear. Some authors do not associate response failure with target modification, as has been observed with other antifungal drugs (11), but with inappropriate concentrations of AMB at the infection site (12). In accordance with this hypothesis, cells exposed to amphotericin B may exhibit different physiological conditions which are related to drug concentration and exposure time (12).…”

“…Some authors do not associate response failure with target modification, as has been observed with other antifungal drugs (11), but with inappropriate concentrations of AMB at the infection site (12). In accordance with this hypothesis, cells exposed to amphotericin B may exhibit different physiological conditions which are related to drug concentration and exposure time (12). In addition, studies of yeast apoptosis have revealed the occurrence of a programmed cell death or a cellular necrotic response depending on the AMB concentration (13,14).…”

“…1B). Previous studies have shown that some yeast cells exposed to amphotericin B demonstrate a capacity for resuscitation, although they are unable to replicate (12,28). It is notable that increasing and then decreasing AMB-L concentrations rapidly evoke compensatory responses by yeast cells, including the recovery of replication ability.…”

“…Metabolic changes induced by AMB-L in S. cerevisiae were evaluated using 5-carboxyfluorescein diacetate (5-CFDA; Sigma-Aldrich, Munich, Germany) at a 10 M final concentration. Antifungal-treated cell suspensions were stained with 5-CFDA and incubated for 45 min at 35°C at 150 rpm in the dark (12). The mean intensity of fluorescence (MIF) was registered at FL1 (530 nm).…”