Drug resistance and apoptosis in Candida biofilms.
PhD thesis, University of Glasgow.
Full text available as:
Candida species are commonly part of the normal flora in humans; however, they are opportunistic fungal pathogens that are capable of causing a variety of infections in hospitalized and immunocompromised individuals. These infections range from superficial to systemic ones. Many Candida infections involve biofilm formation on the surfaces of implanted devices, such as catheters and prostheses, or host tissues. Candida biofilms are resistant to a range of antifungal agents in current clinical use but the basis of this drug resistance is not clear. The aim of this project was to investigate possible resistance mechanisms using two fungicidal agents, amphotericin B and caspofungin, a new drug reported to have anti-biofilm activity.
The activity of amphotericin B and caspofungin at different development phases of Candida biofilms was investigated in vitro. Amphotericin B at two times the MIC (for planktonic culture) had the least effect on Candida biofilms, but at a higher concentration (five times the MIC) it showed relatively high activity against biofilms of C. parapsilosis and C. glabrata, especially at the late development phase. Biofilms of C. albicans were more resistant to amphotericin B throughout development (except for the earliest stage) than the other Candida species. Caspofungin, at two times the MIC, generally exhibited a greater effect on Candida biofilms than amphotericin B although this was not observed with C. parapsilosis biofilms in some development phases. Caspofungin, at five times the MIC, was slightly less effective than at the lower concentration against C. tropicalis in all development phases tested. The species most susceptible to caspofungin throughout biofilm development was C. glabrata. In no case were biofilm cells of any Candida species completely killed by either amphotericin B or caspofungin.
The penetration of caspofungin through biofilms of different Candida species was evaluated using an in vitro filter disc bioassay. Caspofungin penetration through biofilms of C. albicans SC5314 was initially faster than C. albicans GDH2346; however, after 6 h drug diffusion was greater with biofilms of strain GDH2346 (70.8% of the control value). Among other Candida species tested, the highest drug penetration was observed with C. glabrata and C. parapsilosis (81.2% and 73.3% of the control value, respectively), while the lowest was seen with biofilms of C. krusei. Biofilms of C. tropicalis also showed poor penetration. Exposure of biofilms of any Candida species to caspofungin (or amphotericin B) in this assay failed to result in complete killing of biofilm cells. However, evaluation of caspofungin activity against biofilms was complicated by the paradoxical phenomenon (reduced activity of the drug at high concentrations, above the minimum inhibitory concentration). Scanning electron microscopy revealed that caspofungin caused more structural damage to biofilm cells and matrix than did amphotericin B; the highest degree of damage due to caspofungin was observed in biofilms of C. glabrata and C. krusei.
The presence of a small number of drug-tolerant or persister cells is one possible mechanism of biofilm drug resistance. Biofilms and planktonic cells of five Candida species were surveyed for the presence of persister cell populations after exposure to amphotericin B. None of the planktonic cultures (exponential or stationary phase) contained persister cells. However, persisters were found in biofilms of one of two strains of C. albicans tested and in biofilms of C. krusei and C. parapsilosis, but not in biofilms of C. glabrata or C. tropicalis. Live-dead staining with fluorescein diacetate confirmed these results which do, however, suggest that persister cells cannot solely account for drug resistance in Candida biofilms.
If microorganisms exposed to antimicrobial agents undergo a type of programmed cell death or apoptosis, persisters could be variant in which this process has been disabled. Here, specific staining methods were used to investigate the existence of apoptosis in Candida biofilms subjected to different concentrations of amphotericin B. Caspase activity, indicative of apoptosis, was detected with SR-FLICA and D2R fluorochrome-based staining reagents in all of these biofilms. The general inhibitor of mammalian caspases, Z-VAD-FMK, when used at a low concentration (2.5 μM), increased the viability of drug-treated biofilms up to 11.5-fold (P<0.001%). Seven specific caspase inhibitors had different effects on C. albicans biofilm viability, but inhibitors of caspases-1, -9, -5, -3, and -2 all significantly increased cell survival (40-fold, 8-fold, 3.5-fold, 1.9-fold and 1.7-fold, respectively). On the other hand, histone deacetylase (HDA) inhibitors enhanced the activity of amphotericin B against biofilms of all three Candida species. Sodium butyrate and sodium valproate, for example, when added concurrently with amphotericin B, completely eliminated biofilm populations of C. albicans. Overall, these results demonstrate an apoptotic process in amphotericin-treated biofilms of three Candida species. They also indicate that HDA inhibitors can enhance the action of the drug and in some cases even eradicate persister subpopulations, suggesting that histone acetylation might activate apoptosis in these cells.
Actions (login required)