Reduced Biocide Susceptibility in Candida albicans Biofilms

Abstract

Candida biofilm formation is common during infection and environmental growth. We tested the impacts of three biocides (ethanol [EtOH], H₂O₂, and sodium dodecyl sulfate) on Candida albicans, C. parapsilosis, and C. glabrata biofilms. Higher concentrations of the biocides were required for efficacy against biofilms than for efficacy against planktonic controls. A combination study with two biocides (EtOH and H₂O₂) and fluconazole demonstrated that the combination had enhanced efficacy.

Candida species cause a wide spectrum of diseases, including hospital-acquired and device-associated infections (33). In the hospital setting, Candida persists on colonized individuals and medical equipment (10). When it is growing on a surface, such as a medical device, Candida adapts to a biofilm lifestyle (14, 15, 19, 22). Biofilm formation is a common mode of growth during infection and survival in the environment (11, 15, 19, 22, 34). Biofilms consist of cells attached to a surface and embedded in a matrix produced by the organisms (13). Phenotypic changes are associated with biofilm formation, and among these, resistance to antifungal agents has been implicated in the difficulty of treatment of biofilm infections (2, 5, 21, 27, 36-38). In fact, removal of the Candida-infected medical device is nearly always required for cure of the infection (33). Biofilm resistance to antifungals has been well described (3, 4, 8, 16, 20, 37). Comparisons of biofilm cells and planktonic cells, which are the free-floating counterparts of biofilm cells, demonstrate that biofilms have up to a 1,000-fold increased resistance (8, 17, 25, 32). Candida biofilm susceptibility to biocides has received less attention. Biocides are chemical or physical agents that inactivate microorganisms. Because they commonly demonstrate a broad spectrum of activity, the agents are often used as topical therapies for patients or environmental disinfectants (12, 26, 41). Several studies of C. albicans biofilms have examined the activities of chlorhexidine, ethanol (EtOH), hydrogen peroxide, betadine, and sodium hypochlorite, although the findings have not been consistent (7, 9, 23, 42, 43, 45, 46). These investigations have not routinely included comparisons of biofilms with planktonic cell cultures. Also, experimental designs have not accounted for the difference in cell numbers between planktonic cell cultures and intact biofilms.

The purpose of this study was (i) to compare the activities of different biocides against Candida albicans, C. parapsilosis, and C. glabrata biofilm and planktonic cells and (ii) to investigate the impacts of biocides on the activity of an antifungal, fluconazole, for the prevention and treatment of Candida biofilms (C. albicans only). We chose to study EtOH, H₂O₂, and sodium dodecyl sulfate (SDS) because they are relatively commonly used and the have different modes of action (26). Fluconazole was selected for use in the combination therapy investigations due to the resistance of Candida biofilms to this common antifungal (3, 9, 27, 39).

C. albicans (strains DAY 185 and K1), C. parapsilosis (strain 5986), and C. glabrata (strain 5740) biofilms were grown in 96-well polystyrene plates as described previously (30, 37). The wells of the plates were inoculated with either a standard CLSI (formerly NCCLS) inoculum or a higher inoculum in RPMI- morpholinepropanesulfonic acid on the basis of the burden of viable cells in the biofilm assay at the start of therapy (10⁶ to 10⁷ cells/well). After 24 h of incubation at 30°C, the biofilms were washed twice with phosphate-buffered saline. For the prevention assays, the biocides were added at the time of inoculation. For the treatment assays, the biocides and fresh medium were added to mature biofilms after 24 h of biofilm growth. Dilutions of the biocides were studied to include concentrations generally effective against planktonic organisms (26). The concentration ranges studied were as follows; H₂O₂, 1 to 1,000 mM; EtOH, 0.05 to 50%; and SDS, 0.0004 to 0.4%. After a 24-h incubation at 30°C, a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide reduction assay was performed (30, 37).

We determined the drug concentrations associated with a 50% reduction (50% effective concentration [EC₅₀]) and an 80% reduction (EC₈₀) in the optical density compared to that for the no-drug controls. For the studies with planktonic cells, the MICs of the biocides were measured by using CLSI endpoints. To account for the increased number of cells in the biofilm (10⁶ to 10⁷ CFU/well) compared to the number recommended for use in the CLSI method (0.5 × 10³ to 2.5 × 10³ CFU/ml), planktonic cell MICs were adjusted to a similar inoculum (18, 29). The assays were performed in triplicate on two occasions.

The impacts of the biocides in combination with fluconazole (concentration range, 0.0625 to 1,000 μg/ml) on the treatment of mature biofilms were similarly examined by using a checkerboard format. Mature biofilms (24 h) were incubated in the presence of the biocide and antifungal combination for 24 h, and the endpoints were assessed as described above. Fractional inhibitory concentration (FIC) indices were used to estimate the impact of each biocide on the activity of fluconazole, as described previously (30, 44).

EtOH, H₂O₂, and SDS were effective at reducing the metabolic activities of the C. albicans biofilms at concentrations commonly used for disinfection (26). However, the concentrations of the biocides required to inhibit growth were higher for biofilms than for planktonic cell cultures containing similar numbers of cells (Table 1). The concentrations needed to decrease the burden of mature biofilm cells by 50% were from 2- to 10-fold higher for biofilm cell inhibition than for planktonic cell inhibition, as follows: for EtOH, 25 to 35%; for H₂O₂, 125 to 250 mM; and for SDS, 0.05 to 0.15%. Decreasing the biofilm burden by 80% required even higher concentrations of EtOH and H₂O₂ (Table 1) (all P values were <0.05). Similarly high concentrations of EtOH and SDS were needed to prevent C. albicans biofilm formation. However, lower concentrations of H₂O₂ (40 mM) prevented biofilm formation (Table 1). Two of the biocides, EtOH and H₂O₂, potentiated the activity of fluconazole against C. albicans biofilms. However, SDS did not enhance the action of fluconazole. The positive impacts of these cell wall-perturbing agents on the activity of fluconazole along with the changes in the Candida cell wall during biofilm growth suggest a potential role for cell wall integrity in biofilm resistance (24, 30).

TABLE 1.

Effects of biocides on Candida biofilm and planktonic cells^a

Biocide	Strain	Plank. cell MIC	Modified plank. cellsb		Biofilm				Fluc FICc
					Treatment		Prevention
			EC50	EC80	EC50	EC80	EC50	EC80
EtOH	C. albicans DAY 185	3	6	13	35	40	35	35	<0.5
	C. albicans K1	1.5	6	13	25	25	NA	NA	NA
	C. parapsilosis 5986	1.5	6	6	25	25	NA	NA	NA
	C. glabrata 5740	3	13	13	25	25	NA	NA	NA
H2O2	C. albicans DAY 185	16	31	63	175	200	20	40	<0.27
	C. albicans K1	1	63	63	250	250	NA	NA	NA
	C. parapsilosis 5986	1	63	63	125	250	NA	NA	NA
	C. glabrata 5740	16	16	31	125	500	NA	NA	NA
SDS	C. albicans DAY 185	0.01	0.05	0.05	0.15	0.15	0.05	0.1	1
	C. albicans K1	0.01	0.03	0.05	0.05	0.1	NA	NA	NA
	C. parapsilosis 5986	0.01	0.03	0.05	0.05	0.1	NA	NA	NA
	C. glabrata 5740	0.01	0.03	0.03	0.05	0.05	NA	NA	NA

^aPlank., planktonic; Fluc, fluconazole; NA, not applicable. The units for the MICs, EC₅₀s, and EC₈₀s are percent for EtOH and SDS and mM for H₂O₂.

^bThe inoculum used for modified planktonic cell EC₅₀ and EC₈₀ testing was identical to the cell density used for the mature biofilms (10⁶ to 10⁷ cells/well). The endpoint reading was based on the optical density at 550 nm.

^cFIC is equal to [(EC₅₀ of drug A in combination)/(EC₅₀ of drug A alone)] + [(EC₅₀ of drug B in combination)/(EC₅₀ of drug B alone)]; values less than 0.5 indicate an enhanced interaction.

These disinfectants were able to affect Candida cell viability. However, the concentrations of biocides required for efficacy against biofilm cells were greater than those associated with the killing of planktonic cells, suggesting that a reduction in susceptibility is associated with biofilms. These data suggest that concentrations higher than those previously thought necessary may be needed to disinfect contaminated medical devices and equipment. Further studies with biofilm models may be useful to determine the biocide concentrations necessary for disinfection and biofilm eradication.

EtOH and H₂O₂ enhancement of the activity of fluconazole may prove to be useful in the treatment and disinfection of Candida biofilms associated with medical devices and equipment. H₂O₂ has been used for disinfection of oral hygiene devices and contact lenses (6, 28, 40). Also, the utility of ethanol (25 to 70%) as central venous catheter lock therapy is under investigation (1, 31, 35). The current study suggests that azole drugs, such as fluconazole, may act to enhance the activities of these biocides when they are used to prevent or treat fungal biofilms.