Effects of Cetylpyridinium Chloride Resistance and Treatment on Fluconazole Activity versus Candida albicans

Abstract

Mouthwash antiseptic cetylpyridinium chloride (CPC) has potent activity against Candida albicans; however, two of five azole-resistant strains showed reduced CPC susceptibility. To further examine the potential for cross-resistance, CPC-resistant mutants were selected in vitro and their fluconazole susceptibility was tested. MICs were unchanged, and trailing growth generally decreased. With CPC-fluconazole combinations, both antagonism and synergism were observed, which can be explained, in part, by CDR1-CDR2 multidrug transporter upregulation.

Biocides, better known as antiseptics, disinfectants, or preservatives, are commonly added to mouthwashes, toothpastes, hand soaps, and related consumer products. With pathogenic bacteria, the potential for cross-resistance between biocides and antibiotics has been demonstrated; e.g., with triclosan-resistant Pseudomonas aeruginosa and benzalkonium chloride-resistant Staphylococcus aureus (1, 3, 4, 5). In comparison, pathogenic fungi have received little attention as biocide targets. Here, interactions of cetylpyridinium chloride (CPC) with the yeast Candida albicans were studied. CPC is the antiseptic component in the widely used mouthwashes Scope and Cepacol. Its mechanism of action is poorly understood, but structural relatedness to quaternary ammonium compounds is consistent with studies suggesting a membrane target (9, 10, 16). C. albicans is commonly found at low levels among the normal oral flora, but its overgrowth in immunocompromised individuals or following broad-spectrum antibiotic therapy leads to oropharyngeal candidiasis (2). This is typically treated with fluconazole or related azole antifungals, inhibitors of ergosterol biosynthesis (2, 18). However, extended treatment frequently selects for fluconazole-resistant strains that display upregulated expression of multidrug transporters, specifically those encoded by the CDR1, CDR2, and MDR1 genes, along with mutations in the ERG11-encoded target enzyme (13, 18, 20). Although previous studies have documented CPC activity versus C. albicans (6, 7, 11, 12, 14) and several of these have proposed its therapeutic use against candidiasis, there have been no reports on the development of CPC resistance in yeast. Furthermore, no studies have examined CPC-azole interaction, i.e., the potential effects of combination treatment on oropharyngeal candidiasis. A particularly important issue that has not been addressed is the potential for CPC-resistant C. albicans to display azole cross-resistance.

CPC has broad-spectrum anti-Candida activity.

CPC (Sigma-Aldrich, St. Louis, Mo.; stocks prepared in dimethyl sulfoxide) was initially tested for inhibitory activity versus seven strains of C. albicans and two strains each of C. glabrata, C. parapsilosis, and C. krusei with an agar dilution assay (Fig. 1). YPD medium (1% yeast extract, 2% peptone, 2% dextrose) was used since CPC was poorly active in RPMI 1640 medium (data not shown). C. glabrata and C. krusei were the most susceptible to CPC (no growth at 2 μg/ml), followed by C. parapsilosis (4 μg/ml) and C. albicans (4 or 6 μg/ml). An exception was strain HH, one of two fluconazole-resistant C. albicans strains in this experiment, which showed reduced CPC susceptibility (partial growth at 8 μg/ml). To further examine this potential correlation, three additional fluconazole-resistant C. albicans strains were tested, and one (strain 23-79) showed reduced susceptibility (growth on CPC at 12 but not 16 μg/ml; data not shown). Thus, two of five fluconazole-resistant strains showed partial CPC cross-resistance.

FIG. 1.

Agar dilution assay testing Candida species for CPC susceptibility. The chart indicates strain locations, where Ca is C. albicans, Cg is C. glabrata, Cp is C. parapsilosis, and Ck is C. krusei. Strains were obtained from the American Type Culture Collection or were recent clinical isolates (from T. White, University of Washington; J. Rex, University of Texas—Houston Medical School; and E. Reiss, Centers for Disease Control and Prevention). Incubation was at 30°C for 2 days.

Selection of CPC-resistant C. albicans.

To experimentally examine the development of CPC resistance, single-step selection was attempted by plating C. albicans on YPD containing CPC at 16 μg/ml; however, no colonies were obtained after prolonged incubation (data not shown). Therefore, multistep selection in liquid medium was used. Strains LL, 66027, and 2-76 (5 × 10⁶ cells per ml in 4 ml of YPD) were initially cultured in a partially inhibitory CPC concentration of 4 μg/ml. After 3 days, cultures were diluted to the same concentration in fresh medium containing CPC at 5 or 6 μg/ml; this passaging was repeated two additional times to a final CPC concentration of 11 or 12 μg/ml. Cells were then streaked twice for isolated colonies on CPC-free YPD plates. To confirm and quantify CPC resistance, a broth microdilution assay was used (19). For strains LL and 66027, all three of the mutants tested showed twofold CPC resistance with a MIC (≥80% inhibition) of 8 μg/ml (MIC for parent strain = 4 μg/ml). One of the strain 2-76 mutants was similarly resistant (MIC = 8 μg/ml), while the other two appeared to have normal CPC susceptibility in this assay (MIC = 4 μg/ml). Thus, while CPC-resistant mutants were obtained for all three strains, the level of resistance was clearly modest.

Examination of fluconazole cross-resistance.

Broth microdilution assays revealed that for the CPC-resistant mutants described above the fluconazole MICs were unaltered (Fig. 2). In fact, seven of the nine mutants appeared hypersusceptible in terms of reduced trailing growth, which is commonly observed at higher fluconazole concentrations after prolonged incubation (15, 19). Specifically, trailing growth was 40% of the drug-free growth of parent strain 66027, while the trailing growth of one mutant was reduced 10-fold to 4%. In all six of the C. albicans 2-76 and LL mutants, trailing growth was reduced approximately threefold.

FIG. 2.

Broth microdilution assays examining fluconazole susceptibility of CPC-resistant C. albicans mutants and the indicated parent strains. Incubation was for 30 h.

Interactions between CPC and fluconazole.

Since it is not unlikely that CPC and fluconazole treatments may be combined in oropharyngeal candidiasis patients, CPC-fluconazole interactions in wild-type strains were also examined. This was done by checkerboard broth microdilution assay with CPC concentrations of 0 to 8 μg/ml combined with fluconazole that was serially diluted from 8 μg/ml. Representative results are presented for C. albicans strain 2-76 after 24 and 40 h of incubation (Fig. 3). No growth was observed in any well with CPC at 8 μg/ml (data not shown). CPC concentrations between 1 and 4 μg/ml antagonized fluconazole activity fourfold at 24 h (fluconazole MICs = 2 and 0.5 μg/ml with and without CPC, respectively). At 40 h, antagonism was still apparent at the higher CPC concentration; however, synergism was also observed (MICs of ≤4 and >8 μg/ml with and without CPC, respectively) because of a reduction in trailing growth. Similar antagonism of fluconazole activity by CPC was observed in C. albicans strains 66027 and LL, and reduced trailing growth was similarly observed in all three of the additional strains tested (66027, LL, and 24433) (data not shown).

FIG. 3.

Broth microdilution assays examining interactions between CPC and fluconazole in C. albicans strain 2-76. Incubation was for 24 or 40 h as indicated.

CPC-induced upregulation of multidrug transporter genes.

In previous studies, antagonism and synergism of azole activity by unrelated drugs were correlated with effects on expression of the multidrug transporters genes CDR1 and CDR2 (8, 19). Therefore, the effects of CPC treatment on CDR1 and CDR2 expression were similarly examined (19) to potentially shed light on the complex interaction patterns described above. Following treatment with CPC at 4 or 8 μg/ml for 30 min, strong upregulation of CDR1 and CDR2 RNA was observed (Fig. 4). On the basis of this result, C. albicans strains with CDR1 and CDR2 deleted (17) were tested for CPC susceptibility; there was no significant difference compared to control strain CAF2-1 (data not shown). Consistent with this, studies with Saccharomyces cerevisiae have implicated overexpression of the major facilitator gene SGE1 in CPC resistance (unpublished data).

FIG. 4.

Slot blot RNA hybridization assay (19) showing upregulation of CDR1 and CDR2 after CPC treatment (30 min at 4 or 8 μg/ml) in three different C. albicans strains.

Summary and conclusions.

This study was undertaken to explore the possibility that CPC, the antiseptic component in widely used mouthwashes, could adversely affect azole treatment of oropharyngeal candidiasis in immunocompromised patients. This could occur from either cross-resistance to azoles in CPC-resistant mutants or antagonism between CPC and azoles when they are used in combination. In mouthwashes, CPC is present at about 500 μg/ml, or 100 times the MICs presented above. However, this concentration is maintained only briefly in the oral cavity and concentrations near the MIC that could select for resistant C. albicans may be present for several hours. In initial studies, two of five fluconazole-resistant C. albicans strains (uncharacterized with respect to resistance mechanism) exhibited reduced CPC susceptibility, raising concerns that CPC-resistant mutants selected by mouthwash use might be similarly cross-resistant to fluconazole. To test this, CPC-resistant mutants were isolated by multistep selection, which simulates daily use of mouthwash. Mutants were obtained for all three C. albicans strains, although CPC MICs increased only twofold. Fluconazole cross-resistance was not observed; in fact, seven of nine CPC-resistant mutants were fluconazole hypersusceptible in terms of reduced trailing. These data suggest that alternating CPC and fluconazole treatments in oral candidiasis might be beneficial in terms of reducing resistance. With respect to combining treatments, CPC enhanced fluconazole activity by reducing trailing growth, but antagonism was also observed at lower fluconazole concentrations. In vivo interactions between CPC and fluconazole are therefore difficult to predict. Antagonism of fluconazole activity very likely results from CPC induction of CDR1 and CDR2, a mechanism previously reported (8). The basis for CPC inhibition of fluconazole trailing growth remains unclear, although other drugs are known to have the same effect (19).