Patients with Long-Term Oral Carriage Harbor High-Persister Mutants of Candida albicans

Fungal biofilms produce a small number of persister cells which can tolerate high concentrations of fungicidal agents. Persisters form upon attachment to a surface, an important step in the pathogenesis of Candida strains. The periodic application of antimicrobial agents may select for strains with increased levels of persister cells. In order to test this possibility, 150 isolates of Candida albicans and C. glabrata were obtained from cancer patients who were at high risk for the development of oral candidiasis and who had been treated with topical chlorhexidine once a day. Persister levels were measured by exposing biofilms growing in the wells of microtiter plates to high concentrations of amphotericin B and plating for survivors. The persister levels of the isolates varied from 0.2 to 9%, and strains isolated from patients with long-term carriage had high levels of persisters. High-persister strains were isolated from every patient with Candida carriage of more than 8 consecutive weeks but from no patients with transient carriage. All of the high-persister isolates had an amphotericin B MIC that was the same as that for the wild type, indicating that these strains were drug-tolerant rather than drug-resistant mutants. Biofilms of the majority of high-persister strains also showed an increased tolerance to chlorhexidine and had the same MIC for this antimicrobial as the wild type. This study suggests that persister cells are clinically relevant, and antimicrobial therapy selects for high-persister strains in vivo. The drug tolerance of persisters may be a critical but overlooked component responsible for antimicrobial drug failure and relapsing infections.Candida species are opportunistic pathogens that are typically present in the oral cavities of healthy individuals (2, 14, 24). In immunocompromised patients, the severity of Candida infection can range from a superficial annoyance to a life-threatening systemic infection of the organs and sepsis. While superficial infections of the oral or vaginal mucosa are easily treatable with azoles, 5 to 8% resist therapy, producing relapses (11, 30). Systemic invasive fungal infections, characterized by the hyphal growth of Candida albicans, are the cause of high rates of morbidity and mortality, which approach 40% (8). Difficult-to-treat Candida infections also occur on prosthetic devices, such as catheters and heart valves, and an infected prosthesis requires device removal to avoid systemic infection (22).Candida forms biofilms on the surfaces of mucosal tissues and prostheses that are highly tolerant to antifungal agents (18, 26). The recalcitrance of biofilm infections to antimicrobials is not obvious, since planktonic populations of disease-causing strains can be highly susceptible to antifungals, including azoles, echinocandins, and amphotericin B (AMB) (16).We reported that upon attachment, C. albicans forms a small subpopulation (∼1%) of persister cells that are completely tolerant to the currently used systemic antifungals (16) and resemble well-characterized dormant persisters formed by pathogenic bacteria (12, 13, 18, 27, 28, 31, 32). The concentration-dependent killing of a C. albicans biofilm with a fungicidal agent such as AMB shows a sharply biphasic pattern, with the bulk of the cells rapidly dying and a small plateau of surviving persisters being present. Vital staining shows live persister cells present in a biofilm killed by exposure to a high level of AMB, and these cells can be sorted out from the bulk. Surviving persisters produce a new biofilm with a similarly small population of persisters, indicating that these cells are not classical resistant mutants but phenotypic variants of the wild type. Persisters exhibit multidrug tolerance, which is a hallmark of a biofilm infection.Attachment to a surface is an important step in fungal pathogenesis, including pathogenesis resulting in vaginitis, oral thrush, and catheter biofilm infections (6, 10, 15). It seems that persisters, which form upon attachment of the pathogen to a surface, may play an important role in the tolerance of Candida infections to antifungals.In Escherichia coli, the periodic application of a high concentration of a bactericidal antibiotic in vitro leads to the selection of high-persister (hip) mutants (20, 21). Importantly, these mutants have the same MIC as the wild type, but they produce considerably more persister cells. While the persister phenotype itself is not due to a mutation, high-persister strains carry mutations that cause an increased incidence of persisters. One of these mutants was mapped to an allele of a hipA gene coding for a toxin of the hipBA toxin/antitoxin module (13, 21). The mechanistic basis of HipA-dependent persister formation was recently identified (27). HipA is a protein kinase (3) that phosphorylates elongation factor EF-Tu, which leads to the inhibition of protein synthesis. This creates a dormant, persister state. The HipA7 allele, which causes the high persistence of the hip mutant, apparently has decreased binding to the antitoxin HipB, which leads to increased persister production.We reasoned that a similar selection for high-persister mutants is likely to occur in vivo, especially in cases of recalcitrant infections, where pathogens are periodically exposed to high levels of antimicrobial compounds. With this in mind, we tested a collection of 150 clinical isolates of Candida species for their persister levels. The strains were obtained from cancer patients who received daily topical chlorhexidine (CHX) treatment. We report that the strains isolated from patients with long-term Candida carriage had increased levels of surviving persisters.