Candida albicans Biofilms Produce Antifungal-Tolerant Persister Cells

Abstract

Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. The expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that is a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibited a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug-tolerant subpopulation. Interestingly, surviving C. albicans persisters were detected only in biofilms and not in exponentially growing or stationary-phase planktonic populations. Reinoculation of cells that survived killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment, and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters formed only in biofilms, mutants defective in biofilm formation were examined for tolerance of amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug-tolerant persister cells in both planktonic and biofilm populations, and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy that leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.Candida albicans is an opportunistic pathogen that is a common member of the human oral and gastrointestinal microflora. Biofilms of C. albicans often form on indwelling devices, such as blood and urinary catheters and heart valves. These infections are essentially untreatable by antifungals and can develop into life-threatening disease with a mortality rate approaching 40%. The recalcitrance of these infections to antifungals is puzzling, since planktonic populations of the same strain can be susceptible to a range of antifungals, including azole compounds, echinocandins, and am...

Gold nanoparticles enhance methylene blue– induced photodynamic therapy: a novel therapeutic approach to inhibit Candida albicans biofilm

“…10 It became imperative to remove these biofilm-attached implants and devices, otherwise they may disseminate candidiasis infection. 52 Besides the multifactorial phenomenon, [11][12][13][14][15] the unique properties of Candida biofilm matrix materials thwart antimicrobial activity against well-ensconced Candida cells as compared to planktonic cells. The chemical composition of ECM of Candida biofilm has not been not fully elucidated.…”

“…Among the most important factors are biofilm matrix or exopolmeric substance (EPS), high density of cells in biofilm, presence of presister cells, and drug-efflux pumps. [11][12][13][14][15] Ambient matrix or EPS produced by sessile cells of biofilm recently gained the spotlight and imparting the impregnation of drug molecules and putative charge barrier. 16,17 Recently, photodynamic therapy (PDT) emerged as an alternative to conventional treatment of infections caused by C. albicans.…”

Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies

“…However, their importance in chronic infections is becoming increasingly clear, motivating more researchers to tackle the issue of fungal biofilms. LaFleur et al (2006) reported for the first time the presence of a population of antifungal-tolerant cells within a Candida biofilm, much resembling the bacterial persister cells found in P. aeruginosa and E. coli planktonic and biofilm populations. Contrary to bacterial populations, planktonic Candida populations do not produce persister cells refractory to the lethal action of antibiotics.…”

The toxin-antitoxin systemtisB-istR1

“…57 Persistence is not restricted to bacteria since, e.g., it has also been observed in the fungus Candida albicans. 58 It is epigenetic in nature, even though many genes by know are known to affect the frequency by which persisters are formed. 15,59,60,65 Since persisters are extremely slow growing or totally growth arrested, it has been speculated that expression of toxins might stochastically drive a fraction of a bacterial population into the persister state.…”