Candida species are the most frequently isolated human fungal pathogens and cause a wide spectrum of diseases, including superficial mucosal, severe deep, and systemic infections42,44. The increased use of biomaterial implantations, bone marrow and solid organ transplantations, immunosuppressive treatment, and the use of broad-spectrum antibiotics are associated with severe systemic infections44.

One of the most important virulence factors of Candida infections involves the formation of biofilms42. Candida species have differing abilities for biofilm formation and frequently play a role in biofilm-associated infections42. In particular, Candida albicans is an intensely biofilm-forming fungus12,23.

Cells in the biofilm are protected from the external environment, immune system, and antifungal treatment; in fact, the most important characteristic of biofilms is their high antimicrobial resistance42. Different antimicrobials have been used in infections due to C. albicans14,31,43, whose biofilms show high resistance to fluconazole, a frequently preferred treatment2,34,42. Amphotericin B shows dose-dependent activity, while echinocandins have in vitro action against biofilms20,24,42,43. Although most new azoles affect planktonic cells, they do not sufficiently inhibit sessile cells20,24,43.

Because planktonic and sessile forms of microorganisms have different antimicrobial susceptibilities, treatment strategies based on minimal inhibitory concentration (MIC) results of planktonic cells may fail.

The incidence of life-threatening biofilm-related infections has grown with an increasing number of oncological and immunosuppressed patients and the use of indwelling medical equipment; therefore, prophylactic treatment strategies are needed15,16. Few studies have investigated the benefit of administering antifungals at subinhibitory concentrations for infection prophylaxis and their inhibitory effects on Candida biofilm formation.

In this study, we determined the rate of biofilm formation in C. albicans strains that frequently cause systemic infections. We then compared the antifungal susceptibility profiles of planktonic and sessile forms and the efficacy of antifungals at subinhibitory concentrations for preventing biofilm formation in selected isolates.

The increased prevalence of biofilm-related infections in high-risk patients is a significant health concern. In our study, we detected biofilm formation in 90% of 162 C. albicans isolates obtained from blood cultures, which is a higher rate than that observed in previous studies6,7,29,36. The choice of assay may influence this finding, since the Christensen method stains dead cells, living cells, and the extracellular matrix, while the XTT assay only measures the metabolism of living cells.

Mature biofilms are complex structures containing sessile cells with different metabolic activities42. Moreover, studies have shown disagreement regarding the significant correlation between metabolic activity as measured by XTT and cell numbers determined by other methods11,22,23,34. Therefore, previous data, together with our own findings, prompt us to consider if comparing results obtained from different assays can accurately characterize biofilm formation in these strains.

Plates with needle lids are frequently used to detect bacterial biofilms and their antimicrobial susceptibilities; however, few studies utilizing this approach with Candida cells have been published10,28,30,41. Studies reported that the optimal cell number for Candida biofilm formation was 107CFU/ml, and sucrose significantly increased C. albicans biofilm formation on needles23,30. Therefore, we adjusted the yeast inocula to McFarland 1 (30×107CFU/ml for bacteria) and diluted the suspension 1 in 30 with growth medium because yeast cells are larger in size than bacteria and few cells are needed for biofilm formation8. Therefore, we optimized the conditions and determined significant biofilm using the Calgary method9.

In our study, the planktonic forms of all 30 extensive biofilm-forming isolates were susceptible to echinocandins and azoles as reported in previous epidemiological studies26,32.

Several studies have investigated the effect of fluconazole on sessile C. albicans cells and found MBIC ranges of 4–1024μg/ml2,3,25,27,28,34. In contrast, we found higher MBIC50 and MBIC90 values (≥2048μg/ml). Only few studies have tested the effect of itraconazole on sessile cells. We observed similar MBIC values (1–128μg/ml) as indicated in these studies13,27,28. The published MBIC range for voriconazole is 0.5–>265μg/ml20,27,37; however, only a few studies have investigated the efficacy of posaconazole on sessile cells20,43. Similarly to previous results, we found that sessile cells were resistant to voriconazole and posaconazole.

The published amphotericin B MBIC range is 0.015–2μg/ml2,3,28,37. We observed higher MBIC values (4–128μg/ml) and greater amphotericin B resistance among our clinical isolates compared to data in the aforementioned studies.

Many studies have found that echinocandins effectively inhibit C. albicans sessile cells at MBIC of 0.03–4μg/ml of caspofungin, ≤0.03–2μg/ml of micafungin, and 0.015–16μg/ml of anidulafungin3,17,18,20,24,37,38. We determined the MBIC ranges for caspofungin and micafungin as 0.125–16μg/ml and ≤0.06–32μg/ml, respectively. Although these overall ranges are similar to those published in other studies, the maximum values that we observed were higher, with the exception of anidulafungin17,20,38.

Although there are no standard breakpoints for the susceptibility of sessile cells, the MBIC values of echinocandins were partially within the planktonic susceptibility range of CLSI. Among the intense biofilm-forming and standard strains, 11 were susceptible to caspofungin, 9 were susceptible to anidulafungin, and 5 were susceptible to micafungin. In addition, the MBIC50 of caspofungin was within the therapeutic concentration range (2μg/ml) as indicated by Cocuaud et al.5, and caspofungin was effective against 50% of the clinical isolates we tested.

Overall, the MBIC values found in our investigation were higher than those found in some studies2,4,10,13,17,28,34,37 but similar to others13,24,27,28,34. The higher MBIC values we observed may be due to our 48-h biofilm formation period, the relatively short 24-h exposure to each antifungal, and the intense biofilm-forming capabilities of our strains. In addition, methodological differences and the isolation of our strains from blood, as opposed to other culture sources, may be significant factors.

In our study, we compared MBIC/MIC ratios of the antifungal agents to determine their efficacy on biofilm maintenance directly, which has not been widely reported43. The lowest statistically significant MBIC/MIC ratio was determined for amphotericin B, although the MBIC/MIC ratio is based on planktonic MIC values. Although the MBIC/MIC ratio was low for amphotericin B, its MBIC values were above the susceptibility ranges provided by CLSI, highlighting its ineffectiveness at therapeutic concentrations and toxicity at high doses in infections involving planktonic cells33.

Very low planktonic MIC values found for echinocandins in our study resulted in higher MBIC/MIC ratios when compared to amphotericin B. The importance of our finding is that the MBIC values of the echinocandins were closest to the planktonic susceptibility limits of CLSI criteria.

We expected azoles to be less effective on sessile cells, but the MBIC/MIC ratio of fluconazole and voriconazole was >32000 and higher than anticipated. The MBIC/MIC ratio of posaconazole was only 1536, echoing the results of Tobudic et al., who reported decreased efficacy of posaconazole during all biofilm phases of C. albicans43.

Administering lower doses of antifungal agents over a longer period of time in some high-risk patient groups for treatment or prophylaxis is of interest, as blood levels drop below clinical subinhibitory concentrations or in vitro MIC values15,16. To date, only a few studies have explored the inhibitory effect of antifungal agents on Candida biofilm formation and with varying methodologies, such as enabling biofilm formation post-exposure to an antifungal agent or co-incubating the cells with an antifungal agent1,16,24. In our study, C. albicans cells were incubated with MIC/2–MIC/128 sub-MICs of each antifungal agent, and the concentration that inhibited biofilm formation 100% at needle-tips was the BIC. We encountered some methodological difficulties, such as a fluctuating growth density observed in some strain-antifungal combinations, and instead measured the 100% biofilm inhibitory concentration. In general, total inhibition was seen close to MIC values (MIC/2 and MIC/4). We also found that needle lids were not suitable for measuring biofilm formation inhibition due to the fluctuant growth pattern of cells and difficulty in obtaining reproducible results.

The biofilm formation of some strains was not inhibited and sometimes increased close to the MIC of the antifungal agent. Schadow et al. first showed increased Staphylococcus epidermidis biofilm formation at sub-MIC values of rifampin19,35, and since then various bacteria-antibiotic combinations have been observed19. Dose-effect efficacy curves of an agent are usually biphasic and characterized by biofilm formation at low doses and biofilm inhibition at high doses. Some antibiotics act as antagonists for biofilm formation at low or high doses but as agonists at moderate doses. Similarly, we observed significant inhibition of biofilm formation sub-MIC levels in some strain-antifungal combinations but less inhibition at some higher concentrations. This fluctuant growth pattern was seen in several yeast-echinocandin combinations in contrast to amphotericin B or most of the azoles, with the exception of posaconazole.

Strain-specific factors may have influenced our study because this effect was not seen in all strains. Sub-inhibitory concentrations of an antimicrobial agent often cause physiological changes in the microorganism, such as stimulation or inhibition of enzyme or toxin production15,16,45,47. Simitsopoulou et al.38 reported a paradoxical increase in biofilm formation of C. albicans strains at concentrations higher than the MIC, which led to uncertainty regarding the ability of echinocandins to reach sub-inhibitory concentrations in the biofilms, induce chitin formation, and antifungal resistance.

To our knowledge, no previous studies have investigated Candida biofilm capabilities using 8 different antifungal agents with the Calgary method in the number of strains used in our research. However, our methodology could be improved by combining the BioTimer and Calgary approaches to obtain earlier test results and enable more timely clinical decision-making regarding prophylaxis and treatment. We also evaluated the use of needle lids to investigate inhibition of Candida biofilm formation and found that, despite its ease of use, this method does not yield reproducible results due to fluctuant growth patterns. Thus, we recommend needle lids be combined with or replaced by methods that measure metabolism or depend on the morphological analysis of the cells.

Our results indicate that echinocandins are suitable for treating biofilm-related infections, and that itraconazole may be used for prophylaxis of such infections involving C. albicans. The increased prevalence of biofilm-associated fungal infections of C. albicans needs the development of new treatment and prophylaxis strategies, especially in high-risk patients. An antifungal ability to limit biofilm formation should be considered when selecting an appropriate agent. In addition, even agents effective at sub-MIC levels could lead to antifungal resistance and should be used judiciously16. Therefore, further in vivo and in vitro studies exploring the use of sub-MICs of antifungal agents in prophylaxis strategies and the application of standardized biofilm measurement methods are needed to determine the unique dosage and exposure time of an antifungal for its maximum anti-biofilm effectiveness and patient safety.

The authors declare that they have no conflicts of interest.