Investigating Dual-Species Candida auris and Staphylococcal Biofilm Antiseptic Challenge

Abstract

Candida auris can persistently colonize human skin, alongside a diverse bacterial microbiome. In this study we aimed to investigate the efficacy of antiseptic activities on dual-species interkingdom biofilms containing staphylococci to determine if antiseptic tolerance was negatively impacted by dual-species biofilms. Chlorhexidine, povidone iodine, and hydrogen peroxide (H₂O₂), were able to significantly reduce biofilm viable cell counts following exposure at 2%, 10%, and 3%, respectively. Notably, H₂O₂-treated biofilms were able to significantly recover and considerably repopulate following treatment. Fortunately, inter-kingdom interactions in dual-species biofilms of C. auris and staphylococci did not increase the tolerance of C. auris against antiseptics in vitro. These data indicate mixed infections are manageable with chlorhexidine and povidone iodine, but caution should be exercised in the consideration of H₂O₂.

1. Introduction

In the decade since its spontaneous emergence, Candida auris has quickly established itself as a prolific healthcare pathogen. With outbreaks recorded in six continents, C. auris has spread rapidly throughout healthcare environments, displaying worrying characteristics such as multi-drug resistance and the ability to survive on surfaces for at least 2 weeks [1]. Our inability to diagnose this pathogen accurately and consistently contributes to its continued spread within healthcare settings. Within healthcare environments, live C. auris cells have been recovered from the surrounding area of colonised patients such as mattresses, bedside tables, and chairs [2]. There is growing evidence corroborating these findings that also reveal the ability of C. auris to tolerate clinically relevant concentrations of antiseptics such as chlorhexidine (CHX) and hydrogen peroxide (H₂O₂) [3].

C. auris most commonly colonises the skin of both healthy and immunocompromised individuals, and therefore inevitably encounters members of the skin microflora [4]. Staphylococcus has been described as the dominant genus of the skin microflora with increases in the abundance of species such as S. aureus being observed in wound infections [5]. Other pathogenic fungi such as C. albicans have extensively shown their ability to interact with bacteria that comprise part of the skin microbiota [6]. These interactions are commonplace within biofilms and often result in increases of virulence factors, such as adhesins or drug resistance mechanisms [6]. Previous studies have also shown C. auris isolates are capable of forming robust biofilms where cell phenotype significantly influences virulence and biofilm quality [7].

There is a significant lack of information on whether C. auris interacts with other fungi or bacteria despite it occupying the same niche as other fungi and bacteria. It has been shown that C. albicans’ extracellular matrix is capable of coating and protecting S. aureus from the effects of vancomycin treatment [8], so we hypothesised that dual-species interactions with C. auris and S. aureus may mirror this effect and could have implications for skin and wound infections. Therefore, we herein aimed to investigate whether the interaction between C. auris and either S. aureus or S. epidermidis within a dual-species biofilm model influences the tolerance profile of C. auris to the commonly used antiseptics CHX, H₂O₂, and povidone iodine (PVP-I).

2. Results

Bacteria and fungi are commonly isolated together in healthcare environments, and the interactions that take place within these biofilms can drive antimicrobial resistance [6]. However, interactions between C. auris and bacteria are yet to be explored. Therefore, a dual-species biofilm model containing C. auris and the prolific skin pathogens S. aureus and S. epidermidis was developed. Multiple ratios of bacteria to fungi were assessed to ensure that bacteria did not inhibit fungal growth. It was observed that shifting ratios of S. aureus did not significantly alter biofilm growth and biomass (Figure 1A). Interestingly, dual-species biofilms grown with C. auris and S. epidermidis showed a 1.2-fold increase in biomass compared to S. epidermidis alone when bacterial cells were 100-fold fewer and where both C. auris and S. epidermidis were present in equal numbers (Figure 1B). As shifting ratios of bacteria to fungi did not significantly alter the biofilm biomass of dual-species biofilms, a ratio of 1:1 at 1 × 10⁶ cells/mL was selected, which has been used previously in a dual-species biofilm between Candida albicans and S. aureus [6]. Dual-species biofilms at this inoculum concentration supported the growth of both bacteria and fungi, which is visually represented in Figure 2. From these data it is evident that Staphylococcus spp. do not inhibit C. auris growth.

As single and dual-species biofilms of both bacteria and fungi are typically associated with an increased tolerance to antimicrobial challenges, we next sought to test the ability of such biofilms to tolerate antiseptics. In all cases, the mean number of fungi and bacteria in each biofilm was reduced by at least 2 × log₁₀ CFU/mL following treatment (Figure 3). CHX displayed the highest efficiency by significantly reducing viable cells of both S. aureus, S. epidermidis, as well as C. auris by more than 4 × log₁₀ (p < 0.05). H₂O₂ was the least effective against fungi within single and multi-species biofilm environments. H₂O₂ prevented the growth of S. epidermidis biofilms when grown on cellulose matrices (Figure 3D), but all other biofilms survived treatment with more than 1 × 10² CFU/mL remaining within the biofilm. Treatments with 10% PVP-I were able to reduce biofilm viability and also showed a greater efficacy against C. auris biofilms grown on plastic coverslips compared to cellulose matrices (Figure 3A; p < 0.05).

As shown above, each antiseptic was able to reduce the total number of viable cells within each type of biofilm. Therefore, we then assessed their ability to tolerate and regrow following treatment (Figure 4). CHX and PVP-I were again confirmed as the most potent agents against both single and dual-species biofilms, with residual regrowth occurring over 48 h. After 48 h of regrowth, dual-species biofilms treated with 3% (v/v) H₂O₂ recovered considerably. Biofilms containing C. auris and S. aureus, or C. auris and S. epidermidis, were found to be 4.5- and 2.6-fold higher, respectively, than single species C. auris biofilms.

3. Discussion

Interactions between fungi and bacteria have been shown to enhance antimicrobial tolerance [6,8,9]. Due to this, polymicrobial infections are responsible for higher mortality rates than their mono-microbial counterparts [6]. Any interactions that may take place between C. auris and other fungi or bacteria are yet to be characterised. We therefore aimed to begin to identify whether C. auris interacts with staphylococci species within a biofilm and if these interactions influence the resistance profile of the biofilm. C. auris biofilms have recently been shown to tolerate antiseptic treatments at clinically relevant concentrations [3]. Levels of tolerance to antiseptics were dependant on biofilm complexity, with developing biofilms being more susceptible to CHX and H₂O_2. However, C. auris was still recovered from the skin of hospitalised patients receiving daily CHX washes [4]. This decreased susceptibility to CHX in clinical settings may be a result of protective interactions with other non-staphylococcal members of the skin microbiome. In 2017, Moore and colleagues showed that C. auris was more susceptible to 2% (v/v) CHX with 70% (v/v) isopropyl alcohol [10]. Despite these promising results, the fungicidal activity of this antiseptic–alcohol mixture was observed in cells in suspension. Therefore, the activity of this treatment on single and multi-species C. auris biofilms is unknown.

In this study C. auris was shown to be less responsive to PVP-I compared to previous studies [3,10]. As discussed above, Moore and colleagues tested the C. auris response to antiseptic challenge in planktonic cells, which may explain their increased susceptibility [10]. Furthermore, our group reported PVP-I to be highly effective against mature C. auris biofilms, though this was from a different strain [3]. Indeed, we have previously shown that biofilms formed by C. auris NCPF 8973 (the strain used in this study) formed a more robust biofilm than all other strains tested [7]. Levels of glucans and mannans within the C. auris biofilm matrix have recently been characterised and differences between strains were observed [11]. Taken together, strain-specific biofilm heterogeneity can ultimately lead to varying responses of C. auris to antiseptics such as PVP-I.

Herein, we demonstrated a reduced activity of H₂O₂ against single and dual-species biofilms and similar findings have been reported elsewhere [3]. C. auris has been reported to contain catalase genes while both S. aureus and S. epidermidis display catalase activity that can be utilized as a survival factor against osmotic stressors and a H₂O₂ challenge, indicating why H₂O₂ is less effective than CHX and PVP-I [12,13,14].

Although there is no observed increase in biofilm tolerance, growth within a dual-species biofilm appeared to drive C. auris to form small aggregates. The ability of cells to form aggregates has been characterised in cystic fibrosis and chronic wounds [15]. Although not directly adhered to surfaces, these cells display biofilm traits such as recalcitrance to antimicrobial therapies [16]. Therefore, existing within a dual-species environment may increase the ability of non-aggregating C. auris to tolerate disinfection protocols and drive spread throughout healthcare environments.

This small study has only used a single C. auris strain, which is a member of the Indian clade. To fully characterise levels of antiseptic tolerance between C. auris and staphylococci, additional C. auris strains should be used. We also did not include a physical challenge in addition to the chemical action of the antiseptics used (e.g., wiping), which would likely affect cell survival rates. The data discussed herein reveal that interactions between non-aggregating C. auris and Staphylococcus species do not increase biofilm biomass, which is reassuring for the management of these infections. Notably however, the observed increase in cellular regrowth within the H₂O₂ treated dual-species biofilm indicates that bacteria have the potential to influence the ability of C. auris to persist within healthcare environments. Further research is required to fully understand the intercellular interactions that take place between C. auris and other bacterial species.

4. Conclusions

Being frequently isolated from the skin of both healthy and immunocompromised patients, the opportunities for C. auris to interact with bacteria that also occupy the same niche. Data herein show that Staphylococcus spp. do not promote a more tolerant phenotype in C. auris. However, similar interactions between staphylococci and C. albicans increase both fungal and bacterial virulence. Therefore, further studies may reveal other effects of these interactions.

5. Materials and Methods

5.1. Microbial Growth and Standardisation

C. auris isolate NCPF 8973 (Indian clade), S. aureus NCTC 10,833, and S. epidermidis RP62A (ATCC 35984) were used throughout this study. C. auris was grown on Sabouraud’s Dextrose (SAB) agar (Sigma-Aldrich, Dorset, UK) at 30 °C for 48 h and bacterial strains were grown for 24 h at 37 °C on Luria Bertoni (LB) agar (Sigma-Aldrich). All microorganisms on agar were stored at 4 °C. Yeast and bacterial colonies were suspended in yeast peptone dextrose media (YPD; Sigma-Aldrich) or LB broth, respectively. Broths were incubated for 18 h overnight before washing twice with phosphate-buffered saline (PBS; Sigma-Aldrich). Staphylococcal cells were diluted to 0.6 OD₆₀₀ (approx. 1 × 10⁸ cells/mL) and yeast cells were counted using a haemocytometer. Microorganisms were diluted to assay specific concentrations in desired media.

5.2. Biofilm Growth, Quantification, and Visualisation

Fungal and bacterial broths were standardised as described above. C. auris was adjusted to 1 × 10⁶ cells/mL as done so previously [7,17]. Bacteria were diluted to concentrations ranging from 1 × 10⁴ to 1 × 10⁶ CFU/mL. Cell suspensions were prepared as single or dual species inoculums in Todd Hewitt broth (THB; Thermo Fisher, Paisley, UK) supplemented with hemin and menadione, mixed 1:1 with Roswell Parks Memorial Institute (RPMI) media (Sigma-Aldrich). Biofilms were grown in treated 96-well, flat-bottom, microtitre plates (Thermo Fisher) and incubated at 37 °C for 24 h. Biofilm formation was assessed via crystal violet staining as described previously [6].

Bioflims were visualised by fluorescently labelling microorganisms with 1.5 mM hexidium iodide (Fisher Scientific, Loughborough, UK) and 1.5 mM calcofluor white (Thermo Fisher). Biofilms were incubated with the fluorescent dyes at 37 °C for 1 h. Biofilms were washed twice with PBS to remove excess dye and imaged using an EVOS cell imaging system (Thermo Fisher) at 40× magnification. Calcofluor white and hexidium iodide fluorescence was detected at excitation/emission wavelengths of 357/447 and 531/593 nm, respectively, before overlaying the images.

5.3. Hydrogel Preparation and Biofilm Growth on Cellulose Matrix

A 3-dimensional biofilm model was used as described previously by our group [3]. Briefly, this was prepared using 10% 3-sulfopropyl acrylate potassium salt, 0.95% poly(ethylene glycol) deacrylate, 0.01% 1-hydroxycyclohexyl phenyl ketone, and 50% horse serum diluted using 2× PBS pH 7.3. The mixture was added to treated, flat-bottomed, 12-well plates (Thermo Fisher) and gels were polymerised under 365 nm ultraviolet light for 30 min. For biofilm growth, microorganisms were standardised to 1 × 10⁷ cells/mL in PBS and incubated for 2 h with the required number of cellulose matrix pieces (cut to 1.25 cm²) at 37 °C with agitation. Sections of matrix were placed on top of the gels and incubated for a further 24 h at 37 °C.

5.4. Biofilm Treatment and Re-Growth Assessment

Single and multi-species biofilms on coverslips and cellulose matrices were exposed to 2% (v/v) CHX, 10% (w/v) PVP-I, or 3% (v/v) H₂O₂ for 5 min, and neutralised using 5% (w/v) sodium thiosulphate as described previously [3]. Biofilm viability was quantified directly after treatment using 0.01% (w/v) resazurin sodium salt using the method outlined above, and by colony forming unit (CFU) quantification. Biofilm biomass was removed from the growth substrate via sonication in an ultrasonic water bath (Fisher Scientific, Loughborough, UK) for 10 min at 35 kHz. The biomass was subsequently serially diluted 10-fold in an untreated, round-bottom microtitre plate as described elsewhere [17] and plated onto selective agars. C. auris was selected for using SAB agar supplemented with 8 µg/mL amoxicillin and 75 µg/mL erythromycin, and LB agar supplemented with 64 µg/mL amphotericin B was used to select for S. aureus and S. epidermidis growth [18].

To assess biofilm re-growth, fresh growth media was added back to the treated biofilms and then incubated for a further 48 h. Every 24 h biofilm viability was quantified by diluting a 1% (w/v) stock of resazurin sodium salt 1:100 in biofilm growth media. Diluted resazurin salt was incubated with biofilms for 1 h. Media fluorescence was measured at an excitation/emission of 530/590 nm in a microtitre plate reader (FLUOStar Omega, BGM Labtech, Aylesbury, UK).

5.5. Statistical Analysis

All experiments were carried out in triplicate on three independent occasions. Positive untreated controls were included for comparison to treated biofilms, and negative controls were used to test for contamination. Statistical analysis and graph production was performed using GraphPad Prism (Version 8; La Jolla, CA, USA) and SPSS Statistics (Version 26.0; IBM, Chicago, IL, USA). Kruskal–Wallis tests were used to compare means of biofilm metabolic activity and biofilm CFUs before and after treatment. Differences between means were deemed significant where p < 0.05.