Bacteriophages improve the effectiveness of rhamnolipids in combating the biofilm of Candida albicans

doi:10.21203/rs.3.rs-4963945/v1

Download PDF

Research Article

Bacteriophages improve the effectiveness of rhamnolipids in combating the biofilm of Candida albicans

https://doi.org/10.21203/rs.3.rs-4963945/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Biofilms produced by Candida albicans pose significant therapeutic challenges due to their resistance to conventional antimicrobials. In response, the need for the development of more potent strategies to combat such infections persists.

Rhamnolipids (RLs) are biosurfactants with diverse antimicrobial properties. Bacteriophages (phages) are viruses that target specific bacterial strains, although recent studies have shown that they may also potentially affect biofilm formation by fungi and yeasts. This study investigated the combined antimicrobial effect of RLs and bacteriophages against C. albicans biofilms, focusing on their anti-adhesive and inhibitory effects on biofilm development. RT-PCR assays were used to analyze gene modulation in C. albicans biofilm formation in response to RLs and phage treatments. Additionally, hyphae formation in the presence of RLs, phages, and their mixtures was examined using fluorescence microscopy.

The results demonstrated that the combined treatment of RLs and bacteriophages significantly reduced biofilm formation compared to individual treatments. The combination of 200 mg/L rhamnolipid with BF9 phage achieved a 94.8% decrease in biofilm formation. This synergy was confirmed in subsequent models, with rhamnolipids at the same concentration and phage LO5/1f nearly completely inhibiting biofilm formation (~ 96%). Gene expression analysis revealed a profound downregulation of key biofilm-associated genes when Candida cells were treated with 200 mg/L RLs and four phages (BF17, L05/1f, JG004, FD).

The results of this study suggest the potential of combining RLs and bacteriophages in combating C. albicans biofilms, indicating a promising perspective for future therapeutic approaches, offering renewed hope in the battle against resilient infections.

Candida albicans

rhamnolipid

bacteriophages

antimicrobial agents

combined therapy

biofilm assay

Candida is a yeast pathogen that poses a range of threats to human health. Superficial candidiasis, such as oral thrush or vaginal yeast infections, can cause discomfort and affect the quality of life. Moreover, invasive candidiasis, particularly in immunocompromised individuals, can lead to bloodstream infections with high mortality rates. Additionally, the emergence of drug-resistant Candida strains presents challenges in treatment, potentially resulting in limited therapeutic options [1].

Biofilms produced by Candida albicans represent a particular risk to human health. These structures provide protection against host immune responses and antimicrobial agents, leading to persistent infections [2]. Moreover, C. albicans biofilms are associated with medical device-related infections, such as those linked to catheters and prosthetic implants, increasing the risk of complications and treatment failures. Additionally, the complex matrix of biofilms promotes the exchange of genetic material, contributing to the development of drug resistance, thereby further complicating treatment options. Currently applied anti-yeast agents and strategies for eliminating Candida biofilm are depicted in Fig. 1 and Fig. 2, respectively. However, given that Candida biofilms are notoriously resistant to antifungal agents, rendering treatment challenging and increasing the risk of recurrent infections, there is an urgent need for the development of new, effective, and safe strategies to combat infections of this origin [3]. Consequently, surfactants, renowned for their antimicrobial properties, are being investigated as potential solutions [4].

Surfactants are chemical compounds exhibiting surface-active properties [7]. Each surfactant possesses an amphipathic structure, characterized by a hydrophobic fragment (typically a hydrocarbon chain) and a hydrophilic fragment (consisting of anionic, cationic, non-ionic, or amphoteric groups). This structure enables these molecules to assemble into micelles, thereby diminishing interfacial and surface tension [8]. Surfactants can be synthesized through chemical processes, while biosurfactants are naturally produced by plants, animals, and microorganisms [9]. Biosurfactants are considered a more appealing option compared to synthetic surfactants due to their higher biodegradability, lower toxicity, superior emulsifying capacity, and foaming properties [7]. Their lower critical micelle concentrations (CMCs) contribute to enhanced efficiency in comparison to synthetic surfactants. Furthermore, these compounds are well-documented in the literature for their adhesive properties [10]. Biosurfactants find applications across diverse fields such as bioremediation, agriculture, medicine, but also as pharmaceuticals, detergents, and cosmetics [11].

Rhamnolipids (RLs) are a type of glycolipids classified as low molecular weight biosurfactants, alongside sophorolipids and trehalolipids. They consist of one or two rhamnose units acetylated with up to two hydroxy long-chain fatty acids. While predominantly produced by Pseudomonas aeruginosa, documented instances exist of other microorganisms capable of RLs production, such as Burkholderia thailandensis [12], or engineered strains of E. coli and Pseudomonas putida [13]. Typically, these compounds are present in a combination of mono- and di-RLs [14]. The specific composition of the RLs mixture can vary based on factors such as culture conditions, the carbon source utilized, and the bacterial strain involved [15].

On the list of alternatives to conventional antimicrobial strategies, the utilization of bacteriophages (phages, bacterial viruses) is frequently highlighted. Phages' capability to eradicate bacteria has been harnessed almost since their discovery in the early 20th century. Phage therapy, once overshadowed by the advent of penicillin and the rapid advancement of antibiotic-based treatments, has experienced a resurgence in recent years as a response to the antibiotic resistance crisis. Phages possess several attributes that render them attractive for application. In contrast to biosurfactants, which influence the function of bacterial cell membranes, phages infiltrate cells and exploit the bacterial replicative machinery to proliferate, ultimately resulting in bacterial lysis [16]. Additionally, phages exhibit high specificity, targeting only specific types or even species of bacteria. Moreover, phages exhibit a unique characteristic termed "auto-dosing," wherein they replicate at the infection site as long as the bacterial host persists. Finally, bacteriophages are deemed safe and non-toxic to humans and animals [17]. Although bacteriophages are believed to lack natural tropism for eukaryotic cells, interactions of such nature have been frequently reported [18, 19]. Notably, these interactions extend to interference with yeast and fungal biofilm formation [20, 21].

Current research is centered on developing combined systems to enhance pathogen elimination, with studies demonstrating, for instance, the synergistic effects of biosurfactants and antibiotics [22]. Therefore, this study aimed to investigate the combined effect of RLs and bacteriophages against C. albicans biofilms. The research assessed the formulations' capacity to inhibit biofilm growth and prevent adhesion. To validate their efficacy, reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate the expression of genes involved in C. albicans biofilm formation. Additionally, the ability of Candida cells treated with RLs, phages, and combinations of these agents to form hyphae was visualized with fluorescent microscopy.

2.1. Strains, media, and compounds

The wild-type strain Candida albicans ATCC 10231 was used in this study. Frozen glycerol stock culture was regularly revived on YPD agar medium (yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L and agar 20 g/L). The ATCC 10231 strain was cultured overnight in liquid YPD medium with shaking at 28 ℃, and then used for further experiments. RLs were purchased from AGAE Technologies LLC (Corvallis, OR, USA) as a mixture of different mono- and di-RLs homologues. The composition of the RLs sample is listed in Table S1.

2.2. Critical micelle concentration (CMC)

The measurements were carried out on a Krüss, K6 tensiometer (Krüss, Hamburg, Germany) using the du Noüy ring method. A 10 mL solution of RLs dissolved in YPD medium at a concentration of 150 mg/mL was prepared. The surface tension of the YPD medium, RLs solutions, and their series of dilutions were measured. CMC was calculated by measuring the surface tension of RLs solutions prepared in a YPD medium at different concentrations. Subsequently, a curve was plotted based on the obtained results, and two tangents were drawn. The point of intersection between these tangents determines the CMC.

2.3. Bacteriophages

Four bacteriophages as well as their host bacteria (JG004/P. aeruginosa, Felix/Salmonella, T4/E. coli, FD/E. coli) were obtained from DSMZ-German Collection of Microorganisms and Cell Cultures (Leipzig, Germany). Six other bacteriophages (BF9/E. coli; BF15/E. coli; BF17/E. coli, TO1/6f/ Ent. faecalis; TO1/7f/Ent. faecalis; LO5/1f/ Enterobacter cloacae) were derived from Collection of Microorganisms of Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences. Bacterial hosts of these phages are environmental isolates also deposited in the same collection. The basic characteristics of bacteriophages used in the studies are presented in Table S2.

2.4. Amplification of bacteriophages

Bacteriophages were amplified with a two-stage method described previously by Skaradzińska et al [23] with some modifications. Briefly, a single colony of the host strain was transferred to 30 mL of liquid Luria-Bertani (LB) medium (10 g/L bacto-tryptone, 5 g/L bacto-yeast extract, 10 g/L NaCl) medium and incubated for 20 hours (37°C, 150 rpm). After incubation, 10 mL of respective bacteriophage preparation was added and phage-bacteria culture was incubated for the next 24 hours in the same conditions. Then, the culture was centrifuged (2000 g, 5 minutes) and filtered through syringe filters with a diameter of 33 mm and polyethersulfone (PES) membrane pore size of 0.22 µm (Merck-Millipore, USA). The filtrate was added to a 20 hours host bacterial culture prepared analogously as at the previous stage and subsequently incubated for 24 hours (37°C, 150 rpm). The culture was again centrifuged (2000 g, 5 minutes) and filtered through syringe filters with a diameter of 33 mm and PES membrane pore size of 0.22 µm (Merck-Millipore, USA). Phage titer in preparations was determined using the standard routine test dilution (RTD) and double-layer method [24]. All phage preparations used in the experiments had a phage titer brought to a value of 5 × 10⁷ pfu/mL.

2.5. Influence of RLs on the activity of bacteriophages

One hundred microliters of bacteriophage preparation with a previously determined phage titer were introduced into a plastic tube and 100 µl of RLs were added to obtain final concentrations: 500 mg/L, 250 mg/L, 125mg/L. The final volume of the solutions was 200 µL. The mixtures were incubated for 24 h at 37℃ and then phage titer was determined with a standard double-layer method [24].

2.6. Bacterial control lysates

Two bacterial cultures were prepared by transferring two single colonies into each of the two plastic tubes containing 30 mL of LB. The cultures were incubated for 20 hours (37°C, 150 rpm) and after incubation 10 mL of respective bacteriophage preparation was added to one culture. Optical density at 600 nm (OD₆₀₀) of the culture with phage was monitored in 30-min intervals and when it started to drop (amplified phages were released from cells due to bacterial lysis), the other culture was disrupted by ultrasounds with an amplitude of 40 for 10 minutes (Vibra cell, Sonics and Materials, USA). Both cultures were filtered through syringe filters with a diameter of 33 mm and PES membrane pore size of 0.22 µm (Merck-Millipore, USA).

2.7. Effect of rhamnolipid and bacteriophages on the growth of Candida albicans

The experiment examining the effects of rhamnolipid and bacteriophages on C. albicans was carried out in liquid culture using a 96-well plate. Each well initially contained 100 µL of YPD medium. To achieve the desired concentrations (200 mg/L, 100 mg/L, 50 mg/L for RLs and 5 x 10⁷ pfu/mL for phages) 100 µL of rhamnolipid or bacteriophages prepared at twice higher concentrations were added. A control well with 200 µL of YPD medium was included. Each well was inoculated with 1 µL of an overnight culture of C. albicans and the plate was incubated for 24 hours at 37°C. Optical density at 600 nm (OD₆₀₀) was measured using microplate reader TECAN Spark 10 M (Tecan Group Ltd, Männedorf, Switzerland). Each variant was tested in triplicate, then the results were averaged and presented in a graph. A Student's t-test was performed to test the significance of the differences between the control and test samples.

2.8. Anti-adhesion assays

Inhibition of microbial adhesion by RLs, bacteriophages and their combinations was tested in 96-well plates (Sarstedt, Nümbrecht, Germany). Briefly, the wells of a sterile 96-well flat-bottom plate were filled with 100 µL of 50, 100 and 200 mg/mL RLs, bacteriophages (5 × 10⁷ pfu/mL) and their mixtures. The plates were incubated for 2 hours at 37°C on a rotary shaker (MixMate, Eppendorf, Hamburg, Germany) at 300 rpm and subsequently washed twice with sterile water. Control wells contained YPD medium only. The overnight culture of Candida strain was centrifuged, washed twice with sterile water and re-suspended in YPD medium to an optical density OD₆₀₀ = 0.6. A 100 µL aliquot of Candida suspension was added and incubated in the wells. After a 2 hours incubation at 37°C in a rotary shaker at 300 rpm nonadherent cells were removed by three washes with sterile water. Then the plates were stained with 0.1% crystal violet for 5 minutes and again washed three times with sterile water. The adherent Candida cells were permeabilized and the dye was resolubilized with 100 µL of isopropanol per well. Crystal violet optical density readings of each well were taken at 570 nm on the microplate reader TECAN Spark 10 M (Tecan Group Ltd, Männedorf, Switzerland). The assays were carried out in three replicates.

2.9. In vitro anti-biofilm assay

Briefly, 96-well plates were seeded with 100 µL aliquots of C. albicans cells (1.0 × 10⁶ cells/mL) in YPD medium and incubated statically at 37 ℃ for 1.5 hours for initial adhesion. After adhesion of cells to the plate surface, the suspension was gently aspirated and washed with sterile water to remove non-adhering cells. Fresh YPD medium with different concentrations of RLs (50, 100, and 200 mg/L), bacteriophages (5 × 10⁷ pfu/mL) and their mixtures were added, followed by incubation for another 24 hours. After incubation, the supernatant was gently aspirated and each well was washed with sterile water. Then the plates were stained with 0.1% crystal violet for 5 minutes and again washed three times with sterile water. The adherent Candida cells were permeabilized and the dye was resolubilized with 100 µL of isopropanol per well. Crystal violet optical density readings of each well were taken at 570 nm on the microplate reader TECAN Spark 10 M (Tecan Group Ltd, Männedorf, Switzerland). The assays were carried out in three replicates.

2.10. Quantification of gene expression by quantitative real-time PCR (qRT-PCR)

Effect of RLs (200 mg/L) and RLs with bacteriophages (5 × 10⁷ pfu/mL) on the expression of hypha specific genes HWP1 (hyphal wall protein 1), ALS3 (agglutinin-like sequence 3), ECE1 (extent of cell elongation 1) and SAP4 (secreted aspartyl protease 4) was evaluated by two-step quantitative real-time polymerase chain reaction (qRT-PCR). Samples contained C. albicans (5.0 × 10⁶ cells/mL) culture and RLs (200 mg/L) or RLs with bacteriophages (5 × 10⁷ pfu/mL) were incubated at 37°C for 6 hours and then centrifuged at 1000 × g for 5 minutes. Subsequently, the RNA was extracted from cells using a Total RNA Mini Plus kit (A&A Biotechnology, Poland). Each sample was treated with DNase I (Thermo Scientific) for 30 minutes at 37 ℃ and next with EDTA for 10 minutes at 65 ℃. The RNA quantities were measured using a Biochrom WPA Biowave II spectrophotometer (Biochrom Ltd., UK) equipped with a TrayCell system (Hellma Analytics, Germany). The cDNA synthesis was conducted according to Maxima First Strand cDNA. Synthesis kits for qRT-PCR (Thermo Scientific) were used according to the manufacturer’s instructions. The qRT-PCR analyses were carried out using a DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific) and the Eco Real-Time PCR System (Illumina, USA) with 1 ng/µL cDNA in the final reaction. Sequences of all primers (ALS3, ECE1, HWP1, and SAP4) used in the study are listed in Table S3. The results were normalized to the actin gene (ACT1) and analyzed using the ddCT method [25]. The experiment was performed in three biological replicates. Statistical significance was determined using binomial, unpaired Student’s t-test. A P-value of 0.05 was considered to be statistically significant.

2.11. Hypha formation

Hyphal growth assay was performed in 10 mL of RPMI-1640 medium (Sigma-Aldrich, St Louis, MO, USA). Cell suspension was diluted at 1 × 10⁷ cells/mL in medium and incubated with RLs (200 mg/L) and RLs with bacteriophages (5 × 10⁷ pfu/mL) at 37°C for 5 hours. Aliquots of samples were visualized under bright field using 40 objective lens by Zeiss fluorescence microscope and photographed.

3.1. Critical micelle concentration (CMC)

The critical micelle concentration (CMC) is a pivotal parameter that describes surfactant compounds, signifying the minimum concentration of molecules requisite for achieving the lowest surface tension [7]. In elucidating the increase of anti-biofilm properties (section 3.4) at concentrations above 50 mg/L, RLs surface tension was measured in a YPD medium by the du Noüy’s ring method. Through measurements and curve plotting, the CMC for RLs was ascertained to be 44.2 mg/L (Fig. 3). Noteworthy is the fact that biosurfactants remarkably attenuated the surface tension of the YPD medium from 42.1 to 30 mN/m, with concentrations of 5.15 mg/L and 150 mg/L of RLs, respectively, in contrast to the free of RLs YPD medium's surface tension of 51.4 mN/m. Notably, even a minute concentration of rhamnolipid at 5.15 mg/L yielded a surface tension reduction of nearly 10 mN/m. This observation suggests that the mechanisms underpinning the antiadhesive activity of RLs against C. albicans may strongly hinge on micelle formation.

Literature posits that the CMC value of RLs is contingent upon the composition of homologues, particularly the ratio of di-RLs to mono-RLs. Li et al. determined the CMC for RL products derived from soybean oil and glycerol, revealing values of 65 mg/L and 50 mg/L, respectively. They juxtaposed these findings with a standard sample exhibiting a CMC of approximately 110 mg/L, containing almost three and two times fewer di-RLs compared to the test samples [26]. Hence, it can be inferred that the CMC value correlates with the di-RLs content in the mixture.

CMC values exhibit notable variability. For instance, Sharma et al [27] reported a study wherein biosurfactants synthesized by Pseudomonas aeruginosa MTCC7815, utilizing waste cooking oil as the sole carbon source, displayed a composite of mono- and di-RLs with a low CMC of 8.8 ± 0.3 mg/L. Conversely, another study published in 2023 involved the production of rhamnolipid using P. aeruginosa FA1 in solid-state fermentation with peanut meal as the substrate. Rhamnolipid obtained a CMC equal to 70 mg/L [28].

3.2. Influence of RLs on the activity of bacteriophages

To assess the potential inhibitory effects of RLs on bacteriophages, phages were subjected to incubation with varying concentrations of glycolipids (Table 1). Regardless of the concentration of RLs and the type of phage used, in all cases, the phage titer did not drop below the value of 1 x 10⁸ pfu/mL. The most notable reduction recorded was observed for bacteriophage LO5/1f. However, even in this instance, a considerable portion of viable viruses (2,75 x 10⁸ pfu/mL) was detected following incubation with RLs.

While numerous scientific reports highlight the inhibitory properties of biosurfactants against human viruses [29, 30, 31], to the best of our knowledge, there is a dearth of data regarding their impact on bacteriophages. Slightly more information can be gleaned concerning the influence of synthetic surfactants on phage activity. Interestingly, contrary to our observations, most reports suggest an adverse effect of surfactants on phages [32, 33]. Nevertheless, Fister et al [34] conducted an experiment wherein bacteriophage P100 was subjected to incubation with two surfactants, Lutensol (5%) and Sodium Dodecyl Sulfate (SDS) (5%), with the phage titer evaluated after 1 hour, 6 hours, and 24 hours. The results indicated that while Lutensol did not alter the phage titer within a day, SDS caused a drop in phage titer of 1.2 log10 units. These observations suggest that the impact of surfactants on phage viability may depend on the type of molecule employed.

The impact of bacteriophages and rhamnolipid individually on the growth of C. albicans in liquid culture was also assessed (Fig. S1). Based on the Student's t-test, there was no significant difference between the test samples and the control, indicating that neither agent inhibits the growth of C. albicans.

Table 1

Bacteriophage titer (pfu/mL) after incubation with RLs solutions.
Bacteriophage	Phage titer [pfu/mL]	Phage titer after incubation with 125 mg/L RLs	Phage titer after incubation with 250 mg/L RLs	Phage titer after incubation with 500 mg/L RLs
BF9	7,51 x 10⁸	1,55 x 10⁸	1,38 x 10⁸	1,23 x 10⁸
BF15	2,35 x 10⁹	1,91 x 10⁹	1,68 x 10⁹	1,43 x 10⁹
BF17	1,70 x 10⁹	9,51 x 10⁸	1,60 x 10⁹	1,15 x 10⁹
FD	2,5 x 10¹⁰	1,57 x 10¹⁰	9,50 x 10⁹	5,0 x 10⁹
Felix	9,13 x 10⁹	9,10 x 10⁹	8,02 x 10⁹	8,34 x 10⁹
JG004	1,97 x 10⁹	1,02 x 10⁹	9,5 x 10⁸	8,25 x 10⁸
LO5/1f	1,92 x 10⁹	2,55 x 10⁸	2,75 x 10⁸	2,0 x 10⁸
T4	3,50 x 10⁸	3,31 x 10⁸	2,80 x 10⁸	2,75 x 10⁸
TO1/6f	5,10 x 10⁹	2,05 x 10⁹	1,26 x 10⁹	8,35 x 10⁸
TO1/7f	4,97 x 10⁹	4,49 x 10⁹	3,06 x 10⁹	2,02 x 10⁹

3.3. Antiadhesive properties of RLs, bacteriophages and their combinations

The initial attachment of cells to a surface plays a pivotal role in the formation of a biofilm structure [35]. Consequently, many innovative approaches are directed towards thwarting biofilm formation by engineering anti-adhesive surfaces [36]. The findings of this study unveiled that RLs might impede biofilm formation by C. albicans in a concentration-dependent manner (Table 2). These observations align with prior studies demonstrating the antiadhesive prowess of RLs in thwarting biofilm formation by C. albicans [37], as well as other microorganisms, including bacteria [38, 39, 40, 41] and filamentous fungi [42].

Conversely, it is widely recognized that bacteriophages possess the capability to adhere to abiotic surfaces [42]. Furthermore, previous research has illustrated that this particular feature of phages could be involved in developing safeguarding surfaces against bacterial contamination. For instance, phages may be used in designing coating materials aimed at preventing biofilm formation on surfaces such as catheters, materials particularly prone to microbial infections [43]. The study's outcomes indicated that despite the demonstrated inhibitory effect of phages in anti-adhesion treatments against C. albicans cells, the effect is notably weaker compared to that observed for RLs. The least inhibition of biofilm formation, a mere 4.9%, was noted for bacteriophage LO5/1f compared to the control lacking phages. Conversely, the most robust inhibitory effect was observed for bacteriophages T4 and TO1/6f, which decreased biofilm formation by 17.7% and 19.6%, respectively, in comparison to the control.

The primary objective of this study was to assess the antiadhesive properties of mixtures comprising RLs and bacteriophages in C. albicans biofilm formation. It is noteworthy that in all instances, irrespective of the RLs concentration and the type of phage used, combinations of both antimicrobials exhibited a stronger inhibitory effect against biofilm formation by Candida cells compared to when these factors were applied individually. At the highest biosurfactant concentration, the reduction in biofilm formation reached nearly 95% for most phages. Particularly, the combination of 200 mg/L rhamnolipid with phage BF9 emerged as the most efficacious, achieving a 94.8% decrease compared to the control with an untreated surface. However, even at the lowest rhamnolipid concentration (50 mg/L), combination with phages resulted in approximately an 80% reduction in biofilm formation (ranging from 77.8% for phage BF15 to 85% for phage T4).

Significantly, bacteriophage preparations, undergoing the amplification process, may contain bacterial residues stemming from host cell lysis. Therefore, to ascertain whether the observed synergistic effect was not influenced by bacterial fragments, bacterial lysates devoid of phages were prepared. Subsequent experiments were conducted analogously to those with preparations containing bacterial viruses. No inhibitory effect on biofilm formation by C. albicans cells was observed with bacterial lysates, indicating that phages are indeed the agents enhancing the antiadhesive activity of RLs.

Table 2. The percentage of biofilm formation by C. albicans on surfaces pretreated with RLs, bacteriophages, and their combinations. The results represent the averages of triplicate experiments ± standard deviations. The colors red and shades of orange correspond to the most significant inhibition of biofilm formation, yellow corresponds to intermediate inhibition of biofilm formation, while green indicates the weakest inhibition.

a – statistically insignificant results; Student’s t-test was used to compare values obtained from individual experimental models, where the surface was pretreated with RLs, phages, or combinations of these agents before introducing Candida cells. Differences were considered significant at a P-value of < 0.05.

3.4. Anti-biofilm properties of RLs, bacteriophages and their mixtures

In an experimental model where rhamnolipid and bacteriophages were concurrently introduced into the wells alongside C. albicans, the outcomes mirrored those observed when antibacterials were utilized to pre-coat the surface to prevent biofilm formation. Across all concentrations of RLs employed in individual experimental models, inhibition of Candida biofilm formation was evident. However, for concentrations of 50 mg/L and 100 mg/L, the inhibitory effect was comparable, amounting to approximately 70% biofilm inhibition (Table 3). Notably, at the highest RLs concentration tested in the experiment, the inhibition was even more significant, reaching 77%.

Bacteriophages exhibited a more pronounced inhibition of biofilm formation by Candida cells compared to the previous preadhesion model. Nonetheless, the effectiveness varied significantly among different phages. For instance, phages BF5 and Felix inhibited Candida biofilm formation by only 6% and 8.1% respectively, compared to the control. Conversely, phages TO1/6f, FD, and LO5/1f demonstrated stronger inhibitory properties, reducing biofilm formation by 30.4%, 32.1%, and 35.5% respectively, compared to the control. Despite the limited scientific data on phage interactions with yeast cells, previous observations have indicated the inhibition of C. albicans biofilm formation by phages. Namely, Nazik et al [20] demonstrated that phage Pf4, specific to P. aeruginosa, could inhibit C. albicans biofilm formation. This effect was correlated with the phage titer in the preparation, and notably, the phage was also effective in eliminating pre-formed biofilm.

Similar to the previous experimental model, synergy between RLs and phages was evident for each biosurfactant concentration and each phage tested. The most pronounced inhibitory effect was observed with the highest rhamnolipid concentration (200 mg/L) in combination with phage LO5/1f. This combination resulted in nearly complete inhibition of biofilm formation, approximately 96% compared to the control. It is noteworthy that phages BF17, FD, and JG004 also significantly reduced Candida biofilm formation, achieving inhibition rates of 90.5%, 91.7%, and 91.8% respectively.

Table 3. The percentage of biofilm formation by C. albicans with RLs, bacteriophages, and their combinations. The results represent the averages of triplicate experiments ± standard deviations. The colors red and shades of orange correspond to the most significant inhibition of biofilm formation, yellow corresponds to intermediate inhibition of biofilm formation, while green indicates the weakest inhibition.

a – statistically insignificant results; Student’s t-test was used to compare values obtained from individual experimental models, where Candida cells were introduced together with RLs, phages, or combinations of these agents. Differences were considered significant at a P-value of < 0.05.

3.5. Expression of genes responsible for biofilm formation by C. albicans in the presence of RLs, phages and their combinations

Four experimental models demonstrating the most robust inhibition of biofilm formation were selected for the subsequent stage of the study, in which the synergistic effect of RLs and phages was analyzed at the molecular level. Four genes associated with biofilm formation were chosen, and their expression was assessed in the presence of RLs, phages, and their combinations. The HWP1 gene encodes a cell surface protein exclusively expressed on hyphae, playing diverse roles including cell wall assembly, intracellular signaling, hyphal development, and adhesion to epithelial cells. The products of the ALS3, ECE1, and SAP4 genes, along with HWP1, contribute to hyphal growth [44, 45].

The results showed that rhamnolipids may significantly reduce biofilm formation by downregulating the expression of genes related to this structure. This aligns with the findings of Haque et al [46], who demonstrated that sophorolipids can inhibit the expression of hypha-specific genes HWP1, ALS1, ALS3, ECE1, and SAP4 in C. albicans, possibly explaining the inhibitory effect of these compounds on biofilm formation. Although there are no analogous data on rhamnolipids, Saadati et al [47] showed that rhamnolipids reduced the expression of quorum-sensing pathway genes agrA, agrC, icaA, and icaD, which are involved in the biofilm formation of methicillin-resistant Staphylococcus aureus. These data suggest that biosurfactants may influence biofilm formation by downregulating the genes responsible for this structure in various microorganisms.

On the other hand, there are no reports indicating that a similar mechanism operates in the case of bacteriophages, even for bacterial biofilms. The main mechanisms considered responsible for biofilm degradation by phages are: (i) replication within cells and the spread of progeny bacteriophages through the biofilm, progressively removing cell layers; (ii) production of depolymerizing enzymes that degrade the EPS; (iii) induction of depolymerizing enzymes from within the host genome; and (iv) persistence in cells that, upon reactivation, commence a productive infection, thereby destroying the cells [48]. However, in this study, phage LO5/1f reduced the expression of all tested genes, while phage FD downregulated the expression of HWP1 and ALS3. This suggests that influencing the expression of genes related to biofilm formation may be another potential mechanism by which phages interfere with biofilm, at least in some models.

Notably, the expression of each of the selected genes was significantly inhibited when Candida cells were treated with mixtures of RLs and bacteriophages compared to groups where biosurfactants and phages were used separately (Fig. 4a-d).

3.6. Microscopic observation of hyphae formation by Candida cells

The synergistic effect of RLs and phages in inhibiting hyphae formation was subsequently validated through microscopic observation. Similarly, the same four models demonstrating the strongest inhibition of biofilm formation were selected for the experiments. Cells treated with mixtures of RLs and phages exhibited a significant reduction in hyphae formation compared to the control group comprising untreated cells, as well as in groups where RLs and phages were applied separately (Fig. 5).

Eliminating biofilms presents formidable challenges due to their intricate structure and resistance mechanisms. Traditional antifungal treatments often struggle to penetrate the dense biofilm matrix, resulting in incomplete eradication. Consequently, this can foster chronic and recalcitrant infections, particularly among individuals with compromised immune systems. Developing strategies to disrupt biofilm formation stands as a pivotal endeavor for combating persistent infections caused by biofilm-forming microorganisms such as Candida.

New approaches are focused on preventing biofilm formation by the development of antiadhesive surfaces or by the inhibition or reduction of pathogen adhesion. Both RLs and bacteriophages exhibit the potential to impede the formation of Candida biofilm when introduced simultaneously with yeast cells or when the surface is pre-treated with these potent antimicrobials. It is worth noting that the efficacy of biosurfactants surpasses that observed for bacterial viruses. This outcome was anticipated, given that phages lack a natural tropism for eukaryotic cells. Nonetheless, the findings of this study corroborate those of numerous others, suggesting the occurrence of such interactions. Moreover, these findings fall within an uncharted scientific field exploring the mutual interplay between bacteriophages and yeast cells.

In alignment with the prevailing scientific trend of employing combination therapy featuring antibacterials with diverse modes of action to combat infections caused by a myriad of pathogens, this study sought to investigate the potential synergistic effects of RLs and phages in preventing Candida biofilm formation. The results of this study revealed that combined therapy of RLs and bacteriophages may provide a new perspective for eliminating biofilms formed by C. albicans. Furthermore, the inhibitory effect was markedly stronger when antimicrobials were administered together, compared to their separate application. Although the molecular underpinnings of this phenomenon remain elusive, the undeniable synergistic effect between RLs and phages presented in this study delineates novel prospects for the application of these two renowned antibacterial agents.

Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used ChatGPT in order to check grammar and spelling. After using this tool authors reviewed and edited the content as needed and took full responsibility for the content of the publication.

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

J. Talapko, M. Juzbašić, T. Matijević, E. Pustijanac, S. Bekić, I. Kotris, I. Škrlec, (2021). Candida albicans—the virulence factors and clinical manifestations of infection. Journal of Fungi, 7(2), 79. https://doi.org/10.3390/jof7020079
Massey, J., Zarnowski, R., Andes, D. (2023). Role of the extracellular matrix in Candida biofilm antifungal resistance. FEMS Microbiology Reviews, 47(6), fuad059. https://doi.org/10.1093/femsre/fuad059
Ajetunmobi, O. H., Badali, H., Romo, J. A., Ramage, G., Lopez-Ribot, J. L. (2023). Antifungal therapy of Candida biofilms: Past, present and future. Biofilm, 5, 100126. https://doi.org/10.1016/j.bioflm.2023.100126
Ohadi, M., Forootanfar, H., Dehghannoudeh, N., Banat, I. M., Dehghannoudeh, G. (2023). The role of surfactants and biosurfactants in the wound healing process: A review. Journal of Wound care, 32(Sup4a), xxxix-xlvi. https://doi.org/10.12968/jowc.2023.32.Sup4a.xxxix
Pereira, R., Dos Santos Fontenelle, R. O., De Brito, E. H. S., De Morais, S. M. (2021). Biofilm of Candida albicans: formation, regulation and resistance. Journal of Applied Microbiology, 131(1), 11-22. https://doi.org/10.1111/jam.14949
Yin, W., Xu, S., Wang, Y., Zhang, Y., Chou, S. H., Galperin, M. Y., He, J. (2021). Ways to control harmful biofilms: prevention, inhibition, and eradication. Critical reviews in microbiology, 47(1), 57-78. https://doi.org/10.1080/1040841X.2020.1842325
Sarubbo, L. A., Maria da Gloria, C. S., Durval, I. J. B., Bezerra, K. G. O., Ribeiro, B. G., Silva, I. A., Twigg, M. S., Banat, I. M. (2022). Biosurfactants: Production, properties, applications, trends, and general perspectives. Biochemical Engineering Journal, 181, 108377. https://doi.org/10.1016/j.bej.2022.108377
Vieira, I. M. M., Santos, B. L. P., Ruzene, D. S., Silva, D. P. (2021). An overview of current research and developments in biosurfactants. Journal of Industrial and Engineering Chemistry, 100, 1-18. https://doi.org/10.1016/j.jiec.2021.05.017
Sanches, M. A., Luzeiro, I. G., Alves Cortez, A. C., Simplício de Souza, É., Albuquerque, P. M., Chopra, H. K., Braga de Souza, J. V. (2021). Production of biosurfactants by Ascomycetes. International Journal of Microbiology, 2021. https://doi.org/10.1155/2021/6669263
Drakontis, C. E., Amin, S. (2020). Biosurfactants: Formulations, properties, and applications. Current Opinion in Colloid & Interface Science, 48, 77-90. https://doi.org/10.1016/j.cocis.2020.03.013
Giri, S. S., Ryu, E., Sukumaran, V., Park, S. C. (2019). Antioxidant, antibacterial, and anti-adhesive activities of biosurfactants isolated from Bacillus strains. Microbial pathogenesis, 132, 66-72. https://doi.org/10.1016/j.micpath.2019.04.035
Kumar, R., Barbhuiya, R. I., Bohra, V., Wong, J. W., Singh, A., Kaur, G. (2023). Sustainable rhamnolipids production in the next decade–advancing with Burkholderia thailandensis as a potent biocatalytic strain. Microbiological Research, 127386. https://doi.org/10.1016/j.micres.2023.127386
Li, Q. (2017). Rhamnolipid synthesis and production with diverse resources. Frontiers of Chemical Science and Engineering, 11, 27-36. https://doi.org/10.1007/s11705-016-1607-x
Esposito, R., Speciale, I., De Castro, C., D’Errico, G., Russo Krauss, I. (2023). Rhamnolipid Self-Aggregation in Aqueous Media: A Long Journey toward the Definition of Structure–Property Relationships. International Journal of Molecular Sciences, 24(6), 5395. https://doi.org/10.3390/ijms24065395
Mishra, A., Tiwari, P., Pandey, L. M. (2023). Surface, interfacial and thermodynamic aspects of the Rhamnolipid-salt systems. Journal of Molecular Liquids, 384, 122245. https://doi.org/10.1016/j.molliq.2023.122245
Strathdee, S. A., Hatfull, G. F., Mutalik, V. K., Schooley, R. T. (2023). Phage therapy: From biological mechanisms to future directions. Cell, 186(1), 17-31. https://doi.org/10.1016/j.cell.2022.11.017
Jończyk-Matysiak, E., Łodej, N., Kula, D., Owczarek, B., Orwat, F., Międzybrodzki, R., Neuberg, J., Bagińska, N., Weber-Dąbrowska, B., Górski, A. (2019). Factors determining phage stability/activity: Challenges in practical phage application. Expert review of anti-infective therapy, 17(8), 583-606. https://doi.org/10.1080/14787210.2019.1646126
Górski, A., Borysowski, J., Miȩdzybrodzki, R. (2021). Bacteriophage interactions with epithelial cells: therapeutic implications. Frontiers in microbiology, 11, 631161. https://doi.org/10.3389/fmicb.2020.631161
Bichet, M. C., Adderley, J., Avellaneda-Franco, L., Magnin-Bougma, I., Torriero-Smith, N., Gearing, L. J., Deffrasnes, C., David, C., Pepin, G., Gantier, M. P., Lin, R. CY., Patwa, R., Moseley, G. W., Doerig, C., Barr, J. J. (2023). Mammalian cells internalize bacteriophages and use them as a resource to enhance cellular growth and survival. PLoS Biology, 21(10), e3002341. https://doi.org/10.1371/journal.pbio.3002341
Nazik, H., Joubert, L. M., Secor, P. R., Sweere, J. M., Bollyky, P. L., Sass, G., Cegelski, L., Stevens, D. A. (2017). Pseudomonas phage inhibition of Candida albicans. Microbiology, 163(11), 1568-1577. https://doi.org/10.1099/mic.0.000539
Penner, J. C., Ferreira, J. A., Secor, P. R., Sweere, J. M., Birukova, M. K., Joubert, L. M., Haagensen, J. A. J., Garcia, O., Malkovskiy, A. V., Kaber, G., Nazik, H., Manasherob, R., Spormann, A. M., Clemons, K. V., Stevens, D. A., Bollyky, P. L. (2016). Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration. Microbiology, 162(9), 1583-1594. https://doi.org/10.1099/mic.0.000344
Havenga, B., Reyneke, B., Waso-Reyneke, M., Ndlovu, T., Khan, S., Khan, W. (2022). Biological Control of Acinetobacter baumannii: In Vitro and in vivo activity, limitations, and combination therapies. Microorganisms, 10(5), 1052. https://doi.org/10.3390/microorganisms10051052
Skaradzińska, A., Ochocka, M., Śliwka, P., Kuźmińska-Bajor, M., Skaradziński, G., Friese, A., ... & Roesler, U. (2020). Bacteriophage amplification–A comparison of selected methods. Journal of Virological Methods, 282, 113856. https://doi.org/10.1016/j.jviromet.2020.113856
Adams, M. H. (1959). Enumeration of bacteriophage particles. The Bacteriophages, 29.
Schmittgen, T. D., Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nature protocols, 3(6), 1101-1108. https://doi.org/10.1038/nprot.2008.73
Li, Z., Zhang, Y., Lin, J., Wang, W., Li, S. (2019). High-yield di-rhamnolipid production by Pseudomonas aeruginosa YM4 and its potential application in MEOR. Molecules, 24(7), 1433. https://doi.org/10.3390/molecules24071433
Sharma, S., Datta, P., Kumar, B., Tiwari, P., Pandey, L. M. (2019). Production of novel rhamnolipids via biodegradation of waste cooking oil using Pseudomonas aeruginosa MTCC7815. Biodegradation, 30, 301-312. https://doi.org/10.1007/s10532-019-09874-x
Zhao, F., Zheng, M., Xu, X. (2023). Microbial conversion of agro-processing waste (peanut meal) to rhamnolipid by Pseudomonas aeruginosa: solid-state fermentation, water extraction, medium optimization and potential applications. Bioresource Technology, 369, 128426. https://doi.org/10.1016/j.biortech.2022.128426
Vollenbroich, D., Özel, M., Vater, J., Kamp, R. M., Pauli, G. (1997). Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis. Biologicals, 25(3), 289-297. https://doi.org/10.1006/biol.1997.0099
Hegazy, G. E., Abu-Serie, M. M., Abou-Elela, G. M., Ghozlan, H., Sabry, S. A., Soliman, N. A., Teleb, M., Abdel-Fattah, Y. R. (2022). Bioprocess development for biosurfactant production by Natrialba sp. M6 with effective direct virucidal and anti-replicative potential against HCV and HSV. Scientific Reports, 12(1), 16577. https://doi.org/10.1038/s41598-022-20091-0
Balakrishnan, S., Rameshkumar, M. R., Nivedha, A., Sundar, K., Arunagirinathan, N., Valan Arasu, M. (2023). Biosurfactants: An Antiviral Perspective. In Multifunctional Microbial Biosurfactants (pp. 431-454). Cham: Springer Nature Switzerland. https://doi.org/10.1007/978-3-031-31230-4_20
Chattopadhyay, D., Chattopadhyay, S., Lyon, W. G., Wilson, J. T. (2002). Effect of surfactants on the survival and sorption of viruses. Environmental science & technology, 36(19), 4017-4024. https://doi.org/10.1021/es0114097
Vodolazkaya, N., Laguta, A., Farafonov, V., Nikolskaya, M., Balklava, Z., Khayat, R., Stich, M., Mchedlov-Petrossyan, N., Nerukh, D. (2023). Influence of various colloidal surfactants on the stability of MS2 bacteriophage suspension. The charge distribution on the PCV2 virus surface. Journal of Molecular Liquids, 387, 122644. https://doi.org/10.1016/j.molliq.2023.122644
Fister, S., Robben, C., Witte, A. K., Schoder, D., Wagner, M., Rossmanith, P. (2016). Influence of environmental factors on phage–bacteria interaction and on the efficacy and infectivity of phage P100. Frontiers in microbiology, 7, 1152. https://doi.org/10.3389/fmicb.2016.01152
Palmer, J., Flint, S., Brooks, J. (2007). Bacterial cell attachment, the beginning of a biofilm. Journal of Industrial Microbiology and Biotechnology, 34(9), 577-588. https://doi.org/10.1007/s10295-007-0234-4
Gutiérrez, D., Rodríguez-Rubio, L., Martínez, B., Rodríguez, A., García, P. (2016). Bacteriophages as weapons against bacterial biofilms in the food industry. Frontiers in microbiology, 7, 189912. https://doi.org/10.3389/fmicb.2016.00825
Turbhekar, R., Malik, N., Dey, D., & Thakare, D. (2015). Disruption of Candida albicans biofilms by rhamnolipid obtained from Pseudomonas aeruginosa. RT. IJRSB, 3(3), 73-78.
Anjos, I., Bettencourt, A. F., Ribeiro, I. A. (2022). Antimicrobial Biosurfactants Towards the Inhibition of Biofilm Formation. In Urinary Stents: Current State and Future Perspectives (pp. 291-304). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-031-04484-7_23
Tambone, E., Bonomi, E., Ghensi, P., Maniglio, D., Ceresa, C., Agostinacchio, F., Caciagli, P., Nollo, G., Piccoli, F., Caola, I., Fracchia, L., Tessarolo, F. (2021). Rhamnolipid coating reduces microbial biofilm formation on titanium implants: An in vitro study. BMC Oral Health, 21, 1-13. https://doi.org/10.1186/s12903-021-01412-7
Ceresa, C., Tessarolo, F., Maniglio, D., Tambone, E., Carmagnola, I., Fedeli, E., Caola, I., Nollo, G., Chiono, V., Allegrone, G., Rinaldi, M., Fracchia, L. (2019). Medical-grade silicone coated with rhamnolipid R89 is effective against Staphylococcus spp. biofilms. Molecules, 24(21), 3843. https://doi.org/10.3390/molecules24213843
Alara, J. A., Alara, O. R. (2024). Antimicrobial and anti-biofilm potentials of biosurfactants. In Industrial Applications of Biosurfactants and Microorganisms (pp. 307-339). Academic Press. https://doi.org/10.1016/B978-0-443-13288-9.00001-2
Richter, Ł., Księżarczyk, K., Paszkowska, K., Janczuk-Richter, M., Niedziółka-Jönsson, J., Gapiński, J., Łoś, M., Hołyst, R., Paczesny, J. (2021). Adsorption of bacteriophages on polypropylene labware affects the reproducibility of phage research. Scientific reports, 11(1), 7387. https://doi.org/10.1038/s41598-021-86571-x
Costa, B., Barros, J., Costa, F. (2022). Antibiotic-Free Solutions for the Development of Biofilm Prevention Coatings. In Urinary Stents: Current State and Future Perspectives (pp. 259-272). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-031-04484-7_21
Malinovská, Z., Čonková, E., Váczi, P. (2023). Biofilm formation in medically important Candida species. Journal of Fungi, 9(10), 955. https://doi.org/10.3390/jof9100955
Staniszewska, M., Bondaryk, M., Malewski, T., Schaller, M. (2014). The expression of the Candida albicans gene SAP4 during hyphal formation in human serum and in adhesion to monolayer cell culture of colorectal carcinoma Caco-2 (ATCC). Open Life Sciences, 9(8), 796-810. https://doi.org/10.2478/s11535-014-0311-4
Haque, F., Alfatah, M., Ganesan, K., Bhattacharyya, M. S. (2016). Inhibitory effect of sophorolipid on Candida albicans biofilm formation and hyphal growth. Scientific Reports, 6(1), 23575. https://doi.org/10.1038/srep23575
Saadati, F., Shahryari, S., Sani, N. M., Farajzadeh, D., Zahiri, H. S., Vali, H., Noghabi, K. A. (2022). Effect of MA01 rhamnolipid on cell viability and expression of quorum-sensing (QS) genes involved in biofilm formation by methicillin-resistant Staphylococcus aureus. Scientific Reports, 12(1), 14833. https://doi.org/10.1038/s41598-022-19103-w
Harper, D. R., Parracho, H. M., Walker, J., Sharp, R., Hughes, G., Werthén, M., Lehman, S., Morales, S. (2014). Bacteriophages and biofilms. Antibiotics, 3(3), 270-284. https://doi.org/10.3390/antibiotics3030270
Yap, M. L., Rossmann, M. G. (2014). Structure and function of bacteriophage T4. Future microbiology, 9(12), 1319-1327. https://doi.org/10.2217/fmb.14.91
Mesyanzhinov, V. V. (2004). Bacteriophage T4: structure, assembly, and initiation infection studied in three dimensions. Advances in Virus Research, 63, 287-352. https://doi.org/10.1016/S0065-3527(04)63005-3
Garbe, J., Bunk, B., Rohde, M., Schobert, M. (2011). Sequencing and characterization of Pseudomonas aeruginosa phage JG004. BMC microbiology, 11, 1-12. https://doi.org/10.1186/1471-2180-11-102
Whichard, J. M., Weigt, L. A., Borris, D. J., Li, L. L., Zhang, Q., Kapur, V., William Pierson, F., Lingohr, E. J., She, Y. Kropinski, A. M., Sriranganathan, N. (2010). Complete genomic sequence of bacteriophage Felix O1. Viruses, 2(3), 710-730. https://doi.org/10.3390/v2030710
Prisco, A., De Berardinis, P. (2012). Filamentous bacteriophage FD as an antigen delivery system in vaccination. International journal of molecular sciences, 13(4), 5179-5194. https://doi.org/10.3390/ijms13045179
Śliwka, P., Weber-Dąbrowska, B., Żaczek, M., Kuźmińska-Bajor, M., Dusza, I., Skaradzińska, A. (2023). Characterization and comparative genomic analysis of three virulent E. coli bacteriophages with the potential to reduce antibiotic-resistant bacteria in the environment. International journal of molecular sciences, 24(6), 5696. https://doi.org/10.3390/ijms24065696

No competing interests reported.

WYSANYSupplementarymaterial08.07.24.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Bacteriophages improve the effectiveness of rhamnolipids in combating the biofilm of Candida albicans

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Strains, media, and compounds

2.2. Critical micelle concentration (CMC)

2.3. Bacteriophages

2.4. Amplification of bacteriophages

2.5. Influence of RLs on the activity of bacteriophages

2.6. Bacterial control lysates

2.7. Effect of rhamnolipid and bacteriophages on the growth of Candida albicans

2.8. Anti-adhesion assays

2.9. In vitro anti-biofilm assay

2.10. Quantification of gene expression by quantitative real-time PCR (qRT-PCR)

2.11. Hypha formation

3. Results and discussion

3.1. Critical micelle concentration (CMC)

3.2. Influence of RLs on the activity of bacteriophages

3.3. Antiadhesive properties of RLs, bacteriophages and their combinations

3.4. Anti-biofilm properties of RLs, bacteriophages and their mixtures

3.6. Microscopic observation of hyphae formation by Candida cells

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1