A Re-Purposing Strategy: Sub-Lethal Concentrations of an Eicosanoid Derived from the Omega-3-Polyunsaturated Fatty Acid Resolvin D1 Affect Dual Species Biofilms

Abstract

The fungal species Candida parapsilosis and the bacterial species Staphylococcus aureus may be responsible for hospital-acquired infections in patients undergoing invasive medical interventions or surgical procedures and often coinfect critically ill patients in complicating polymicrobial biofilms. The efficacy of the re-purposing therapy has recently been reported as an alternative to be used. PUFAs (polyunsaturated fatty acids) may be used alone or in combination with currently available traditional antimicrobials to prevent and manage various infections overcoming antimicrobial resistance. The objectives of the study were to evaluate the effects of Resolvin D1 (RvD1) as an antimicrobial on S. aureus and C. parapsilosis, as well as the activity against the mixed biofilm of the same two species. Microdilution assays and time-kill growth curves revealed bacterial and fungal inhibition at minimum concentration values between 5 and 10 μg mL^-1. In single-species structures, an inhibition of 55% and 42% was reported for S. aureus and C. parapsilosis, respectively. Moreover, RvD1 demonstrated an eradication capacity of 60% and 80% for single- and mixed-species biofilms, respectively. In association with the inhibition activity, a downregulation of genes involved in biofilm formation as well as ROS accumulation was observed. Eradication capability was confirmed also on mature mixed biofilm grown on silicone platelets as shown by scanning electron microscopy (SEM). In conclusion, RvD1 was efficient against mono and polymicrobial biofilms in vitro, being a promising alternative for the treatment of mixed bacterial/fungal infections.

Keywords: Candida parapsilosis; Staphylococcus aureus; mixed biofilm; polyunsaturated fatty acids; resolvin.

Figure 1

Killing kinetics of S. aureus (A) and C. parapsilosis (B) cultured in the presence of 1/2 × MIC, 1 × MIC, and 2 × MIC concentrations of RvD1 over a period of 24 h. Data reported are the mean of three independent experiments ± SD.

Figure 2

(A) Standard optical density biofilm CV assay of single-species and dual-species biofilms. (B) XTT metabolic activity assay of single-species and dual-species biofilms. (C) Cell enumeration in dual-species biofilms. Statistical significance: ** = p < 0.01; **** = p < 0.0001 (Dunnett’s test). Data reported are the mean of three independent experiments ± SD.

Figure 3

Biofilm inhibitory and eradication effect of RvD1 towards single- and mixed-species biofilms. (A) Inhibition effect of RvD1 on mono-microbial and poly-microbial biofilm formations in 96-well microplates. Single and mixed species cells were co-incubated with various concentrations (1.25, 2.5, and 5 µg mL⁻¹) of RvD1 for 24 h; and their biofilm production was compared to that of cells incubated without RvD1 (CV assay). (B) Eradication effect of RvD1 on mono- and poly-microbial mature biofilms in 96-well microtiter plates. Mature biofilms were treated with various concentrations (15, 20, and 25 µg mL⁻¹) of RvD1 for 24 h. Values obtained are given as the percentage of biofilm formation or eradication. The results shown represent the means and standard deviations (error bars) of three independent experiments.

Figure 4

Accumulation of ROS in S. aureus and C. parapsilosis mixed biofilms following RvD1 supplementation. Values are averages of triplicate experiments and error bars indicate the standard deviation. * = significantly different from control (p < 0.01).

Figure 5

Relative fold-change in gene expression of S. aureus and C. parapsilosis. (A) The chart represents changes in expression of bap, icaA, and icaD genes in mono and poly-microbial biofilms of S. aureus, in the presence of sub-inhibitory concertation of RvD1 (2.5 µg mL⁻¹) compared to untreated samples and normalized to the value of 16S rRNA. (B) The chart represents relative changes in expression of the ALS3, EFG1, and ERG11 genes in mono and poly-microbial biofilms of C. parapsilosis during inhibition with 2.5 µg mL⁻¹ RvD1 compared to untreated C. parapsilosis as a control and normalized to the value of actin. Asterisk marked fold changes significantly different from 1 (p-value < 0.05). Red broken lines indicate fold change thresholds of 2 and 0.5, respectively.

Figure 6

Eradication of mixed biofilm by RvD1 on medical grade silicone surface. Polymicrobial biofilms were grown on medical grade silicone surface for 72 h and treated with two concentrations (20; 25 µg mL⁻¹) of RvD1. (A) CFUs of each microorganism in the mixed biofilm treated compared with untreated; (B) total biomass detected by CV of the mixed biofilm treated with two concentrations of RvD1. (C) Crystal violet staining of silicone platelets colonized by S. aureus–C. parapsilosis biofilm and treated with 20 and 25 µg mL⁻¹ RvD1. ctrl = control (untreated), B = blank (without cultures), R1 = replies 1, R2 = replies 2; *** = p < 0.001; **** = p < 0.0001 (Dunnett’s test).

Figure 7

SEM images of polymicrobial biofilm formations on medical grade silicone surface with media supplemented without (A) or with RvD1 (B). Magnification, ×1000. Bar corresponds to 10 µm. Red arrows indicate S. aureus (S.a.), blue arrows indicate C. parapsilosis (C.p).

Figure 8

Cytotoxic effect (%) of RvD1 on the HaCaT cell line after 24 h of treatment with concentrations ranging from 1.25 to 10 μg mL⁻¹. Data represent mean ± standard deviation (SD) of three independent experiments.

Figure 9

Effect of RvD1 on the adhesion of S. aureus and C. parapsilosis mono- and dual-specie culture to HaCaT cells expressed by CFU mL⁻¹. The results were obtained from two independent experiments: ** = p < 0.01; *** = p < 0.001, **** = p < 0.0001 (Tukey’s test).