Coexistence of Pseudomonas aeruginosa With Candida albicans Enhances Biofilm Thickness Through Alginate-Related Extracellular Matrix but Is Attenuated by N-acetyl-l-cysteine

Abstract

Bacteria and Candidaalbicans are prominent gut microbiota, and the translocation of these organisms into blood circulation might induce mixed-organism biofilms, which warrants the exploration of mixed- versus single-organism biofilms in vitro and in vivo. In single-organism biofilms, Acinetobacter baumannii and Pseudomonas aeruginosa (PA) produced the least and the most prominent biofilms, respectively. C. albicans with P. aeruginosa (PA+CA) induced the highest biofilms among mixed-organism groups as determined by crystal violet straining. The sessile form of PA+CA induced higher macrophage responses than sessile PA, which supports enhanced immune activation toward mixed-organism biofilms. In addition, Candida incubated in pre-formed Pseudomonas biofilms (PA>CA) produced even higher biofilms than PA+CA (simultaneous incubation of both organisms) as determined by fluorescent staining on biofilm matrix (AF647 color). Despite the initially lower bacteria during preparation, bacterial burdens by culture in mixed-organism biofilms (PA+CA and PA>CA) were not different from biofilms of PA alone, supporting Candida-enhanced Pseudomonas growth. Moreover, proteomic analysis in PA>CA biofilms demonstrated high AlgU and mucA with low mucB when compared with PA alone or PA+CA, implying an alginate-related mucoid phenotype in PA>CA biofilms. Furthermore, mice with PA>CA biofilms demonstrated higher bacteremia with more severe sepsis compared with mice with PA+CA biofilms. This is possibly due to the different structures. Interestingly, l-cysteine, a biofilm matrix inhibitor, attenuated mixed-organism biofilms both in vitro and in mice. In conclusion, Candida enhanced Pseudomonas alginate-related biofilm production, and Candida presentation in pre-formed Pseudomonas biofilms might alter biofilm structures that affect clinical manifestations but was attenuated by l-cysteine.

Keywords: Candida albicans; Pseudomonas aeruginosa; alginate; biofilms; l-cysteine.

Figure 1

Diagram of the experimental design demonstrated lower bacteria in the biofilm preparation of mixed bacteria and Candida (bacteria + C. albicans) when compared with single-organism biofilms (A), crystal violet–stained biofilm in 96-well plates from single- and mixed-organisms in the time course evaluation (B), and in the biofilms at 48 h post-incubation with representative pictures of crystal violet stained 96-well plates and the color with acetic acid elution (C) are demonstrated. Mean ± SE was used for data presentation, and the differences between groups were examined for statistical significance by one-way ANOVA followed by Tukey’s analysis for comparisons of multiple groups or 2 groups (B, C). A p-value of < 0.05 was considered statistically significant. (Independent triplicate experiments were performed.) (*p<0.05; ^# p<0.05).

Figure 2

Intensity of fluorescent stains from 48 h biofilm on cover glasses of P. aeruginosa alone, Candida alone, and P. aeruginosa + C. albicans (P. aeruginosa + C. albicans) for ECM by AF647 (red color fluorescence), bacterial nucleic acid by SYTO9 (green color fluorescence), and fungal cell wall by calcofluor white (CW2MR; blue color fluorescence) (A) with the representative fluorescent images (B) are demonstrated. There was different depth on biofilms in images of the P. aeruginosa + C. albicans group because of the prominent biofilm thickness. Mean ± SE was used for data presentation, and the differences between groups were examined by one-way ANOVA followed by Tukey’s analysis for comparisons of multiple groups or 2 groups (A). A p-value of < 0.05 was considered statistically significant. (Independent triplicate experiments were performed.) (*p<0.05; ^# p<0.05).

Figure 3

Biofilm thickness from 48 h biofilm on cover glasses of P. aeruginosa alone, Candida alone, and P. aeruginosa + C. albicans (P. aeruginosa + C. albicans) as evaluated by z-stack analysis of fluorescent images (A) and the burdens of organisms by culture for bacteria (TSA) and for fungi (SDA) (B, C) are demonstrated. Mean ± SE was used for data presentation, and the differences between groups were examined by one-way ANOVA followed by Tukey’s analysis for comparisons of multiple groups or 2 groups, (A–C). A p-value of < 0.05 was considered statistically significant. (Independent triplicate experiments were performed.)

Figure 4

Diagram of the experimental design demonstrated the lower bacteria during biofilm preparation of P. aeruginosa and C. albicans (P. aeruginosa + C. albicans) compared with P. aeruginosa alone with the score of fluorescent intensity for ECM (AF647), bacterial nucleic acid (SYTO9), and fungal cell wall (CW2MR) together with representative fluorescent images (right side) with eye visualization biofilms (below) of the catheter biofilms after 48 h in an incubator (A) and in mice (B) are demonstrated. Mean ± SE was used for data presentation, and the differences between groups were examined by Student’s t-test for comparisons of multiple groups or 2 groups (A, B). A p-value of < 0.05 was considered statistically significant. (Independent triplicate experiments were performed for A and n = 5/group for B.)

Figure 5

Characteristics of RAW264.7 cells (macrophages) after 6 h incubation of P. aeruginosa, C. albicans, or P. aeruginosa with C. albicans (combined) in sessile (biofilm) and planktonic form (free-living) as determined by supernatant cytokines (TNF-α, IL-6, IL-10) (A–C), gene expression of macrophage polarization of M1 (iNOS, IL-1β, TNF-α) and M2 (Arg-1, FIZZ-1, TGF-β) (D–I) are demonstrated. Mean ± SE was used for data presentation, and the differences between groups were examined by one-way ANOVA followed by Tukey’s analysis for comparisons of multiple groups or 2 groups (A–I). A p-value of < 0.05 was considered statistically significant. (Independent triplicate experiments were performed.) (*p<0.05; ^# p<0.05).

Figure 6

Diagram of the experimental design demonstrated lower bacteria in the biofilm preparation of simultaneous incubation of P. aeruginosa and Candida (P. aeruginosa + C. albicans) or the Candida addition in 24 h biofilm-formed bacteria (P. aeruginosa > C. albicans) in comparison with P. aeruginosa biofilms (A), crystal violet–stained biofilm in 96-well plates with representative pictures (B), fluorescent intensity of ECM by AF647 (red), bacterial nucleic acid by SYTO9 (green), and fungal cell wall by calcofluor white (CW2MR; blue) induced on cover glass biofilms and catheters (in incubator) (C, D) and organism burdens from biofilms using TSA for bacteria (E) and SDA for fungi (F) are demonstrated. Mean ± SE was used for data presentation, and the differences between groups were examined by one-way ANOVA followed by Tukey’s analysis for comparisons of multiple groups or 2 groups, respectively (B–F). A p-value of < 0.05 was considered statistically significant. (Independent triplicate experiments were performed.) (*p<0.05; ^# p<0.05).

Figure 7

Representative fluorescent images of biofilms induced on cover glasses and catheters (in incubator) (A, B) stained for ECM by AF647 (red), bacterial nucleic acid by SYTO9 (green), and fungal cell wall by calcofluor white (CW2MR; blue) are demonstrated.

Figure 8

Proteomic analysis of biofilms from P. aeruginosa alone (PA) and PA with Candida by simultaneous incubation of Candida together with bacteria (P. aeruginosa + C. albicans; PA+CA) or the Candida addition in 24 h biofilm-formed bacteria (P. aeruginosa > C. albicans; PA>CA) as determined by the peak intensity of proteins in the ECM production process (AlgU, RNA polymerase sigma H factor; mucA, sigma factor AlgU negative regulatory protein; and mucB, negative regulator of the sigma factor AlgU) (A) and those of biofilms from Candida albicans alone (CA) and CA simultaneously incubated together with P. aeruginosa (P. aeruginosa + C. albicans; PA+CA) or the Candida addition in 24 h biofilm-formed bacteria (P. aeruginosa > C. albicans; PA>CA) as determined by the peak intensity of proteins in the ECM production process (ADH2, alcohol dehydrogenase) (B). Mean ± SE was used for data presentation, and the differences between groups were examined by one-way ANOVA followed by Tukey’s analysis (A, B). A p-value of < 0.05 was considered statistically significant. (Independent duplicate experiments were performed.)

Figure 9

Effect of the different concentrations of l-cysteine and β-defensin 3 on bacterial burdens in TSB culture of planktonic P. aeruginosa after 24 h incubation (A) and crystal violet–stained biofilms on 96-well plates after 48 h incubation (B, C) are demonstrated. Mean ± SE was used for data presentation. The differences between groups were examined by Student’s t-test (A) and by one-way ANOVA followed by Tukey’s analysis (B, C). The differences between the individual time points versus the baselines were examined by repeated-measures ANOVA analysis (A–C). A p-value of < 0.05 was considered statistically significant. (Independent triplicate experiments were performed.)

Figure 10

Diagram of some important genes that regulate ECM protein formation (A) demonstrates i) Pseudomonas produces 3-oxo-HSL and several proteins to initiate biofilms (yellow highlights are selected genes to explore in this study) and secretes pyocyanin and C₁₂-HSL to inhibit Candida growth (Kerr et al., 1999), ii) Candida produces Tyrosol and Farnasol for fungal biofilms (Pierce, 2005; McAlester et al., 2008), iii) bacterial attachment on Candida pseudohyphae on mixed-organism biofilms (Fourie et al., 2016), and iv) biofilm inhibition by mucB in planktonic bacteria (left side) and alginate synthesis by AlgU in sessile bacteria (right side) (Schurr et al., 1996). Additionally, expression of some genes from biofilms of P. aeruginosa alone (PA) and PA with Candida by initially simultaneous incubation (P. aeruginosa + C. albicans) or the Candida addition at 24 h biofilm-formed bacteria (P. aeruginosa > C. albicans) with or without l-cysteine (anti-biofilm) (B–E) are demonstrated. Mean ± SE was used for data presentation, and the differences between groups were examined by one-way ANOVA followed by Tukey’s analysis for comparisons of multiple groups or 2 groups (B–E). A p-value of < 0.05 was considered statistically significant. (Independent triplicate experiments were performed.) (*p<0.05; ***p<0.001).

Figure 11

Diagram of the preparation of catheter biofilms of P. aeruginosa alone (PA) and with Candida by simultaneous incubation of Candida together with bacteria (P. aeruginosa + C. albicans) or the Candida addition in 1.5 h biofilm-formed bacteria (P. aeruginosa > C. albicans) before 48 h of subcutaneous implantation in mice (A) are indicated. Characteristics of the catheter insertion mouse model as determined by survival analysis (B), kidney and liver injury (serum creatinine and alanine transaminase) (C, D), bacteremia (E), serum cytokines (F), and organism burdens of catheter biofilms (G) are demonstrated. Additionally, characteristics of mice with P. aeruginosa > C. albicans biofilm catheters with or without l-cysteine incubation as evaluated by these parameters (H–P) are demonstrated. The survival analysis (B, H) and the differences between groups were examined by Log-rank test and one-way ANOVA followed by Tukey’s analysis, respectively (C–G, I–P). A p-value of < 0.05 was considered statistically significant. (n = 12/group for survival analysis and n = 6–8/group for other parameters.)

Figure 12

Representative fluorescent z-stack lateral-view images of bacterial nucleic acid by SYTO9 (green) and fungal cell wall by calcofluor white (CW2MR; blue) from biofilms of single (P. aeruginosa or C. albicans) and mixed organisms that initially incubated together and incubated for 24 h (P. aeruginosa + C. albicans) or the Candida addition at 12 h biofilm-formed bacteria (P. aeruginosa > C. albicans) and further incubated for another 12 h on cover glasses (A) is demonstrated (arrows indicate the possible Candida germ tubes that grow from the top into the bottom of biofilms). In addition, thickness of the biofilms (B) and the working hypothesis (left side, catheter during the preparation; right side, the proposed situations in catheter biofilms in vivo) show the enhanced bacterial dissemination in P. aeruginosa > C. albicans biofilms from Candida germ-tube elongation (C) are demonstrated. Mean ± SE was used for data presentation, and the differences between groups were examined by one-way ANOVA followed by Tukey’s analysis for comparisons of multiple groups or 2 groups (B). A p-value of < 0.05 was considered statistically significant. (Independent triplicate experiments were performed for A, B.)