Yonsei Med J.  2017 Nov;58(6):1135-1143. 10.3349/ymj.2017.58.6.1135.

Proteus vulgaris and Proteus mirabilis Decrease Candida albicans Biofilm Formation by Suppressing Morphological Transition to Its Hyphal Form

• Lee KH
• Park SJ
• Choi SJ
• Park JY

• KMID: 2418895
• DOI: http://doi.org/10.3349/ymj.2017.58.6.1135

Abstract

PURPOSE
Candida albicans (C. albicans) and Proteus species are causative agents in a variety of opportunistic nosocomial infections, and their ability to form biofilms is known to be a virulence factor. In this study, the influence of co-cultivation with Proteus vulgaris (P. vulgaris) and Proteus mirabilis (P. mirabilis) on C. albicans biofilm formation and its underlying mechanisms were examined.
MATERIALS AND METHODS
XTT reduction assays were adopted to measure biofilm formation, and viable colony counts were performed to quantify yeast growth. Real-time reverse transcriptase polymerase chain reaction was used to evaluate the expression of yeast-specific genes (rhd1 and rbe1), filament formation inhibiting genes (tup1 and nrg1), and hyphae-related genes (als3, ece1, hwp1, and sap5).
RESULTS
Candida biofilm formation was markedly inhibited by treatment with either living or heat-killed P. vulgaris and P. mirabilis. Proteus-cultured supernatant also inhibited Candida biofilm formation. Likewise, treatment with live P. vulgaris or P. mirabilis or with Proteus-cultured supernatant decreased expression of hyphae-related C. albicans genes, while the expression of yeast-specific genes and the filament formation inhibiting genes of C. albicans were increased. Heat-killed P. vulgaris and P. mirabilis treatment, however, did not affect the expression of C. albicans morphology-related genes.
CONCLUSION
These results suggest that secretory products from P. vulgaris and P. mirabilis regulate the expression of genes related to morphologic changes in C. albicans such that transition from the yeast form to the hyphal form can be inhibited.

Keyword

Candida albicans; Proteus vulgaris; Proteus mirabilis; biofilm

MeSH Terms

Biofilms/*growth & development
Candida albicans/genetics/isolation & purification/*physiology
Gene Expression Regulation, Fungal
Hyphae/*genetics/growth & development
Microbial Interactions/genetics
Proteus mirabilis/*physiology
Proteus vulgaris/*physiology
Real-Time Polymerase Chain Reaction

Figure

• 

  Fig. 1 The effect of P. vulgaris or P. mirabilis on C. albicans biofilm formation. (A) The co-culturing effect of live or heat-killed P. vulgaris and P. mirabilis on C. albicans biofilm formation. The effects of P. vulgaris and P. mirabilis cultured supernatant on biofilm formation (B) and growth (C) of C. albicans. The amount of biofilm formed was assessed with the XTT reduction assay. C. albicans cell counts were measured as colony forming units (CFUs) on a plate. The values of absorbance are the mean±SEM of three independent assays. **p<0.01 compared with biofilm formation of C. albicans only.

• 

  Fig. 2 The effects of live P. vulgaris or P. mirabilis cells on expression of C. albicans morphology-related genes. Relative quantitation of hyphae-specific (A), filament formation inhibiting (B), and yeast-specific (C) gene expression. Expression of genes was determined by quantitative real-time reverse transcriptase polymerase chain reaction. The data represent the average and standard deviation of three separate experiments. *p<0.05, **p<0.01 compared with biofilm formation of C. albicans only.

• 

  Fig. 3 The effects of killed P. vulgaris or P. mirabilis cells on expression of C. albicans morphology-related genes. Relative quantitation of expression of hyphae-specific (A), filament formation inhibiting (B), and yeast-specific (C) genes. Expression of genes was determined by quantitative real-time reverse transcriptase polymerase chain reaction. The data represent the average and standard deviation of three separate experiments.

• 

  Fig. 4 The effects of cultured P. vulgaris and P. mirabilis supernatants on expression of C. albicans morphology-related genes. Relative quantitation of hyphae-specific (A), filament formation inhibiting (B), and yeast-specific (C) gene expression. Expression of genes was determined by quantitative real-time reverse transcriptase polymerase chain reaction. The data represent the average and standard deviation of three separate experiments. *p<0.05, **p<0.01 compared with the biofilm formation of C. albicans only.

• 

  Fig. 5 Scanning electron microscopy of biofilms. C. albicans biofilm (A, ×500; B, ×4000). Biofilm of co-culture with C. albicans and live P. vulgaris (C) or heat-killed P. vulgaris (D); biofilm of co-culture with C. albicans and live P. mirabilis (E) or heat-killed P. mirabilis (F). Arrows point to examples of heat-killed bacteria. Magnifications are ×500 (scale bar; 20 µm) or ×4000 (scale bar; 2 µm).

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