Aims
Cells limit the cell number of dense biofilms by releasing self‐inhibitory molecules. Here, we aim to assess the effectiveness of yeast quorum sensing (QS) molecules and the antifungal agent natamycin against yeast biofilms of strains commonly isolated from fruit juice ultrafiltration membranes.
Methods and Results
Yeast QS molecules, such as tyrosol, 2‐phenylethanol and farnesol, were detected by solvent extraction and HS‐SPME GC‐MS in Candida tropicalis cultures. The effect of QS molecules on mono‐ and multispecies biofilms formed by Rhodotorula mucilaginosa, C. tropicalis, Candida krusei and Candida kefyr was evaluated by plate count and epifluorescence microscopy. Farnesol caused a decrease in cell number and disrupted mono‐ and multispecies yeast biofilms during adhesion (0·6 mmol l−1). 2‐phenyl ethanol 1·2 mmol l−1 stimulated biofilm density and increased cell number in both mono‐ and multispecies biofilms, while tyrosol did not show effects when tested against C. tropicalis biofilms (0·05–1·2 mmol l−1). Natamycin caused a strong decrease in cell number and disruption of biofilm structure in C. tropicalis biofilms at high concentrations (0·3–1·2 mmol l−1). The combination of farnesol 0·6 mmol l−1 and natamycin at 0·01 mmol l−1, the maximum concentration of natamycin accepted for direct addition into fruit juices, effectively reduced cell counts and disrupted the structure of C. tropicalis biofilms.
Conclusion
Farnesol 0·6 mmol l−1 significantly increased the inhibition exerted by natamycin 0·01 mmol l−1 (~5 ppm) reducing biofilm development from juice on stainless steel surfaces.
Significance and Impact of the Study
These results support the use of QS molecules as biofilm inhibitors in beverages and would certainly inspire the design of novel preservative and cleaning products for the food industry based on combinatory approaches.
Biofilms are often tolerant towards routine cleaning and disinfection processes. As they can grow on fabrics in household or healthcare settings, resulting in odors and serious health problems, it is necessary to contain biofilms through eradication strategies. The current study proposes a novel test model for the growth and removal of biofilms on textiles with Pseudomonas fluorescens and the opportunistic nosocomial pathogen Pseudomonas aeruginosa as model organisms. To assess the biofilm removal on fabrics, (1) a detergent-based, (2) enzyme-based, and (3) combined formulation of both detergent and enzymes (F1/2) were applied. Biofilms were analyzed microscopically (FE-SEM, SEM, 3D laser scanning- and epifluorescence microscopy), via a quartz crystal microbalance with mass dissipation monitoring (QCM-D) as well as plate counting of colonies. This study indicated that Pseudomonas spp. form robust biofilms on woven cellulose that can be efficiently removed via F1/2, proven by a significant reduction (p < 0.001) of viable bacteria in biofilms. Moreover, microscopic analysis indicated a disruption and almost complete removal of the biofilms after F1/2 treatment. QCM-D measurements further confirmed a maximal mass dissipation change after applying F1/2. The combination strategy applying both enzymes and detergent is a promising antibiofilm approach to remove bacteria from fabrics.
When testing conditions that prevail in fruit juice industry it was found that Listeria monocytogenes ATCC 7644 and a wild strain of Listeria innocua were highly hydrophobic, expressed swimming and twitching, co‐aggregated with yeast cells and produced exopolysaccharide, all characteristics that would favor the adhesive process. In mono‐species biofilms, L. innocua adhered on stainless steel at significantly higher counts than L. monocytogenes achieving values of 6.64 ± 0.01 and 5.80 ± 0.21 log CFU/cm2. In dual species biofilms with resident yeasts, L. innocua cells counts increase significantly in the presence of Candida tropicalis and Candida krusei and decrease significantly in the presence of Rhodotorula mucilaginosa and Candida kefyr. When cocultured with Candida tropicalis, the cell numbers of L. monocytogenes had a significant increase. These results revealed synergic and antagonistic interactions among species.
Practical applications
The study supports the plausibility that interactions between L. monocytogenes and members of resident microbiota, such as C. tropicalis, C. krusei, C. kefyr, and R. mucilaginosa, might play an important role for the survival and dissemination of L. monocytogenes. Apple juice processing conditions was used as simulation of the real condition in fruit juice processing environment and these results will alert to fruit juice industry to adopt the best cleaning and disinfecting practices against Listeria.
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