Cell density and extracellular matrix composition mitigate bacterial biofilm sensitivity to UV-C LED irradiation.

Ultraviolet-C light-emitting diodes (UV-C LEDs) are an emerging technology for decontamination applications in different sectors. In this study, the inactivation of bacterial biofilms was investigated by applying an UV-C LED emitting at 280 nm and by measuring both the influence of the initial cell density (load) and presence of an extracellular matrix (biofilm). Two bacterial strains exposing diverging matrix structures and biochemical compositions were used: Pseudomonas aeruginosa and Leuconostoc citreum. UV-C LED irradiation was applied at three UV doses (171 to 684 mJ/cm2) on both surface-spread cells and on 24-h biofilms and under controlled cell loads, and bacterial survival was determined. All surface-spread bacteria, between 105 and 109 CFU/cm2, and biofilms at 108 CFU/cm2 showed that bacterial response to irradiation was dose-dependent. The treatment efficacy decreased significantly for L. citreum surface-spread cells when the initial cell load was high, while no load effect was observed for P. aeruginosa. Inactivation was also reduced when bacteria were grown under a biofilm form, especially for P. aeruginosa: a protective effect could be attributed to abundant extracellular DNA and proteins in the matrix of P. aeruginosa biofilms, as revealed by Confocal Laser Scanning Microscopy observations. This study showed that initial cell load and exopolymeric substances are major factors influencing UV-C LED antibiofilm treatment efficacy. KEY POINTS: • Bacterial cell load (CFU/cm2) could impact UV-C LED irradiation efficiency • Characteristics of the biofilm matrix have a paramount importance on inactivation • The dose to be applied can be predicted based on biofilm properties.

Response of Controlled Cell Load Biofilms to Cold Atmospheric Plasma Jet: Evidence of Extracellular Matrix Contribution.

AIM: Study of the biocidal effect of a cold atmospheric-pressure plasma in ambient air on single-species bacterial biofilms with controlled cell density, characterized by different extracellular matrices. METHODS AND RESULTS: Two bacterial strains were chosen to present different Gram properties and contrasted extracellular matrices: Pseudomonas aeruginosa ATCC 15442 (Gram-negative), and Leuconostoc citreum NRRL B-1299 (Gram-positive). P. aeruginosa biofilm exhibits a complex matrix, rich in proteins while L. citreum presents the specificity to produce glucan-type exopolysaccharides when grown in the presence of sucrose. Plasma was applied on both surface-spread cells and 24-h grown biofilms with controlled cell loads over 5, 10, or 20 min. Surface-spread bacteria showed a time dependent response, with a maximal bacterial reduction of 2.5 log after 20 min of treatment. On the other hand, in our experimental conditions, no bactericidal effect could be observed when treating biofilms of P. aeruginosa and glucan-rich L. citreum. CONCLUSIONS: For biofilms presenting equivalent cell loads, the response to plasma treatment seemed to depend on the properties of the extracellular matrix characterized by infrared spectroscopy, scanning electron microscopy, or dry weight. SIGNIFICANCE AND IMPACT OF STUDY: Both cell load standardization and biofilm characterization are paramount factors to consider the biocide effect of plasma treatments. The extracellular matrix could affect the plasma efficacy by physical and/or chemical protective effects.

A dynamic resazurin microassay allowing accurate quantification of cells and suitable for acid-forming bacteria.

A resazurin micro-assay was developed to quantify acidifying bacteria. The resorufin fluorescent signal was measured over time and the determined time to reach the max slope (TMS) was plotted against CFU (colony forming unit) counts. This dynamic assay enabled to quantify nine lactic acid bacteria and a Bacillus licheniformis strain despite the increasing acidity of the medium.