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.

Comparison of extraction methods for the characterization of extracellular polymeric substances from aggregates of three biofilm-forming phototrophic microorganisms.

This paper aims to define a robust procedure to extract extracellular polymeric substances (EPS) from aggregates of three benthic phototrophic microorganisms: the cyanobacterium Phormidium autumnale, the diatom Nitzschia palea, and the green alga Uronema confervicolum. This study focuses on the extraction efficiency of polysaccharide and protein EPS by using two physical methods (sonication, cation exchange resin) and three chemical methods (formamide, EDTA, Tween 20) with minimum cell lysis. Cell lysis was evaluated by monitoring chlorophyll a release. The results indicated that sonication or incubation of the algae aggregates with 0.25% Tween 20 induced a high level of cell lysis. A combined extraction approach, with an initial dispersing pretreatment (Ultra-Turrax, 13 500 r·min-1, 1 min), followed by formamide addition (0.22%) and then incubation with Dowex cation exchange resin (50 g per g of dry biomass), provided the highest amount of extracted EPS (mostly proteins), with low cell lysis. Furthermore, extracted EPS were characterized by size exclusion chromatography, and the obtained fingerprints revealed similar profiles for the three benthic microorganisms with a majority of low molecular weight polymers (400 to 11 300 Da). However, additional EPS of high (>600 000 Da) and intermediate (20 000 to 80 000 Da) molecular sizes were specifically detected in the diatom extracts.

Anaerobic Digestion Alters Copper and Zinc Speciation.

Anaerobic digestion is a widely used organic waste treatment process. However, little is known on how it could alter the speciation of contaminants in organic waste. This study was focused on determining the influence of anaerobic digestion on the speciation of copper and zinc, two metals that generally occur at high concentration in organic waste. Copper and zinc speciation was investigated by X-ray absorption spectroscopy in four different raw organic wastes (predigestion) and their digested counterparts (postdigestion, i.e., digestates). The results highlighted an increase in the digestates of the proportion of amorphous or nanostructured copper sulfides as well as amorphous or nanostructured zinc sulfides and zinc phosphate as compared to raw waste. We therefore suggest that the environmental fate of these elements would be different when spreading either digestates or raw waste on cropland.

Quantification of amyloid fibrils using size exclusion chromatography coupled with online fluorescence and ultraviolet detection.

An amyloid fibrils investigation within biofilm samples requires distinguishing the amyloid ß-sheet structure of these proteins and quantifying them. In this study, the property of amyloids to incorporate the fluorescent dye Thioflavin T has been exploited to propose a method of quantification. The experimental protocol includes the preparation of amyloids from commercial κ-casein (κCN) and their fractionation through size exclusion chromatography (SEC) to provide calibration curves from fluorescence and absorbance signals. Finally, a bacterial biofilm extract was injected into SEC, and the amyloid fibrils could be expressed as equivalent κCN, representing approximately 21% of the total proteins.

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.

Development and validation of a colorimetric assay for simultaneous quantification of neutral and uronic sugars.

A colorimetric assay based on the conventional anthrone reaction was investigated for specific quantification of uronic acids (UA) in the presence of neutral sugars and/or proteins. Scanning of glucose (Glu) and glucuronic acid (GlA) was performed after the reaction with anthrone and a double absorbance reading was made, at 560 nm and at 620 nm, in order to quantify the UA and neutral sugars separately. The assay was implemented on binary or ternary solutions containing Glu, GlA and bovine serum albumin (BSA) in order to validate its specificity towards sugars and check possible interference with other biochemical components such as proteins. Statistical analysis indicated that this assay provided correct quantification of uronic sugars from 50 to 400 mg/l and of neutral sugars from 20 to 80 mg/l, in the presence of proteins with concentrations reaching 600 mg/l. The proposed protocol can be of great interest for simultaneous determination of uronic and neutral sugars in complex biological samples. In particular, it can be used to correctly quantify the Extracellular Polymeric Substances (EPS) isolated from the biological matrix of many bacterial aggregates, even in the presence of EPS extractant such as EDTA.

New direct contact approach to evaluate soil genotoxicity using the Vicia faba micronucleus test.

A method to assess micronucleus (MN) induction in Vicia faba roots by direct contact exposure to a solid matrix was developed. The procedure comprised a 5-d germination period, as in the well-known method using aqueous extracts. However, the seeds were here sown directly into the test soil whereas a culture period is necessary before exposing seedlings to a liquid medium. One soil under forest and two contaminated soils from areas affected by industrial installations and a coke works were used. Three durations of direct exposure were tested: 2, 5 and 7 d. The optimal duration was evaluated at 2 d to observe maximal MN induction without observing toxicity symptoms. The methodology using aqueous extracts was applied to the same three soils: MN frequency was higher than in the direct contact assay but the ratios of MN frequencies from tested soils in comparison to the negative control were lower. However, for each soil, both the direct contact method and the aqueous extract exposure led to the same risk assessment diagnosis. The evaluation of a concentration range of a polycyclic aromatic hydrocarbons (PAH)-contaminated soil showed a dose-dependent MN frequency when the seeds were allowed to germinate before sowing in the soil: the soil genotoxicity was the highest at intermediate doses. The direct contact method was found to be rapid, sensitive and well suited to the evaluation of soil quality.