Susceptibility and transcriptomic response to plasma-activated water of Listeria monocytogenes planktonic and sessile cells.

Plasma-Activated Water (PAW) was generated from tap water using a surface dielectric barrier discharge at different discharge power (26 and 36 W) and activation time (5 and 30 min). The inactivation of a three-strain Listeria monocytogenes cocktail in planktonic and biofilm state was evaluated. PAW generated at 36 W-30 min showed the lowest pH and the highest hydrogen peroxide, nitrates, nitrites contents and effectiveness against cells on planktonic state, resulting in 4.6 log reductions after a 15-min treatment. Although the antimicrobial activity in biofilms formed on stainless steel and on polystyrene was lower, increasing the exposure time to 30 min allowed an inactivation >4.5 log cycles. The mechanisms of action of PAW were investigated using chemical solutions that mimic its physico-chemical characteristics and also RNA-seq analysis. The main transcriptomic changes affected carbon metabolism, virulence and general stress response genes, with several overexpressed genes belonging to the cobalamin-dependent gene cluster.

The background microbiota and sanitization agent drive the fate of Listeria monocytogenes in multispecies biofilms formed on a plasma-polymerized coating applied on stainless steel.

The present study evaluates the anti-biofilm activity of a coating applied with an atmospheric-pressure plasma jet system on AISI 316 stainless steel (SS) against multispecies biofilms containing Listeria monocytogenes (using background microbiota from three different meat industries) using culture-dependent and culture-independent approaches. Also, the disinfection effectiveness and biofilm evolution after sanitization with two food industry biocides were assessed. The anti-biofilm activity of the coating against L. monocytogenes, observed on mono-species biofilms (p < 0.05), was lost on the multispecies biofilms developed for 7 days at 12 °C (p > 0.05), with L. monocytogenes counts ranging from 5.5 ± 0.7 to 6.1 ± 0.5 CFU/cm2 on the uncoated SS and from 4.4 ± 0.2 to 6.4 ± 0.5 CFU/cm2 on the coated SS. The taxonomic composition of the formed biofilms was highly dependent on the industry but not affected by the artificial inoculation with L. monocytogenes and the nature of the surface (coated vs uncoated SS). When L. monocytogenes was artificially inoculated, its growth was partially controlled in the biofilms developed, with the magnitude of this effect being lower (p < 0.05 on coated SS) for the industry with the lowest taxonomy richness and diversity (3.8 ± 0.2 CFU/cm2), as compared the other two sampled industries (2.4 ± 0.4 and 1.6 ± 0.2 CFU/cm2). The 15-min disinfection treatments with either sodium hypochlorite or peracetic acid at 0.5 % resulted in total viable and L. monocytogenes counts below the limit of detection in most cases, immediately after treatment. The subsequent incubation of the sanitized plates for another 7 days at 12 °C in fresh BHI media led to the development of biofilms with lower bacterial richness and alpha diversity, and higher beta diversity. Even though sodium hypochlorite was in general slightly less effective than peracetic acid immediately after application, it caused a stronger growth control (p < 0.05) of the naturally present L. monocytogenes on the multispecies biofilms developed. This finding highlights the importance of understanding the interspecific competitive relationships between the members of the background microbiota and L. monocytogenes for the long-term control of this pathogen in food processing facilities.

Development and characterization of anti-biofilm coatings applied by Non-Equilibrium Atmospheric Plasma on stainless steel.

Biofilm-mediated microbial persistence of pathogenic and spoilage bacteria is a serious problem in food industries. Due to the difficulty of removing mature biofilms, great efforts are being made to find new strategies to prevent bacterial adherence to surfaces, the first step for biofilm development. In this study, coatings of (3-aminopropyl)triethoxysilane (APTES), tetraethyl orthosilicate (TEOS) and acrylic acid (AA) were applied by Non-Equilibrium Atmospheric Plasma on stainless steel (SS) AISI 316, the SS most commonly used in food industry equipment. Their anti-biofilm activity was assessed against Listeria monocytogenes CECT911 and Escherichia coli CECT515 after incubation at 37 °C. The best results were obtained for L. monocytogenes, with coatings consisting of a base coating of APTES and a functional coating of TEOS (AP10 + TE6) or AA (AP10 + AA6) that reduced biofilm production by 45% and 74%, respectively, when compared with the uncoated SS. These coatings were further characterized, together with a variation of the best one that replaced the acrylic acid with succinic acid (AP10 + SA6). Their anti-biofilm activity was assessed under different incubation conditions, including two strains of L. monocytogenes isolated from processing environments of a meat industry. The coating AP10 + AA6 reduced the biofilm formation by 90% after incubation at 12 °C, a temperature more representative of those commonly found in food processing environments. The morphological and physico-chemical characterization of the selected coatings showed that the coating with the highest anti-biofilm activity (i.e., AP10 + AA6) had lower surface roughness and higher hydrophilicity. This suggests that the formation of a hydration layer prevents the adherence of L. monocytogenes, an effect that seems to be enhanced by low temperature conditions, when the wettability of the strains is increased.

Heterogeneity in biofilm formation and identification of biomarkers of strong biofilm formation among field isolates of Pseudomonas spp.

The biofilm formation ability of a collection of thirty-three Pseudomonas spp. isolates from food processing facilities was investigated in order to find biomarkers of strong biofilm production, a characteristic that can determine persistence in food processing environments. The strains were classified according to the colony pigmentation on solid media as green, brown or not pigmented. The biofilm production on stainless steel and polystyrene was assessed by spectrometric determination of the fixed crystal violet, and the biofilm formed on glass, through confocal laser scanning microscopy. Besides, pyoverdine production, catalase activity, RpoS status and cellular hydrophobicity were also monitored. A significantly higher biofilm production level on stainless steel and polystyrene was observed for green-pigmented strains as compared to brown or not pigmented strains. The influence of iron availability on biofilm formation on stainless steel was studied through the addition of the iron scavenger 2,2-bipyridine resulting in a decrease of 40 % in biofilm formation for the not pigmented strains. For most of the potential biomarkers studied (i.e., pyoverdine production, catalase activity, cellular hydrophobicity), the phenotypic heterogeneity observed among strains was mainly dependent on the Pseudomonas species and no strong associations with the biofilm formation capacity were detected. However, the green colony pigmentation on solid media showed good potential as a biomarker of strong biofilm formation on stainless steel and polystyrene both in P. aeruginosa and Pseudomonas spp.

The role of the general stress response regulator RpoS in Cronobacter sakazakii biofilm formation.

The relationship between biofilm formation and RpoS status was assessed in nine field isolates of C. sakazakii. Their ability to form biofilms was studied in BHI and minimum media with different pH values and supplemented or not with the amino acids arginine, lysine and glutamic acid. Biofilm formation, both on polystyrene and stainless steel, was measured by spectrometric determination of the fixed crystal violet and the biofilms were visualized by confocal laser scanning microscopy and scanning electron microscopy. Despite the existing heterogeneity among the different strains, biofilm formation was generally higher in buffered minimum media (pH 7.0) supplemented with lysine than in other culture media and on stainless steel plates than on polystyrene. The results showed a lower ability to form biofilms for a strain with a loss-of-function mutation in the rpoS gene, the general stress response regulator of Gram-negative bacteria, when compared to the rest of the strains, which harboured a functional rpoS. The complementation of this strain with a functional rpoS gene resulted in an increase in its biofilm formation ability up to levels comparable to those observed for strains with a functional rpoS. However, the differences were markedly reduced when the incubation time was increased from 24 to 48 h, indicating that the loss of RpoS caused a delay in the development of mature biofilms, rather than a complete inhibition of biofilm production in C. sakazakii.

Dynamics of Listeria monocytogenes colonisation in a newly-opened meat processing facility.

This study determined the colonisation scenario of Listeria monocytogenes in a newly-opened ready-to-eat meat processing facility using a combination of classical microbiology and molecular biology techniques. Samples (n=183), including food contact surfaces, non-food contact surfaces, raw materials and food samples, collected on four sampling occasions, were analysed for L. monocytogenes by the ISO 11290:1996 standard method and by real-time PCR applied to the second enrichment broth from the ISO method. No L. monocytogenes were detected on the first sampling occasion, but by the second sampling occasion a persistent clone had colonised the facility. Analysis of the second enrichment of the ISO method by real-time PCR was more sensitive for the detection of L. monocytogenes than the ISO method alone. In order to reduce the risk of cross contamination and the public health risk, awareness and proactive measures are required to control L. monocytogenes from the first days of production in a newly opened meat processing facility.