Biofilm formation by microorganisms of interest in food industriesintraspecies variability, biomarkers and control strategies based on non-thermal atmospheric plasma technologies

  1. Paula Fernández Gómez
Supervised by:
  1. Miguel Prieto Maradona Director
  2. Avelino Álvarez Ordóñez Director
  3. Mercedes López Fernández Director

Defence university: Universidad de León

Fecha de defensa: 17 June 2022

  1. Romain Briandet Chair
  2. Daniel Berdejo Secretary
  3. Bélen Orgaz Martin Committee member

Type: Thesis


Microbial communities colonizing food processing environments in the form of biofilms have a major impact on food quality and safety. Therefore, the understanding of factors influencing the formation, ecology and architecture of biofilms from pathogenic and spoilage microorganisms is key for the development of novel biofilm control or removal strategies. In the present PhD Thesis, the intraspecific variability regarding biofilm formation ability and other phenotypic characteristics was examined in a wide collection of isolates from several microorganisms of interest in the food industry. The importance of the general stress response regulator of Gram-negative bacteria RpoS on biofilm formation was assessed in nine field isolates of Cronobacter sakazakii. Their biofilm formation ability was screened in BHI and minimum media with different pH values and supplemented or not with the amino acids arginine, lysine and glutamic acid, resulting generally higher in buffered minimum media (pH 7.0) supplemented with lysine, despite the existing heterogeneity among the different strains. Biofilm formation was visualized by confocal laser scanning microscopy and scanning electron microscopy and was measured by spectrometric determination of the fixed crystal violet, with higher biofilm formation levels being obtained on stainless steel plates than on polystyrene. A lower ability to form biofilms was found for a strain with a loss-of-function mutation in the rpoS gene compared to the rest of the strains, which harboured a functional rpoS. The complementation of this strain with a functional rpoS gene increased it biofilm formation ability up to levels similar to the strains with a naturally functional rpoS gene after 24 h of incubation. However, the differences observed were reduced when a 48 h incubation was used. These results indicate 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. Also, the RpoS status and biofilm formation ability was evaluated in a collection of thirty-one extended-spectrum β-lactamase (ESBL)-producing Escherichia coli strains isolated from foods of animal origin and from human patients, and reference collection strains, together with their tolerance to food-associated stresses (heat, acid, non-thermal atmospheric plasma (NTAP) and UV-C light). Only minor differences were associated to the carriage of the ESBL genes blaTEM, blaCTX-M and blaSHV and no loss-of-function mutations were found in the rpoS gene. The most relevant phenotypic differences among strains were observed for biofilm formation and heat resistance, with food isolates being significantly more resistant to heat treatments at 58 oC for 1 and 2 min than clinical isolates. Also, the biofilm formation ability on stainless steel was significantly higher for the field isolates, both clinical- and food-related, than for the reference strains. A collection of thirty-three Pseudomonas spp. isolates from food processing facilities was also investigated regarding their RpoS status, catalase activity, pigmentation on solid media, pyoverdine production, cellular hydrophobicity and biofilm formation on stainless steel, polystyrene and glass, in order to find biomarkers of strong biofilm production. The biofilm formation levels on stainless steel and polystyrene were significantly higher for the green-pigmented strains as compared to brown or not pigmented strains. Possible relations between pyoverdine production, colony pigmentation and iron availability were studied using an 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, the phenotypic heterogeneity observed among strains was mainly dependent on the Pseudomonas species (P. aeruginosa vs other Pseudomonas species). However, the green colony pigmentation on solid media was identified as a potential biomarker of strong biofilm formation. During this PhD thesis, two plasma-based biofilm control strategies were evaluated. The first one focussed on the development of anti-biofilm coatings that prevent bacterial adherence to the surfaces through physico-chemical modifications. Different coatings made of (3 aminopropyl)triethoxysilane (APTES), tetraethyl orthosilicate (TEOS) and acrylic acid (AA) were applied by Non-Equilibrium Atmospheric Plasma on stainless steel AISI 316. Their anti-biofilm activity was assessed against biofilms of L. monocytogenes CECT911 and E. coli CECT515 after incubation at 37 oC. The two best coatings, AP10+TE6 and AP10+AA6, that reduced L. monocytogenes biofilm production by 45 % and 74 %, respectively, when compared with the uncoated stainless steel, were selected for a further characterization together with a modification of those, AP10+SA6. The anti-biofilm activity was studied against two L. monocytogenes strains isolated from a meat industry and at lower incubation temperatures, more representative of food processing environments, obtaining the best results for the coating AP10+AA6, that reduced the biofilm formation by 90 % after incubation at 12 oC. The morphological and physico-chemical characterization of the selected coatings showed that the most anti-biofilm coating, AP10+AA6, presented the lower surface roughness and higher hydrophilicity, a characteristic that was enhanced by low temperature conditions, when the wettability of the strains was increased. Altogether, these results suggest the formation of a hydration layer that prevents the adherence of L. monocytogenes cells to the coated surface. The characterization of the coating AP10+AA6 continued through the study of its anti-biofilm activity against multispecies biofilms containing L. monocytogenes (developed using indigenous microbiota from three different meat industries) and by assessing the disinfection effectiveness and biofilm evolution after sanitization with two food industry biocides, using culture-dependent and culture-independent approaches. A lower effectiveness of the coating against L. monocytogenes when inoculated in the multispecies biofilms developed for 7 days at 12 oC was observed, together with a partial and industry dependent control of the growth of the pathogen. The biofilm taxonomic composition was highly dependent on the industry, but it was not affected by the nature of the surface (uncoated vs coated stainless steel) and the artificial inoculation with L. monocytogenes. The biofilms formed after 7 days at 12 oC following the 15-min disinfection treatments showed reduced taxonomic diversity and, although sodium hypochlorite was slightly less effective than peracetic acid immediately after application, it caused a stronger growth control of the naturally present L. monocytogenes on the multispecies biofilms developed. This suggested that that a sanitization able to preserve interspecific competitive relationships between the members of the indigenous microbiota and L. monocytogenes might be more favourable for the long-term control of this pathogen in food processing facilities. The second plasma-based technology evaluated in the current PhD Thesis was the use of plasma-activated water (PAW) as an alternative control strategy for L. monocytogenes inactivation. Different PAWs were generated through the activation of tap water using a surface dielectric barrier discharge (SDBD) plasma working at different conditions of discharge power (26 and 36 W) and activation time (5 and 30 min). The study of their physico-chemical composition (pH and levels of hydrogen peroxide, nitrates and nitrites) and antimicrobial efficacy against a cocktail of three L. monocytogenes strains on planktonic state allowed the identification of the best PAW generation conditions, i.e. 36 W of discharge power with 30 min of activation time (PAW HM30). The antimicrobial activity of this PAW against three-strain L. monocytogenes biofilms was lower than in the planktonic state, where 4.6 log reductions were achieved, but still able to obtain 1.9 and 1.8 log reductions after 15 min of exposure of biofilms formed on stainless steel and on polystyrene, respectively. The mechanisms of PAW inactivation were investigated through RNA-seq analysis of L. monocytogenes after its treatment with PAW HM30. The main transcriptomic changes affected carbon metabolism, some virulence genes and the general stress response, with several of the most overexpressed genes belonging to the cobalamin-dependent gene cluster, previously related to L. monocytogenes pathogenicity and its response to multiple stressors.