A b s t r a c tThe influence of the shear stress (rw) under which biofilms were formed was assessed on their susceptibility to removal when exposed to chemical and mechanical stresses. A rotating cylinder reactor was used to form biofilms, allowing the simulation of rw conditions similar to those found in industrial settings, particularly in areas with low rw like elbows, corners, valves and dead zones. Bacillus cereus was used as a model bacterium for biofilm formation.Biofilms were formed on AISI316 stainless steel cylinders under different rw (estimated at 0.02, 0.12 and 0.17 Pa) for 7 days. Some phenotypic characteristics, including thickness, biomass production, cellular density and extracellular proteins and polysaccharides content were assessed. Biofilm density was found to increase significantly with rw while the thickness decreased. Also, biofilms formed at 0.02 Pa had lowest biomass content, cell density and extracellular polysaccharide content. Those characteristics were not statistically different for the biofilms formed under 0.12 and 0.17 Pa.Ex situ tests were performed by treating the biofilms with the biocide benzyldimethyldodecyl ammonium chloride (BDMDAC), followed by exposure to increasing rw conditions, up to 1.84 Pa (whereas the maximum rw used during growth was 0.17 Pa). The biofilms formed under low rw were more resistant to removal caused by the BDMDAC action alone. Those formed under higher rw were more resistant to the mechanical and the combined chemical and mechanical treatments. The amount of biofilm remaining on the cylinders, after both treatments was statistically similar for biofilms formed under 0.12 and 0.17Pa. The resistance of biofilms to removal by mechanical treatment (alone and combined with BDMDAC) was related to the amount of matrix polysaccharides.However, none of the methods investigated were able to remove all the biofilm from the cylinders. IntroductionThere is a lack of efficient strategies to clean stagnant zones in industrial plants (Brooks and Flint, 2008). Crevices, corners, dead zones, valves or areas where the mixing rate is low are almost inevitable. Stagnation promotes bacterial accumulation, ultimately leading to biofouling (Manuel et al., 2010). Biofouling is a damaging problem, affecting the energetic efficiency of industrial processes, causing corrosion of the surfaces, decreasing product quality and eventually promoting the spread of pathogens and resistant infectious diseases (Costerton et al., 1999;Ludensky, 2003;Srey et al., 2013). In industrial settings, surface disinfection is usually focused on the use of biocides, aiming to inactivate the microorganisms (Cloete et al., 1998;Faille et al., 2013). Since biofilms are complex biological structures adhered to a sur-face, these strategies often fail, as the removal of biomass is neglected. Hence, cleaning the biomass from the surfaces is fundamental for controlling biofilm development (Flemming, 2011).In the biofilm formation process, the hydrodynamic conditions define the transport of the cells, oxyg...

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.

Biofilms are a problem to food industries, causing equipment damage, increased energy costs, and food spoilage, and they are a potential harbour of pathogenic microorganisms. Their extreme anti-microbial resistance means that novel control strategies are necessary. Plant secondary metabolites (phyto-chemicals) have demonstrated promising antimicrobial properties when applied against planktonic cells and biofilms. The aim of this study was to test the effectiveness of two phenolic acids: ferulic (FA) and salicylic (SA), alone and in combination (FSA) on the prevention and control of Bacillus cereus and Pseudomonas fluorescens biofilms. Additional tests were performed to ascertain the effects of FA and SA on bacterial motility, surface properties (physicochemical properties and surface charge), and quorum sensing inhibition (QSI). The effects of a concentration two times the minimum inhibitory concentration (500 g mL -1 ) were assessed on single-and dual-species biofilms. The results demonstrated that only swimming was affected by FA and SA and no clear relationship was obtained between the effects of phenolic acids on motility and biofilm prevention. The bacterial physico-chemical surface properties and charge were affected by the phenolic acids. Salicylic acid demonstrated capacity for QSI. However, both bacteria were able to form single-and dual-species biofilms in the presence of the phenolic acids. The application of FA and SA (single and combined) to biofilms caused low to moderate inactivation and removal. However, dual-species biofilms formed in the presence of phenolic acids were highly susceptible to a second exposure to the chemicals. The continuous exposure of dual-species biofilms to the phenolic acids decreased their resilience and resistance to inactivation and removal. This study clarifies the role of FA and SA in the prevention and control of biofilms formed by two important food spoilage bacteria.2

Standard cleaning processes may not remove all the soiling typically found in food industry, such as carbohydrates, fats, or proteins. Contaminants have a high impact in disinfection as their presence may reduce the activity of disinfectants. The influence of alginic acid, bovine serum albumin, yeast extract, and humic acids was assessed on the antimicrobial activities of benzalkonium chloride and cetyltrimethyl ammonium bromide against Bacillus cereus vegetative cells and Pseudomonas fluorescens. The bacteria (single and consortium) were exposed to surfactants (single and combined) in the absence and presence of potential disinfection interfering substances. The antimicrobial effects of the surfactants were assessed based on the bacterial respiratory activity measured by oxygen uptake rate due to glucose oxidation. The tested surfactants were efficient against both bacteria (single and consortium) with minimum bactericidal concentrations ranging from 3 to 35 mg·L−1. The strongest effect was caused by humic acids that severely quenched antimicrobial action, increasing the minimum bactericidal concentration of the surfactants on P. fluorescens and the consortium. The inclusion of the other interfering substances resulted in mild interferences in the antibacterial activity. This study clearly demonstrates that humic acids should be considered as an antimicrobial interfering substance in the development of disinfection strategies.

The aim of this work was to assess the effectiveness of the biocide benzyldimethyldodecyl ammonium chloride (BDMDAC) on the removal of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens formed in a rotating cylinder reactor (RCR), using AISI316 stainless steel (SS) and polymethyl methacrylate (PMMA) as adhesion surfaces. Additional tests were performed to understand the adhesion of B. cereus and P. fluorescens to the selected surfaces.Predictions of the adhesion potential according to the thermodynamic theory showed more favourable adhesion on SS than on PMMA, for both species.Thermodynamically, adhesion was more favourable for B. cereus. After BDMDAC treatment, thermodynamic adhesion ability was favoured for P. flurescens and decreased for B. cereus, mainly on PMMA. Both bacteria had negative surface charge and the exposure to BDMDAC increased the charge to less negative values. In vitro adhesion results were, for most cases, contradictory to those predicted by the thermodynamic theory. Single and dual species biofilms were formed in the RCR for 7 days. Afterwards, the biofilms were exposed to the chemical (use of BDMDAC) and to hydrodynamic stresses (use of increasing Reynolds number of agitation), alone and combined. The applications of BDMDAC or hydrodynamic stress, when applied alone, were insufficient to remove the biofilms from the surfaces. The combined effects of BDMDAC with a series of increasing Reynolds number of agitation promoted additional biofilm removal. This effect was dependent on the surface used. For PMMA, the hydrodynamic stress was more effective on the removal of BDMDAC-treated dual species biofilms. For SS, the synergy of the chemical and hydrodynamic stresses removed more B. cereus and dual species biofilms. The overall results 2 demonstrate that the species association was not advantageous in biofilm resistance to removal when compared with the single species biofilms, particularly those of P. fluorescens. In general, removal by hydrodynamic stress, alone and preceded by the BDMDAC treatment, was higher for biofilms formed on SS. However, even the combined action of BDMDAC and the exposure to a series of increasing Reynolds number of agitation were not effective to obtain biofilm-free surfaces.

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l(-1) caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.

The presence of biofilms in drinking water distribution systems (DWDS) is a global public health concern as they can harbor pathogenic microorganisms. Sodium hypochlorite (NaOCl) is the most commonly used disinfectant for microbial growth control in DWDS. However, its effect on biofilm removal is still unclear. This work aims to evaluate the effects of the combination of chemical (NaOCl) and mechanical stresses on the removal of single and dual species biofilms of two bacteria isolated from DWDS and considered opportunistic, Acinectobacter calcoaceticus and Stenotrophomonas maltophilia. A rotating cylinder reactor was successfully used for the first time in drinking water biofilm studies with polyvinyl chloride as substratum. The single and dual species biofilms presented different characteristics in terms of metabolic activity, mass, density, thickness and content of proteins and polysaccharides. Their complete removal was not achieved even when a high NaOCl concentrations and an increasing series of shear stresses (from 2 to 23Pa) were applied. In general, NaOCl pre-treatment did not improve the impact of mechanical stress on biofilm removal. Dual species biofilms were colonized mostly by S. maltophilia and were more susceptible to chemical and mechanical stresses than these single species. The most efficient treatment (93% biofilm removal) was the combination of NaOCl at 175mg·l with mechanical stress against dual species biofilms. Of concern was the high tolerance of S. maltophilia to chemical and mechanical stresses in both single and dual species biofilms. The overall results demonstrate the inefficacy of NaOCl on biofilm removal even when combined with high shear stresses.

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