Disrupting Irreversible Bacterial Adhesion and Biofilm Formation with an Engineered Enzyme.

Biofilm formation is often attributed to postharvest bacterial persistence on fresh produce and food handling surfaces. In this study, a predicted glycosyl hydrolase enzyme was expressed, purified, and validated for the removal of microbial biofilms from biotic and abiotic surfaces under conditions used for chemical cleaning agents. Crystal violet biofilm staining assays revealed that 0.1 mg/ml of enzyme inhibited up to 41% of biofilm formation by Escherichia coli O157:H7, E. coli 25922, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. Furthermore, the enzyme was effective at removing mature biofilms, providing a 35% improvement over rinsing with a saline solution alone. Additionally, a parallel-plate flow cell was used to directly observe and quantify the impact of enzyme rinses on E. coli O157:H7 cells adhering to spinach leaf surfaces. The presence of 1 mg/liter enzyme resulted in nearly 6-times-higher detachment rate coefficients than a deionized (DI) water rinse, while the total cells removed from the surface increased from 10% to 25% over the 30-min rinse time, reversing the initial phases of biofilm formation. Enzyme treatment of all 4 cell types resulted in significantly reduced cell surface hydrophobicity and collapse of negatively stained E. coli 25922 cells imaged by electron microscopy, suggesting potential polysaccharide surface modification of enzyme-treated bacteria. Collectively, these results point to the broad substrate specificity and robustness of the enzyme for different types of biofilm stages, solution conditions, and pathogen biofilm types and may be useful as a method for the removal or inhibition of bacterial biofilm formation. IMPORTANCE In this study, the ability of an engineered enzyme to reduce bacterial adhesion and biofilm formation of several foodborne pathogens was demonstrated, representing a promising option for enhancing or replacing chlorine and other chemical sanitizers in food processing applications. Specifically, significant reductions of biofilms of the pathogens Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes are observed, as are reductions in initial adhesion. Enzymes have the added benefits of being green, sustainable alternatives to chemical sanitizers, as well as having a minimal impact on food properties, in contrast to many alternative antimicrobial options such as bleach that aim to minimize food safety risks.

Escherichia coli O157:H7 and Salmonella Typhimurium adhesion to spinach leaf surfaces: Sensitivity to water chemistry and nutrient availability.

This study investigated the effects of solution chemistry and growth conditions on bacterial deposition on spinach leaf surfaces using a parallel plate flow cell. Two food safety pathogens of concern and two non-pathogen bacterial surrogates (environmental E. coli isolates) were grown in ideal (LB media) and nutrient-restricted (M9 media) conditions. Bacterial attachment was quantified as mass transfer rate coefficients for cells suspended in 10 mM KCl, CaCl2 and artificial groundwater, and cell and leaf surfaces were extensively characterized (zeta potential, hydrophobicity, extracellular polymer (EPS) composition). Between the pathogens, E. coli O157:H7 attachment was greater than that of Salmonella Typhimurium, attributed to measurable variability in cell surface charge and hydrophobicity. When grown in M9 media, both pathogens were significantly more adhesive to spinach surfaces (p < 0.01) than when grown in LB media. Surrogates did not follow this trend and showed minimal changes in adhesion kinetics and surface properties between growth conditions. EPS sugar/protein ratios were reduced in some of the highest attachment scenarios, suggesting that changes in EPS composition in favor of proteins may play a role. These results show the importance of growth conditions and solution complexities in understanding mechanisms of aqueous bacterial adhesion to food surfaces.

Using the agricultural environment to select better surrogates for foodborne pathogens associated with fresh produce.

Despite continuing efforts to reduce foodborne pathogen contamination of fresh produce, significant outbreaks continue to occur. Identification of appropriate surrogates for foodborne pathogens facilitates relevant research to identify reservoirs and amplifiers of these contaminants in production and processing environments. Therefore, the objective of this study was to identify environmental Escherichia coli isolates from manures (poultry, swine and dairy) and surface water sources with properties similar to those of the produce associated foodborne pathogens E. coli O157:H7 and Salmonella enterica serotype Typhimurium. The most similar environmental E. coli isolates were from poultry (n=3) and surface water (n=1) sources. The best environmental E. coli surrogates had cell surface characteristics (zeta potential, hydrophobicity and exopolysaccharide composition) that were similar (i.e., within 15%) to those of S. Typhimurium and/or formed biofilms more often when grown in low nutrient media prepared from lettuce lysates (24%) than when grown on high nutrient broth (7%). The rate of attachment of environmental isolates to lettuce leaves was also similar to that of S. Typhimurium. In contrast, E. coli O157:H7, a commonly used E. coli quality control strain and swine isolates behaved similarly; all were in the lowest 10% of isolates for biofilm formation and leaf attachment. These data suggest that the environment may provide a valuable resource for selection of surrogates for foodborne pathogens.

Efficacy of post-harvest rinsing and bleach disinfection of E. coli O157:H7 on spinach leaf surfaces.

Attachment and detachment kinetics of Escherichia coli O157:H7 from baby spinach leaf epicuticle layers were investigated using a parallel plate flow chamber. Mass transfer rate coefficients were used to determine the impact of water chemistry and common bleach disinfection rinses on the removal and inactivation of the pathogen. Attachment mass transfer rate coefficients generally increased with ionic strength. Detachment mass transfer rate coefficients were nearly the same in KCl and AGW rinses; however, the detachment phase lasted longer in KCl than AGW (18 ± 4 min and 4 ± 2 min, respectively), indicating that the ions present during attachment play a significant role in the cells' ability to remain attached. Specifically, increasing bleach rinse concentration by two orders of magnitude was found to increase the detachment mass transfer rate coefficient by 20 times (from 5.7 ± 0.7 × 10-11 m/s to 112.1 ± 26.8 × 10-11 m/s for 10 ppb and 1000 ppb, respectively), and up to 88 ± 4% of attached cells remained alive. The spinach leaf texture was incorporated within a COMSOL model of disinfectant concentration gradients, which revealed nearly 15% of the leaf surface is exposed to almost 1000 times lower concentration than the bulk rinse solution.