H7 Biofilm Research Articles

Inhibition of Escherichia coli O157:H7 biofilm on vegetable surface by solid liposomes of clove oil

Escherichia coli O157:H7 (E. coli O157:H7) easily forms biofilms on pores and veins of plant, which poses a serious hazard to safety of ready-to-eat vegetables. Therefore, this study was designed to evaluate inhibition mechanism of clove oil on E. coli O157:H7 biofilm and apply it to vegetable for reduce risk of contamination. The results of crystal violet staining showed that clove oil could effectively remove biofilm. After treatment of biofilm with clove oil (2 MBIC), the results of resazurin method showed that bacterial metabolic activity decreased by 61.48%, and results of phenol-sulfuric acid method and Coomassie brilliant blue method showed that extracellular protein and polysaccharide content decreased by 62.76% and 47.5%, respectively. This indicates that clove oil can affect biofilm through above aspects. Finally, solid liposomes (SLPs) were prepared and applied to vegetables, which was first time that SLPs were used to remove biofilms. The results showed that clove oil SLPs exhibited excellent anti-biofilm activity at 4 °C and 25 °C without affecting senses and quality of vegetables. Therefore, the clove oil SLPs can exert a long-lasting antibacterial effect, effectively removing E. coli O157:H7 biofilm on vegetable surface, and prolonging the shelf life.

Bio-enzymes for inhibition and elimination of Escherichia coli O157:H7 biofilm and their synergistic effect with sodium hypochlorite

Escherichia coli O157:H7 is one of the most important pathogens worldwide. In this study, three different kinds of enzymes, DNase I, proteinase K and cellulase were evaluated for inhibitory or degrading activity against E. coli O157:H7 biofilm by targeting extracellular DNA, proteins, and cellulose, respectively. The cell number of biofilms formed under proteinase K resulted in a 2.43 log CFU/cm2 reduction with an additional synergistic 3.72 log CFU/cm2 reduction after NaClO post-treatment, while no significant reduction occurred with NaClO treatment alone. It suggests that protein degradation could be a good way to control the biofilm effectively. In preformed biofilms, all enzymes showed a significant reduction of 16.4–36.7% in biofilm matrix in 10-fold diluted media (p < 0.05). The sequential treatment with proteinase K, cellulase, and NaClO showed a significantly higher synergistic inactivation of 2.83 log CFU/cm2 compared to 1.58 log CFU/cm2 in the sequence of cellulase, proteinase K, and NaClO (p < 0.05). It suggests that the sequence of multiple enzymes can make a significant difference in the susceptibility of biofilms to NaClO. This study indicates that the combination of extracellular polymeric substance-degrading enzymes with NaClO could be useful for the efficient control of E. coli O157:H7 biofilms.

Technological Activities and Antibiofilm Effect of Staphylococcus Equorum Isolated from Fermented Sausage

The present study aimed at evaluating the technological properties and the ability of Staphylococcus equorum to inhibit biofilm formation by Staphylococcus aureus and Escherichia coli O157:H7. Seventeen S. equorum strains were isolated from fermented sausage and examined for their ability to grow at 10 or 15 ºC in the presence of 10 or 15% NaCl at pH values of 4.5, 5.0 or 5.5. Technological properties including catalases, superoxide dismutase, lipolytic and proteolytic activities of S. equorum strains were determined. All strains grew under the studied conditions and showed variable activities. The strain S. equorum HMA4 showed good characters of all the studied metabolic properties and had ability to grow under all studied conditions. The antagonistic effect of S. equorum HMA4 against S. aureus or E. coli O157:H7 biofilms was studied under the above conditions. There was a significant increasing in the reduction percentage of biofilm formation at pH 5.0 and 5.5 comparing with those obtained at pH 4.5. The antibiofilm effect of S. equorum HMA4 against E. coli O157:H7 was higher than its effect on biofilm of S. aureus. In conclusion, beside its role in the developing flavor and enhancing the color of the fermented foods, S. equorum HMA4 could be used as a protective starter against some foodborne pathogens in fermented foods.

Estimation of inactivation effects against Escherichia coli O157:H7 biofilm by different plasma-treated solutions and post-treatment storage

This study investigated the optimum conditions to maximize the inactivation of biofilms using both plasma-treated solutions and post-treatment storage conditions. Chemical properties of plasma-treated solutions were also analyzed to identify their possible biofilm inactivation mechanisms. Escherichia coli O157:H7 biofilms on stainless steel were prepared and immersed in distilled water (DW), 100 ppm of sodium chloride solution (NaCl), or 100 ppm of sodium hypochlorite solution (NaOCl), followed by container-type plasma treatment for 10 min (15 kHz and 250 W). After plasma discharge was switched off, biofilms immersed in the plasma-treated solution were stored for 10 min with the plasma apparatus closed or open. The log reduction of E. coli O157:H7 was NaOCl (3.58) &amp;gt; NaCl (2.06) = DW (1.95) in the closed storage condition. In addition, the bactericidal effect of NaOCl was higher in the closed condition than in the open condition. Concentrations of the hydroxyl radical (OH·) and peroxynitrite (ONOO−) were also the highest for NaOCl in the closed condition. Consequently, plasma treatment using NaOCl solution in the closed condition was found to be the optimum method for inactivation of E. coli O157:H7 biofilms, due to high hydroxyl radical and peroxynitrite concentrations. This method also has the merit of using a lower concentration of NaOCl than that used typically in industry.

Novel surfactant peptide for removal of biofilms.

Conventional chemical surfactants attach on blots randomly, accompanied with health and environmental issues. To address this, a surfactant peptide was designed to mimic chemical surfactants with an affinity binding peptide as a hydrophobic tail for the cleanup of biofilm contaminations. The micelle forming and structural changes of the peptide in aqueous solution were systematically investigated. More importantly, the biofilm removal efficiency toward Escherichia coli O157:H7 biofilm reached 75% in neutral aqueous solutions at the concentration of 125 mg/L (critical micelle concentration 91 mg/L), a significant improvement in comparison to conventional surfactants and random surfactant peptide. The dynamic removal process reported by confocal laser scanning microscope (CLSM) also displayed the different constituents of biofilm blots, which associated with surfactant peptide binding efficiency. Hopefully, this surfactant strategy will eventually provide new scopes in the design of surface active biological agents.

Inhibition and Inactivation of Escherichia coli O157:H7 Biofilms by Selenium

The present study investigated the efficacy of selenium (Se) in reduction of enterohemorrhagic Escherichia coli (EHEC) exopolysaccharide (EPS) synthesis, inhibition of biofilm formation at 25 and 4°C on polystyrene surface, and inactivation of mature EHEC biofilms in combination with hot water. Sterile 96-well polystyrene plates inoculated with EHEC (∼6.0 log CFU per well) were treated with a subinhibitory concentration (SIC) of Se, and biofilms were allowed to mature at 4 and 25°C for 96 h. Biofilm-associated bacterial population was determined by scraping and plating, whereas the extent of EPS production was determined using ruthenium red staining assay. Solid surface assay was used to study the effect of Se on early attachment of EHEC cells to polystyrene. The efficacy of Se in rapid inactivation of preformed, mature EHEC biofilm was investigated by treating biofilms on polystyrene plates with the MBC of Se in combination with hot water at 80°C with a contact time of 0 min, 30 s, 2 min, and 5 min. Furthermore, the effect of Se on EHEC biofilm architecture was visualized using confocal microscopy, whereas the effect of Se on EHEC biofilm genes was determined using real-time quantitative PCR (RT-qPCR). Finally, the potential feasibility of coating stainless steel surfaces with Se nanoparticles to inhibit EHEC biofilm formation was studied. Se reduced early attachment of planktonic cells, biofilm formation, and EPS synthesis in EHEC ( P < 0.05). Se in combination with hot water reduced biofilm-associated bacterial counts by 3 to 4 log CFU/mL at 5 min of exposure compared with the control ( P < 0.05). However, hot water treatment alone decreased biofilm-associated bacterial counts by only 1.0 log CFU/mL. RT-qPCR results revealed that Se down-regulated the transcription of critical genes associated with biofilm synthesis in EHEC ( P < 0.05). The results collectively suggest that Se could potentially be used to control EHEC biofilms in food processing environments, but appropriate applications need to be validated.

Sequential effect of phages and cold nitrogen plasma against Escherichia coli O157:H7 biofilms on different vegetables

Escherichia coli O157:H7 (E. coli O157:H7) is one of the most common pathogens in fresh vegetables and fruits, and most of the diseases produced by E. coli O157:H7 are associated with biofilms. Cold nitrogen plasma (CNP) is a cold sterilization technique which has no residue. However to completely eliminate the biofilm on the surface of vegetables the processing power and time of CNP have to be enhanced, which will impact on the quality of fruits and vegetables. Thus the sequential treatment of CNP and phage techniques was engineered in this study. Compared to treatment performed separately, sequential treatment not only had more mild treatment conditions as 400W CNP treatment for 2min and 5% phage treatment for 30min, but also exhibited more remarkable effect on eradicating E. coli O157:H7 biofilms in vitro and on vegetables. The population of E. coli O157:H7 was approximately reduced by 2logCFU/cm2 after individual treatment of 5% phages for 30min or 500W CNP for 3min. While the sequential treatment of CNP (400W, 2min) and phages (5%, 30min) reduced the E. coli O157:H7 viable count in biofilm by 5.71logCFU/cm2. Therefore, the sequential treatment holds a great promise to improve the current treatment systems of bacterial contamination on different vegetable surfaces.

The antibacterial activity of clove oil/chitosan nanoparticles embedded gelatin nanofibers against Escherichia coli O157:H7 biofilms on cucumber

This study aims to evaluate the antibacterial activity of clove oil-loaded chitosan nanoparticles (CO@CNPs) and gelatin electrospun nanofibers against Escherichia coli O157:H7 (E. coli O157:H7) biofilms on cucumbers. The optimal CO@CNPs were prepared when the initial concentration of clove oil (CO) was 2.5mg/mL according to the ionic crosslinking method. CO@CNPs showed high antibacterial activity against E. coli O157:H7 biofilms. After 8h treatment, almost 99.98% reduction in E. coli O157:H7 population was achieved when CO@CNPs were applied at 30% (w/v). Subsequently, the prepared CO@CNPs were incorporated successfully within gelatin nanofibers by electrospinning. After 9mg/mL gelatin/CO@CNPs treatment for 24h, the population of E. coli O157:H7 biofilm reduced by about 99.99% in vitro. Further, the application of gelatin/CO@CNPs nanofibers on cucumber against E. coli O157:H7 biofilm was evaluated as well. After 6mg/mL and 9mg/mL gelatin/CO@CNPs nanofibers treatment at 12°C for 4days, 4.28 and 4.97 log10 reductions of E. coli O157:H7 biofilm in population were observed, respectively. Finally, the sensory evaluation results implied that the gelatin/CO@CNPs nanofibers treatment could maintain the color and flavor of cucumber well for >4days.

Antibiofilm Effect of Poly(Vinyl Alcohol-co-Ethylene) Halamine Film against Listeria innocua and Escherichia coli O157:H7.

Bacterial biofilm formation is linked to several infections and foodborne disease outbreaks. To address this challenge, there is an unmet need to develop rechargeable antimicrobial materials that can provide continuous sanitation of contact surfaces, especially in the food industry. This study was aimed at evaluating a novel rechargeable antimicrobial polymer formed using poly(vinyl alcohol-co-ethylene) (PVA-co-PE) with halamine functionality to prevent biofilm formation with repeated exposure to high loads of bacteria and organic content and also to aid in inactivation of preformed biofilms upon contact with this novel material. The antibiofilm activity of this rechargeable antimicrobial material was evaluated using a combination of fluorescence and scanning electron microscopy techniques and biofilm metabolic activity analyses. The results determined on the basis of imaging and metabolic activity measurements demonstrated that halamine-functionalized polymer films significantly reduced Listeria innocua and Escherichia coli O157:H7 biofilm formation. This novel polymeric material maintained its antibiofilm activity with repeated cycles of extended exposure to high levels of bacterial load. These polymeric films were recharged using bleach and cleaned using mechanical sonication after each cycle of extended incubation with bacteria. Halamine-functionalized polymeric material also exhibited significant antibacterial activity against preformed biofilms on a model surface. In summary, our results demonstrate the potential of this antimicrobial material to provide continuous sanitation of surfaces and applications for inactivating preformed biofilms without extensive use of resources, including water and heat. This polymeric material may be used as a replacement for existing polymeric materials or as a coating on diverse materials.IMPORTANCE Conventional sanitizers can have limited efficacy in inactivating biofilms in areas with limited accessibility and buildup of organic biomass. Furthermore, none of the current approaches provide continuous sanitation of surfaces. There is a significant unmet need to develop and validate materials that can prevent biofilm formation as well as inactivate preformed biofilms. In this study, the efficacy of a copolymer film containing N-halamine against biofilms of L. innocua and E. coli O157:H7 was evaluated. The polymer film showed strong inhibitory activity against pregrown biofilm or prevented the growth of a new biofilm. The polymer film also maintained its antibiofilm activity after multiple cycles of exposure to high titers of bacterial load with recharging of the polymer film using bleach at intermediate steps between the cycles. Overall, the results demonstrate the potential of a novel antimicrobial material to inhibit and treat biofilms in food industry applications.

Inactivation of bacterial biofilms using visible-light-activated unmodified ZnO nanorods

Various zinc oxide (ZnO) nanostructures are widely used for photocatalytic antibacterial applications. Since ZnO possesses a wide bandgap, it is believed that only UV light may efficiently assist bacterial inactivation, and diverse crystal lattice modifications should be applied in order to narrow the bandgap for efficient visible-light absorption. In this work we show that even unmodified ZnO nanorods grown by an aqueous chemical growth technique are found to possess intrinsic defects that can be activated by visible light (λ = 405 nm) and successfully applied for total inactivation of various highly resistant bacterial biofilms rather than more sensitive planktonic bacteria. Time-resolved fluorescence analysis has revealed that visible-light excitation creates long-lived charge carriers (τ > 1 μs), which might be crucial for destructive biochemical reactions achieving significant bacterial biofilm inactivation. ZnO nanorods covered with bacterial biofilms of Enterococcus faecalis MSCL 302 after illumination by visible light (λ = 405 nm) were inactivated by 2 log, and Listeria monocytogenes ATCL3C 7644 and Escherichia coli O157:H7 biofilms by 4 log. Heterogenic waste-water microbial biofilms, consisting of a mixed population of mesophilic bacteria after illumination with visible light were also completely destroyed.

Strategies for Biofilm Inhibition and Virulence Attenuation of Foodborne Pathogen-Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (E. coli) O157:H7, a gram-negative bacteria identified as a foodborne pathogen causing severe disease is of great concern worldwide. The pathogenicity of E. coli O157:H7 is due to the presence of some virulence factors and its ability to form biofilm which resist antimicrobial compounds, withstand harsh environmental condition and protects from the host immune responses. Formation of biofilm is a multistep process such as adhesion, cellular aggregation and productions of extracellular matrix in which colonies are embedded. There are high numbers of research in the discovery of natural and synthetic compounds which can attenuate the E. coli O157:H7 biofilm formation as well as suppress virulence-related genes. The present review article focuses on the steps involved in E. coli O157:H7 biofilm formation, factors associated with virulence and attenuation.

Essential Oils-Based Antibacterial Agent Against Escherichia coli O157:H7 Biofilm on Cucumber

Guided by the principle of hurdle technology, a high-efficiency antibacterial agent (AA) had been formulated, which contained six kinds of essential oils, three pieces of food additives and ethyl alcohol. The antibacterial effects of AA against E. coli O157:H7 biofilm on simulated and real cucumbers were testified by MTT staining method, colony-forming unit (CFU) counting method, scanning electron microscopy (SEM) analysis and confocal laser scanning microscopy (CLSM) assay. The remarkable anti-biofilm activity of AA was achieved under low concentration and short treatment duration. The results also revealed that AA inhibited the multiplication of E. coli O157:H7 via destroying the cell membrane integrity. Besides, color, texture and sensory evaluation illustrated that AA could be applied as a disinfectant to eliminate the biofilm formed on cucumber without impact on food quality. Practical Applications As a kind of high-efficiency antimicrobials, AA has significant inhibition and elimination effects on biofilm under a low concentration and short incubation time. Especially, AA was effective to eliminate the formed E. coli O157:H7 biofilm on the surface of cucumber without impact on the quality and flavor of the product. On the basis of the results, we believe that AA can be applied in vegetables or fruits processing and preservation.

Quorum sensing is crucial to Escherichia coli O157:H7 biofilm formation under static or very slow laminar flow conditions

Quorum sensing (QS) is defined as a cellto- cell signaling process that collectively regulates the gene expression of bacteria via small signaling molecules called autoinducers (AIs). It was reported that QS-regulated gene expression in Pseudomonas aeruginosa failed to occur at a high Reynolds number (Re= 3,000), since AI-2, a secreted interspecies signaling molecule, was washed away and so could not reach the minimum concentration required for QS. In this study, we describe the effects of flow speed on QS-stimulated biofilm formation in Escherichia coli O157:H7 inside a very thin microchannel (3 cm×1 cm×45 μm). In microtiter plates, the wild-type strain produced high amounts of exopolysaccharide, whereas its QS mutants ΔluxS and ΔlsrK, defective in AI-2 production and phosphorylation, made less exopolysaccharide. Confocal laser scanning microscopy showed that at a flow rate of 1 μL/min the wild-type strain formed rounded biofilms, whereas such biofilms formed by the QS mutants were fewer and thinner. At a flow rate of 10 μL/min, none of the tested strains formed mature biofilms. Our results suggest that QS is essential in the biofilm maturation under static or very slow laminar flow conditions where the biofilm signal can be easily accumulated and transported to the sessile cells.

Antimicrobial effect of synergistic interaction between UV-A light and gallic acid against Escherichia coli O157:H7 in fresh produce wash water and biofilm

Abstract A synergistic interaction between gallic acid (GA) and UV-A light (UV-A + GA) to inactivate E. coli O157:H7 in spinach wash water and in biofilm was evaluated. A 30-min exposure to UV-A light in presence of 10 mM GA had relevant biological effect in the inactivation of E. coli O157:H7 in suspension in the absence (> 5 log(CFU/mL) ) and the presence of organic content (> 3-log(CFU/mL) in 2000 mg O2/L COD (Chemical Oxygen Demand) organic load), and resulted in ~ 80% decrease in the metabolic activity of E. coli O157:H7 biofilm. GA solutions could be recycled through at least 3-cycles of UV-A treatment without a significant loss in antibacterial effect. Catalase reduced the extent of E. coli O157:H7 inactivation from the UV-A + GA treatment suggesting that generation of hydrogen peroxide was partially responsible for the observed antimicrobial effect. The UV-A + GA treatment was also found to be effective in causing > 3 log(CFU/mL) reductions in E. coli O157:H7 on the surface of spinach leaves. UV-A + GA treatment can serve as an effective intervention in the fresh produce sanitation. Industrial relevance The results of this study show that a synergistic interaction between gallic acid (GA) and UV-A (365 nm) light is an effective treatment for sanitation of fresh produce and water used to wash fresh produce. It was also found to be effective against the E.coli O157:H7 biofilm. The attractive attributes of this technology include a relatively low cost; specific, light-triggered activity; non-toxic nature and scalability. Thus, this technology has potential to replace conventional chemical sanitizer-based sanitation approaches.

Synergistic bactericidal action of phytic acid and sodium chloride against Escherichia coli O157:H7 cells protected by a biofilm

The food industry must prevent the build-up of strong Escherichia coli O157:H7 biofilms in food processing environments. The present study examined the bactericidal action of phytic acid (PA), a natural extract from rice bran and the hulls/peels of legumes, against E. coli O157:H7 biofilms. The synergistic bactericidal effects of PA plus sodium chloride (NaCl) were also examined. E. coli O157:H7 biofilms were allowed for form on stainless steel coupons by culture in both rich (tryptic soy broth, TSB) and minimal (M9) medium at 22°C for 6days. Bacterial cells within biofilms grown in M9 medium were significantly more resistant to PA than those grown in TSB (p<0.05); thus M9 medium was selected for further experiments. The anti-biofilm effect of PA was significantly increased by addition of NaCl (2–4%) (p<0.05); indeed, the combination of 0.4% PA plus 3–4% NaCl completely inactivated E. coli O157:H7 biofilms without recovery (a>6.5logCFU/cm2 reduction). Neither PA nor NaCl alone were this effective (PA, 1.6–2.7logCFU/cm2 reduction; NaCl, <0.5logCFU/cm2 reduction). Confocal laser scanning microscopy images of propidium iodide-treated cells showed that PA (0.4%) plus NaCl (2–4%) had marked membrane permeabilizing effects. These results suggest that a sanitizer that combines these two naturally occurring antimicrobial agents may be useful to food safety managers who encounter thick biofilm formation in food processing environments.

Synergetic antibacterial efficacy of cold nitrogen plasma and clove oil against Escherichia coli O157:H7 biofilms on lettuce

This study aims to evaluate the antimicrobial effects of cold nitrogen plasma (CNP) and clove oil against Escherichia coli O157:H7 (E. coli O157:H7) biofilm on lettuce. Both clove oil (1 mg/mL, 2 mg/mL and 4 mg/mL) and CNP (400–600 W) displayed significant eradication effect on E. coli O157:H7 biofilms in vitro (p < 0.001). Subsequently, the antibiofilm effect of combined treatment was studied as well. Compared with the respective treatment, combined treatment exhibited remarkable synergistic effect on eradicating E. coli O157:H7 biofilms. The confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) had also visually testified that the antibacterial effects of clove oil on E. coli O157:H7 biofilms (in vitro and on lettuce) were enhanced by CNP at 400 W for short treatment duration. The results of sensory evaluation indicated that combined treatment has mild negative effect on lettuce quality. Moreover, the synergetic antibacterial mechanism of clove oil and CNP against E. coli O157:H7 was concluded as that they could damage the bacterial cell wall and the outer membrane, leading to leakage of cellular components, such as nucleic acid and ATP.

Curli fimbriae are conditionally required in Escherichia coli O157:H7 for initial attachment and biofilm formation

Several species of enteric pathogens produce curli fimbriae, which may affect their interaction with surfaces and other microbes in nonhost environments. Here we used two Escherichia coli O157:H7 outbreak strains with distinct genotypes to understand the role of curli in surface attachment and biofilm formation in several systems relevant to fresh produce production and processing. Curli significantly enhanced the initial attachment of E. coli O157:H7 to spinach leaves and stainless steel surfaces by 5-fold. Curli was also required for E. coli O157:H7 biofilm formation on stainless steel and enhanced biofilm production on glass by 19–27 fold in LB no-salt broth. However, this contribution was not observed when cells were grown in sterile spinach lysates. Furthermore, both strains of E. coli O157:H7 produced minimal biofilms on polypropylene in LB no-salt broth but considerable amounts in spinach lysates. Under the latter conditions, curli appeared to slightly increase biofilm production. Importantly, curli played an essential role in the formation of mixed biofilm by E. coli O157:H7 and plant-associated microorganisms in spinach leaf washes, as revealed by confocal microscopy. Little or no E. coli O157:H7 biofilms were detected at 4 °C, supporting the importance of temperature control in postharvest and produce processing environments.

A study on prophagic and chromosomal sodC genes involvement in Escherichia coli O157:H7 biofilm formation and biofilm resistance to H2O2.

Escherichia coli O157:H7 possesses one chromosomal and two prophagic sodC genes encoding for Cu,Zn superoxide dismutases. We evaluated the contribution of sodC genes in biofilm formation and its resistance to hydrogen peroxide. The biofilm of sodC deletion mutants has been studied, in presence or absence of hydrogen peroxide, by crystal violet in 96-well plates and Scanning Electron Microscopy on glass coverslips. Deletion of prophagic sodC genes had no effect on biofilm construction, in contrast to the chromosomal gene deletion. Hydrogen peroxide treatment showed higher cell mortality and morphological alterations in sodC deletion mutants respect to wild type. These effects were related to the biofilm development stage. The role of the three SodCs is not redundant in biofilm formation and the resistance to oxidative damage. The stage of biofilm development is a crucial factor for an effective sanitization.

Transcriptional response of selected genes of Salmonella enterica serovar Typhimurium biofilm cells during inactivation by superheated steam

Superheated steam (SHS), produced by the addition of heat to saturated steam (SS) at the same pressure, has great advantages over conventional heat sterilization due to its high temperature and accelerated drying rate. We previously demonstrated that treatment with SHS at 200°C for 10 sec inactivated Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes biofilm cells on the surface of stainless steel to below the detection limit. However, bacteria withstanding heat stress become more resistant to other stress conditions, and may be more virulent when consumed by a host. Herein, we studied the transcriptional regulation of genes important for stress resistance and virulence in Salmonella biofilms after SHS treatments. Genes encoding heat shock proteins and general stress resistance proteins showed transcriptional surges after 1 sec of SHS treatment at 200°C, with parallel induction of stress-related regulator genes including rpoE, rpoS, and rpoH. Interestingly, Salmonella biofilm cells exposed to SHS showed decreased transcription of flagella and Salmonella pathogenicity island-1 (SPI-1) genes required for motility and invasion of host cells, respectively, whereas increased transcription of SPI-2 genes, important for bacterial survival and replication inside host cells, was detected. When the transcriptional response was compared between cells treated with SHS (200°C) and SS (100°C), SHS caused immediate changes in gene expression by shorter treatments. Understanding the status of Salmonella virulence and stress resistance induced by SHS treatments is important for wider application of SHS in controlling Salmonella biofilm formation during food production.

Inactivation of Escherichia coli O157:H7 in biofilm on food-contact surfaces by sequential treatments of aqueous chlorine dioxide and drying

We investigated the efficacy of sequential treatments of aqueous chlorine and chlorine dioxide and drying in killing Escherichia coli O157:H7 in biofilms formed on stainless steel, glass, plastic, and wooden surfaces. Cells attached to and formed a biofilm on wooden surfaces at significantly (P≤0.05) higher levels compared with other surface types. The lethal activities of sodium hypochlorite (NaOCl) and aqueous chlorine dioxide (ClO2) against E. coli O157:H7 in a biofilm on various food-contact surfaces were compared. Chlorine dioxide generally showed greater lethal activity than NaOCl against E. coli O157:H7 in a biofilm on the same type of surface. The resistance of E. coli O157:H7 to both sanitizers increased in the order of wood>plastic>glass>stainless steel. The synergistic lethal effects of sequential ClO2 and drying treatments on E. coli O157:H7 in a biofilm on wooden surfaces were evaluated. When wooden surfaces harboring E. coli O157:H7 biofilm were treated with ClO2 (200μg/ml, 10min), rinsed with water, and subsequently dried at 43% relative humidity and 22°C, the number of E. coli O157:H7 on the surface decreased by an additional 6.4CFU/coupon within 6h of drying. However, when the wooden surface was treated with water or NaOCl and dried under the same conditions, the pathogen decreased by only 0.4 or 1.0logCFU/coupon, respectively, after 12h of drying. This indicates that ClO2 treatment of food-contact surfaces results in residual lethality to E. coli O157:H7 during the drying process. These observations will be useful when selecting an appropriate type of food-contact surfaces, determining a proper sanitizer for decontamination, and designing an effective sanitization program to eliminate E. coli O157:H7 on food-contact surfaces in food processing, distribution, and preparation environments.