Influence of Cell Surface Components of Shiga Toxin Producing Escherichia Coli on their Abilities to Form Biofilms and their Tolerance to Cleaning and Sanitizing Treatments

Non-Technical Summary:<br/>
Shiga toxin producing Escherichia coli (STEC) is a group of foodborne bacterial pathogens that can cause different clinical manifestations, ranging from mild diarrhea to severe complications such as hemorrhagic colitis and hymolytic uremic syndrome. Although more than 200 serotypes of STEC have been identified, the predominant serotypes of STEC that are implicated in human illness include O157, O26, O111, O103, O121, O45, O145 and O104. Cattle are the primary reservoirs of STEC, but E. coli O157 and non-O157 strains have been isolated from not only retail beef but also many other food products. Human STEC infections are usually transmitted via the consumption of food contaminated with STEC during food production, transport and preparation. Therefore, studying the ability of STEC to survive in the environments is critical for control and elimination of the pathogens from food. Cells of E. coli tend to adhere to surfaces and form biofilms in order to sustain their stability in the environments. Cell surface appendages of STEC including curli and cellulose play important roles in the biofilm development process. Curli is adhesive fimbriae located on the surface of enteric bacteria cells. It mediates cell aggregation and in some cases virulence. Curli expressed by E. coli enhance the ability of cells to adhere to their contact surfaces. Cellulose is, nevertheless a long chain polymer of glucose with b-1, 4 glycosidic bonds. It is highly insoluble, inelastic and has extreme strength. Cellulose confers mechanical, chemical or biological protection to bacterial cells. The polymer maintains its structural stability through a high degree of polymerization and crystallinity and the ability to form hydrogen bonding networks. Co-expression of cellulose and curli confers hydrophobic properties to E. coli cell surface and mediates the interaction between bacterial cells and their contact surfaces. Previous studies have demonstrated that the cellulose and curli protected E. coli from environmental stress. However, their influence on cell susceptibility to cleaning and sanitizing treatments and biofilm control measures has not been adequately investigated. The overall goals of this project are to investigate the influence of cell surface components of STEC including cellulose and/or curli on cell susceptibility to cleaning and sanitizing treatments, and to evaluate the efficacies of selected cleaners and sanitizers in degrading/removing cellulose or curli, controlling biofilms, and inactivating the cells of STEC. Specific objectives of the project include: 1) To determine the amounts of cellulose and curli produced by the cells of selected STEC strains; 2) To determine the efficacies of selected cleaning and sanitizing treatments in degrading/removing the cellulose and curli produced by selected STEC strains; 3) To determine the survival of STEC as influenced by cellulose and curli production as well as treatments with selected cleaners and sanitizers; 4) To quantify the biofilms formed by the cells of selected STEC; 5) To determine the efficacy of selected sanitizing treatments on biofilm control.
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Approach:<br/>
Objective 1. The amounts of cellulose and curli produced by the cells of selected STEC strains. Six wild type strains of STEC (6-8 (O5:H), 6-35 (O103:H2), 7-17 (O26:H11), 7-49 (O103:H2, 7-50 (O103:H2) and 7-51 (O103:H2)], all from our laboratory culture collection, will be grown on LBNS agar at 28 C for 72 h. Cellulose produced by the STEC cultures will be quantified using a previous published protocol. Curli-expressing and non curli-expressing variants will be isolated from STEC strain 5-11, 7-52 and 7-57 on LBNS agar at 28 C. Variants of the three strains will be used as separate cultures. The cultures will be grown on LBNS agar at 28 C for 72 h for qualitative assessment of curli that are produced by the STEC cells.
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Objective 2: The efficacy of selected cleaning and sanitizing treatments in degrading/removing the cellulose and curli produced by selected STEC strains. The STEC cultures will be treated with 15 ml of 0.05 M sodium acetate buffer (pH 5.0) containing different concentrations of cellulase (0.51, 2.12, or 3.83 U/15 ml) at 37 C for 2 h, with 15 ml of 10 mM sodium acetate buffer with 5 mM calcium acetate (pH 7.5) containing different concentrations of protease (1 or 2 U/ml) at 37 C for 30 min, with 15 ml of 2 or 4% acetic or lactic acid at room temperature for 20 min, and with an appropriate concentration of an alkaline (Ecolab) or acidic (ZEP Manufacturing Company) sanitizer at room temperature for 7 and 15 min, respectively. After the treatments, cellulose and curli remained on the surface of STEC will be quantified using previously published procedures. Objective 3: Survival of STEC as influenced by cellulose and curli production as well as treatments with selected cleaners and sanitizers. Immediately after the treatments described above, surviving cells of STEC will be enumerated and the correlation coefficients between residual amounts of cellulose or curli on STEC and surviving STEC populations after each treatment will be calculated using the JMP software (JMP, 2008). Objective 4: Biofilms formed by the cells of selected STEC as influenced by cellulose and curli production. Diluted cell cultures of STEC described above will be allowed to form biofilms on polystyrene microtiter plates and stainless steel coupons, respectively. The biofilm mass will be measured using the crystal violet binding assay (Pawar et al., 2005). Objective 5: The efficacy of selected sanitizing treatments on biofilm control. Biofilms formed on polystyrene and stainless steel surfaces will be treated with 2% acetic or lactic acid, the acidic or alkaline sanitizers described previously. All treatments will be conducted at room temperature for 15 min with gentle shaking at 50 rpm. After treatments, the biofilm remaining on the tissue culture plates and stainless steel surfaces will be determined using the crystal violet binding assay. All experiments will have appropriate duplication and replication, and statistical tools will be used to design the experiments and analyze results.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: A group of STEC strains were grown on Luria-Bertani no salt agar (LBNS) agar supplemented with Congo red and Coomassie brilliant blue at 28C for 72 h. Cellulose or curli expressed by the cells of resulting cultures were quantified. For cellulose quantification, 10 ml of each cell culture was placed in glass centrifuge tubes with conical bottoms. The cultures were centrifuged at 3,400 g for 25 min. The cell pellet of each culture was collected and three ml of an acetic-nitric reagent was added and mixed properly. The test tubes were covered with aluminum foil and placed in a boiling water bath for 30 min, after which the samples were re-centrifuged at 3,400 g for 15 min. Following centrifugation the pellets were washed twice with sterile distilled water and then suspended in 1 ml of 67% sulfuric acid. The samples were allowed to stand for 1 h at room temperature before being diluted with 4 ml distilled water and 10 ml refrigerated anthrone reagent. The centrifuge tubes were inverted gently, placed in a boiling water bath for 16 min, and then cooled rapidly in the ice bath. The absorbance of each sample at 620 nm (A620) was recorded. A standard curve of absorbance as a function of cellulose concentration was prepared. The quantities of cellulose produced by STEC cells were calculated by comparing the absorbance values of the standard with the values of the tested samples. For curli quantification, cultures of STEC strains were harvested and pelleted by centrifugation for 10 min at 12,000 g. Cell pellets were suspended in 1.5 ml of 2% sodium dodecyl sulfate, and the samples were then boiled for 45 min. Boiled samples were centrifuged for 5 min at 7,000 rpm, and harvested pellets were washed three times each with 1.5 ml of distilled water. After each washing, sample was centrifuged as described above. The pellet was stained with 1.5 ml 0.02% Congo red solution for 10 min at room temperature. Following staining, the sample was centrifuged again at 12,000 g for 10 min. The optical density of unbound Congo red in the supernatant was measured against a saline background at 500 nm, and the optical density of 0.02% Congo red solution was also measured for the calculation of Congo red binding.
<br/>PARTICIPANTS: Nothing significant to report during this reporting period.
<br/>TARGET AUDIENCES: Nothing significant to report during this reporting period.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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IMPACT: The six wild type STEC strains used in the study expressed different amounts of cellulose on their surfaces. The amounts of cellulose on 6-8, 7-49, 7-50 and 7-51 were significantly (P < 0.05) higher than the amount of cellulose produced by 7-17. Cells of 6-35 produced an average of 1.32 ug cellulose per 1010 cells, which was significantly (P < 0.05) lower than the amounts of cellulose produced by the cells of 6-8, 7-49, 7-50 and 7-51. Cells of 7-51, the greatest cellulose producer among strains evaluated in this study, produced approximately 5.58-time more cellulose than did the cells of 7-17. STEC 7-57+ produced the greatest amounts of curli compare to the other strains. In addition, the 2 variants of STEC pairs, 7-52 and 7-57, showed the significant difference (P < 0.05) in curli production after 72 h incubation. The curli on the cells of STEC strains, 7-52+ and 7-57+, were significantly higher in the Congo red binding units compared to that by 7-52- and 7-57- (P < 0.05). Furthermore, cells of STEC 5-11+ produced numerically (P > 0.05) higher level of curli than their counterpart, 5-11-.