Progress 05/01/12 to 04/30/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
See last progress report
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Researchers and other personel in the university, government and food industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has trained Ph.D. level graduate students. How have the results been disseminated to communities of interest?Yes. The results have been presented in the annual meeting of professional societies. What do you plan to do during the next reporting period to accomplish the goals?We will continuously make presentations at the annual meetings of professional societies and publish papers in peer reviewed professional journals.
Impacts
What was accomplished under these goals?
Bacterial strains and growth conditions. Two groups of STEC and a total of twelve strains from our laboratory collection were used in the study (Table 1). The first group (Group I) consisted of six STEC strains, 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). These six STEC strains were known to produce different amounts of cellulose. The second group (Group II) was comprised of three pairs of STEC, and each pair has a curli producer (C+), and a non-curli producer (C-), O111:H- 7-57C+ and 7-57C-, O157:H7 5-11C+ and 5-11C-, and O103:H2 7-52C+ and 7-52C. Confluent cell cultures of the STEC strains were grown on Luria Bertani no salt (LBNS) agar plates at 28oC for 72 h. Preparation of STEC cultures and abiotic surfaces for biofilm development. Each STEC culture grown on LBNS agar was transferred into 9 ml of LBNS broth, and the inoculated cultures were incubated for 18 h at 28°C. The resulting cultures were diluted (1: 40) with fresh LBNS broth, and the diluted cultures were used in the following experiment. Polystyrene tissue culture plates (Falcon 3847, Becton Dickinson Labware, Frankline Lakes, NJ; growth area 2 cm2) and stainless steel coupons (2 x 5 cm2, 14- gauge and 2B finish; Impulse Manufacturing, Inc; Dawsonville, GA) were used for biofilm development. Purchased polystyrene tissue culture plates were sterile and ready-to-use. The stainless steel coupons were soaked prior to the experiment in diluted SparKLEEN solution (1:10; Fisher Scientific, Pittsburgh, PA) at room temperature for 18 h. The coupons were then cleaned with a Cascade dish-washing detergent (Cincinnati, OH) and rinsed thoroughly with deionized water before being autoclaved at 121°C for 30 min. Biofilm formation on polystyrene surfaces. Diluted cell cultures of STEC described above (2 ml) were inoculated into individual wells of the tissue culture plates. The plates with un-inoculated broth were used as controls. Following an incubation period of 7 d at 28°C, the broth was withdrawn and loosely attached bacterial cells were removed by washing the plates twice, each with 2 ml of sterile water. The plates with biofilms were air-dried at 60°C for 2 h before the biofilm mass was measured using the crystal violet binding assay. Briefly, the tissue culture plates were passed over the flame of a burner (Fisher Scientific) several times. Fixed biofilms on the polystyrene surface were stained with 2 ml of 1% crystal violet (Fisher Scientific) at room temperature for 15 min and then rinsed with running deionized water until the wash water contained no visible stains. After drying for 2 h at 60°C and cooling to room temperature, the crystal violet in the biofilms were extracted using 2 ml of ethanol-acetone solution (80:20). The concentrations of extracted crystal violet were determined by measuring the absorbance of the ethanol-acetone solutions at the wavelength of 550 nm. Biofilm formation on stainless steel surfaces. Stainless steel coupons were placed horizontally in Petri plates (100 X15 mm; Fisher Scientific). Each Petri plate had one stainless coupon and 30 ml of a diluted STEC culture described above. Petri plates with the same number of coupon and same volume of un-inoculated LBNS broth served as controls. At the end of the 7 day incubation period at 28°C, the broth was withdrawn and the stainless steel coupons were rinsed once with 30 ml of sterile water for 10 sec at room temperature to remove loosely attached cells. Biofilm mass on the stainless steel coupons were determined using the crystal violet binding assay. Specifically, stainless steel coupons with biofilms were rinsed, air-dried, and fixed as described above. Fixed biofilms on the stainless steel surface were stained with 5 ml of 1% crystal violet at room temperature for 15 min, and then rinsed with running deionized water until no visible stain in the rinsing water. The coupons were dried for 2 h at 60ºC, and then cooled to room temperature. Individual coupon in a Petri plate was washed with 5 ml of ethanol-acetone solution (80:20) with gentle shaking at 50 rpm to extract the crystal violet. The concentrations of extracted crystal violet were determined as described above. Results. Research showed that strain 7-49 and 7-50 formed significantly more biofilms than the other Group I STEC strains on polystyrene surface (P < 0.05). The amounts of biofilm formed by 6-8, 6-35, and 7-51 were significantly lower than those of 7-49 and 7-50, but significantly (P < 0.05) higher than the amount of biofilms formed by 7-17 (P < 0.05). The trend of biofilm formation by Group I STEC on stainless steel coupons was similar to that on polystyrene surfaces. Cells of 7-50 formed the greatest amount of biofilm followed by the cells of 7-49. The amounts of biofilms formed by 6-8, 6-35 and 7-51 were significantly (P < 0.05) higher than the amount of biofilm formed by 7-17. Cells of 7-52+ and 7-57+ formed significantly (P < 0.05) more biofilms than their non curli expressing counterparts, 7-52- and 7-57- on polystyrene surface. The amounts of biofilm formed by 5-11+ and 5-11- were however, only numerically (P > 0.05) different. Similar to the biofilms formed on polystyrene surface, cells of 7-52+ and 7-57+ formed significantly (P < 0.05) more biofilms than those of their non curli expressing counterparts, 7-52- and 7-57-. The amounts of biofilm formed by the cells of 5-11+ and 5-11- were only numerically (P > 0.05) different.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Park, Y. J., and J. Chen. 2015. Control of biofilms of Shiga toxin-producing Escherichia coli using treatments with organic acids and commercial sanitizers. J. Food Prot. 78:990-995.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Researchers and other personnel in the academia, government and food industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has trained Ph.D. level graduate students. How have the results been disseminated to communities of interest?Yes. The results have been presented in the annual meeting of professional societies. What do you plan to do during the next reporting period to accomplish the goals?We will continuously make presentations at the annual meetings of professional societies and publish papers in peer reviewed professional journals.
Impacts
What was accomplished under these goals?
The biofilm-forming ability of two groups of STEC strains capable of producing different amounts of curli and cellulose was assessed in polystyrene and stainless surfaces in LBNS broth. Polystyrene tissue culture plates were purchased sterile and ready to use. The stainless steel coupons were soaked before the experiment in diluted SparKLEEN solution (1:10; Fisher Scientific, Pittsburgh, PA) at room temperature for 18 h. The coupons were then cleaned with a dishwashing detergent (Cascade, Cincinnati, OH), rinsed thoroughly with deionized water, and autoclaved at 121oC for 30 min. Diluted STEC cell cultures (2 ml) were inoculated into individual wells of the tissue culture plates. Plates with uninoculated broth were used as controls. After incubation for 7 days at 28oC, the broth culture was withdrawn, and bacterial cells loosely attached to the plate were removed by washing the plates twice with 2 ml of sterile water each time. The plates with biofilms were air dried at 60oC for 2 h, and the biofilm mass was measured using the crystal violet binding assay previously described by Pawar et al. Stainless steel coupons were placed horizontally in petri plates (100 by 15 mm; Fisher Scientific). Each plate contained one stainless coupon and30 ml of a diluted STEC culture. Plates with a coupon and the same volume of un-inoculated LBNS broth served as controls. At the end of the 7-day incubation period at 28oC, the broth was withdrawn and the stainless steel coupons were rinsed once with 30 ml of sterile water for 10 s at room temperature to remove loosely attached cells. The mass of the biofilm formed on the stainless steel coupons was determined using the crystal violet binding assay of Pawar et al. Three replicates of each experiment were performed, and each experiment was conducted in duplicate. The data collected in the study were analyzed using Fisher's least significant difference design of the Statiscal Analysis Software. Results of the research indicated that compared with their counterpartscells expressing a greater amount of cellulose or curli formed more biofilm mass. Cells of STEC formed more biofilms on polystyrene than on stainless steel surface.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Park, Y. J., and J. Chen. 2015. Control of biofilms of Shiga toxin-producing Escherichia coli using treatments with organic acids and commercial sanitizers. J. Food Prot. 78:990-995.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:Researchers and other personel in the university, government and food industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has trained Ph.D. level graduate students. How have the results been disseminated to communities of interest?Yes. The results have been presented in the annual meeting of professional societies. What do you plan to do during the next reporting period to accomplish the goals?We will continuously make presentations at the annual meetings of professional societies and publish papers in peer reviewed professional journals.
Impacts
What was accomplished under these goals?
A group of STEC cultures, capable of producing cellulose or curli were 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 37ºC 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 37ºC for 30 min, with 15 ml of 2 or 4% acetic or lactic acid at room temperature for 20 min, and with a manufacture-recommended concentration of an alkaline (Ecolab) or acidic (ZEP Manufacturing Company) sanitizer at room temperature for 7 and 15 min, respectively. Immediately after the treatments described above, 1 ml of each treated STEC cell suspension and untreated control were mixed with 9 ml of double strength Dey-Engley (DE) buffer. The cell suspensions were left in the DE buffer at room temperature for 15 min before serial dilutions were made in 0.1% buffered peptone water. The last three dilutions of each cell suspension (0.1 ml) or the undiluted samples (0.5 ml) were plated in duplicate on tryptic soy agar plates. The colonies on the surface of TSA plates were enumerated after 24 h incubation at 37°C. The detection limit was2 CFU/ml or 0.30 log CFU/ml. All samples were tested in duplicates and had appropriate controls. Each experiment was repeated in three independent trials. The influence of the treatments on the survival of the STEC was determined using the studentt-test and general linear model of the Statistical Analysis Software at a 95% confidence level. Treatments with cellulase and proteasedid not have any influence on the survival of STEC cells. However, treatments with 2 or 4% acetic and lactic acid significantly (P < 0.05) reduced the average populations of the six STEC strains as well as the populations of individual STEC strains used in the study except for STEC strain 6-35 and 7-51 treated by 2% acetic acid. Treatments with the two sanitizers reduced the average cell populations of the six STEC strains to undetectable levels.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Park, Y. J., and J. Chen. Inactivation of Shiga toxin-producing Escherichia coli (STEC) and degradation/removal of cellulose from STEC surfaces using selected enzymatic and chemical treatments. Appl. Environ. Microbiol. 77:8532-8537.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Researchers and other personel in the university, government and food industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Yes. The results have been presented in the annual meetings of prefessional societies. What do you plan to do during the next reporting period to accomplish the goals? Will continuously make presentation at the annual meetings of professional societies and publish papers in peer-reviewed prefessional journals.
Impacts
What was accomplished under these goals?
A group of STEC cultures, capable of producing cellulose or curli were 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 37ºC 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 37ºC for 30 min, with 15 ml of 2 or 4% acetic or lactic acid at room temperature for 20 min, and with a manufacture-recommended 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 were quantified. For cellulose quantification, 10 ml of treated cell 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 remained on STEC surfaces were calculated by comparing the absorbance values of the standard with the values of the tested samples. For curli quantification, treated 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. Treatments with cellulose, lactic acid and selected commercial sanitizers significantly decreased the amounts of cellulose on STEC. Treatments with acetic and lactic organic acid as well as selected commercial sanitizers also significantly reduced the amounts of curli on STEC surface. Treatments with protease were effective in degrading/removing curli proteins from STEC surface. Results suggest that these selected enzymatic and chemical agents could be used to to remove cellulose and curli from STEC surfaces.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Inactivation of Shiga toxin-producing Escherichia coli (STEC) and degradation/removal of cellulose from STEC surfaces using selected enzymatic and chemical treatments. Appl. Environ. Microbiol. 77:8532-8537.
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Progress 01/01/12 to 12/31/12
Outputs
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. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
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-.
Publications
- No publications reported this period
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