Scott L. Burnett

Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing.

Confocal scanning laser microscopy (CSLM) was used to determine the location of Escherichia coli O157:H7 cells on the surface and in tissue of bruised Red Delicious cv. apples. Undamaged and bruised apples were inoculated by immersing in a suspension of E. coli O157:H7 cells transformed with a plasmid that encodes for the production of a green fluorescent protein. Apples were then washed in 0.1% (wt/vol) peptone water and/or rubbed with a polyester cloth and examined to determine if these treatments removed or introduced cells into lenticels, cutin, and cracks on the skin surface. Optical slices of the apples obtained using CSLM were examined to determine the depth at which colonization or attachment of cells occurred. Populations of E. coli O157:H7 on the surface of apples were determined to assess the effectiveness of washing and rubbing in physically removing cells. The location of cells on or in undamaged and bruised areas of apples that were not washed or rubbed did not differ significantly. However, washing apples resulted in an approximate 2-log reduction in CFU of E. coli O157:H7 per cm2 of apple surface. On unwashed apples, cells were detected at depths up to 30 microm below the surface. No E. coli O157:H7 cells were detected at locations more than 6 microm below the surface of washed apples. Cells that remained on the surface of rubbed apples appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets. These cells may be protected from disinfection and subsequently released when apples are eaten or pressed for cider production.

Comparison of methods for fluorescent detection of viable, dead, and total Escherichia coli O157:H7 cells in suspensions and on apples using confocal scanning laser microscopy following treatment with sanitizers.

The influence of treating Escherichia coli O157:H7 cells labeled with an enhanced green fluorescent protein (EGFP) plasmid with 20 microg/ml active chlorine, 100 mg/ml hydrogen peroxide, and 80 mg/ml acetic acid on fluorescence intensity was determined. In addition, fluorescent staining methods to differentiate viable and dead E. coli O157:H7 cells on the cuticle of Red Delicious cv. apples following treatment with water or 200 microg/ml active chlorine were evaluated. Suspensions of E. coli O157:H7 EGFP+ cells were exposed to chemical treatment solutions for 0, 30, 60, 120, or 300 s before populations (log10 cfu/ml) were determined by surface plating, and fluorescence intensities of suspensions and individual cells were measured using spectrofluorometry and confocal scanning laser microscopy (CSLM), respectively. The relative fluorescence intensity of suspensions and individual cells changed upon exposure to various treatments. Results indicate that the use of EGFP to tag E. coli O157:H7 may not be appropriate for investigations seeking to microscopically differentiate viable and dead cells on produce following surface treatment with sanitizers. SYTOX Orange and SYTOX Green nucleic acid stains fluorescently labeled dead E. coli O157:H7 cells attached to apple cuticles more intensely than did propidium iodide. A cross-signal occurred between CSLM photomultipliers when examining tissues treated with SYTOX Orange to detect dead cells and antibody labeled with Alexa Fluor 488 to detect total (dead and viable) cells. Because of the possibility of cross-signal resulting in an overestimation of the number of dead cells on apples and, perhaps, other produce treated with these stains, SYTOX Green is preferred to detect dead cells and antibody labeled with Alexa Fluor 594 is preferred to detect the total number of cells on apple surfaces following treatment with sanitizers. The performance of SYTOX Green in combination with Alexa Fluor 594 to detect dead and total cells of E. coli O157:H7 on other produce remains to be determined.

Use of octanoic acid as a postlethality treatment to reduce Listeria monocytogenes on ready-to-eat meat and poultry products

The antilisterial efficacy and organoleptic impact of an octanoic acid (OA)-based treatment for ready-to-eat (RTE) meat and poultry products were investigated. Whole-muscle and comminuted RTE products were inoculated with a five-strain mixture of Listeria monocytogenes. The OA treatments were applied to the surface of RTE products by dispensing a specific volume of solution directly into the final package prior to vacuum sealing. Once sealed, the vacuum-packaged RTE products containing OA were immersed in water heated to 93.3 degrees C (200 degrees F) for 2 s to effect adequate film shrinkage. Extending the time at which the packaged, treated RTE products were exposed to water heated to 93.3 degrees C was also evaluated with a commercial cascading shrink tunnel fitted with a modified drip pan. Once treated, RTE products were examined for survivor populations of L. monocytogenes after 24 h of storage at 5 degrees C. Sensory evaluation was conducted with a 60-member trained panel on 11 uninoculated, treated RTE products. The OA treatment of RTE products reduced L. monocytogenes numbers to between 0.85 log CFU per sample (oil-browned turkey) and 2.89 log CFU per sample (cured ham) when compared with controls. The antilisterial activity of OA was improved by increasing the duration of the heat shrink exposure. Specifically, reductions of L. monocytogenes ranged from 1.46 log CFU per sample (oil-browned turkey) to 3.34 log CFU per sample (cured ham). Results from the sensory evaluation demonstrated that 10 of the 11 treated RTE products were not perceived as different (P < or = 0.05) from the untreated controls. Panelists detected reduced (P < or = 0.05) smoke flavor intensity with treated mesquite turkey, although the treated product was viewed as acceptable. Results demonstrate the effectiveness of OA as a postlethality treatment meeting U.S. Food Safety and Inspection Service regulatory guidelines for RTE meat and poultry products with minimal impact on sensory quality.

Differentiation of viable and dead Escherichia coli O157:H7 cells on and in apple structures and tissues following chlorine treatment

Confocal scanning laser microscopy (CSLM) was used to differentiate viable and nonviable cells of Escherichia coli O157:H7 on and in raw apple tissues following treatment with water and 200 or 2,000 ppm active chlorine solution. Whole unwaxed Red Delicious cultivar apples at 25 degrees C were inoculated by dipping in a suspension of E. coli O157:H7 (8.48 log10 CFU/ml) at 4 degrees C, followed by treatment in water or chlorine solution at 21 degrees C for 2 min. The dead cells on and in apples were distinguished from live cells by treating tissue samples with SYTOX green nucleic acid stain. Viable and dead cells were then labeled with an antibody conjugated with a fluorescent dye (Alexa Fluor 594). The percentage of viable cells on the apple surface, as well as at various depths in surface and internal structures, was determined. The mean percentages of viable cells located at the sites after treatment with water or chlorinated water were in the following order, which also reflects the order of protection against inactivation: floral tube wall (20.5%) > lenticels (15.0%) > damaged cuticle surrounding puncture wounds (13.0%) > intact cuticle (8.1%). The location of viable cells within tissues was dependent on the structure. Except for lenticels, the percentage of viable cells increased as depth into the CSLM stacks increased, indicating that cells attached to subsurface structures were better protected against inactivation with chlorine than were cells located on exposed surfaces. Further research is warranted to investigate the efficacy of other chemical sanitizers. as well as that of surfactants and solvents in combination with sanitizers, in removing or killing E. coli O157:H7 lodgedin protective structures on the surface and within tissues of apples.