Shiga toxin-producing Escherichia coli (STEC) are pathogens of concern across various products within the food industry, as they have been connected to a wide variety of outbreaks and recalls. Most of the scientific literature concerning the removal of attached STEC cells focuses on E. coli O157:H7, as it was the first STEC to be considered an adulterant in nonintact beef products in the United States after a large outbreak from undercooked ground beef patties in 1982.[1]

Worldwide, non-O157 STEC strains are estimated to cause 20 to 50 percent of STEC-related infections.[2] A review of outbreaks from 1983 through 2002 found six serogroups (O26, O111, O103, O121, O145 and O45) to be the most common non-O157 STECs causing human illness in the United States.[3] With an estimated 70 percent of non-O157 STEC infections being caused by these serogroups, the U.S. Department of Agriculture Food Safety and Inspection Service has included these serogroups along with E. coli O157:H7 as adulterants in nonintact beef products.[4]

The Biofilm-Stainless Steel Connection
Biofilms are communities of microorganisms that can form on both living and nonliving surfaces, including those found in food processing plants. Biofilm formation depends on the microorganisms present and can be affected by a variety of environmental conditions, including nutrient availability, temperature, the cleanliness of the surface and the presence of other microorganisms.[5–8] Previous studies have determined that E. coli O157:H7 can attach and form biofilms on surfaces such as stainless steel and plastic.[6,7,9] A series of studies, including two conducted in our laboratory, have shown STEC attachment is strain dependent.[4] This finding was important because it shows assumptions cannot be made about the entire serogroup in terms of attachment to and biofilm formation on these surfaces.

A complete sanitation program including the removal of solids and utilization of both detergents and sanitizers within a food processing environment is essential to producing safe, wholesome products for consumers. Once formed, biofilms have an increased resistance to sanitizers.[4,5,10] However, only a few studies have utilized a combination of detergents and sanitizers to determine their effectiveness against biofilms containing pathogens like STECs attached to commonly used surfaces like stainless steel. The objective of this study was to determine the effectiveness of a detergent and a quaternary ammonium sanitizer to remove STEC cells attached to stainless steel. Quaternary ammonium is a commonly used sanitizer within the food industry that is effective in killing pathogens but doesn’t cause corrosion of equipment.

Mimicking food processing environments where these STEC cells could be found was an important aspect of this study. Multiple strains from all seven STEC serogroups (O157:H7, O26, O45, O103, O111, O121 and O145) were screened for their ability to attach to stainless steel in full and minimal nutrient media over time at 25 °C in previous studies. Attachment to stainless steel was strain dependent, and we found that attachment of STEC strains was higher under minimal nutrient conditions (data not shown).

For the objective of determining the effectiveness of the detergent/sanitizer combination, we used one strain from each serogroup that showed a high affinity to attach to stainless steel in minimal nutrient media (Table 1). For each strain (n = 7), five pieces (coupons) of stainless steel were incubated in minimal nutrient media for 24 hours at 25 °C to allow the STEC to attach to the surface. After 24 hours of attachment, the loose cells were gently removed by rinsing with water.

After the loose cells were removed, the stainless steel coupons were subjected to one of five treatments: detergent only (detergent/water), sanitizer only (water/sanitizer), detergent/sanitizer combination (detergent/sanitizer), control (water/water) or untreated control (inoculated with no treatment). Each combination was tested separately for each strain and replications were conducted in triplicate.

The detergent/sanitizer combination was prepared according to the manufacturer’s instructions, with a target sanitizer concentration of 200 ppm. Treatment solutions were put into separate foaming hand soap dispensers to simulate foaming application of the chemicals in a food processing environment. The coupons were exposed for 5 minutes to the treatments, then rinsed with water and transferred to new wells to prevent continued contact with the previous treatment. All coupons were in contact with the treatments for 5 minutes, then rinsed with water and transferred to a clean well for a colorimetric assay. Coupons were exposed through immersion only, and no mechanical action was applied to the coupons upon application of treatment. The colorimetric assay was used to determine the amount of STEC remaining on the coupons after treatment by measuring absorbance of the solution at 590 nm. Statistical analysis was performed to determine the least squared mean with an α of 0.05.

Detergent/Sanitizer Combination Effectiveness
Significant (p < 0.001) differences were found among treatments as well as strains. Untreated stainless steel coupons had a significantly (p < 0.0002) higher OD₅₉₀ absorbance value compared with the other treatments, indicating the treatments removed a large number of attached bacteria, as noted in Table 2. The most effective treatment was the detergent/sanitizer combination, with an overall reduction of over 0.023 in absorbance from the untreated stainless steel coupons, although the reduction was not significant (p > 0.05) when compared with the control (water only) and detergent-only treatments. The differences can be visually noted in Figure 1.

A complete cleaning and sanitation program, including the application of both detergent and sanitizer at manufacturer-recommended concentrations, can significantly reduce the amount of STEC bacteria attached to stainless steel. Because the STEC populations were not enumerated, we cannot confirm that all of the attached bacteria were removed. However, a study to determine the reduction of attached STEC bacteria using a complete sanitation program is in progress. Others have found a complete cleaning and sanitation program was more efficient in removing bacterial numbers and attributed the findings to the action of the detergent.[11] These conclusions were made in part because the sanitizer became less effective as the soil residue increased over time within their testing system. Further, Listeria monocytogenes biofilms were more effectively removed from high-density polyethylene cutting boards when both a rinse with distilled water and chemical inactivation with sanitizer were applied.[12] In our study, STECs were allowed to attach in laboratory media, so no food residue was present to reduce the effectiveness of the sanitizer, but our results still found decreased bacterial removal for sanitizer-only applications compared with applying both detergent and sanitizer.

Key Piece of Research
Through an extensive literature review at the design stage of this experiment, we believe this to be one of the first studies to examine a complete cleaning and sanitation program in the removal of attached STECs, as many of the studies published look only at the efficacy of sanitizers applied to the biofilms. We chose to include detergent to more closely mimic what occurs in food processing facilities. The food industry commonly uses quaternary ammonium-based sanitizers. In one study, 77 food production facilities in the UK were surveyed, and 55 percent of them used a quaternary ammonium compound as their sanitizer, making it the most commonly used.[13] Because research on the removal of STECs from equipment is limited, we also chose to use the manufacturer’s recommended concentrations to prepare our sanitizer treatment. Further research, including the enumeration of these STEC strains after treatment, is warranted to understand the total number of viable cells left on the equipment surfaces after the cleaning and sanitation program is complete.

Dourou and others[14] determined E. coli O157:H7 biofilm studies performed using laboratory media only were not adequate to mimic what would happen when the bacteria were exposed to food residues in a processing environment. As research about non-O157 STECs, especially serogroups O45 and O121, and their ability to attach and form biofilms is limited, we chose to perform these studies under laboratory conditions. Additional research is needed to determine how these bacteria act when exposed to food residues and other microorganisms present, and how those residues may impact the efficacy of cleaning and sanitation programs to remove STECs from equipment surfaces. In conclusion, our study shows that a complete cleaning and sanitation program administered within food production facilities is more effective at removing STEC bacteria from stainless steel in laboratory media when the chemicals are applied using manufacturers’ recommendations.

Amy R. Parks, Ph.D., is a postdoctoral research associate at Texas Tech University specializing in food safety research. Amy has 15 years of food microbiology and quality assurance experience.

Mindy M. Brashears, Ph.D., is a professor and director of the International Center for Food Industry Excellence at Texas Tech University. Her research focuses on interventions in pre- and postharvest environments and on the emergence of antimicrobial drug resistance. She teaches courses in food microbiology and food safety and offers industry training in food sanitation, recalls and food security. She holds a B.Sc. in food technology from Texas Tech and M.Sc. and Ph.D. degrees in food science from Oklahoma State University.

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