Editor’s Note: This research originally published in Food Safety Magazine’s April/May 2015 issue, a portion of which is reprinted here with permission. To read the entire article, please visit http://bit.ly/MeatSciReview0715.

Shiga toxin-producing Escherichia coli (STEC) are pathogens of concern across various food products 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 non-intact beef products in the United States after a large outbreak from undercooked ground beef patties in 1982 (6).

Worldwide, non-O157 STEC strains are estimated to cause 20 to 50% of STEC-related infections (5). 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 an estimated 70% of non-O157 STEC infections in the United States (1). The United States Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) has included these serogroups along with E. coli O157:H7 as adulterants in non-intact beef products (9).

Biofilms are communities of microorganisms that can form on both living and non-living 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 (4, 7, 8, 10). Previous studies have determined that E. coli O157:H7 can attach and form biofilms on surfaces such as stainless steel and plastic (2, 7, 8). A series of studies, including two conducted in our laboratory, have shown STEC attachment is strain-dependent (9) showing that assumptions cannot be made about the entire serogroup in terms of attachment to and biofilm formation on 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 to enjoy. However, only a few studies have utilized a combination of detergents and sanitizers to determine their effectiveness against biofilms containing pathogens like STECs that are attached to commonly used surfaces like stainless steel.  Mimicking food-processing environments where STEC cells could be found was an important aspect of this study. 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.

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). One strain from each serogroup that showed a high affinity to attach to stainless steel in minimal nutrient media was used. For each strain (n=7), 5 pieces (coupons) of stainless steel were incubated in minimal nutrient media for 24 h at 25°C to allow the STEC to attach to the surface. After 24 h 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.

Detergent and sanitizer 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 min, then rinsed with water, and transferred to a clean well for the 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 means (LSMs) with an aof 0.05.

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 as compared to 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 and 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 to 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 currently in progress. Others have found a complete cleaning and sanitation program was more efficient in removing bacteria numbers and contributed the findings to the action of the detergent (3). These conclusions were made in part because the sanitizer became less effective as the soil residue increased over time within their testing system.

In our study, the 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 as compared to applying both detergent and sanitizer. Because research on the removal of STECs from equipment is limited, we also chose to use the manufacturer’s recommended concentrations for sanitizer concentrations. Further research, included 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.

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 on the removal of 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. NP

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