Shiga-toxigenic E. Coli Persistence Mechanisms and Surface Biofilm Detection Using Near-Infrared Spectroscopy on Beef Processing Facilities

Background

A great deal of food safety research has focused on E. coli O157:H7, even though it is only one of thousands of different Shiga toxin -producing E. coli (STEC). Although all are potentially harmful, they may differ in their ability to withstand sanitizers commonly used to clean meat processing equipment. They may also differ in their ability to form biofilms that enable bacteria to fasten themselves to surfaces and survive exposure to sanitizers, heat, steam, etc. The ability to form biofilms greatly increase the risk that bacteria will be able establish more permanent colonies in or on meat processing equipment or surfaces that can go on to contaminate beef. These biofilms can also be very difficult to remove and clean (e.g. dental plaque is a bacterial biofilm), or even detect. 

Objectives

• Determine the capacity of different STEC to survive and transfer from single and multispecies biofilms (wet or dry) formed on different materials (stainless steel and polyurethane) and environmental conditions (at 15 and 25°C) onto fresh beef surfaces (adipose and lean tissue).

• Test the effectiveness of chemical sanitizers to eliminate wet and dry single and multispecies STEC biofilms.
• Test the ability of near-infrared spectroscopy (NIRS) to detect STEC biofilms on beef processing equipment. 

What they Did

The study focused on understanding how STEC interacts with spoilage bacteria in the food industry, and if these interactions can help STEC to persist. How these spoilage bacteria could affect STEC survival, transfer to beef, and susceptibility to different sanitizers was investigated.  Environment factors such as temperature (10°C and 25°C) and humidity (wet and dry) on how they can affect biofilms and STEC persistence were also tested.

Scientists also looked at the genomes of these STEC related to outbreaks to explore if these bacteria carry genes that allow them to form biofilms and perhaps explain if these biofilm formation abilities could be helping STEC to cause disease. They identified genes related to biofilm formation, but bacteria like generic E. coli that is not pathogenic also carry some of biofilm related genes. However it was found that STEC carry different genes related to virulence (the ability of the bacteria to adapt to stressful conditions) that might be influencing biofilm formation.

NIRS spectroscopy was tested as a potential way to detect biofilms on stainless steel and plastic surfaces (polyurethane). It was found that NIRS was able to detect biofilms on polyurethane surfaces, often used to manufacture conveyor belts). This technology could be developed for surface testing at a commercial beef processing facility.

What They Learned

Bacteria commonly found in the food industry have been found to play a big role in the survival and persistence of the harmful bacteria STEC. The researchers looked at different combinations of bacteria in slimy layers called biofilms and found that a mixture of Pseudomonas and Comamonas was particularly effective at fighting STEC within the biofilm. Interestingly, another type of bacteria including lactic acid bacteria didn’t seem to reduce STEC within the biofilms however but showed to be more susceptible when treated with sanitizers.

The conditions in which these biofilms form, such as humidity, the surface they stick to, and how long they are stored, also play a significant role in how easily STEC contaminates beef. In particular, when beef comes into contact with fresh and moist biofilms on plastic surfaces contamination is more severe.

Researchers also discovered that the performance of the sanitizers varied based on factors like temperature, surface type, and the specific combination of bacteria present in the biofilm.

The results showed that all sanitizers effectively removed single-species biofilms (only STEC) formed at lower temperatures (10°C) due to the weaker structure of these biofilms. It was found that when the biofilms were formed at higher temperatures (25°C), sodium hypochlorite was not very effective.

 A sanitizer composed of hydrogen peroxide, sulphonic acid, Acetic acid and peroxyacetic acid developed to eradicate biofilms proved to be the most effective sanitizer against all biofilm combinations.

The study also emphasized the significance of thorough surface scrubbing during cleaning and sanitation procedures. The specific combination of bacteria within the biofilm had a substantial impact on its resilience, underscoring the importance of proper scrubbing to reduce bacterial levels.

ATP testing (test for organic material on surfaces) was employed to assess the cleanliness of surfaces after cleaning. The findings demonstrated that different combinations of bacteria within the biofilm resulted in varying levels of cleanliness after scrubbing, with some combinations achieving better cleaning efficacy than others.

Furthermore, the researchers explored the use of near-infrared (NIR) technology for detecting biofilms on food contact surfaces. NIR proved effective in detecting biofilms on thermoplastic polyurethane (TPU) (plastic) surfaces but not on stainless steel surfaces. This information is valuable as TPU surfaces were found to be more prone to biofilm formation.

Genomics analysis enabled the identification of specific genes associated with biofilm formation and resistance to some sanitizers. However, further research is needed to fully comprehend the complex processes involved in biofilm formation and bacterial persistence.

What It Means

This research highlights the significant role of bacteria commonly found in the food industry in the survival and persistence of harmful bacteria, such as STEC. The conditions under which biofilms form, including humidity, surface type, and storage duration, greatly influence STEC contamination of beef, with fresh and moist biofilms on plastic surfaces leading to more severe contamination. Biofilms can be formed at 25 and 10°C, and STEC can survive within those biofilms, survival depends on what type of bacteria resides in the biofilm community. The performance of sanitizers varied based on temperature, surface type, and the specific combination of bacteria in the biofilm; sanitizers are more effective on weaker biofilms formed at 10 °C, and when they are with biofilms formed by lactic acid bacteria, however, some STEC cells can still survive depending on the sanitizer type.  Thorough surface scrubbing is crucial to reduce bacterial levels. ATP testing revealed varying levels of cleanliness after scrubbing surfaces containing biofilms and it depends on the bacteria combination. Near-infrared (NIR) technology effectively detected biofilms on plastic surfaces, which are more prone to biofilm formation.