Battling Biofilms Using Novel Strategies

Background

Shiga toxin-producing E. coli (STEC; including E. coli O157) – and other food safety pathogens or spoilage organisms – are especially problematic when they form biofilms. Biofilms are structures that bacteria build to help them persist in the environment. Biofilms are really difficult to detect and to clean. Sometimes cleaning efforts only end up dispersing them and starting new biofilms elsewhere.

Biofilms form when bacteria communicate using “quorum sensing” compounds. Some non-toxic “quorum-sensing inhibitors” like essential oils have been identified that may be able to help prevent biofilm formation. Some bacteria naturally found in meat processing facilities (MSB) may help inhibit or enable biofilm formation.

Objectives

• Using meat-processing surface bacteria (MSB) and Shiga toxin-producing E. coli (STEC) evaluated for biofilm-producing ability in previous studies, select mixtures of STEC and MPB which can form extremely strong multi-species biofilms on polystyrene surfaces
• Evaluate non-toxic quorum sensing inhibitors (QSI) to determine if they can prevent inclusion of STEC in multi-species biofilms or completely inhibit biofilm formation using combinations of isolates capable of forming extremely strong biofilms identified in part one of the study
• Evaluate the impacts of a biofilm-inhibiting MSB on the efficacy of QSI for preventing the biofilm formation in mixtures of E. coli and biofilm-enhancing MPB capable of forming extremely strong biofilms identified in part one of the study
• Use RNA sequence analyses to compare gene expression by STEC before and after exposure to increasing concentrations of the different QSI
• To evaluate the efficacy of amphiphilic molecules as delivery vehicles for antimicrobials / enzymes / QSI on conveyor belts and stainless-steel surfaces
• To assess the effectiveness of pulsed electric fields in disrupting biofilms formed on conveyor belt surfaces

What they will do

Building on previous work, these researchers will study whether quorum sensing compounds or bacteria can help prevent biofilm formation on polystyrene surfaces (foam and hard plastic) and study STEC gene expression to figure out the mechanisms involved. Also, the hot water used in cleaning may increase protein adhesion to packing plant surfaces – this would help biofilms stick to equipment and surfaces. Hydrophobic compounds (like essential oils) don’t mix with water, so better detergents (like micelles or solid lipid nanoparticles) and an “electric pulse” that makes bacterial cells leaky may help deliver QSI into biofilms and combat them on stainless steel and conveyor belt surfaces more effectively and help to reduce water and sanitizer use and combat biofilms.

1. Gram positive meat plant bacteria that form extremely strong biofilms with STEC (Lueteococcus japonica, Microbacterium phyllosphaerae, Plantibacter spp.) will be mixed (singly or in combinations of two or three other species) with each of 10 different STEC isolates (5 that are known to form isolates, 5 that don’t). This will make seven meat plant bacteria combinations x 10 STEC isolates, plus a negative control. Optical density / absorbance will be used to determine biofilm strength. qPCR targeting the stx1 gene will evaluate STEC concentrations. This will tell them whether biofilm strength is mostly due to the STEC or the meat plant bacteria. Pseudomonas will be studied to see whether it can inhibit biofilm formation.

2. QSI: various concentrations of thymol-carvacrol, cinnamon (essential oils shown to prevent biofilm formation) and lactic acid (widely used but unknown effectiveness) will be added to cultures with the strongest biofilms to find out which of them can inhibit STEC incorporation or prevent biofilm formation.

3. Five strains of commensal Pseudomonas bacteria (and/or other inhibitory meat plant bacteria, such as Aeromonas, J. lividum, Serratia spp., or H. alvei) will be tested to identify the concentrations that inhibit biofilms.

4. Gene expression will be measured to see how STEC respond to the various concentrations of biofilm inhibitors in both traditional and a beef juice-containing culture media

5. micelles (structures that allow oils to dissolve in water) and solid lipid nanoparticles will be designed using modelling software to see if the nanocarriers are compatible with a large range of different amphiphilic molecules. Strong biofilms (containing E. coli O157, Raoultella, Comonas) will be formed at 10 and 25^oC on stainless steel coupons and exposed to the new micelles and nanoparticles for 2, 5 or 10 minutes to see which of them most effectively combat established biofilms. The most promising combinations will be tested (compared to negative and positive controls) to see which can most effectively prevent biofilm formation.

6. A device will be designed to deliver varying intensity and durations of a pulsed electrical field to moist thermoplastic polyurethane coupons carrying biofilms. Temperature will be monitored to avoid melting the coupons. Bacterial reductions and surface damage will be evaluated.

Implications

Finding effective ways to inhibit or prevent the formation of biofilms in meat processing facilities will further improve food safety and support confidence in Canadian beef among domestic and international customers and consumers.