Are E. Coli Really Becoming More Heat Resistant?

Background

Shiga toxin-producing E. coli (STEC) is a serious human pathogen, with cattle being an important reservoir. To reduce the risk of beef contamination with STEC and other related pathogens, various antimicrobial interventions have been implemented in beef processing plants in Canada. Some of these interventions include pasteurization of carcasses with hot water and spraying carcasses/cuts with organic acid solutions. These interventions have resulted in significant improvement in the microbiological condition of beef carcasses and beef products produced at federally inspected beef processing facilities. Meat processing equipment is also routinely cleaned and sanitized. However, there are concerns that regular use of hot water and organic acids may lead to bacteria that can withstand these physical/chemical treatments. In addition, studies previously funded by BCRC demonstrated that some E. coli strains can survive and persist on equipment surfaces, and at times are the primary source of E. coli on meat. Thus, a better understanding of the mechanisms of survival and transmission would be of value for the eventual control of STEC on beef. 

Objectives

The overarching goal of this project was to determine whether E. coli are gaining resistance to antimicrobial hurdles used in meat plants and identify mechanisms by which E. coli survive and transfer in meat plants. The objectives were to:  

• Determine if E. coli from cattle are increasing in resistance to heat, acid, and sanitizers and in biofilm forming potential;  
• Determine mechanisms by which E. coli persist in beef packing facilities;  
• Identify genetic elements that confer resistance to E. coli using whole genome sequence approach;  
• Determine the role and mechanisms of biofilms in the transfer of pathogens from various surfaces to meat;  
• Explore novel sanitization technologies to control food-borne pathogens. 

What They Did

E. coli is a diverse species and as such we took a population-based approach to address Obj. 1-3, to reduce potential bias. For meat plant E. coli, 700 isolates from two beef packing plants were isolated from seven sources: carcasses before and after the application of antimicrobial interventions; carcasses before and after a dry chilling process; cuts and trimmings; meat processing equipment before and after cleaning. For cattle E. coli, 750 isolates belonging to the “Top 7 STEC” serogroups O26, O45, O103, O111, O121, O145 and O157 were used. The E. coli isolates were tested for their resistance to heat, by assessing the decimal reduction at 60°C (D_60°C), the time it takes to kill 90% of a bacterial population at 60°C and determination of the genetic marker for heat resistance (locus of heat resistance, or LHR); for resistance to lactic acid; for susceptibility to two commonly used sanitizers by assessing minimal inhibitory concentrations (MIC); and for their biofilm forming potential on plastic surfaces. Selected isolates were whole genome sequenced and analyzed for genetic determinants for phenotypes along with E. coli genomes in the public database. For Obj. 4, the transfer to beef of cells in biofilms developed on stainless steel by STEC with different biofilm forming potential under various conditions (temperature, growth time and ageing time) were examined. For Obj. 5, bactericidal activities against 7 LHR-positive E. coli strains of rechargeable N-chloramine coating on stainless coupons were assessed. 

What They Learned

Most (97.2%) E. coli were heat sensitive, with D_60°C values ≤ 2 min. No increase in heat resistance in cattle-derived E. coli on preintervention carcasses, for populations along the beef production or over time, as reflected by D_60°C and the prevalence of LHR. A large-scale genomic analysis using over 18,000 E. coli genomes showed incompatibility of Shiga toxin genes and LHR. For lactic acid treatments, E. coli from preintervention carcasses were slightly more resistant than E. coli from equipment, with the former group having a higher prevalence of genes coding for the metabolism of long-chain sugar acids and short-chain fatty acids. For sanitizers, the MIC values were ≤ 9 ppm, well below the in-use concentration of 200 ppm, and no differences in MICs were observed for different populations of meat plant E. coli. For cattle E. coli, slightly higher MICs, up to 25 ppm, for sanitizers, and lower reductions by the lactic acid treatment were observed, although all E. coli evaluated would be sensitive to concentrations of sanitizers used in meat plants. The proportion of E. coli that could form biofilms varied among different isolation sources, with the highest (87.3%) and lowest (7.1%) being for equipment and cattle E. coli populations, respectively. Isolates from equipment after sanitation had a greater biofilm-forming capacity than those before sanitation. Genomic analysis revealed a higher prevalence of genes involved in novel substrate utilization and iron acquisition in persistent E. coli but failed to identify genes associated with the biofilm forming phenotype for E. coli of any origin. Population structures of E. coli differed between those from animals and equipment, with the latter better suited for environmental survival as revealed by genomic analysis. The post dry chilling carcass E. coli population had a smaller proportion of biofilm-forming isolates, lower prevalence of LHR, and greater reduction by lactic acid treatment compared to their pre-chilling counterpart. The inactivation of LHR positive E. coli strains by N-chloramine coatings ranged from 2 to 7 log units and variation in cell reduction for the same E. coli strain was noted between different recharge cycles. The potential and extent of biofilm formation by the STEC strains were affected by strain, temperature, and ageing time. The rate of transfer to beef of bacterial cells in biofilms increased with growth time and can be detectable up to 30 days albeit decreasing with ageing time.   

What It Means

The antimicrobial hurdles assessed would unlikely lead to increased heat or sanitizer resistance in E. coli in beef processing environments. The likelihood of STEC harboring the heat resistance marker LHR is very low. The survival and persistence of E. coli strains in meat processing environments are likely caused by their biofilm formation and ability to utilize novel substrates, rather than resistance to antimicrobial hurdles. Effective cleaning and sanitization protocols which could address biofilms are critical in meat processing facilities to reduce STEC contamination. A less laborious means of predicting biofilm formation in E. coli instead of direct evaluation of biofilm growth would be useful in this regard. Dry chilling could be further explored as a green technology for controlling pathogens on meat, including the difficult to control biofilm formers. N-chloramine coating has potential for controlling E. coli on equipment surface, with further refinement in coating development.