In-plant validation of harvest process controls, environmental monitoring of Shiga toxin-producing Escherichia coli (STEC) and Salmonella and antimicrobial resistance and virulence factor analysis of isolated STECs from meat processing facilities in Honduras

Salmonella and Shiga Toxin producing Escherichia coli (STEC) can cause severe illness in adults and children alike. Salmonella and STECs together cause over 1 million illnesses in the United States. Life threatening complications can occur if infected by these pathogens. As food safety is becoming more important worldwide, research in low and middle-income countries is required. In Honduras the government has implemented an aggressive plan for cattle repopulation, which led to an increase interest in assessment of food safety conditions inside beef processing plants. These studies were realized to assess food safety needs in meat processing plants in Honduras and improve food safety conditions in the country. The first study sought to validate the beef harvest HACCP and food safety programs of two beef processing plants in Honduras operating under United States (US) equivalency standards by evaluating the presence of Salmonella and STEC on hides, their transfer from hide to carcass detected by pre-evisceration sampling, the mitigation of transferred pathogens by 50-100 ppm peracetic acid spray in plant A and 2.0-2.5% lactic acid intervention in plant B and Salmonella prevalence in lymph nodes. In plant A, prevalence of Salmonella on hides (n = 30/687; 4.4%) was significantly higher (p < 0.10) than on carcasses swabbed at pre-evisceration (n = 7/687; 1.0%) and post-intervention (n = 13/678; 1.9%), and in lymph nodes (n = 14/691; 2.0%). No significant difference in pathogen prevalence among the last three processing sites was found (p = 0.2751). In plant B, STEC prevalence on hides (n = 21/85; 24.7%) was higher (p < 0.10) than in carcasses at pre-evisceration (n = 3/85; 3.5%) and post-intervention (n = 1/85; 1.2%). Pathogen prevalence did not differ (p = 0.306) between carcasses in pre-evisceration and post-intervention but the initial presence was very low. Salmonella spp. and STEC prevalence in beef carcasses can be mitigated by process controls implemented in Honduran plants. Overall sanitary conditions, good manufacturing practices and HACCP interventions on the harvest floor, effectively control and reduce pathogen prevalence on processed beef carcasses. Hide-to-carcass pathogen transfer is significantly mitigated and the resulting pathogen prevalence in carcasses after microbial intervention with lactic acid is substantially low. Upon due validation of their harvest HACCP programs, both plants were granted their respective US equivalency for beef exporting purposes. A follow-up validation study must be conducted during the following year to demonstrate that the HACCP program is still effective for reducing pathogen prevalence in beef carcasses. The second study focused on evaluation of reduction of STEC and Salmonella in two small beef processing plants in Honduras after training and implementation of sanitary standard operating procedures (SSOP) and good manufacturing practices (GMP). In Plant A, STEC occurred in 10%, 12.5%, 0% and 5% of the environmental samples respective to each month, indicating a significant reduction of STEC (p < 0.10) by the third sampling month. This coincided with the completion of GMP, SSOP and HACCP training, providing customized GMP manuals, and the use of SSOP for disinfection of targeted equipment, surfaces, and utensils found with positive pathogen presence in previous sampling dates. In plant A, after implementation of SSOPs for targeted positive sampling areas, samples were not positive in subsequent sampling months after SSOP implementation, indicating effectiveness of recommended SSOPs. In Plant A, Salmonella environmental presence was at 0% except at one sampling interval and thus was not adequate to validate effective implementation of interventions. Salmonella environmental presence for each month in Plant B was of 0.0%, 5.6%, 6.3%, 27.3% and 0.0% respectively. In plant B, persistence of Salmonella was observed in the stuffer, where confirmed Salmonella isolates were found in January and March. After second positive sample was found, in-plant training of stuffer SSOP was made, resulting in absence of Salmonella in the stuffer in June. The highest Salmonella presence was observed by the third sampling month. Therefore, additional actions were taken to reinforce implementation of SSOP and GMP manual instructions, and verification procedures, which collectively resulted in a Salmonella reduction to 0% (p < 0.10) by the fourth sampling month. STEC presence in this plant was too low to be an indication of validation of the effectiveness of implementation of interventions. These experiences demonstrate the feasibility of fully implementing SSOP with verification procedures and compliance with the instructions of a customized GMP manual. The third study had as an objective to determine virulence factors and acquired antimicrobial resistance genes patterns of presumptive STEC isolates from meat processing plants in Honduras. The extraction of DNA of 69 presumptive STEC isolates was performed. DNA libraries were prepared using the Nextera XT DNA Library preparation kit. These libraries were paired-ended and sequenced on an Ilumina MiSew platform. Raw reads were assembled using SPADES Genome Assembler. Virulence Factors and Acquired antimicrobial resistance genes were determined using the Center for Genomic Epidemiology pipelines. Resistance genes to 15 different antimicrobial classes were evaluated, 36.7% ± 8.8% of the isolates were deemed resistant to at least 1 antimicrobial class. From the resistant isolates, 45.5% ±1 5.0% were resistant to at least 2 or more antimicrobial classes. Beta-Lactam, fosfomycin and fluoroquinolone genotypic resistance were significantly higher than all antimicrobial classes. Thirty-two different virulence factors were found in the isolates. Presence of gad, iss, and IpfA was significantly higher than the rest of virulence factors. Virulence factors found are involved in attachment and adhesion, known and predicted effectors, type III secretion system, locus of enterocyte effacement island non-effectors and regulators. Understanding the antimicrobial resistance patterns and infection systems of the pathogens can be key for adequate treatment of infection. Proper monitoring procedures of STEC prevalence in meat processing environments in Honduras is required to mitigate the risks of their presence in finished products.