Antimicrobial Tolerance in Salmonella: Contributions to Survival and Persistence in Processing Environments.

Salmonella remains a top bacterial pathogen implicated in several food-borne outbreaks, despite the use of antimicrobials and sanitizers during production and processing. While these chemicals have been effective, Salmonella has shown the ability to survive and persist in poultry processing environments. This can be credited to its microbial ability to adapt and develop/acquire tolerance and/or resistance to different antimicrobial agents including oxidizers, acids (organic and inorganic), phenols, and surfactants. Moreover, there are several factors in processing environments that can limit the efficacy of these antimicrobials, thus allowing survival and persistence. This mini-review examines the antimicrobial activity of common disinfectants/sanitizers used in poultry processing environments and the ability of Salmonella to respond with innate or acquired tolerance and survive exposure to persists in such environments. Instead of relying on a single antimicrobial agent, the right combination of different disinfectants needs to be developed to target multiple pathways within Salmonella.

Controlling Salmonella: strategies for feed, the farm, and the processing plant.

Controlling Salmonella in poultry is an ongoing food safety measure and while significant progress has been made, there is a need to continue to evaluate different strategies that include understanding Salmonella-poultry interaction, Salmonella-microbiota interactions, Salmonella genetics and response to adverse conditions, and preharvest and postharvest parameters that enable persistence. The purpose of this symposium is to discuss different strategies to consider from feed milling to the farm to the processing environment. This Poultry Science Association symposium paper is divided into 5 different sections that covers 1) immunological aspects of Salmonella control, 2) application of Salmonella genetics for targeted control strategies in poultry production, 3) improving poultry feed hygienics: utilizing feed manufacture techniques and equipment to improve feed hygienics, 4) practical on farm interventions for controlling Salmonella-what works and what may not work, and 5) monitoring and mitigating Salmonella in poultry. These topics elucidate the critical need to establish control strategies that will improve poultry gut health and limit conditions that exposes Salmonella to stress causing alterations to virulence and pathogenicity both at preharvest and postharvest poultry production. This information is relevant to the poultry industry's continued efforts to ensure food safety poultry production.

Combined Quantification and Deep Serotyping for Salmonella Risk Profiling in Broiler Flocks.

Despite a reduction of Salmonella contamination on final poultry products, the level of human salmonellosis cases attributed to poultry has remained unchanged over the last few years. There needs to be improved effort to target serovars which may survive antimicrobial interventions and cause illness, as well as to focus on lessening the amount of contamination entering the processing plant. Advances in molecular enumeration approaches allow for the rapid detection and quantification of Salmonella in pre- and postharvest samples, which can be combined with deep serotyping to properly assess the risk affiliated with a poultry flock. In this study, we collected a total of 160 boot sock samples from 20 broiler farms across four different integrators with different antibiotic management programs. Overall, Salmonella was found in 85% (68/80) of the houses, with each farm having at least one Salmonella-positive house. The average Salmonella quantity across all four complexes was 3.6 log10 CFU/sample. Eleven different serovars were identified through deep serotyping, including all three key performance indicators (KPIs; serovars Enteritidis, Infantis, and Typhimurium) defined by the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS). There were eight multidrug resistant isolates identified in this study, and seven which were serovar Infantis. We generated risk scores for each flock based on the presence or absence of KPIs, the relative abundance of each serovar as calculated with CRISPR-SeroSeq (serotyping by sequencing the clustered regularly interspaced palindromic repeats), and the quantity of Salmonella organisms detected. The work presented here provides a framework to develop directed processing approaches and highlights the limitations of conventional Salmonella sampling and culturing methods. IMPORTANCE Nearly one in five foodborne Salmonella illnesses are derived from chicken, making it the largest single food category to cause salmonellosis and indicating a need for effective pathogen mitigation. Although industry has successfully reduced Salmonella incidence in poultry products, there has not been a concurrent reduction in human salmonellosis linked to chicken consumption. New efforts are focused on improved control at preharvest, which requires improved Salmonella surveillance. Here, we present a high-resolution surveillance approach that combines quantity and identity of Salmonella in broiler flocks prior to processing which will further support improved Salmonella controls in poultry. We developed a framework for this approach, indicating that it is possible and important to harness deep serotyping and molecular enumeration to inform on-farm management practices and to minimize risk of cross-contamination between flocks at processing. Additionally, this framework could be adapted to Salmonella surveillance in other food animal production systems.

Differences in biofilm formation of Salmonella serovars on two surfaces under two temperature conditions.

AIMS: Salmonella is extremely diverse, with >2500 serovars that are genetically and phenotypically diverse. The aim of this study was to build a collection of Salmonella isolates that are genetically diverse and to evaluate their ability to form biofilm under different conditions relevant to a processing environment. METHODS AND RESULTS: Twenty Salmonella isolates representative of 10 serovars were subtyped using Clustered regularly interspaced short palindromic repeats (CRISPR)-typing to assess the genetic diversity between isolates of each serovar. Biofilm formation of the isolates on both plastic and stainless-steel surfaces at 25 and 15°C was assessed. At 25°C, 8/20 isolates each produced strong and moderate biofilm on plastic surface compared to stainless-steel (3/20 and 13/20 respectively). At 15°C, 5/20 produced strong biofilm on plastic surface and none on stainless-steel. Several isolates produced weak biofilm on plastic (11/20) and stainless-steel (16/20) surfaces. Serovar Schwarzengrund consistently produced strong biofilm while serovars Heidelberg and Newport produced weak biofilm. CONCLUSION: These results suggest that Salmonellae differ in their attachment depending on the surface and temperature conditions encountered, which may influence persistence in the processing environment. SIGNIFICANCE AND IMPACT OF STUDY: These differences in biofilm formation could provide useful information for mitigation of Salmonella in processing environments.

Prevalence of Salmonella enterica on poultry processing equipment after completion of sanitization procedures.

Salmonella is a poultry-borne pathogen that causes illness throughout the world. Consequently, it is critical to control Salmonella during the process of converting broilers to poultry meat. Sanitization of a poultry processing facility, including processing equipment, is a crucial control measure that is utilized by poultry integrators. However, prevalence of Salmonella on equipment after sanitization and its potential risk to food safety has not been evaluated thoroughly. Therefore, the objective of this study was to evaluate the persistence of Salmonella on poultry processing equipment before and following cleaning and sanitization procedure. A total of 15 locations within 6 commercial processing plants were sampled at 3 time points: (A) after processing; (B) after cleaning; and (C) after sanitization, on 3 separate visits for a total of 135 samples per plant. Salmonella-positive isolates were recovered from samples using the United States Department of Agriculture MLG 4.09 conventional method. Presumptive Salmonella colonies were subjected to biochemical tests for confirmation. Salmonella isolates recovered after sanitization were serotyped and tested for the presence of specific virulence genes. A completely randomized design with a 6 × 3 × 15 factorial arrangement was utilized to analyze the results for Salmonella prevalence between processing plants. Means were separated using Fishers protected least significant difference when P ≤ 0.05. For Salmonella prevalence between processing plants, differences (P < 0.0001) were observed in the 6 plants tested where the maximum and minimum prevalence was 29.6 and 7.4%, respectively. As expected, there was a difference (P < 0.0001) in the recovery of Salmonella because of sampling time. Salmonella prevalence at time A (36%) was significantly higher, whereas there was no difference between time B (12%) and C (9%). There was a location effect (P < 0.0001) for the prevalence of Salmonella with the head puller, picker, cropper, and scalder having a significantly higher prevalence when compared with several other locations. At sampling time C, a trend toward a difference (P = 0.0899) was observed for Salmonella prevalence between the 6 plants, whereas significant differences were observed because of location (P = 0.0031). Five prominent Salmonella enterica serovars were identified, including Kentucky, Schwarzengrund, Enteritidis, Liverpool, and Typhimurium with S. Kentucky being the most prevalent. PCR analysis of 8 Salmonella virulence genes showed that the invA, sipB, spiA, sseC, and fimA were detected in all isolates, whereas genes carried on plasmids and/or fimbriae varied remarkably among all isolates. This study established Salmonella prevalence and persistence in poultry processing facilities after antimicrobial application through sanitization procedures which could result in contamination of poultry carcasses and food safety risks because of poultry meat.

Homologous stress adaptation, antibiotic resistance, and biofilm forming ability of Salmonella enterica serovar Heidelberg ATCC8326 on different food-contact surfaces following exposure to sublethal chlorine concentrations1.

Salmonella enterica serovar Heidelberg (American Type Culture Collection; ATCC 8326) was examined for the ability to adapt to the homologous stress of chlorine through exposure to increasing chlorine concentrations (25 ppm daily increments) in tryptic soy broth (TSB). The tested strain exhibited an acquired tolerance to chlorine in TSB with the tolerant cells growing in concentrations up to 400 ppm. In addition, the chlorine stressed cells displayed rugose morphology on tryptic soy agar (TSA) plates at 37°C. The minimum inhibitory concentration (MIC) of chlorine for adapted (rugose and smooth) cells was determined to be 550 ppm and 500 ppm, respectively whereas the MIC for the control was 450 ppm. The biofilm forming ability of the adapted and control cells were examined on both plastic and stainless steel surface at room temperature and 37°C. The rugose variant, in contrast to the smooth (adapted and control) showed the ability to form strong biofilms (P ≤ 0.05) on a plastic surface at room temperature and 37°C. Rugose cells compared to smooth and control attached more (P ≤ 0.05) to steel surfaces as well. The possibility of cross-adaptation was examined by exposing the adapted and control cells to different antibiotics according to the Clinical & Laboratory Standards Institute guidelines. Adapted cells exhibited reduced susceptibility to some of the antibiotics tested as compared to control. The findings of this study suggest that exposure to sublethal chlorine concentration during the sanitization procedure can result in tolerant Salmonella cells. Chlorine may confer cross-protection that aids in the survival of the tolerant population to other environmental stresses.