Increase in temperature facilitates Campylobacter jejuni biofilm formation under both aerobic and microaerobic incubation.

The formation of Campylobacter jeuni biofilms on processing surfaces is a significant concern in poultry processing, contributing to food safety risks. This study focused on assessing the biofilm forming capabilities of 12 field isolates of C. jejuni of different aerotolerance categories on stainless steel surfaces, a prevalent material in poultry processing environments. Working cultures of each isolate were prepared to approximately 6 log CFU/mL and incubated on stainless steel coupons under microaerobic or aerobic conditions at room temperature or 42°C for 72 h. Biofilm attached cells were enumerated using direct plating and biofilm density was measured using a crystal violet assay by measuring the optical density (OD600) a. Data analysis was conducted using the PROC GLIMMIX procedure in SAS 9.4 with a significance level of 0.05. The study revealed a notable interaction between aerotolerance categories and temperature (P < 0.039) impacting the number of biofilms attached C. jejuni cells on stainless steel coupons. All isolates had significantly higher counts when incubated at 42°C compared to room temperature, regardless of oxygen level (P < 0.001). Furthermore, stronger biofilm density was observed at 42°C compared to room temperature, regardless of oxygen level. These findings underscore the influence of temperature on the biofilm forming ability of C. jejuni. The ability of these field isolates to form biofilms under various environmental conditions suggests a heightened potential for surface colonization and increased infection risk in poultry processing facilities.

Aerotolerance and Multi-Locus Sequence Typing of Campylobacter jejuni Isolated from Commercial Broiler Processing Plants.

Campylobacter jejuni is one of the leading causes of acute diarrhea in the United States. Despite being a microaerophilic pathogen, C. jejuni continues to endure within the domain of food production, especially in poultry processing. Recent research on aerotolerance indicates that close monitoring of this pathogen is necessary. A total of 40 C. jejuni isolates previously obtained from commercial broiler processing plants were analyzed for aerotolerance and genetic diversity. In addition, the effect of aerotolerance and storage time (days) on the survival of C. jejuni on broiler drumsticks at refrigeration (4 °C) and freezing conditions (-20 °C) was also evaluated. Out of 40 isolates, 25 (62.5%) were aero-sensitive (AS), 10 (25%) were intermediately aerotolerant (IAT), and 5 (12.5%) were hyper aerotolerant (HAT). The isolates belonged to four clonal complexes (CCs) and six sequence types, with the majority of isolates assigned to the CC-353 clonal complex. C. jejuni counts were reduced by 0.40 log CFU/g after 7 days at 4 °C and by 1.50 log CFU/g after 14 days at -20 °C, respectively, irrespective of aerotolerance (p < 0.001). At both refrigeration (p < 0.013) and freezing (p < 0.001), HAT showed greater reductions as compared to AS and IAT. These findings suggest that both refrigeration and freezing reduce C. jejuni counts.

Salmonella Biofilm Formation under Fluidic Shear Stress on Different Surface Materials.

This study characterized biofilm formation of various Salmonella strains on common processing plant surface materials (stainless steel, concrete, rubber, polyethylene) under static and fluidic shear stress conditions. Surface-coupons were immersed in well-plates containing 1 mL of Salmonella (6 log CFU/mL) and incubated aerobically for 48 h at 37 °C in static or shear stress conditions. Biofilm density was determined using crystal violet assay, and biofilm cells were enumerated by plating on tryptic soy agar plates. Biofilms were visualized using scanning electron microscopy. Data were analyzed by SAS 9.4 at a significance level of 0.05. A surface-incubation condition interaction was observed for biofilm density (p < 0.001). On stainless steel, the OD600 was higher under shear stress than static incubation; whereas, on polyethylene, the OD600 was higher under static condition. Enumeration revealed surface-incubation condition (p = 0.024) and surface-strain (p < 0.001) interactions. Among all surface-incubation condition combinations, the biofilm cells were highest on polyethylene under fluidic shear stress (6.4 log/coupon; p < 0.001). Biofilms of S. Kentucky on polyethylene had the highest number of cells (7.80 log/coupon) compared to all other strain-surface combinations (p < 0.001). Electron microscopy revealed morphological and extracellular matrix differences between surfaces. Results indicate that Salmonella biofilm formation is influenced by serotype, surface, and fluidic shear stress.

Roles of Aerotolerance, Biofilm Formation, and Viable but Non-Culturable State in the Survival of Campylobacter jejuni in Poultry Processing Environments.

Campylobacter jejuni is one of the most common causes of foodborne human gastroenteritis in the developed world. This bacterium colonizes in the ceca of chickens, spreads throughout the poultry production chain, and contaminates poultry products. Despite numerous on farm intervention strategies and developments in post-harvest antimicrobial treatments, C. jejuni is frequently detected on broiler meat products. This indicates that C. jejuni is evolving over time to overcome the stresses/interventions that are present throughout poultry production and processing. The development of aerotolerance has been reported to be a major survival strategy used by C. jejuni in high oxygen environments. Recent studies have indicated that C. jejuni can enter a viable but non-culturable (VBNC) state or develop biofilm in response to environmental stressors such as refrigeration and freezing stress and aerobic stress. This review provides an overview of different stressors that C. jejuni are exposed to throughout the poultry production chain and the genotypic and phenotypic survival mechanisms, with special attention to aerotolerance, biofilm formation, and development of the VBNC state.

The Prevalence of Salmonella and Campylobacter on Broiler Meat at Different Stages of Commercial Poultry Processing.

In poultry processing, Salmonella and Campylobacter contaminations are major food safety concerns. Peracetic acid (PAA) is an antimicrobial commonly used in commercial poultry processing to reduce pathogen prevalence so as to meet the USDA-FSIS performance standards. The objective of this study was to determine the prevalence of Salmonella and Campylobacter on broiler meat in various steps of commercial poultry processing in plants that use PAA. Post-pick, pre-chill, post-chill, and drumstick chicken samples were collected from three processing plants and mechanically deboned meat (MDM) was collected from two of the three plants. Each plant was sampled thrice, and 10 samples were collected from each processing step during each visit. Among the 420 samples, 79 were contaminated with Salmonella and 155 were contaminated with Campylobacter. Salmonella and Campylobacter contamination on the post-pick samples averaged 32.2%. Significant reductions in Salmonella and Campylobacter were observed in pre-chill to post-chill samples, where the prevalence was reduced from 34% and 64.4% to nondetectable limits and 1.1%, respectively (p < 0.001). Salmonella and Campylobacter remained undetectable on the drumstick samples in all three processing plants. However, the prevalence of Salmonella and Campylobacter on MDM was similar to the post-pick prevalence, which suggests substantial cross-contamination from post-chill to MDM.

Spoilage Bacteria Counts on Broiler Meat at Different Stages of Commercial Poultry Processing Plants That Use Peracetic Acid.

In poultry processing, spoilage microbes are persistent microorganisms, which affect the quality of broiler meat. Peracetic acid (PAA) is the most common antimicrobial used by commercial processing plants, which can reduce the prevalence of these microbes. The goal of this study was to determine the concentrations of aerobic bacteria, coliforms, lactic acid bacteria, and Pseudomonas on broiler meat in processing plants that use peracetic acid in various concentrations as the primary antimicrobial. Samples were collected from three processing plants at five processing steps: post-pick (defeathering), pre-chill, post-chill, mechanically deboned meat (MDM), and drumsticks. Samples were rinsed in buffered peptone water for bacteria isolation. Over six log CFU/sample of aerobic plate counts (APC), lactic acid bacteria, and coliforms were detected on post-pick samples. All spoilage bacteria were reduced to nondetectable levels on post-chill samples (p < 0.001). However, the presence of all bacteria on mechanically deboned meat (MDM) samples indicated varying degrees of cross contamination from post-chill and MDM samples. These results suggest PAA effectively reduces spoilage microbes in chilling applications irrespective of differences in PAA concentrations. However, due to the levels of spoilage microbes detected in MDM, it may be worth investigating the potential interventions for this stage of processing.

Prevalence and Molecular Characteristics of Avian Pathogenic Escherichia coli in "No Antibiotics Ever" Broiler Farms.

Avian pathogenic Escherichia coli (APEC) causes significant economic and welfare concerns to the broiler industry. For several decades, prophylactic supplementation of antimicrobial growth promoters was the primary method to control APEC; however, the recent shift to no antibiotics ever (NAE) production has increased colibacillosis incidence. The objectives of this study were to determine the influence of season, flock age, and sample type on the prevalence and virulence of E. coli and to identify the serogroups and antimicrobial susceptibility of virulent and nonvirulent E. coli in NAE broiler farms. Litter, feces, cloacal swabs, and tracheal swabs were collected from 4 NAE farms during spring and summer seasons, and E. coli was isolated and confirmed by PCR. Confirmed E. coli isolates were tested for 5 APEC-virulence-associated genes (VAGs) using quantitative PCR (qPCR). Further, E. coli isolates with all five VAGs (100 isolates) and E. coli isolates without any VAGs (87 isolates) were screened against 11 antimicrobials through Kirby-Bauer disk diffusion assay, and their serogroups were tested using PCR. Data were analyzed using the GLIMMIX procedure of SAS 9.4, and statistical significance was determined at a P value of ≤0.05. Overall, the prevalence of E. coli was not affected by season, flock age, or sample type. However, the prevalence of all tested VAGs decreased from spring to summer (P ≤ 0.002). The frequency of resistance was highest for tetracycline, and serogroups O8 (31%) and O78 (11%) were most frequent in virulent E. coli. In conclusion, there is a high prevalence of virulent E. coli in NAE farms, especially in the spring season. IMPORTANCE Avian pathogenic Escherichia coli causes one of the most detrimental bacterial diseases to the United States poultry industry, colibacillosis. Colibacillosis leads to decreased performance, early mortality, and subsequent production loss. Previously, colibacillosis was largely mitigated by the use of antimicrobial growth promoters. Due to concerns about antimicrobial resistance, the use of these promoters has been largely removed from the broiler industry. With recent shifts in the poultry industry to NAE broiler production, there is an increase in bacterial disease and mortality. We do not know how this shift to NAE affects APEC prevalence within broiler farms. Therefore, in the current study, we attempted to assess the prevalence and virulence of E. coli within an antibiotic-free broiler environment, assessed antimicrobial susceptibility, and identified the serogroups of virulent and nonvirulent E. coli.

Research Note: Prevalence and molecular characteristics of Clostridium perfringens in "no antibiotics ever" broiler farms.

Clostridium perfringens (C. perfringens) is the etiological agent of necrotic enteritis and gangrenous dermatitis; 2 diseases that cause significant economic and welfare concerns to the broiler industry. Previously, Clostridium-related diseases were managed with the use of antimicrobial growth promoters fed to broilers that improved gut health and performance. The recent shift to no antibiotics ever (NAE) production has increased the incidence of Clostridium-related diseases. The objective of this study was to identify C. perfringens prevalence and toxinotypes in NAE farms. Samples of litter, feces, and cloacal swabs were collected from 4 NAE broiler farms in the summer of 2019, on d 28 and d 56 of one flock cycle. A total of 734 presumptive isolates were obtained from 192 samples collected in the study. Irrespective of the age of flock and sample type, all 192 samples contained at least one colony presumptively identified as C. perfringens on Perfringens agar plate with morphology as a single, round colony with opaque ring and black center. All isolates were further screened using PCR for confirmation, toxinotyping, and identification of virulence-associated genes. Only 9 isolates among the 734 presumptive isolates were confirmed as C. perfringens and all confirmed isolates were toxinotype A with variation in presence of netB, cpb2, and tpeL. More extensive studies are required to assess the prevalence and virulence of C. perfringens in NAE farms.

A Review of Salmonella and Campylobacter in Broiler Meat: Emerging Challenges and Food Safety Measures.

Poultry is one of the largest sources of animal-based protein in the United States. Poultry processing has grown from a small local network of plants to nearly 500 plants nationwide. Two of the most persistent bacteria in poultry processing are Salmonella and Campylobacter. It was not until the introduction of Hazard Analysis and Critical Control Point systems in 1996 that major efforts to reduce bacterial contamination were developed. Traditionally, chlorine has been the industry standard for decontaminating chicken meat. However, antimicrobials such as peracetic acid, cetylpyridinium chloride, and acidified sodium chlorite have replaced chlorine as primary antimicrobials. Despite current interventions, the emergence of stress-tolerant and biofilm-forming Salmonella and Campylobacter is of primary concern. In an effort to offset growing tolerance from microbes, novel techniques such as cold plasma treatment, electrostatic spraying, and bacteriophage-based applications have been investigated as alternatives to conventional treatments, while new chemical antimicrobials such as Amplon and sodium ferrate are investigated as well. This review provides an overview of poultry processing in the United States, major microbes in poultry processing, current interventions, emerging issues, and emerging technologies in antimicrobial treatments.