Exploiting the microbiota of organic and inorganic acid-treated raw poultry products to improve shelf-life.

Introduction: Targeted amplicon sequencing of the 16S rRNA delineates the complex microbial interactions that occur during food spoilage, providing a tool to intensively screen microbiota response to antimicrobial processing aids and interventions. The current research determines the microbiota and spoilage indicator (total aerobes and lactic acid bacteria; LAB) response to inorganic and organic antimicrobial intervention use on the shelf-life of fresh, never-frozen, skin-on, bone-in chicken wings. Methods: Wings (n=200) were sourced from local processor and either not treated (NT) or treated with 15-s dips of tap water (TW), organic (peracetic acid; PAA), inorganic acids (sodium bisulfate; SBS), and their combination (SBS + PAA). Wings were stored (4°C) and rinsed in neutralizing Buffered Peptone Water (BPW) for 1 min on d 0, 7, 14, and 21 post-treatment. Spoilage indicators, aerobic mesophiles and LAB, were quantified from rinsates. Genomic DNA of d 14 and 21 rinsates were extracted, and V4 of 16S rRNA gene was sequenced. Sequences were analyzed using QIIME2.2019.7. APC and LAB counts were reported as Log10 CFU/g of chicken and analyzed in R Studio as a General Linear Model using ANOVA. Pairwise differences were determined using Tukey's HSD (P£0.05). Results: Spoilage was indicated for all products by day 21 according to APC counts (>7 Log10 CFU/g); however, wings treated with SBS and SBS + PAA demonstrated a 7-day extended shelf-life compared to those treated with NT, TW, or PAA. The interaction of treatment and time impacted the microbial diversity and composition (p < 0.05), with those treated with SBS having a lower richness and evenness compared to those treated with the controls (NT and TW; p < 0.05, Q < 0.05). On d 14, those treated with SBS and SBS + PAA had lower relative abundance of typical spoilage population while having a greater relative abundance of Bacillus spp. (~70 and 50% of population; ANCOM p < 0.05). By d 21, the Bacillus spp. populations decreased below 10% of the population among those treated with SBS and SBS + PAA. Discussion: Therefore, there are differential effects on the microbial community depending on the chemical intervention used with organic and inorganic acids, impacting the microbial ecology differently.

Long-term impacts of conservation pasture management in manuresheds on system-level microbiome and antibiotic resistance genes.

Animal manure improves soil fertility and organic carbon, but long-term deposition may contribute to antibiotic resistance genes (ARGs) entering the soil-water environment. Additionally, long-term impacts of applying animal manure to soil on the soil-water microbiome, a crucial factor in soil health and fertility, are not well understood. The aim of this study is to assess: (1) impacts of long-term conservation practices on the distribution of ARGs and microbial dynamics in soil, and runoff; and (2) associations between bacterial taxa, heavy metals, soil health indicators, and ARGs in manures, soils, and surface runoff in a study following 15 years of continuous management. This management strategy consists of two conventional and three conservation systems, all receiving annual poultry litter. High throughput sequencing of the 16S ribosomal RNA was carried out on samples of cattle manure, poultry litter, soil, and runoff collected from each manureshed. In addition, four representative ARGs (intl1, sul1, ermB, and blactx-m-32) were quantified from manures, soil, and runoff using quantitative PCR. Results revealed that conventional practice increased soil ARGs, and microbial diversity compared to conservation systems. Further, ARGs were strongly correlated with each other in cattle manure and soil, but not in runoff. After 15-years of conservation practices, relationships existed between heavy metals and ARGs. In the soil, Cu, Fe and Mn were positively linked to intl1, sul1, and ermB, but trends varied in runoff. These findings were further supported by network analyses that indicated complex co-occurrence patterns between bacteria taxa, ARGs, and physicochemical parameters. Overall, this study provides system-level linkages of microbial communities, ARGs, and physicochemical conditions based on long-term conservation practices at the soil-water-animal nexus.

Complete Genome Sequence of Terrisporobacter glycolicus Strain WW3900, Isolated from Influent Wastewater at a Research Center with Multiple-Species Research Animal Facilities.

Terrisporobacter glycolicus is an emerging obligate anaerobic pathogen. We report the 3.9-Mbp genome sequence of T. glycolicus strain WW3900, which was isolated from wastewater at a research center with laboratory animal facilities. The genome sequence predicted a biosynthetic gene cluster encoding an S-adenosylmethionine enzyme and other synthetic genes associated with potential antimicrobial producers.

Consequences of Implementing Neutralizing Buffered Peptone Water in Commercial Poultry Processing on the Microbiota of Whole Bird Carcass Rinses and the Subsequent Microbiological Analyses.

In 2016, the United States Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) established guidelines which modified the Buffered Peptone Water (BPW) rinsate material to include additional compounds that would better neutralize residual processing aids and allow for better recovery of sublethal injured Salmonella spp. cells. While the added compounds improved the recovery of Salmonella spp., specific data to understand how the new rinse agent, neutralizing Buffered Peptone Water (nBPW), impacts the recovery of other microorganisms such as Campylobacter spp. and indicator microorganisms are lacking. Therefore, this study evaluated the impact of rinse solutions (BPW or nBPW) used in Whole Bird Carcass rinsate (WBCR) collections on the subsequent microbiome and downstream culturing methodologies. Carcasses exiting a finishing chiller were rinsed in 400 ml of BPW or nBPW. Resulting rinsates were analyzed for Enterobacteriaceae (EB), Salmonella, and Campylobacter spp. prevalence and total aerobic bacteria (APC) and EB load. The 16S rDNA of the rinsates and the matrices collected from applied microbiological analyses were sequenced on an Illumina MiSeq®. Log10-transformed counts were analyzed in JMP 15 using ANOVA with means separated using Tukey's HSD, and prevalence data were analyzed using Pearson's χ2 (P ≤ 0.05). Diversity and microbiota compositions (ANCOM) were analyzed in QIIME 2.2019.7 (P ≤ 0.05; Q ≤ 0.05). There was an effect of rinsate type on the APC load and Campylobacter spp. prevalence (P < 0.05), but not the quantity or prevalence of EB or Salmonella spp. prevalence. There were differences between the microbial diversity of the two rinsate types and downstream analyses (P < 0.05). Additionally, several taxa, including Streptococcus, Lactobacillus, Aeromonas, Acinetobacter, Clostridium, Enterococcaceae, Burkholderiaceae, and Staphylococcaceae, were differentially abundant in paired populations. Therefore, the rinse buffer used in a WBCR collection causes proportional shifts in the microbiota, which can lead to differences in results obtained from cultured microbial populations.

Comparison of ready-to-eat "organic" antimicrobials, sodium bisulfate, and sodium lactate, on Listeria monocytogenes and the indigenous microbiome of organic uncured beef frankfurters stored under refrigeration for three weeks.

Listeria monocytogenes has been implicated in several ready-to-eat (RTE) foodborne outbreaks, due in part to its ability to survive under refrigerated conditions. Thus, the objective of this study was to evaluate the effects of sodium bisulfate (SBS), sodium lactate (SL), and their combination as short-duration antimicrobial dips (10-s) on L. monocytogenes and the microbiome of inoculated organic frankfurters (8 Log10 CFU/g). Frankfurters were treated with tap water (TW), SBS0.39%, SBS0.78%, SL0.78%, SL1.56%, SBS+SL0.39%, SBS+SL0.78%. In addition, frankfurters were treated with frankfurter solution water (HDW)+SBS0.78%, HDW+SL1.56%, and HDW+SBS+SL0.78%. After treatment, frankfurters were vacuum packaged and stored at 4°C. Bacterial enumeration and 16S rDNA sequencing occurred on d 0, 7, 14, 21. Counts were Log10 transformed and calculated as growth potential from d 0 to d 7, 14, and 21. Data were analyzed in R using mixed-effects model and One-Way ANOVA (by day) with differences separated using Tukey's HSD at P ≤ 0.05. The 16S rDNA was sequenced on an Illumina MiSeq and analyzed in Qiime2-2018.8 with significance at P ≤ 0.05 and Q ≤ 0.05 for main and pairwise effects. An interaction of treatment and time was observed among the microbiological plate data with all experimental treatments reducing the growth potential of Listeria across time (P < 0.0001). Efficacy of treatments was inconsistent across time; however, on d 21, SBS0.39% treated franks had the lowest growth potential compared to the control. Among diversity metrics, time had no effect on the microbiota (P > 0.05), but treatment did (P < 0.05). Thus, the treatments potentially promoted a stable microbiota across time. Using ANCOM, Listeria was the only significantly different taxa at the genus level (P < 0.05, W = 52). Therefore, the results suggest incorporating SBS over SL as an alternative antimicrobial for the control of L. monocytogenes in organic frankfurters without negatively impacting the microbiota. However, further research using multiple L. monocytogenes strains will need to be utilized in order to determine the scope of SBS use in the production of RTE meat.

Yeast Fermentate-Mediated Reduction of Salmonella Reading and Typhimurium in an in vitro Turkey Cecal Culture Model.

Salmonella Reading is an ongoing public health issue in the turkey industry, leading to significant morbidity in humans in the United States. Pre-harvest intervention strategies that contribute to the reduction of foodborne pathogens in food animals, such as the yeast fermentation metabolites of Original XPCTM (XPC), may become the key to multi-hurdle farm to fork strategies. Therefore, we developed an anaerobic in vitro turkey cecal model to assess the effects of XPC on the ceca of commercial finisher tom turkeys fed diets void of XPC and antibiotics. Using the in vitro turkey cecal culture method, ceca were tested with and without XPC for their anti-Salmonella Reading and the previously defined anti-Typhimurium (ST97) effects. Ultimately, the anti-Salmonella effects were independent of serovar (P > 0.05). At 0 h post inoculation (hpi), Salmonella levels were equivalent between treatments at 7.3 Log10 CFU/mL, and at 24 hpi, counts in XPC were reduced by 5 Log10 CFU/mL, which was 2.1 Log10 lower than the control (P < 0.05). No differences in serovar prevalence existed (P > 0.05), with a 92% reduction in Salmonella positive XPC-treated ceca cultures by 48 hpi (P < 0.05). To evaluate changes to the microbiota independent of the immune response, the 16S rDNA was sequenced using the Illumina MiSeq platform. Data indicated a profound effect of time and treatment for the reduction of Salmonella irrespective of serovar. XPC sustained diversity metrics compared to the control, demonstrating a reduction in diversity over time (Q < 0.05).

The biological effects of microencapsulated organic acids and botanicals induces tissue-specific and dose-dependent changes to the Gallus gallus microbiota.

BACKGROUND: Microencapsulated organic acids and botanicals have the potential to develop into important tools for the poultry industry. A blend of organic acids and botanicals (AviPlus®P) has previously shown to reduce Salmonella and Campylobacter in chickens; however, changes to the microbiota of the jejunum and ileum have not been evaluated. Microbiota diversity is linked to, but not correlated with, the efficacy of natural products; therefore, understanding the effects on the microbiota is necessary for evaluating their potential as an antibiotic alternative. RESULTS: Ileal and jejunal segments from control and supplement-fed chickens (300 and 500 g/metric ton [MT]) were subjected to alpha diversity analysis including Shannon's diversity and Pielou's Evenness. In both analytics, the diversity in the ileum was significantly decreased compared to the jejunum irrespective of treatment. Similarly, beta diversity metrics including Bray-Curtis dissimilarity index and Weighted Unifrac Distance Matrix, were significant (Q < 0.05) for both tissue and treatments comparisons. Alpha and beta diversity analytics indicated compartmentalization effects between the ileum and jejunum. Additionally, analysis of communities in the microbiota (ANCOM) analysis showed Lactobacilliaceae predominated the total operational taxonomic units (OTU), with a stepwise increase from 53% in the no treatment control (NTC) to 56% in the 300 g/MT and 67% in the 500 g/MT group. Staphylococcaceae were 2% in NTC and 2 and 0% in 300 and 500 g/MT groups. Enterobacteriaceae decreased in the 500 g/MT (31%) and increased in the 300 g/MT (37%) compared to the NTC (35%). Aerococcaceae was 0% for both doses and 7% in NTC. Ruminococcaceae were 0% in NTC and 2 and 1% in the 300 and 500 g/MT. These changes in the microbial consortia were statistically (Q < 0.05) associated with treatment groups in the jejunum that were not observed in the ileum. Least discriminant analysis effect size (LEfSE) indicated different changes directly corresponding to treatment. Enterobacteriaceae demonstrated a stepwise decrease (from NTC onward) while Clostridiaceae, were significantly increased in the 500 g/MT compared to NTC and 300 g/MT (P < 0.05). CONCLUSION: The bioactive site for the microencapsulated blend of organic acids and botanicals was the jejunum, and dietary inclusion enhanced the GIT microbiota and may be a viable antibiotic alternative for the poultry industry.

Aerobic plate count, Salmonella and Campylobacter loads of whole bird carcass rinses from pre-chillers with different water management strategies in a commercial poultry processing plant.

Salmonella and Campylobacter are significant issues for poultry processors because of increasing regulatory standards as well as public health concerns. The goal of this study is to report the effects of two different pre-chiller systems that utilize different temperatures and water recirculation systems on whole bird carcass rinsates. Both pre-chiller tanks were contained within a single poultry processing facility and operated at different temperatures and water systems. The incidence of Campylobacter spp. and Salmonella spp., as well as the aerobic plate counts on whole bird carcass rinses are reported in this study from each pre-chiller system. The results from this study reveal that there are significant differences in how microbial populations and pathogens change over time in each pre-chiller system. Furthermore, we identify that these patterns are different per system. Such data are impactful as it indicates that measuring carcasses within a plant must consider both temperature and water recirculation as it may prevent comparability of different lines within a single processing facility.

The Reduction of Pathogen Load on Ross 708 Broilers when Using Different Sources of Commercial Peracetic Acid Sanitizers in a Pilot Processing Plant.

Peracetic acid (PAA) in poultry processing is not necessarily the same from company to company. Anecdotal evidence suggests that PeraClean may be more stable compared to the competition; however, it is not known what impact potential differences in chemical stability may have. In order to evaluate the antimicrobial effects of PAA, one PAA (PeraClean, P) was qualitatively compared against two competitor products (Competitors 1 and 2, C1 and C2) at the University of Arkansas Pilot Processing Plant. A total of 150 Ross 708 broilers (42 d) were used in the current study. Briefly, prior to treatment, 10 birds were sampled post-evisceration (C). Then, one of four treatment groups per PAA were applied (A1, A2, B1, and B2). The birds were dipped in either 400 ppm or 600 ppm PAA (A or B), chilled in either 25 ppm or 45 ppm PAA (1 or 2), and then manually agitated in 400 mL of nBPW for 1 min. There were 10 birds per treatment group in total. The resulting rinsates were transported to the Center for Food Safety and assessed for total microbiological load with total aerobic plate counts (Trypticase Soy Agar; APC), coliforms, (Eosin Methylene Blue Media; EMB), Salmonella (Xylose Lysine Deoxycholate agar, XLD), and Campylobacter (modified Charcoal Cefoperazone Deoxycholate Agar, mCCDA). The microbiological plates were incubated as per manufacturer's directions. Statistical analyses were calculated in JMP 14.0, with a significance level of p ≤ 0.05. Data indicate that all three sources of PAA are effective sanitizers for poultry processing applications compared within treatment. Qualitatively, there were differences in efficacy between the treatments. However, additional studies will be required to determine if those differences are quantitatively distinctive and if they are attributable to differences in product stability.

Acute systemic inflammatory response to lipopolysaccharide stimulation in pigs divergently selected for residual feed intake.

BACKGROUND: It is unclear whether improving feed efficiency by selection for low residual feed intake (RFI) compromises pigs' immunocompetence. Here, we aimed at investigating whether pig lines divergently selected for RFI had different inflammatory responses to lipopolysaccharide (LPS) exposure, regarding to clinical presentations and transcriptomic changes in peripheral blood cells. RESULTS: LPS injection induced acute systemic inflammation in both the low-RFI and high-RFI line (n = 8 per line). At 4 h post injection (hpi), the low-RFI line had a significantly lower (p = 0.0075) mean rectal temperature compared to the high-RFI line. However, no significant differences in complete blood count or levels of several plasma cytokines were detected between the two lines. Profiling blood transcriptomes at 0, 2, 6, and 24 hpi by RNA-sequencing revealed that LPS induced dramatic transcriptional changes, with 6296 genes differentially expressed at at least one time point post injection relative to baseline in at least one line (n = 4 per line) (|log2(fold change)| ≥ log2(1.2); q < 0.05). Furthermore, applying the same cutoffs, we detected 334 genes differentially expressed between the two lines at at least one time point, including 33 genes differentially expressed between the two lines at baseline. But no significant line-by-time interaction effects were detected. Genes involved in protein translation, defense response, immune response, and signaling were enriched in different co-expression clusters of genes responsive to LPS stimulation. The two lines were largely similar in their peripheral blood transcriptomic responses to LPS stimulation at the pathway level, although the low-RFI line had a slightly lower level of inflammatory response than the high-RFI line from 2 to 6 hpi and a slightly higher level of inflammatory response than the high-RFI line at 24 hpi. CONCLUSIONS: The pig lines divergently selected for RFI had a largely similar response to LPS stimulation. However, the low-RFI line had a relatively lower-level, but longer-lasting, inflammatory response compared to the high-RFI line. Our results suggest selection for feed efficient pigs does not significantly compromise a pig's acute systemic inflammatory response to LPS, although slight differences in intensity and duration may occur.

A Historical Review on Antibiotic Resistance of Foodborne Campylobacter.

Campylobacter is one of the most commonly reported foodborne human bacterial gastrointestinal pathogens. Campylobacter is the etiological agent of campylobacteriosis, which is generally a self-limited illness and therefore does not require treatment. However, when patients are immunocompromised or have other co-morbidities, antimicrobial treatment may be necessary for clinical treatment of campylobacteriosis, macrolides and fluoroquinolones are the drugs of choices. However, the increase in antimicrobial resistance of Campylobacter to clinically important antibiotics may become insurmountable. Because of the transmission between poultry and humans, the poultry industry must now allocate resources to address the problem by reducing Campylobacter as well as antimicrobial use, which may reduce resistance. This review will focus on the incidence of antibiotic-resistant Campylobacter in poultry, the clinical consequences of this resistance, and the mechanisms of antibiotic resistance associated with Campylobacter.

A Microbiomic Analysis of a Pasture-Raised Broiler Flock Elucidates Foodborne Pathogen Ecology Along the Farm-To-Fork Continuum.

While conventionally grown poultry continues to dominate the U. S. poultry industry, there is an increasing demand for locally-grown, "all natural" alternatives. The use of next generation sequencing allows for not only the gross (e.g., community structure) but also fine-scale (e.g., taxa abundances) examination of these complex microbial communities. This data provides a better understanding of how a pasture flock's microbiome changes throughout the production life cycle and how that change in microbial ecology changes foodborne pathogens in alternative poultry production systems. In order to understand this ecology better, pooled broiler samples were taken during the entire flock life cycle, from pre-hatch gastrointestinal samples (N = 12) to fecal samples from the brood (N = 5), and pasture (N = 10) periods. Additional samples were taken during processing, including skin and feather rinsates (N = 12), ceca (N = 12), and whole carcass rinses (N = 12), and finally whole carcasss rinsates of final products (N = 3). Genomic DNA was extracted, 16S rDNA microbiome sequencing was conducted (Illumina MiSeq), and microbiomes were analyzed and compared using QIIME 1.9.1 to determine how microbiomes shifted throughout production continuum, as well as what environmental factors may be influencing these shifts. Significant microbiome shifts occurred during the life cycle of the pasture broiler flock, with the brood and pasture fecal samples and cecal samples being very distinct from the other pre-hatch, processing, and final product samples. Throughout these varied microbiomes, there was a stable core microbiome containing 13 taxa. Within this core microbiome, five taxa represented known foodborne pathogens (Salmonella, Campylobacter) or potential/emerging pathogens (Pseudomonas, Enterococcus, Acinetobacter) whose relative abundances varied throughout the farm-to-fork continuum, although all were more prevalent in the fecal samples. Additionally, of the 25 physiochemical and nutrient variables measured from the fecal samples, the carbon to nitrogen ratio was one of the most significant variables to warrant further investigations because it impacted both general fecal microbial ecology and Campylobacter and Enterococcus taxa within the core fecal microbiomes. These findings demonstrate the need for further longitudinal, farm-to-fork studies to understand the ecology of the microbial ecology of pasture production flocks to improve animal, environmental, and public health.

Short Communication: Preliminary Differences Identified in Genes Responsible for Biofilm Formation in Poultry Isolates of Salmonella enterica Heidelberg, Enteritidis, and Kentucky.

Salmonella enterica is one of the most prevalent foodborne pathogens. The large quantity of serovar types results in the colonization of a large spectrum of hosts, with different environmental conditions and hazards. The aim of this study was to evaluate the differences in gene expression (bcsA and csgD) of Salmonella enterica serovars Heidelberg, Kentucky, and Enteritidis during biofilm formation using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Overall, there appeared to be differences in expression between the different serovars with high variation between strains. These data are important as they demonstrate considerable variability in gene expression between serovars and strains of poultry isolates of Salmonella enterica.

Comparison of 16S rDNA Next Sequencing of Microbiome Communities From Post-scalder and Post-picker Stages in Three Different Commercial Poultry Plants Processing Three Classes of Broilers.

Poultry processing systems are a complex network of equipment and automation systems that require a proactive approach to monitoring in order to protect the food supply. Process oversight requires the use of multi-hurdle intervention systems to ensure that any undesirable microorganisms are reduced or eliminated by the time the carcasses are processed into final products. In the present study, whole bird carcass rinses (WBCR) collected at the post-scalder and post-picker locations from three different poultry processing facilities (Plant A: mid-weight broiler processing, B: large-weight broiler processing, C: young broiler (Cornish) processing) were subjected to next generation sequencing (NGS) and microbial quantification using direct plating methods to assess the microbial populations present during these stages of the poultry process. The quantification of aerobic plate counts (APC) and Enterobacteriaceae (EB) demonstrated that reductions for these microbial classes were not consistent between the two sampling locations for all facilities, but did not provide a clear picture of what microorganism(s) may be affecting those shifts. With the utilization of NGS, a more complete characterization of the microbial communities present including microorganisms that would not have been identified with the employed direct plating methodologies were identified. Although the foodborne pathogens typically associated with raw poultry, Salmonella and Campylobacter, were not identified, sequence analysis performed by Quantitative Insights of Microbiology Ecology (QIIME) indicated shifts of Erwinia, Serratia, and Arcobacter, which are microorganisms closely related to Salmonella and Campylobacter. Additionally, the presence of Chryseobacterium and Pseudomonas at both sampling locations and at all three facilities provides evidence that these microorganisms could potentially be utilized to assess the performance of multi-hurdle intervention systems.

Saccharomyces cerevisiae Fermentation Products That Mitigate Foodborne Salmonella in Cattle and Poultry.

Prior studies revealed that yeast fermentation products, specifically XPC™ and related products (Diamond V, Cedar Rapids, IA), serve as viable food safety tools across multiple food animal species including cattle and poultry. Providing this supplement in feed leads to reduced prevalence, load, virulence, and antibiotic resistance of foodborne pathogens such as Salmonella and Escherichia coli O157:H7. These findings are worthy of further study, especially when coupled with the enhanced growth and performance observed with these products. Mechanistically, XPC appears to modulate these effects through the immune system and gut microbiome. Herein we further investigated this product and demonstrate that XPC mediates an enhancement of immunocyte killing of Salmonella in calves fed the product. Additionally, these studies reveal that XPC reduces the lymph node infiltration, invasiveness, and antibiotic resistance of Salmonella in dairy calves fed the product-consistent with findings observed in poultry and adult beef cattle. Furthermore, the reduction in invasiveness does not lead to a rebound hyperinvasive phenotype in Salmonella obtained from XPC-fed animals. In summary, these studies suggest that XPC reduces the invasion of Salmonella and may alter various phenotypic characteristics of the pathogen.

The Efficacy of Sodium Bisulfate Salt (SBS) Alone and Combined With Peracetic Acid (PAA) as an Antimicrobial on Whole Chicken Drumsticks Artificially Inoculated With Salmonella Enteritidis.

The presence of Salmonella spp. on poultry products is one of the leading causes of foodborne illness in the United States. Therefore, novel antimicrobial substances are being explored as potential interventions in poultry processing facilities. The objective of the current study was to evaluate the efficacy of varying concentrations of sodium bisulfate salt, SBS, alone or in combination with peracetic acid, PAA, in 15 s whole part dips. Ninety six drumsticks (4 replications, 8 treatments, 3 days) were inoculated separately in a 400 mL solution of nalidixic resistant (NA) Salmonella Enteritidis (107 CFU/mL) and allowed to adhere for 60 to 90 min at 4°C for a final concentration of 106 CFU/g. The experimental treatments included: a no treatment (control), and 15 s dips in 300 mL of tap water alone (TW) or with the addition of 1; 2; and 3% SBS; 1; 2; and 3% SBS+PAA. After treatment, drumsticks were stored at 4°C until microbial sampling was conducted. On d 0, l, and 3, drumsticks were rinsed in 150 mL of nBPW for 1 min, 100 µL of rinsate was serially diluted, spread plated on XLT4+NA (20 µg/mL), and incubated aerobically at 37°C for 24 h. Log-transformed counts were analyzed using a randomized complete block design (day) using One-Way ANOVA, polynomial contrasts, and pairwise comparisons with means being separated by Tukey's HSD with a significance level of P ≤ 0.05. A treatment by day interaction (P = 0.14071) was not substantial. Thus, the treatment effect was investigated separately by days. Over time, a linear trend was observed in S. Enteritidis concentration when SBS was increased (1 < 2 < 3%). The concentration of S. Enteritidis was different between 1% SBS and 1% SBS+PAA on d 0. However, the level of S. Enteritidis was not different among drumsticks treated in 2 and 3% SBS and 2 and 3% SBS+PAA across d 0, 1, 3. The application of 3% SBS alone or in combination with 200 ppm of PAA is capable of reducing the presence of Salmonella over a 3-d refrigeration period; potentially increasing the safety of poultry products for consumers.

Establishment of a Standardized 16S rDNA Library Preparation to Enable Analysis of Microbiome in Poultry Processing Using Illumina MiSeq Platform.

The standardization of the microbiome sequencing of poultry rinsates is essential for generating comparable microbial composition data among poultry processing facilities if this technology is to be adopted by the industry. Samples must first be acquired, DNA must be extracted, and libraries must be constructed. In order to proceed to library sequencing, the samples should meet quality control standards. Finally, data must be analyzed using computer bioinformatics pipelines. This data can subsequently be incorporated into more advanced computer algorithms for risk assessment. Ultimately, a uniform sequencing pipeline will enable both the government regulatory agencies and the poultry industry to identify potential weaknesses in food safety. This chapter presents the different steps for monitoring the population dynamics of the microbiome in poultry processing using 16S rDNA sequencing.

The Preliminary Development of an in vitro Poultry Cecal Culture Model to Evaluate the Effects of Original XPCTM for the Reduction of Campylobacter jejuni and Its Potential Effects on the Microbiota.

Poultry is a major reservoir for the pathogen Campylobacter jejuni. C. jejuni inhabits the poultry gastrointestinal tract as a part of the gut microbiota. The objective of this study was to evaluate both the survival of C. jejuni and the changes in the population dynamics of the cecal microbiome during an in vitro C. jejuni inoculation in the presence or absence of the functional metabolites of Diamond V Original XPCTM (XPC). Two independent trials were conducted. Broiler chickens (n = 6 per Trial 1 and n = 3 per Trial 2) were raised according to standard industry guidelines and euthanized on Day 41. The ceca were collected aseptically, their contents removed independently and then used in an in vitro microaerobic model with 0.1% cecal contents + Campylobacter with or without 1% XPC (w/v). Before the inoculation with a chloramphenicol resistant marker strain of C. jejuni, the cecal contents were pre-incubated with XPC at 42°C for 24 h, in a shaking incubator (200 rpm) under microaerobic conditions, then experimentally inoculated with 108/ml of C. jejuni into the appropriate treatment groups. At 0 and 24 h for Trial 1, and 48 h for Trial 2, sub-samples of the culture (n = 3 ceca, two technical replicates per ceca, XPC alone or ceca culture alone) were enumerated using a Petroff-Hausser counter, and the DNA was extracted for microbiome analysis. DNA was isolated using the Qiagen QIAamp Fast Stool DNA Mini Kit and sequenced using the Illumina MiSeq platform. The reads were filtered, normalized, and assigned taxonomical identities using the QIIME2 pipeline. The relative microbiota populations were identified via ANCOM. Altogether, evidence suggests that XPC alters the microbiome, and in turn reduces Campylobacter survival.

Semi-Quantification of Total Campylobacter and Salmonella During Egg Incubations Using a Combination of 16S rDNA and Specific Pathogen Primers for qPCR.

Rapid molecular techniques that evaluate eggs for the presence of foodborne pathogens is an essential component to poultry food safety monitoring. Interestingly, it is not just table eggs that contribute to outbreaks of foodborne disease. Broiler layer production actively contributes to sustaining of foodborne pathogens within a flock. The surface contamination of production eggs with invasive pathogens such as Salmonella enterica, Campylobacter jejuni, and Listeria monocytogenes during embryogenesis results in gastrointestinal tract (GIT) colonization. Pathogens that secure a niche within the GIT during embryonic development are nearly impossible to eradicate from the food chain. Therefore, current monitoring paradigms are not comprehensive because they fail to capture the presence of invasive pathogens within the embryonic GIT rapidly. By developing tools to recognize the pathogens' presence in the GIT during embryogenesis, producers are then able to spot evaluate broiler eggs for their potential risk as carriers of foodborne pathogens. In this study a novel qPCR assay was developed to semi-quantify pathogen load relative to total bacterial burden. Eggs sampled from three independent production broiler flocks of different ages were assayed for S. enterica (invA), C. jejuni (HipO), and L. monocytogenes (HlyA) against total microbial load (16s). The eggs were sampled at 1-day post-set within each flock, 2 weeks post-set, after vaccination (at 2.5 weeks) and 1-day post-hatch. The eggs were washed, and the yolk and embryonic chick GIT were collected. The DNA was extracted and subjected to a qPCR assay. The results confirm a novel technique for pathogen monitoring relative to total bacterial load and a unique method for monitoring the dynamics of foodborne pathogen invasion throughout broiler egg production.

Developments in Rapid Detection Methods for the Detection of Foodborne Campylobacter in the United States.

The accurate and rapid detection of Campylobacter spp. is critical for optimal surveillance throughout poultry processing in the United States. The further development of highly specific and sensitive assays to detect Campylobacter in poultry matrices has tremendous utility and potential for aiding the reduction of foodborne illness. The introduction and development of molecular methods such as polymerase chain reaction (PCR) have enhanced the diagnostic capabilities of the food industry to identify the presence of foodborne pathogens throughout poultry production. Further innovations in various methodologies, such as immune-based typing and detection as well as high throughput analyses, will provide important epidemiological data such as the identification of unique or region-specific Campylobacter. Comparable to traditional microbiology and enrichment techniques, molecular techniques/methods have the potential to have improved sensitivity and specificity, as well as speed of data acquisition. This review will focus on the development and application of rapid molecular methods for identifying and quantifying Campylobacter in U.S. poultry and the emergence of novel methods that are faster and more precise than traditional microbiological techniques.