Co-exposure to polyethylene fiber and Salmonella enterica serovar Typhimurium alters microbiome and metabolome of in vitro chicken cecal mesocosms.

Humans and animals encounter a summation of exposures during their lifetime (the exposome). In recent years, the scope of the exposome has begun to include microplastics. Microplastics (MPs) have increasingly been found in locations, including in animal gastrointestinal tracts, where there could be an interaction with Salmonella enterica serovar Typhimurium, one of the commonly isolated serovars from processed chicken. However, there is limited knowledge on how gut microbiomes are affected by microplastics and if an effect would be exacerbated by the presence of a pathogen. In this study, we aimed to determine if acute exposure to microplastics in vitro altered the gut microbiome membership and activity. The microbiota response to a 24 h co-exposure to Salmonella enterica serovar Typhimurium and/or low-density polyethylene (PE) microplastics in an in vitro broiler cecal model was determined using 16S rRNA amplicon sequencing (Illumina) and untargeted metabolomics. Community sequencing results indicated that PE fiber with and without S. Typhimurium yielded a lower Firmicutes/Bacteroides ratio compared with other treatment groups, which is associated with poor gut health, and overall had greater changes to the cecal microbial community composition. However, changes in the total metabolome were primarily driven by the presence of S. Typhimurium. Additionally, the co-exposure to PE fiber and S. Typhimurium caused greater cecal microbial community and metabolome changes than either exposure alone. Our results indicate that polymer shape is an important factor in effects resulting from exposure. It also demonstrates that microplastic-pathogen interactions cause metabolic alterations to the chicken cecal microbiome in an in vitro chicken cecal mesocosm. IMPORTANCE: Researching the exposome, a summation of exposure to one's lifespan, will aid in determining the environmental factors that contribute to disease states. There is an emerging concern that microplastic-pathogen interactions in the gastrointestinal tract of broiler chickens may lead to an increase in Salmonella infection across flocks and eventually increased incidence of human salmonellosis cases. In this research article, we elucidated the effects of acute co-exposure to polyethylene microplastics and Salmonella enterica serovar Typhimurium on the ceca microbial community in vitro. Salmonella presence caused strong shifts in the cecal metabolome but not the microbiome. The inverse was true for polyethylene fiber. Polyethylene powder had almost no effect. The co-exposure had worse effects than either alone. This demonstrates that exposure effects to the gut microbial community are contaminant-specific. When combined, the interactions between exposures exacerbate changes to the gut environment, necessitating future experiments studying low-dose chronic exposure effects with in vivo model systems.

General media over enrichment media supports growth of Campylobacter jejuni and maintains poultry cecal microbiota enabling translatable in vitro microbial interaction experiments.

AIM: This study aimed to assess the suitability of two media types, Bolton enrichment broth (BEB) and anaerobic dilution solution (ADS), in replicating the poultry cecal environment to investigate metabolic interactions and Campylobacter presence within poultry ceca. METHODS: Using an anaerobic in vitro poultry cecal model, cecal contents (free of culturable Campylobacter) were diluted in BEB and ADS, inoculated with 105 CFU of Campylobacter jejuni, and incubated for 48 h at 42°C under microaerophilic conditions. Samples were collected at 0, 24, and 48 h. Genomic DNA was extracted, amplified, and sequenced on Illumina MiSeq platform. Data underwent analysis within QIIME2-2021.11, including alpha and beta diversity assessments, ANOVA, ADONIS, ANCOM, and Bradford assay for protein concentration. RESULTS: ADS supported a more diverse microbial population than BEB, influencing C. jejuni presence. ANCOM highlighted dominant genera in BEB (Lactobacillus and Campylobacter) and affirmed C. jejuni growth in ADS. Core microbiota analysis revealed unique associations with each media type, while the Bradford assay indicated ADS consistently yielded more uniform microbial growth. CONCLUSIONS: ADS was identified as a preferred diluent for faithfully replicating cecal microbial changes in the presence of Campylobacter.

Salmonella in eggs and egg-laying chickens: pathways to effective control.

Eggs contaminated with Salmonella have been internationally significant sources of human illness for several decades. Most egg-associated illness has been attributed to Salmonella serovar Enteritidis, but a few other serovars (notably S. Heidelberg and S. Typhimurium) are also sometimes implicated. The edible interior contents of eggs typically become contaminated with S. Enteritidis because the pathogen's unique virulence attributes enable it to colonize reproductive tissues in systemically infected laying hens. Other serovars are more commonly associated with surface contamination of eggshells. Both research and field experience have demonstrated that the most effective overall Salmonella control strategy in commercial laying flocks is the application of multiple interventions throughout the egg production cycle. At the preharvest (egg production) level, intervention options of demonstrated efficacy include vaccination and gastrointestinal colonization control via treatments such as prebiotics, probiotics, and bacteriophages, Effective environmental management of housing systems used for commercial laying flocks is also essential for minimizing opportunities for the introduction, transmission, and persistence of Salmonella in laying flocks. At the postharvest (egg processing and handling) level, careful regulation of egg storage temperatures is critical for limiting Salmonella multiplication inside the interior contents.

Identification of bacterial isolates from commercial poultry feed via 16S rDNA.

The study's objective was to identify typical aerobic isolates from commercial, corn-soybean meal poultry diets utilizing 16S rDNA, assign them their corresponding taxonomy, and compare the data with the previously published WGS analysis of these same isolates. Ten grams of a commercial corn-soybean meal poultry diet was homogenized in 100 mL of tryptic soy broth for 2 min, serially diluted, plated onto tryptic soy agar (TSA), and incubated aerobically for 24 h at 37 °C. Subsequently, 20 unique colonies were streaked for isolation on TSA and incubated aerobically for 24 h at 37 °C. This process was repeated three consecutive times for purification of isolates until only 11 morphologically distinct colonies were obtained. DNA was extracted using Qiagen's DNeasey® Blood and Tissue Kit. The 16S rRNA V4 region was targeted using an Illumina MiSeq and analyzed via QIIME2-2020.2. Alpha diversity and Beta diversity metrics were generated, and taxa were aligned using Silva in Qiime2-2020.2. Twenty-five distinct genera were identified within the 11 different colonies. Because 16S rDNA identification can provide an understanding of pathogen associations and microbial niches within an ecosystem, the information may present a potential method to establish and characterize the hygienic indicator microorganisms associated with poultry feed.

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.

Non-molecular characterization of pellicle formation by poultry Salmonella Kentucky strains and other poultry-associated Salmonella serovars in Luria Bertani broth.

There is limited research concerning the biofilm-forming capabilities of Salmonella Kentucky, a common poultry isolate. The objective was to quantitate pellicle formation of S. Kentucky versus better-characterized Salmonella strains of Enteritidis and Heidelberg. In separate experiments, Salmonella strains and serovars were tested for their biofilm-forming abilities in different Luria-Bertani (LB) broths (1); pellicle formation in different volumes of LB without salt (2); and the potential priming effects on formation after pellicles were transferred three consecutive times (3). Data were analyzed using One-Way ANOVA with means separated using Tukey's HSD (P ≤ 0.05). In the first experiment, there was no significant effect between strain and serovars (P > 0.05), but media type affected pellicle formation significantly with LB Miller and LB minus NaCl plus 2% glucose resulting in no pellicle formation (P < 0.001). When grown in 50 mL, Kentucky 38-0085 produced larger pellicles than Kentucky 38-0055, and Heidelberg strain 38-0127 (P < 0.0001). Serial transfers of pellicles did not significantly affect pellicle formation (P > 0.05); however, Kentucky 38-0084, 38-0085 and 38-0086 produced larger pellicles than Kentucky 38-0055 and 38-0056 and Heidelberg 38-0126, 38-0127 and 38-0152. The current study demonstrates the consistent biofilm forming capabilities of Kentucky and may explain why Kentucky is frequently isolated in poultry processing facilities.

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.

Alternative Additives for Organic and Natural Ready-to-Eat Meats to Control Spoilage and Maintain Shelf Life: Current Perspectives in the United States.

Food additives are employed in the food industry to enhance the color, smell, and taste of foods, increase nutritional value, boost processing efficiency, and extend shelf life. Consumers are beginning to prioritize food ingredients that they perceive as supporting a healthy lifestyle, emphasizing ingredients they deem acceptable as alternative or "clean-label" ingredients. Ready-to-eat (RTE) meat products can be contaminated with pathogens and spoilage microorganisms after the cooking step, contributing to food spoilage losses and increasing the risk to consumers for foodborne illnesses. More recently, consumers have advocated for no artificial additives or preservatives, which has led to a search for antimicrobials that meet these demands but do not lessen the safety or quality of RTE meats. Lactates and diacetates are used almost universally to extend the shelf life of RTE meats by reducing spoilage organisms and preventing the outgrowth of the foodborne pathogen Listeria monocytogenes. These antimicrobials applied to RTE meats tend to be broad-spectrum in their activities, thus affecting overall microbial ecology. It is to the food processing industry's advantage to target spoilage organisms and pathogens specifically.

Adaptation of a Commercial Qualitative BAX® Real-Time PCR Assay to Quantify Campylobacter spp. in Whole Bird Carcass Rinses.

Poultry is the primary reservoir of Campylobacter, a leading cause of gastroenteritis in the United States. Currently, the selective plating methodology using selective agars, Campy Cefex and Modified Charcoal Cefoperazone Deoxycholate agar, is preferentially used for the quantification of Campylobacter spp. among poultry products. Due to the specific nature of Campylobacter, this methodology is not sensitive, which can lead to skewed detection and quantification results. Therefore, Campylobacter detection and quantification methods are urgently needed. The objective was to develop a shortened enrichment-based quantification method for Campylobacter (CampyQuant™) in post-chill poultry rinsates using the BAX® System Real-Time PCR assay for Campylobacter. The specificity and sensitivity for the detection of C. jejuni, C. coli, and C. lari in pure culture were determined. The BAX® System Real-Time PCR assay consistently detected and identified each species 100% of the time with an enumeration range of 4.00 to 9.00 Log10 CFU/mL. Enrichment time parameters for low-level concentrations (0.00, 1.00, and 2.00 Log10 CFU/mL) of Campylobacter using the BAX® System Real-Time PCR assay were elucidated. It was determined that an enrichment time of 20 h was needed to detect at least 1.00 Log10 CFU/mL of Campylobacter spp. Using the BAX® System Real-Time PCR assay for Campylobacter. As a result, time of detection, detection limits, and enrichment parameters were used to develop the CampyQuant™ linear standard curve using the detected samples from the BAX® System Real-Time PCR assay to quantify the levels in post-chill poultry rinsates. A linear fit equation was generated for each Campylobacter species using the cycle threshold from the BAX® System Real-Time PCR assay to estimate a pre-enrichment of 1.00 to 4.00 Log10 CFU/mL of rinsates detected. The statistical analyses of each equation yielded an R2 of 0.93, 0.76, and 0.94 with a Log10 RMSE of 0.64, 1.09, and 0.81 from C. jejuni, C. coli, and C. lari, respectively. The study suggests that the BAX® System Real-Time PCR assay for Campylobacter is a more rapid, accurate, and efficient alternative method for Campylobacter enumeration.

Co-exposure to Polyethylene Fiber and Salmonella enterica Typhimurium Alters Microbiome and Metabolome of in vitro Chicken Cecal Mesocosms.

Humans and animals encounter a summation of exposures during their lifetime (the exposome). In recent years, the scope of the exposome has begun to include microplastics. Microplastics (MPs) have increasingly been found in locations where there could be an interaction with Salmonella enterica Typhimurium, one of the commonly isolated serovars from processed chicken. In this study, the microbiota response to a 24-hour co-exposure to Salmonella enterica Typhimurium and/or low-density polyethylene (PE) microplastics in an in vitro broiler cecal model was determined using 16S rRNA amplicon sequencing (Illumina) and untargeted metabolomics. Community sequencing results indicated that PE fiber with and without S. Typhimurium yielded a lower Firmicutes/Bacteroides ratio compared to other treatment groups, which is associated with poor gut health, and overall had greater changes to the cecal microbial community composition. However, changes in the total metabolome were primarily driven by the presence of S. Typhimurium. Additionally, the co-exposure to PE Fiber and S. Typhimurium caused greater cecal microbial community and metabolome changes than either exposure alone. Our results indicate that polymer shape is an important factor in effects resulting from exposure. It also demonstrates that microplastic-pathogen interactions cause metabolic alterations to the chicken cecal microbiome in an in vitro chicken cecal model.

Impact of a Blend of Microencapsulated Organic Acids and Botanicals on the Microbiome of Commercial Broiler Breeders under Clinical Necrotic Enteritis.

Previously, the supplementation of a microencapsulated blend of organic acids and botanicals improved the health and performance of broiler breeders under non-challenged conditions. This study aimed to determine if the microencapsulated blend impacted dysbiosis and necrotic enteritis (NE) in broiler breeders. Day-of-hatch chicks were assigned to non-challenge and challenge groups, provided a basal diet supplemented with 0 or 500 g/MT of the blend, and subjected to a laboratory model for NE. On d 20-21, jejunum/ileum content were collected for microbiome sequencing (n = 10; V4 region of 16S rRNA gene). The experiment was repeated (n = 3), and data were analyzed in QIIME2 and R. Alpha and beta diversity, core microbiome, and compositional differences were determined (significance at p ≤ 0.05; Q ≤ 0.05). There was no difference between richness and evenness of those fed diets containing 0 and 500 g/MT microencapsulated blend, but differences were seen between the non-challenged and challenged groups. Beta diversity of the 0 and 500 g/MT non-challenged groups differed, but no differences existed between the NE-challenged groups. The core microbiome of those fed 500 g/MT similarly consisted of Lactobacillus and Clostridiaceae. Furthermore, challenged birds fed diets containing 500 g/MT had a higher abundance of significantly different phyla, namely, Actinobacteriota, Bacteroidota, and Verrucomicrobiota, than the 0 g/MT challenged group. Dietary supplementation of a microencapsulated blend shifted the microbiome by supporting beneficial and core taxa.

Campylobacter jejuni Response When Inoculated in Bovine In Vitro Fecal Microbial Consortia Incubations in the Presence of Metabolic Inhibitors.

Infection with the foodborne pathogen Campylobacter is the leading bacterial cause of human foodborne illness in the United States. The objectives of this experiment were to test the hypothesis that mixed microbial populations from the bovine rumen may be better at excluding Campylobacter than populations from freshly voided feces and to explore potential reasons as to why the rumen may be a less favorable environment for Campylobacter than feces. In an initial experiment, C. jejuni cultures inoculated without or with freshly collected bovine rumen fluid, bovine feces or their combination were cultured micro-aerobically for 48 h. Results revealed that C. jejuni grew at similar growth rates during the first 6 h of incubation regardless of whether inoculated with the rumen or fecal contents, with rates ranging from 0.178 to 0.222 h-1. However, C. jejuni counts (log10 colony-forming units/mL) at the end of the 48 h incubation were lowest in cultures inoculated with rumen fluid (5.73 log10 CFUs/mL), intermediate in cultures inoculated with feces or both feces and rumen fluid (7.16 and 6.36 log10 CFUs/mL) and highest in pure culture controls that had not been inoculated with the rumen or fecal contents (8.32 log10 CFUs/mL). In follow-up experiments intended to examine the potential effects of hydrogen and hydrogen-consuming methanogens on C. jejuni, freshly collected bovine feces, suspended in anaerobic buffer, were incubated anaerobically under either a 100% carbon dioxide or 50:50 carbon dioxide/hydrogen gas mix. While C. jejuni viability decreased <1 log10 CFUs/mL during incubation of the fecal suspensions, this did not differ whether under low or high hydrogen accumulations or whether the suspensions were treated without or with the mechanistically distinct methanogen inhibitors, 5 mM nitrate, 0.05 mM 2-bromosulfonate or 0.001 mM monensin. These results suggest that little if any competition between C. jejuni and hydrogen-consuming methanogens exists in the bovine intestine based on fecal incubations.

Chlortetracycline Concentration Impact on Salmonella Typhimurium Sustainability in the Presence of Porcine Gastrointestinal Tract Bacteria Maintained in Continuous Culture.

Concern exists that the continued use of antibiotics in animal feeds may lead to an increased prevalence of resistant bacteria within the host animal's gastrointestinal tract. To evaluate the effect of chlortetracycline on the persistence of Salmonella enterica serotype Typhimurium within a diverse population of porcine cecal bacteria, we cultured a mixed population of cecal bacteria without or with added chlortetracycline. When grown at a 24 h vessel turnover rate, chlortetracycline-susceptible S. Typhimurium exhibited more than 2.5 times faster (p < 0.05) disappearance rates than theoretically expected (0.301 log10 colony-forming unit/mL per day) but did not differ whether treated or not with 55 mg of chlortetracycline/L. Chlortetracycline-resistant S. Typhimurium was not recovered from any of these cultures. When the mixed cultures were inoculated with a chlortetracycline-resistant S. Typhimurium, rates of disappearance were nearly two times slower (p < 0.05) than those observed earlier with chlortetracycline-susceptible S. Typhimurium, and cultures persisted at >2 log10 colony-forming units/mL for up to 14 days of treatment with 110 mg of chlortetracycline/L. Under the conditions of this study, chlortetracycline-resistant S. Typhimurium was competitively enabled to persist longer within the mixed populations of porcine gut bacteria than chlortetracycline-susceptible S. Typhimurium, regardless of the presence or absence of added chlortetracycline.

Mitigating the attachment of Salmonella Infantis on isolated poultry skin with cetylpyridinium chloride.

To provide the poultry industry with effective mitigation strategies, the effects of cetylpyridinium chloride (CPC) on the reduction of Salmonella Infantis, hilA expression, and chicken skin microbiota were evaluated. Chicken breast skins (4×4 cm; N = 100, n = 10, k = 5) were inoculated with Salmonella (Typhimurium or Infantis) at 4°C (30min) to obtain 108 CFU/g attachment. Skins were shaken (30s), with remaining bacteria being considered firmly attached. Treatments were applied as 30s dips in 50 mL: no inocula-no-treatment control (NINTC), no treatment control (NTC), tap water (TW), TW+600 ppm PAA (PAA), or TW+0.5% CPC (CPC). Excess fluid was shaken off (30s). Samples were homogenized in nBPW (1 min). Samples were discarded. Salmonella was enumerated and Log10 transformed. Reverse transcriptase-qPCR (rt-qPCR) was performed targeting hilA gene and normalized using the 2-ΔΔCt method. Data were analyzed using one-way ANOVA in RStudio with means separated by Tukey's HSD (P≤0.05). Genomic DNA of rinsates was extracted, 16S rRNA gene (V4) was sequenced (MiSeq), and data analyzed in QIIME2 (P≤0.05 and Q≤0.05). CPC and PAA affected Salmonella levels differently with CPC being effective against S. Infantis compared to TW (P<0.05). Treatment with CPC on S. Infantis-infected skin altered the hilA expression compared to TW (P<0.05). When inoculated with S. Typhimurium, there was no difference between the microbiota diversity of skins treated with PAA and CPC; however, when inoculated with S. Infantis, there was a difference in the Shannon's Entropy and Jaccard Dissimilarity between the two treatments (P<0.05). Using ANCOM at the genus level, Brochothrix was significant (W = 118) among skin inoculated with S. Typhimurium. Among S. Infantis inoculated, Yersiniaceae, Enterobacterales, Lachnospiraceae CHKCI001, Clostridia vadinBB60 group, Leuconostoc, Campylobacter, and bacteria were significant (408). CPC and PAA-treated skins had lowest relative abundance of the genera. In conclusion, CPC mitigated Salmonella Infantis, altered hilA expression, and influenced the chicken skin microbiota.

Microbiome applications for laying hen performance and egg production.

Management of laying hens has undergone considerable changes in the commercial egg industry. Shifting commercial production from cage-based systems to cage-free has impacted the housing environment and created issues not previously encountered. Sources of microorganisms that become established in the early stages of layer chick development may originate from the hen and depend on the microbial ecology of the reproductive tract. Development of the layer hen GIT microbiota appears to occur in stages as the bird matures. Several factors can impact the development of the layer hen GIT, including pathogens, environment, and feed additives such as antibiotics. In this review, the current status of the laying hen GIT microbial consortia and factors that impact the development and function of these respective microbial populations will be discussed, as well as future research directions.

Impact of the gastrointestinal microbiome and fermentation metabolites on broiler performance.

Optimal broiler performance is dependent on several factors such as bird genetics, environment management, and nutrition. The gastrointestinal tract microbial ecology and metabolic activities have long been considered factors contributing to broiler performance responses. However, until recently, it was difficult to define the impact of the gastrointestinal microorganisms on the broiler host. With advances in microbiome sequencing technology, there has been a rapid increase in data generated using both experimental and commercial broiler operations. As the gastrointestinal microbiome data becomes more in-depth, opportunities to link microbiota composition to broiler performance metrics such as broiler growth rate and feed conversion efficiency have emerged. In parallel, with the increased understanding of the microbiota, there has been a shift to modulate the microbiome in order to alter metabolic patterns such as fermentation products. In this review, fermentation pathways and metabolites and the relationship with the microbiome will be discussed. Additionally, this review will connect these patterns and interpretations with current broiler performance data and the potential future directions these relationships could take the broiler industry.

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.

Practical opportunities for microbiome analyses and bioinformatics in poultry processing.

Poultry processing is undergoing changes both in operations as well as microbial methodologies. Traditionally, microbial data has been gathered through a series of culturing methods using liquid media and plating for isolation and enumeration. Both foodborne pathogens and nonpathogenic bacterial populations are estimated to assess food safety risks as well as the potential for spoilage. Bacterial loads from carcasses are important for estimating processing control and the effectiveness of antimicrobial applications. However, these culture-based approaches may only provide part of the microbial ecology landscape associated with chicken carcasses and the subsequent changes that occur in these populations during processing. Newer molecular-based approaches, such as 16S sequencing of the microbiota, offer a means to retrieve a more comprehensive microbial compositional profile. However, such approaches also result in large data sets which must be analyzed and interpreted. As more data is generated, this will require not only bioinformatic programs to process the data but appropriate educational forums to present the processed data to a broad audience.

Application of microbial analyses to feeds and potential implications for poultry nutrition.

Poultry nutrition and feed manufacturing are interrelated for a variety of reasons. Diet formulation is essential for optimizing bird growth and feed conversion, but compositional differences and the presence of certain feed additives can alter the gastrointestinal microbial composition and functionality. Not only does dietary composition and digestibility influence poultry performance, but specific physical characteristics such as feed particle size and thermal treatments can impact the avian gastrointestinal tract (GIT) microbiota. Poultry feeds also have a characteristic microbial ecology consisting of pathogenic and nonpathogenic microorganisms. Some feed-borne pathogens such as Salmonella are well studied and linked with the colonization of birds consuming the feed. However, much less is known about the nonpathogenic feed microbiome and what impact that might have on the bird's GIT. This review discusses the potential interaction between poultry feed and the GIT microbiome, microbial ecology of feed, application of microbiome analyses to feed, and approaches for communicating these complex data sets to the poultry industry.

Communicating the utility of the microbiome and bioinformatics to small flock poultry producers.

The use of "omics" has become widespread across poultry production, from breeding to management to bird health to food safety and everywhere in between.  While the conventional poultry industry has become more exposed to the power and utility of "omic" technologies, smaller poultry flock producers typically do not have this same level of experience. Because smaller, nonconventional poultry production is a growing portion of the overall poultry market, it is important that they also have educational access to these research tools and the resultant data. While small flock producers are dedicated and knowledgeable farmers, their knowledge of these newer technologies may be limited at best, and it is the task of academic researchers to communicate the importance of these "omic" tools and how the omic data can improve a variety of different aspects of their operations. This review discusses ways to effectively communicate complex microbiota and microbial genome sequence data to small flock producers and transforming this data into meaningful and applicable information that they can utilize to inform beneficial management decisions.