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.

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.

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.

Impact of Acidulants on Salmonella and Escherichia coli O157:H7 in Water Microcosms Containing Organic Matter.

As demands for fresh water become more competitive between the processing plant and other consumers of water such as municipalities, interest has grown in recycling or reusing water for food processing. However, recycling the processing water from a poultry plant, for example, represents challenges due to increased organic loads and the presence of bacterial contaminants including foodborne pathogens. The objective in the current study was to evaluate the inactivation of Salmonella and E. coli O157:H7 using combinations (0.5% and 1%) of sodium bisulfate (SBS) and 1% lactic acid (LA) in water and water with organic matter in the form of horse blood serum (0.3%) with exposure times of 1 min and 5 min. Pathogen reductions after a 5 min exposure time were greater than corresponding reductions after a 1 min exposure time for all acid solutions. The Salmonella counts were significantly reduced (i.e., ≥1 log-unit) in all acid solutions after a 5 min exposure time with the combination of LA + SBS acid solutions being more effective than the corresponding 2% LA solutions. None of the acid solutions were effective in reducing the E. coli O157:H7 after a 1 min exposure time. The 1% LA + 1% SBS solution was the most effective acid solution against both pathogens and was the only acid solution effective in reducing E. coli O157:H7 by at least one log unit after 5 min of exposure.

Natural Antimicrobials for Listeria monocytogenes in Ready-to-Eat Meats: Current Challenges and Future Prospects.

Listeria monocytogenes, an intra-cellular, Gram-positive, pathogenic bacterium, is one of the leading agents of foodborne illnesses. The morbidity of human listeriosis is low, but it has a high mortality rate of approximately 20% to 30%. L. monocytogenes is a psychotropic organism, making it a significant threat to ready-to-eat (RTE) meat product food safety. Listeria contamination is associated with the food processing environment or post-cooking cross-contamination events. The potential use of antimicrobials in packaging can reduce foodborne disease risk and spoilage. Novel antimicrobials can be advantageous for limiting Listeria and improving the shelf life of RTE meat. This review will discuss the Listeria occurrence in RTE meat products and potential natural antimicrobial additives for controlling Listeria.

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.

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.

Probiotics and potential applications for alternative poultry production systems.

Concerns over animal welfare continue to be a critical component of law and policies associated with commercial food animal production. Social and market pressures are the driving forces behind the legislation and result in the change of poultry production management systems. As a result, the movement toward cage-free and aviary-based egg production systems has become standard practices. Cage-based systems being replaced by alternative methods that offer a suitable housing environment to meet or exceed poultry welfare needs and require different management, including the ban of antibiotics in poultry diets. For broiler production, pasture- raised and free-range management systems have become more popular. However, challenges remain from exposure to disease-causing organisms and foodborne pathogens in these environments. Consequently, probiotics can be supplemented in poultry diets as commercial feed additives. The present review discusses the impacts of these probiotics on the performance of alternative poultry production systems for improving food safety and poultry health by mitigating pathogenic organisms and improving egg and meat quality and production.

An overview of health challenges in alternative poultry production systems.

Due to consumer demand and changing welfare standards on health, ecology, equity, and safety concepts, poultry production has changed markedly over the past 20 y. One of the greatest changes to poultry production standards is now offering poultry limited access to the outdoors in alternative and organic poultry production operations. Although operations allowing access to the outdoors are still only a small portion of commercial poultry production, it may impact the gastrointestinal (GIT) health of the bird in different ways than birds raised under conventional management systems. The present review describes current research results in alternative systems by identifying how different poultry production operations (diet, environmental disruptive factors, diseases) impact the ecology and health of the GIT. Various research efforts will be discussed that illustrate the nutritional value of free-range forages and how forages could be beneficial to animal health and production of both meat and eggs. The review also highlights the need for potential interventions to limit diseases without using antibiotics. These alternatives could enhance both economics and sustainability in organic and free-range poultry production.

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).

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.

Application of Bacteriophages to Limit Campylobacter in Poultry Production.

Campylobacter is a major foodborne pathogen with over a million United States cases a year and is typically acquired through the consumption of poultry products. The common occurrence of Campylobacter as a member of the poultry gastrointestinal tract microbial community remains a challenge for optimizing intervention strategies. Simultaneously, increasing demand for antibiotic-free products has led to the development of several alternative control measures both at the farm and in processing operations. Bacteriophages administered to reduce foodborne pathogens are one of the alternatives that have received renewed interest. Campylobacter phages have been isolated from both conventionally and organically raised poultry. Isolated and cultivated Campylobacter bacteriophages have been used as an intervention in live birds to target colonized Campylobacter in the gastrointestinal tract. Application of Campylobacter phages to poultry carcasses has also been explored as a strategy to reduce Campylobacter levels during poultry processing. This review will focus on the biology and ecology of Campylobacter bacteriophages in poultry production followed by discussion on current and potential applications as an intervention strategy to reduce Campylobacter occurrence in poultry production.