Microbiome analyses of poultry feeds: Part II. Comparison of different poultry feeds.

Within the realm of poultry feed mill operations, the persistent concern over microbial feed quality necessitates the establishment of a robust baseline for enhancing and sustaining the standards of commercial feeds. This dual-phase investigation, comprising Parts I, was previously published, and the current study presented here as Part II aimed to illuminate this baseline using 16S rRNA gene sequencing. In Part II, nine distinct commercial poultry feeds formulated as starters, growers, starter/growers, or supplements, the selected feeds underwent genomic DNA extraction, amplification with custom dual-indexed primers, and subsequent Illumina MiSeq sequencing. Through data analysis in QIIME2-2021.4 and R Studio, the study unveils alpha (Kruskal-Wallis) and beta (ANOSIM) diversity, taxonomic differences (ANCOM), and core microbiomes (core_members), deeming main and pairwise effects statistically significant at p < 0.05 and Q < 0.05. Notably, the investigation identified 30% common core microbial members across the nine feed types, shedding light on potential foodborne poultry pathogens such as Helicobacter and Campylobacter. Probiotic-associated feeds exhibited distinct microbial communities, emphasizing the need to explore their impact on the early poultry gastrointestinal tract (GIT) further.

Comparison of optical density-based growth kinetics for pure culture Campylobacter jejuni, coli and lari grown in blood-free Bolton broth.

Campylobacter growth kinetic parameters can be used to refine the sensitivity and efficiency of microbial growth-based methods. Therefore, the aim of this study was to construct growth curves for C. jejuni, C. coli, and C. lari in pure culture and calculate growth kinetics for each Campylobacter species in the same environmental conditions. Campylobacter jejuni, C. coli and C. lari were grown over 48 h and inoculated into 15 mL Hungate tubes (N = 3 trials per species; 5 biological replicates per trial; 3 species; 1 strain per species). Absorbance measurements were taken in 45 min intervals over 24 h. Optical density readings were plotted versus time to calculate growth kinetic parameters. C. jejuni exhibited the longest lag phase (p < 0.001) at 15 h 20 min ± 30 min, versus C. coli at 11 h 15 min ± 17 min, and C. lari at 9 h 27 min ± 15 min. The exponential phase duration was no longer than 5 h for all species, and doubling times were all less than 1h 30 min. The variation in growth kinetics for the three species of Campylobacter illustrates the importance of determining individual Campylobacter spp. growth responses for optimizing detection based on low bacterial levels. This study provides kinetics and estimates to define enrichment times necessary for low concentration Campylobacter detection.

Litter Commensal Bacteria Can Limit the Horizontal Gene Transfer of Antimicrobial Resistance to Salmonella in Chickens.

Fostering a "balanced" gut microbiome through the administration of beneficial microbes that can competitively exclude pathogens has gained a lot of attention and use in human and animal medicine. However, little is known about how microbes affect the horizontal gene transfer of antimicrobial resistance (AMR). To shed more light on this question, we challenged neonatal broiler chicks raised on reused broiler chicken litter-a complex environment made up of decomposing pine shavings, feces, uric acid, feathers, and feed-with Salmonella enterica serovar Heidelberg (S. Heidelberg), a model pathogen. Neonatal chicks challenged with S. Heidelberg and raised on reused litter were more resistant to S. Heidelberg cecal colonization than chicks grown on fresh litter. Furthermore, chicks grown on reused litter were at a lower risk of colonization with S. Heidelberg strains that encoded AMR on IncI1 plasmids. We used 16S rRNA gene sequencing and shotgun metagenomics to show that the major difference between chicks grown on fresh litter and those grown on reused litter was the microbiome harbored in the litter and ceca. The microbiome of reused litter samples was more uniform and enriched in functional pathways related to the biosynthesis of organic and antimicrobial molecules than that in fresh litter samples. We found that Escherichia coli was the main reservoir of plasmids encoding AMR and that the IncI1 plasmid was maintained at a significantly lower copy per cell in reused litter compared to fresh litter. These findings support the notion that commensal bacteria play an integral role in the horizontal transfer of plasmids encoding AMR to pathogens like Salmonella. IMPORTANCE Antimicrobial resistance spread is a worldwide health challenge, stemming in large part from the ability of microorganisms to share their genetic material through horizontal gene transfer. To address this issue, many countries and international organizations have adopted a One Health approach to curtail the proliferation of antimicrobial-resistant bacteria. This includes the removal and reduction of antibiotics used in food animal production and the development of alternatives to antibiotics. However, there is still a significant knowledge gap in our understanding of how resistance spreads in the absence of antibiotic selection and the role commensal bacteria play in reducing antibiotic resistance transfer. In this study, we show that commensal bacteria play a key role in reducing the horizontal gene transfer of antibiotic resistance to Salmonella, provide the identity of the bacterial species that potentially perform this function in broiler chickens, and also postulate the mechanism involved.

Using E. coli population to predict foodborne pathogens in pastured poultry farms.

Escherichia coli shows the potential of indicating foodborne pathogens. The purpose of this study is to investigate the association between E. coli and foodborne pathogens such as Campylobacter, Salmonella, and Listeria in pastured poultry farms, as well as in related processing facilities. Five different sample types: (i) feces, (ii) soil, (iii) whole carcass rinse during processing (WCR-P), (iv) whole carcass rinse of final product after chilling and storage (WCR-F), and (v) ceca were measured for E. coli populations. A logistic regression model for pathogen presence was developed for each sample type. The E. coli population significantly increased the predicted probability of Salmonella presence for soil and WCR-P samples (p = 0.0011 and p = 0.0157 respectively). For Campylobacter, the initial prevalence in feces and ceca were high and a decreasing trend of detecting Campylobacter was observed as E. coli concentration increased. In soil and WCR-P models, the probability of the presence of Campylobacter significantly increased as E. coli population increased. These models provide a practical and effective way of evaluating the relationship between E. coli and foodborne pathogens and enable prediction of foodborne pathogen presence based on E. coli prevalence within the pastured poultry farm-to-fork continuum.

Urea Hydrolysis and Calcium Carbonate Precipitation in Gypsum-Amended Broiler Litter.

Broiler () litter is subject to ammonia (NH) volatilization losses. Previous work has shown that the addition of gypsum to broiler litter can increase nitrogen mineralization and decrease NH losses due to a decrease in pH, but the mechanisms responsible for these effects are not well understood. Therefore, three laboratory studies were conducted to evaluate the effect of gypsum addition to broiler litter on (i) urease activity at three water contents, (ii) calcium carbonate precipitation, and (iii) pH. The addition of gypsum to broiler litter increased ammonium concentrations ( < 0.0033) and decreased litter pH by 0.43 to 0.49 pH units after 5 d ( < 0.0001); however, the rate of urea hydrolysis in treated litter only increased on Day 0 for broiler litter with low (0.29 g HO g) and high (0.69 g HO g) water contents, and on Day 3 for litter with medium (0.40 g HO g) water content ( < 0.05). Amending broiler litter with gypsum also caused an immediate decrease in litter pH (0.22 pH units) due to the precipitation of calcium carbonate (CaCO) from gypsum-derived calcium and litter bicarbonate. Furthermore, as urea was hydrolyzed, more urea-derived carbon precipitated as CaCO in gypsum-treated litter than in untreated litter ( < 0.001). These results indicate that amending broiler litter with gypsum favors the precipitation of CaCO, which buffers against increases in litter pH that are known to facilitate NH volatilization.

In-Package Inactivation of Pathogenic and Spoilage Bacteria Associated with Poultry Using Dielectric Barrier Discharge-Cold Plasma Treatments.

The goal of this study was to test the efficacy of in-package dielectric barrier discharge-cold plasma (DBD-CP) treatment to inactivate poultry-associated spoilage (Pseudomonas fluorescens) and pathogenic (Salmonella enterica Typhimurium, Campylobacter jejuni) bacteria. Liquid cultures of the bacterial isolates were sealed within packages containing ambient air (Trial 1) or modified air (65% O2:30% CO2:5% N2; Trial 2). The packages were subjected to treatment times ranging from 30 to 180 s, and after 24 h incubation at 4 °C, bacterial titers were determined. The DBD-CP system completely inactivated the four isolates tested, although the in-package gas composition and treatment times were isolate-specific. Both C. jejuni isolates were completely inactivated between 30 s (modified air) and 120 s (ambient air), while modified air was required for the complete inactivation of S. typhimurium (90 s) and P. fluorescens (180 s). This DBD-CP system is effective for inactivating major poultry-associated spoilage and pathogenic bacteria in liquid culture, and through this study, system parameters to optimize inactivation were determined. This study demonstrates the potential for DBD-CP treatment to inactivate major bacteria of economic interest to the poultry industry, thus potentially allowing for reduced spoilage (e.g., longer shelf life) and increased safety of poultry products.

Antibiotic Resistance Patterns of Major Zoonotic Pathogens from All-Natural, Antibiotic-Free, Pasture-Raised Broiler Flocks in the Southeastern United States.

The use of antibiotics in agroecosystems has been implicated in the rise in antibiotic resistance (AR), which can affect environmental, animal, and human health. To determine the environmental impact of antibiotic use in agroecosystems, appropriate background levels of AR in agricultural environments in the absence of antibiotic application must be determined. Therefore, to determine background levels of AR in broiler production, four target microbes (, , , and ) were isolated from 15 all-natural, antibiotic-free, pasture-raised broiler flocks from six farms within the southeastern United States. The AR profiles of these isolates were characterized using the CDC National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), and these resistance patterns were compared across target microbes and farms and throughout the life cycle of the flocks along the farm-to-fork continuum. Antibiotic resistances were most prevalent in and and least prevalent in . Although and were isolated from the same farms and characterized using the same NARMS plates, they exhibited distinct AR profiles, with demonstrating clear farm-specific resistance patterns. Multidrug resistance rates (three or more antibiotics), in order of prevalence, were (63.9%), (36.0%), (12.7%), and (1.4%). The results of this study demonstrate the variability in background AR among major food safety-related microbes, even when isolated from similar production and processing samples from the same farms, and indicate the need for the proper design of future broiler production studies to account for this highly dynamic background AR.

How Should We Be Determining Background and Baseline Antibiotic Resistance Levels in Agroecosystem Research?

Although historically, antibiotic resistance has occurred naturally in environmental bacteria, many questions remain regarding the specifics of how humans and animals contribute to the development and spread of antibiotic resistance in agroecosystems. Additional research is necessary to completely understand the potential risks to human, animal, and ecological health in systems altered by antibiotic-resistance-related contamination. At present, analyzing and interpreting the effects of human and animal inputs on antibiotic resistance in agroecosystems is difficult, since standard research terminology and protocols do not exist for studying background and baseline levels of resistance in the environment. To improve the state of science in antibiotic-resistance-related research in agroecosystems, researchers are encouraged to incorporate baseline data within the study system and background data from outside the study system to normalize the study data and determine the potential impact of antibiotic-resistance-related determinants on a specific agroecosystem. Therefore, the aims of this review were to (i) present standard definitions for commonly used terms in environmental antibiotic resistance research and (ii) illustrate the need for research standards (normalization) within and between studies of antibiotic resistance in agroecosystems. To foster synergy among antibiotic resistance researchers, a new surveillance and decision-making tool is proposed to assist researchers in determining the most relevant and important antibiotic-resistance-related targets to focus on in their given agroecosystems. Incorporation of these components within antibiotic-resistance-related studies should allow for a more comprehensive and accurate picture of the current and future states of antibiotic resistance in the environment.

Antibiotics in Agroecosystems: Introduction to the Special Section.

The presence of antibiotic drug residues, antibiotic resistant bacteria, and antibiotic resistance genes in agroecosystems has become a significant area of research in recent years and is a growing public health concern. While antibiotics are used in both human medicine and agricultural practices, the majority of their use occurs in animal production where historically they have been used for growth promotion, in addition to the prevention and treatment of disease. The widespread use of antibiotics and the application of animal wastes to agricultural lands play major roles in the introduction of antibiotic-related contamination into the environment. Overt toxicity in organisms directly exposed to antibiotics in agroecosystems is typically not a major concern because environmental concentrations are generally lower than therapeutic doses. However, the impacts of introducing antibiotic contaminants into the environment are unknown, and concerns have been raised about the health of humans, animals, and ecosystems. Despite increased research focused on the occurrence and fate of antibiotics and antibiotic resistance over the past decade, standard methods and practices for analyzing environmental samples are limited and future research needs are becoming evident. To highlight and address these issues in detail, this special collection of papers was developed with a framework of five core review papers that address the (i) overall state of science of antibiotics and antibiotic resistance in agroecosystems using a causal model, (ii) chemical analysis of antibiotics found in the environment, (iii) need for background and baseline data for studies of antibiotic resistance in agroecosystems with a decision-making tool to assist in designing research studies, as well as (iv) culture- and (v) molecular-based methods for analyzing antibiotic resistance in the environment. With a focus on the core review papers, this introduction summarizes the current state of science for analyzing antibiotics and antibiotic resistance in agroecosystems, discusses current knowledge gaps, and develops future research priorities. This introduction also contains a glossary of terms used in the core reivew papers of this special section. The purpose of the glossary is to provide a common terminology that clearly characterizes the concepts shared throughout the narratives of each review paper.

Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase.

Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition.

The characterization of Salmonella enterica serotypes isolated from the scalder tank water of a commercial poultry processing plant: Recovery of a multidrug-resistant Heidelberg strain.

The recent multistate outbreak of a multidrug-resistant (MDR) Salmonella Heidelberg strain from commercial poultry production highlights the need to better understand the reservoirs of these zoonotic pathogens within the commercial poultry production and processing environment. As part of a larger study looking at temporal changes in microbial communities within the major water tanks within a commercial processing facility, this paper identifies and characterizes Salmonella enterica isolated from the water in a final scalder tank at 3 times during a typical processing day: prior to the birds entering the tank (start), halfway through the processing day (mid), and after the final birds were scalded (end). Over 3 consecutive processing days, no Salmonella were recovered from start-of-day water samples, while a total of 56 Salmonella isolates were recovered from the mid-day and end-of-day scalder water samples. Traditional and newer PCR-based serotyping methods eventually identified these isolates as either group C3 S. Kentucky (n=45) and group B S. Heidelberg (n=11). While none of the S. Kentucky isolates possessed any resistances to the antimicrobials tested, all S. Heidelberg isolates were found to be multidrug resistant to 5 specific antimicrobials representing 3 antimicrobial classes. Due to the potential public health impact of S. Heidelberg and the recent nationwide poultry-associated outbreak of multidrug-resistant S. Heidelberg, future studies should focus on understanding the transmission and environmental growth dynamics of this serotype within the commercial poultry processing plant environment.

Exploring Pathogen Presence Prediction in Pastured Poultry Farms through Transformer-Based Models and Attention Mechanism Explainability.

In this study, we explore how transformer models, which are known for their attention mechanisms, can improve pathogen prediction in pastured poultry farming. By combining farm management practices with microbiome data, our model outperforms traditional prediction methods in terms of the F1 score-an evaluation metric for model performance-thus fulfilling an essential need in predictive microbiology. Additionally, the emphasis is on making our model's predictions explainable. We introduce a novel approach for identifying feature importance using the model's attention matrix and the PageRank algorithm, offering insights that enhance our comprehension of established techniques such as DeepLIFT. Our results showcase the efficacy of transformer models in pathogen prediction for food safety and mark a noteworthy contribution to the progress of explainable AI within the biomedical sciences. This study sheds light on the impact of effective farm management practices and highlights the importance of technological advancements in ensuring food safety.

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.

Ruminal Microbiome Differences in Angus Steers with Differing Feed Efficiencies during the Feedlot Finishing Phase.

The catabolic activity of the ruminal microbial community of cattle enables the conversion of low-quality feedstuffs into meat and milk. The rate at which this conversion occurs is termed feed efficiency, which is of crucial importance given that feed expenses account for up to 70% of the cost of animal production. The present study assessed the relationship between cattle feed efficiency and the composition of their ruminal microbial communities during the feedlot finishing period. Angus steers (n = 65) were fed a feedlot finishing diet for 82 days and their growth performance metrics were evaluated. These included the dry matter intake (DMI), average daily gain (ADG), and residual feed intake (RFI). Steers were rank-ordered based upon their RFI, and the five lowest RFI (most efficient) and five highest RFI (least efficient) steers were selected for evaluations. Ruminal fluid samples were collected on days 0 and 82 of the finishing period. Volatile fatty acids (VFA) were quantified, and microbial DNA was extracted and the 16S rRNA gene was sequenced. The results showed that the ADG was not different (p = 0.82) between efficiency groups during the 82-day feedlot period; however, the efficient steers had lower (p = 0.03) DMI and RFI (p = 0.003). Less-efficient (high RFI) steers developed higher (p = 0.01) ruminal Methanobrevibacter relative abundances (p = 0.01) and tended (p = 0.09) to have more Methanosphaera. In high-efficiency steers (low RFI), the relative abundances of Ruminococcaceae increased (p = 0.04) over the 82-day period. The molar proportions of VFA were not different between the two efficiency groups, but some changes in the concentration of specific VFA were observed over time. The results indicated that the ruminal microbial populations of the less-efficient steers contained a greater relative abundance of methanogens compared to the high-efficiency steers during the feedlot phase, likely resulting in more energetic waste in the form or methane and less dietary energy being harvested by the less-efficient animals.

Characterization of the Campylobacter jejuni cryptic plasmid pTIW94 recovered from wild birds in the southeastern United States.

The complete nucleotide sequence was determined for a cryptic plasmid, pTIW94, recovered from several Campylobacter jejuni isolates from wild birds in the southeastern United States. pTIW94 is a circular molecule of 3860 nucleotides, with a G+C content (31.0%) similar to that of many Campylobacter spp. genomes. A typical origin of replication, with iteron sequences, was identified upstream of DNA sequences that demonstrated similarity to replication initiation proteins. A total of five open reading frames (ORFs) were identified; two of the five ORFs demonstrated significant similarity to plasmid pCC2228-2 found within Campylobacter coli. These two ORFs were similar to essential replication proteins RepA (100%; 26/26 aa identity) and RepB (95%; 327/346 aa identity). A third identified ORF demonstrated significant similarity (99%; 421/424 aa identity) to the MOB protein from C. coli 67-8, originally recovered from swine. The other two identified ORFs were either similar to hypothetical proteins from other Campylobacter spp., or exhibited no significant similarity to any DNA or protein sequence in the GenBank database. Promoter regions (-35 and -10 signal sites), ribosomal binding sites upstream of ORFs, and stem-loop structures were also identified within the plasmid. These results demonstrate that pTIW94 represents a previously un-reported small cryptic plasmid with unique sequences as well as highly similar sequences to other small plasmids found within Campylobacter spp., and that this cryptic plasmid is present among Campylobacter spp. recovered from different genera of wild birds.

Assessing the Risk of Seasonal Effects of Campylobacter Contaminated Broiler Meat Prepared In-Home in the United States.

Campylobacter has consistently posed a food safety issue in broiler meat. This study aimed to create a quantitative microbial risk assessment model from retail to consumption, designed to evaluate the seasonal risk of campylobacteriosis associated with broiler meat consumption in the United States. To achieve this, data was gathered to build distributions that would enable us to predict the growth of Campylobacter during various stages such as retail storage, transit, and home storage. The model also included potential fluctuations in concentration during food preparation and potential cross-contamination scenarios. A Monte Carlo simulation with 100,000 iterations was used to estimate the risk of infection per serving and the number of infections in the United States by season. In the summer, chicken meat was estimated to have a median risk of infection per serving of 9.22 × 10-7 and cause an average of about 27,058,680 infections. During the winter months, the median risk of infection per serving was estimated to be 4.06 × 10-7 and cause an average of about 12,085,638 infections. The risk assessment model provides information about the risk of broiler meat to public health by season. These results will help understand the most important steps to reduce the food safety risks from contaminated chicken products.

Predicting foodborne pathogens and probiotics taxa within poultry-related microbiomes using a machine learning approach.

BACKGROUND: Microbiomes that can serve as an indicator of gut, intestinal, and general health of humans and animals are largely influenced by food consumed and contaminant bioagents. Microbiome studies usually focus on estimating the alpha (within sample) and beta (similarity/dissimilarity among samples) diversities. This study took a combinatorial approach and applied machine learning to microbiome data to predict the presence of disease-causing pathogens and their association with known/potential probiotic taxa. Probiotics are beneficial living microorganisms capable of improving the host organism's digestive system, immune function and ultimately overall health. Here, 16 S rRNA gene high-throughput Illumina sequencing of temporal pre-harvest (feces, soil) samples of 42 pastured poultry flocks (poultry in this entire work solely refers to chickens) from southeastern U.S. farms was used to generate the relative abundance of operational taxonomic units (OTUs) as machine learning input. Unique genera from the OTUs were used as predictors of the prevalence of foodborne pathogens (Salmonella, Campylobacter and Listeria) at different stages of poultry growth (START (2-4 weeks old), MID (5-7 weeks old), END (8-11 weeks old)), association with farm management practices and physicochemical properties. RESULT: While we did not see any significant associations between known probiotics and Salmonella or Listeria, we observed significant negative correlations between known probiotics (Bacillus and Clostridium) and Campylobacter at the mid-time point of sample collection. Our data indicates a negative correlation between potential probiotics and Campylobacter at both early and end-time points of sample collection. Furthermore, our model prediction shows that changes in farm operations such as how often the houses are moved on the pasture, age at which chickens are introduced to the pasture, diet composition and presence of other animals on the farm could favorably increase the abundance and activity of probiotics that could reduce Campylobacter prevalence. CONCLUSION: Integration of microbiome data with farm management practices using machine learning provided insights on how to reduce Campylobacter prevalence and transmission along the farm-to-fork continuum. Altering management practices to support proliferation of beneficial probiotics to reduce pathogen prevalence identified here could constitute a complementary method to the existing but ineffective interventions such as vaccination and bacteriophage cocktails usage. Study findings also corroborate the presence of bacterial genera such as Caloramator, DA101, Parabacteroides and Faecalibacterium as potential probiotics.

Relationship of the Poultry Microbiome to Pathogen Colonization, Farm Management, Poultry Production, and Foodborne Illness Risk Assessment.

Despite the continuous progress in food science and technology, the global burden of foodborne illnesses remains substantial, with pathogens in food causing millions of infections each year. Traditional microbiological culture methods are inadequate in detecting the full spectrum of these microorganisms, highlighting the need for more comprehensive detection strategies. This review paper aims to elucidate the relationship between foodborne pathogen colonization and the composition of the poultry microbiome, and how this knowledge can be used for improved food safety. Our review highlights that the relationship between pathogen colonization varies across different sections of the poultry microbiome. Further, our review suggests that the microbiome profile of poultry litter, farm soil, and farm dust may serve as potential indicators of the farm environment's food safety issues. We also agree that the microbiome of processed chicken samples may reveal potential pathogen contamination and food quality issues. In addition, utilizing predictive modeling techniques on the collected microbiome data, we suggest establishing correlations between particular taxonomic groups and the colonization of pathogens, thus providing insights into food safety, and offering a comprehensive overview of the microbial community. In conclusion, this review underscores the potential of microbiome analysis as a powerful tool in food safety, pathogen detection, and risk assessment.

Genotypic characterization, antimicrobial susceptibility and virulence determinants of Campylobacter jejuni and Campylobacter coli isolated from pastured poultry farms.

Aim: Campylobacter is the leading bacterial pathogen that causes foodborne illnesses worldwide. Pasture farming is regarded as an important source of agricultural production for small farming communities. Consumer preference for pasture-raised animal products has increased; however, there is a paucity of information on the microbiological quality of pasture-raised poultry products. The purpose of this study was to explore genetic relatedness of thermophilic Campylobacter isolates, to assess antibiotic resistance phenotypically and genotypically, and to screen the presence of virulence determinants of Campylobacter isolates from pasture-raised poultry farms from southeastern United States. Methods: Ninety-seven Campylobacter isolates previously identified by Q7 BAX® System Real-Time PCR were genotyped by multilocus sequence typing (MLST). Campylobacter isolates were then evaluated for their phenotypic antimicrobial susceptibility against nine antimicrobial agents using Sensititre plates. Additionally, Campylobacter isolates were tested for the presence of antimicrobial resistance-associated elements. Furthermore, Campylobacter isolates were screened for the presence of 13 genes encoding putative virulence factors by PCR. These included genes involved in motility (flaA and flhA), adhesion and colonization (cadF, docC, racR, and virB11), toxin production (cdtA, cdtB, cdtC, wlaN, and ceuE) and invasion (ciaB and iamA). Results: Among 97 Campylobacter isolates, Campylobacter jejuni (n = 79) and Campylobacter coli (n = 18) were identified. By MLST, C. jejuni isolates were assigned to seven clonal complexes. Among them, ST-353, ST-607 and ST-21 were the most common STs recognized. All C. coli (n = 18) isolates were included in CC-828. Interestingly, eight STs identified were not belonging any previous identified clonal complex. Campylobacter isolates displayed a high resistance rate against tetracycline (81.4%), while a low rate of resistance was observed against macrolides (azithromycin and erythromycin), quinolones and fluoroquinolones (nalidixic acid and ciprofloxacin), aminoglycosides (gentamicin), ketolide (telithromycin), amphenicol (florfenicol) and lincomycin (clindamycin). Thirteen isolates (13.54%) were pan-susceptible to all tested antibiotics, while nine isolates were multi-antimicrobial resistant (MAR; resist to three or more antimicrobial classes). Interestingly, there were no isolates resistant to all antimicrobial classes. Thr86Ile mutation was identified in all quinolones resistant strains. Erythromycin encoding gene (ermB) was identified in 75% of erythromycin resistant isolates. The A2075 mutation was detected in one erythromycin resistant strain, while A2074 could not be identified. The tetO gene was identified in 93.7% of tetracycline resistant isolates and six tetracycline susceptible isolates. In conclusion, the results of this study revealed that Campylobacter isolates from pasture-raised poultry farms showed the ST relatedness to Campylobacter isolates commonly associated with humans, indicating pasture-raised broiler flocks, similar to conventionally-reared broiler flocks, as a potential vector for antibiotic-resistant and pathogenic strains of thermophilic Campylobacter to humans.

Listeria Occurrence in Conventional and Alternative Egg Production Systems.

Listeria continues to be a persistent foodborne pathogen that is responsible for human cases of listeriosis when contaminated food products are consumed. Human subjects considered to be most susceptible include the elderly, immunocompromised, and pregnant women. Listeria is characterized as a saprophytic organism with the capability of responding and adapting to constantly changing environments because it possesses multiple stress response mechanisms to overcome varying temperatures, salt concentrations, and pH, among others. Primary foods and food products associated with listeriosis include dairy products and ready-to-eat meats such as turkey products. Historically, chicken eggs have not been identified as a primary source of Listeria, but the potential for contamination during egg production and processing does exist. Listeria species have been isolated from egg-processing plant equipment and are presumed to occur in egg-processing plant environments. Whether Listeria is consistently disseminated onto eggs beyond the egg-processing plant is a risk factor that remains to be determined. However, research has been conducted over the years to develop egg wash solutions that generate combinations of pH and other properties that would be considered inhibitory to Listeria. Even less is known regarding the association of Listeria with alternative egg production systems, but Listeria has been isolated from pasture flock broilers, so it is conceivable, given the nature of the outdoor environments, that layer birds under these conditions would also be exposed to Listeria and their eggs become contaminated. This review focuses on the possibility of Listeria occurring in conventional and alternative egg-laying production and processing systems.