A Novel Slope-Matrix-Graph Algorithm to Analyze Compositional Microbiome Data.

Networks are widely used to represent relationships between objects, including microorganisms within ecosystems, based on high-throughput sequencing data. However, challenges arise with appropriate statistical algorithms, handling of rare taxa, excess zeros in compositional data, and interpretation. This work introduces a novel Slope-Matrix-Graph (SMG) algorithm to identify microbiome correlations primarily based on slope-based distance calculations. SMG effectively handles any proportion of zeros in compositional data and involves: (1) searching for correlated relationships (e.g., positive and negative directions of changes) based on a "target of interest" within a setting, and (2) quantifying graph changes via slope-based distances between objects. Evaluations on simulated datasets demonstrated SMG's ability to accurately cluster microbes into distinct positive/negative correlation groups, outperforming methods like Bray-Curtis and SparCC in both sensitivity and specificity. Moreover, SMG demonstrated superior accuracy in detecting differential abundance (DA) compared to ZicoSeq and ANCOM-BC2, making it a robust tool for microbiome analysis. A key advantage is SMG's natural capacity to analyze zero-inflated compositional data without transformations. Overall, this simple yet powerful algorithm holds promise for diverse microbiome analysis applications.

16S amplicon-based microbiome biomapping of a commercial broiler hatchery.

Hatcheries, where eggs from multiple breeder farms are incubated and hatched before being sent to different broiler farms, represent a nexus point in the commercial production of broilers in the United States. Considering all downstream microbial quality and safety aspects of broiler production (live production, processing, consumer use) can be potentially affected by the hatchery, a better understanding of microbial ecology within commercial hatcheries is essential. Therefore, a commercial broiler hatchery was biomapped using 16S rRNA amplicon-based microbiome analyses of four sample type categories (Air, Egg, Water, Facility) across five different places in the pre-hatch, hatch, and post-hatch areas. While distinct microbiota were found for each sample type category and hatchery area, microbial community analyses revealed that Egg microbiota trended towards clustering with the facility-related samples when moving from the prehatch to post-hatch areas, highlighting the potential effect of the hatchery environment in shaping the pre-harvest broiler-related microbiota. Prevalence analyses revealed 20 ASVs (Core20) present in the core microbiota of all sample types and areas, with each ASV possessing a unique distribution throughout the hatchery. Interestingly, three Enterobacteriaceae ASVs were in the Core20, including Salmonella. Subsequent analyses showed that Salmonella, while a minor prehatch and hatch Core20ASV, dominated the Enterobacteriaceae niche and total microbiota in the chick pad feces in the post-hatch area of the hatchery, and the presence of this Salmonella ASV in the post-hatch feces was associated with swabs of breakroom tables. These findings highlight the complexity of commercial hatchery microbiota, including identifying chick pad feces and breakroom tables as potentially important sampling or disinfection targets for hatchery managers to focus their Salmonella mitigation efforts to reduce loads entering live production farms.

Salmonella Biomapping of a Commercial Broiler Hatchery.

Poultry-associated salmonellosis results in significant costs to poultry producers and consumers. Given the vertically integrated nature of the United States poultry industry, a better understanding of Salmonella ecology throughout all levels of poultry production is essential. One nexus point is the hatchery, where eggs from multiple broiler breeder farms are incubated and hatched, with the chicks being sent to numerous farms; therefore, the hatchery represents an ideal area to understand preharvest Salmonella ecology and flow. To achieve this, a commercial broiler hatchery was biomapped, focusing on Salmonella prevalence and serotype diversity among four major sample type categories (Air, Egg, Water, Facility) across five different places in the prehatch, hatch, and posthatch areas. Following two sets of eggs from broiler breeder farms over two production days, the overall Salmonella prevalence was 26% (48/184). Of the positive samples, the highest prevalence was observed in swabs taken from the floor drains in the facility and transport truck (56%), as well as in the hatch and posthatch hatchery areas (50%). Kentucky (n = 17), Gaminara (n = 12), and Alachua (n = 11) were the dominant Salmonella serotypes, with serotypes of greatest outbreak concern from chickens (Enteritidis) representing only 6.25% (3/48) of all recovered Salmonella isolates. The posthatch transport area, including the underfloor reservoirs of the transport trucks, not only harbored Enteritidis but also the enrichment broths from these Salmonella-positive samples also possessed sequences matching the commercial live-attenuated vaccine Typhimurium strain according to CRISPR SeroSeq analyses. These findings highlight the complex diversity of commercial hatchery Salmonella populations, including identifying facility floor drains and transport trucks as potentially important critical control points for hatchery managers to focus their Salmonella mitigation efforts to reduce loads and serotypes entering live production farms.

Genetic Characteristics of Salmonella Isolates Recovered From Reused Broiler Litter Over Three Successive Flocks.

Salmonella infections are a leading cause of bacterial food-borne illness worldwide. Infections are highly associated with the consumption of contaminated food, and in particular, chicken meat. The severity of Salmonella infections depends on the presence of antimicrobial resistance genes and virulence factors. While there are many studies which have investigated Salmonella strains isolated from postharvest chicken samples, there is a gap in our understanding of the genetic properties that influence the persistence of Salmonella in preharvest and in particular their makeup of antimicrobial resistance genes and virulence factors. We used whole genome sequencing and hierarchical clustering to characterize and classify the genetic diversity of Salmonella enterica isolates (n = 55) recovered from the litter of commercial broiler chicken raised in four colocated broiler houses of one integrated farm over three consecutive flocks. The chicken were raised under a newly adopted "No Antibiotics Ever" program, and copper sulfate was administered via drinking water. In-silico serovar prediction identified three S. enterica serovars: Enteritidis (n = 12), Kentucky (n = 40), and Senftenberg (n = 3). Antimicrobial susceptibility testing revealed that only one S. Kentucky isolate was resistant to streptomycin, while the remaining isolates were susceptible to all antibiotics tested. Metal resistance operons, including copper and silver, were identified chromosomally and on plasmids in serovar Senftenberg and Kentucky isolates, respectively, while serovar Enteritidis carried several virulence factors on plasmids. Serovar Kentucky isolates harboring metal resistance operons were the only Salmonella isolates recovered from the litter of third flock cohort. These results suggest that there might be environmental selection for Salmonella strains carrying plasmid-associated metal resistance and virulence genes, which could play a role in their persistence in litter.

Virulence factors and antimicrobial resistance profiles of Campylobacter isolates recovered from consecutively reused broiler litter.

IMPORTANCE: Campylobacter is a leading cause of foodborne illness in the United States due to consumption of contaminated or mishandled food products, often associated with chicken meat. Campylobacter is common in the microbiota of avian and mammalian gut; however, acquisition of antimicrobial resistance genes (ARGs) and virulence factors (VFs) may result in strains that pose significant threat to public health. Although there are studies investigating the genetic diversity of Campylobacter strains isolated from post-harvest chicken samples, there are limited data on the genome characteristics of isolates recovered from preharvest broiler production. Here, we show that Campylobacter jejuni and Campylobacter coli differ in their carriage of antimicrobial resistance and virulence factors may also differ in their ability to persist in litter during consecutive grow-out of broiler flocks. We found that presence/absence of virulence factors needed for evasion of host defense mechanisms and gut colonization played an integral role in differentiating Campylobacter strains.

Temporal dynamics of the cecal and litter microbiome of chickens raised in two separate broiler houses.

In this study, we investigated the dynamics of the ceca and litter microbiome of chickens from post-hatch through pre-harvest. To achieve this, six hundred one-day old Cobb 500 broiler chicks were raised on floor pens for 49 days in two separate houses. We performed short-read and full-length sequencing of the bacterial 16S rRNA gene present in the meconium and in cecal and litter samples collected over the duration of the study. In addition, we determined the antimicrobial resistance (AMR) phenotype of Escherichia coli and Enterococcus spp. isolated from the meconium and the ceca of 49-day old chickens. We monitored the relative humidity, temperature, and ammonia in each house daily and the pH and moisture of litter samples weekly. The overall microbial community structure of the ceca and litter consistently changed throughout the course of the grow-out and correlated with some of the environmental parameters measured (p < 0.05). We found that the ceca and litter microbiome were similar in the two houses at the beginning of the experiment, but over time, the microbial community separated and differed between the houses. When we compared the environmental parameters in the two houses, we found no significant differences in the first half of the growth cycle (day 0-21), but morning temperature, morning humidity, and ammonia significantly differed (p < 0.05) between the two houses from day 22-49. Lastly, the prevalence of AMR in cecal E. coli isolates differed from meconium isolates (p < 0.001), while the AMR phenotype of cecal Enterococcus isolates differed between houses (p < 0.05).

Broiler house environment and litter management practices impose selective pressures on antimicrobial resistance genes and virulence factors of Campylobacter.

Campylobacter infections are a leading cause of bacterial diarrhea in humans globally. Infections are due to consumption of contaminated food products and are highly associated with chicken meat, with chickens being an important reservoir for Campylobacter. Here, we characterized the genetic diversity of Campylobacter species detected in broiler chicken litter over three consecutive flocks and determined their antimicrobial resistance and virulence factor profiles. Antimicrobial susceptibility testing and whole genome sequencing were performed on Campylobacter jejuni (n = 39) and Campylobacter coli (n = 5) isolates. All C. jejuni isolates were susceptible to all antibiotics tested while C. coli (n =4) were resistant to only tetracycline and harbored the tetracycline-resistant ribosomal protection protein (TetO). Virulence factors differed within and across grow houses but were explained by the isolates' flock cohort, species and multilocus sequence type. Virulence factors involved in the ability to invade and colonize host tissues and evade host defenses were absent from flock cohort 3 C. jejuni isolates as compared to flock 1 and 2 isolates. Our results show that virulence factors and antimicrobial resistance genes differed by the isolates' multilocus sequence type and by the flock cohort they were present in. These data suggest that the house environment and litter management practices performed imposed selective pressures on antimicrobial resistance genes and virulence factors. In particular, the absence of key virulence factors within the final flock cohort 3 isolates suggests litter reuse selected for Campylobacter strains that are less likely to colonize the chicken host.

Chicken Production and Human Clinical Escherichia coli Isolates Differ in Their Carriage of Antimicrobial Resistance and Virulence Factors.

Contamination of food animal products by Escherichia coli is a leading cause of foodborne disease outbreaks, hospitalizations, and deaths in humans. Chicken is the most consumed meat both in the United States and across the globe according to the U.S. Department of Agriculture. Although E. coli is a ubiquitous commensal bacterium of the guts of humans and animals, its ability to acquire antimicrobial resistance (AMR) genes and virulence factors (VFs) can lead to the emergence of pathogenic strains that are resistant to critically important antibiotics. Thus, it is important to identify the genetic factors that contribute to the virulence and AMR of E. coli. In this study, we performed in-depth genomic evaluation of AMR genes and VFs of E. coli genomes available through the National Antimicrobial Resistance Monitoring System GenomeTrackr database. Our objective was to determine the genetic relatedness of chicken production isolates and human clinical isolates. To achieve this aim, we first developed a massively parallel analytical pipeline (Reads2Resistome) to accurately characterize the resistome of each E. coli genome, including the AMR genes and VFs harbored. We used random forests and hierarchical clustering to show that AMR genes and VFs are sufficient to classify isolates into different pathogenic phylogroups and host origin. We found that the presence of key type III secretion system and AMR genes differentiated human clinical isolates from chicken production isolates. These results further improve our understanding of the interconnected role AMR genes and VFs play in shaping the evolution of pathogenic E. coli strains. IMPORTANCE Pathogenic Escherichia coli causes disease in both humans and food-producing animals. E. coli pathogenesis is dependent on a repertoire of virulence factors and antimicrobial resistance genes. Food-borne outbreaks are highly associated with the consumption of undercooked and contaminated food products. This association highlights the need to understand the genetic factors that make E. coli virulent and pathogenic in humans and poultry. This research shows that E. coli isolates originating from human clinical settings and chicken production harbor different antimicrobial resistance genes and virulence factors that can be used to classify them into phylogroups and host origins. In addition, to aid in the repeatability and reproducibility of the results presented in this study, we have made a public repository of the Reads2Resistome pipeline and have provided the accession numbers associated with the E. coli genomes analyzed.

Management and environmental factors influence the prevalence and abundance of food-borne pathogens and commensal bacteria in peanut hull-based broiler litter.

In this study, we conducted a longitudinal sampling of peanut hull-based litter from a farm under a "no antibiotics ever" program. Our objective was to determine broiler management practices and environmental factors that are associated with the occurrence of food-borne pathogens (Salmonella and Campylobacter) and the abundance of commensal bacteria (Escherichia coli, Enterococcus spp., and Staphylococcus spp.). Litter (n = 288) was collected from 4 broiler houses over three consecutive flocks, starting with a complete house cleanout and fresh peanut hull. Litter was sampled at the beginning of each grow-out cycle and at the end of the cycle. Logistic and linear regression models were used to model the relationships between pathogen prevalence, commensal abundance and management practices, and environmental factors. The number of flocks raised on litter, grow-out period, broiler house, litter pH, litter moisture, and house temperature were associated with the prevalence of pathogens and the abundance of commensal bacteria in litter. The final logistic model for pathogens showed that a higher probability of detecting Salmonella in litter was associated with the number of flocks raised on litter and the grow-out period. A higher probability of detecting Campylobacter in litter was associated with the number of flocks raised on litter, broiler house and the sections of the house, and the pH of litter. Our results suggest that management practices and environmental factors affect Salmonella and Campylobacter differently and suggest that each pathogen will require its own tailored intervention to stop their persistence in broiler litter.

Monitoring Behaviors of Broiler Chickens at Different Ages with Deep Learning.

Animal behavior monitoring allows the gathering of animal health information and living habits and is an important technical means in precision animal farming. To quickly and accurately identify the behavior of broilers at different days, we adopted different deep learning behavior recognition models. Firstly, the top-view images of broilers at 2, 9, 16 and 23 days were obtained. In each stage, 300 images of each of the four broilers behaviors (i.e., feeding, drinking, standing, and resting) were segmented, totaling 4800 images. After image augmentation processing, 10,200 images were generated for each day including 8000 training sets, 2000 validation sets, and 200 testing sets. Finally, the performance of different convolutional neural network models (CNN) in broiler behavior recognition at different days was analyzed. The results show that the overall performance of the DenseNet-264 network was the best, with the accuracy rates of 88.5%, 97%, 94.5%, and 90% when birds were 2, 9, 16 and 23 days old, respectively. In addition, the efficient channel attention was introduced into the DenseNet-264 network (ECA-DenseNet-264), and the results (accuracy rates: 85%, 95%, 92%, 89.5%) confirmed that the DenseNet-264 network was still the best overall. The research results demonstrate that it is feasible to apply deep learning technology to monitor the behavior of broilers at different days.

Transferability of ESBL-encoding IncN and IncI1 plasmids among field strains of different Salmonella serovars and Escherichia coli.

OBJECTIVES: This study aimed to sequence, assemble, and annotate three plasmids (two IncN and one IncI1) carrying the blaCTX-M-1 gene and assess their transferability rates between homologous and heterologous serovars and/or species of bacteria. METHODS: First, the plasmids were sequenced, assembled, and annotated. They were then transferred from three donor strains (Escherichia coli/IncN, S. Heidelberg/IncN, and S. Heidelberg/IncI1) into nine recipient strains (S. Enteritidis, S. Heidelberg, S. Saintpaul, S. Cero, S. Infantis, S. Braenderup, E. coli 50, and E. coli 2010). The blaCTX-M-1 gene polymerase chain reaction (PCR), plasmid isolation, and antimicrobial susceptibility testing were used on the transconjugants to confirm the successful transfer of extended-spectrum beta lactamase (EBSL) plasmids into the recipient strains. RESULTS: Both IncN plasmids were 42,407 bp in size and showed >99.4% similarity to the S. Bredeney pET1.2-IncN (GenBank accession CP043224.1), whereas the IncI1 plasmid was 107,635 bp in size and demonstrated >99.9% similarity to the E. coli pCOV33 plasmid (GenBank accession MG649046.1). Successful plasmid transfer was observed between donor ​E. coli (IncN) and all recipient strains except for E. coli 50 and between donor S. Heidelberg (IncN) and all recipient strains. Successful plasmid transfer was also observed between S. Heidelberg (IncI1) and E. coli 50. CONCLUSION: Transfer of the blaCTX-M-1 encoding IncN and IncI1 plasmids via conjugation is possible and yet occurs at different frequencies depending on the donor strain of bacteria, with S. Heidelberg (IncN) having the highest donor-dependent transfer frequency, followed by E. coli 9079 (IncN) and S. Heidelberg (IncI1).

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.

AT Homopolymer Strings in Salmonella enterica Subspecies I Contribute to Speciation and Serovar Diversity.

Adenine and thymine homopolymer strings of at least 8 nucleotides (AT 8+mers) were characterized in Salmonella enterica subspecies I. The motif differed between other taxonomic classes but not between Salmonella enterica serovars. The motif in plasmids was possibly associated with serovar. Approximately 12.3% of the S. enterica motif loci had mutations. Mutability of AT 8+mers suggests that genomes undergo frequent repair to maintain optimal gene content, and that the motif facilitates self-recognition; in addition, serovar diversity is associated with plasmid content. A theory that genome regeneration accounts for both persistence of predominant Salmonella serovars and serovar diversity provides a new framework for investigating root causes of foodborne illness.

Escaping the fate of Sisyphus: assessing resistome hybridization baits for antimicrobial resistance gene capture.

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

Unveiling the Gut Microbiota and Resistome of Wild Cotton Mice, Peromyscus gossypinus, from Heavy Metal- and Radionuclide-Contaminated Sites in the Southeastern United States.

The prevalence of antibiotic resistance genes (ARGs) can be driven by direct selection from antibiotic use and indirect selection from substances such as heavy metals (HMs). While significant progress has been made to characterize the influence of HMs on the enrichment and dissemination of ARGs in the environment, there is still much we do not know. To fill this knowledge gap, we present a comprehensive analysis of gut bacteria associated with wild cotton mice (Peromyscus gossypinus) trapped from several areas affected by legacies of HM and radionuclide contamination. We explore how these contaminants affect gut microbial community (GMC) composition and diversity and the enrichment of antibiotic, biocide, and metal resistance genes. Although we were able to identify that a myriad of co-occurring antimicrobial and HM resistance genes appear in mice from all areas, including those without a history of contamination, the proportions of co-occurring ARGs and metal resistance genes (MRGs) are higher in sites with radionuclide contamination. These results support those from several previous studies and enhance our understanding of the coselection process, while providing new insights into the ubiquity of antimicrobial resistance in the resistome of wild animals. IMPORTANCE Antimicrobial resistance is a serious global public health concern because of its prevalence and ubiquitous distribution. The rapid dissemination of antibiotic resistance genes is thought to be the result of the massive overuse of antibiotics in agriculture and therapeutics. However, previous studies have demonstrated that the spread of antibiotic resistance genes can also be influenced by heavy metal contamination. This coselection phenomenon, whereby different resistance determinants are genetically linked on the same genetic element (coresistance) or a single genetic element provides resistance to multiple antimicrobial agents (cross-resistance), has profound clinical and environmental implications. In contrast to antibiotics, heavy metals can persist in the environment as a selection pressure for long periods of time. Thus, it is important to understand how antibiotic resistance genes are distributed in the environment and to what extent heavy metal contaminants may be driving their selection, which we have done in one environmental setting.

Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar Heidelberg.

The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicrobial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S. Heidelberg strain using various routes of inoculation and determined if S. Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S. Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S. Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoir of an IncI1 plasmid acquired by S. Heidelberg. The carriage of this IncI1 plasmid posed no fitness cost to S. Heidelberg but increased its fitness when exposed to acidic pH in vitro. These results suggest that HGT of plasmids carrying AR shaped the evolution of S. Heidelberg and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella enterica. IMPORTANCE The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, which in turn will allow the currently available antibiotic arsenal to be more effective. This change in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic-susceptible Salmonella enterica serovar Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggest that simply removing antibiotics from food animal production might not be sufficient to limit the spread of antimicrobial resistance.

Diversity of Plasmids and Genes Encoding Resistance to Extended-Spectrum ß-Lactamase in Escherichia coli from Different Animal Sources.

Antimicrobial resistance associated with the spread of plasmid-encoded extended-spectrum ß-lactamase (ESBL) genes conferring resistance to third generation cephalosporins is increasing worldwide. However, data on the population of ESBL producing E. coli in different animal sources and their antimicrobial characteristics are limited. The purpose of this study was to investigate potential reservoirs of ESBL-encoded genes in E. coli isolated from swine, beef, dairy, and poultry collected from different regions of the United States using whole-genome sequencing (WGS). Three hundred isolates were typed into different phylogroups, characterized by BOX AIR-1 PCR and tested for resistance to antimicrobials. Of the 300 isolates, 59.7% were resistant to sulfisoxazole, 49.3% to tetracycline, 32.3% to cephalothin, 22.3% to ampicillin, 20% to streptomycin, 16% to ticarcillin; resistance to the remaining 12 antimicrobials was less than 10%. Phylogroups A and B1 were most prevalent with A (n = 92, 30%) and B1 (87 = 29%). A total of nine E. coli isolates were confirmed as ESBL producers by double-disk synergy testing and multidrug resistant (MDR) to at least three antimicrobial drug classes. Using WGS, significantly higher numbers of ESBL-E. coli were detected in swine and dairy manure than from any other animal sources, suggesting that these may be the primary animal sources for ESBL producing E. coli. These isolates carry plasmids, such as IncFIA(B), IncFII, IncX1, IncX4, IncQ1, CollRNAI, Col440I, and acquired ARGs aph(6)-Id, aph(3″)-Ib, aadA5, aph(3')-Ia, blaCTX-M-15, blaTEM-1B, mphA, ermB, catA1, sul1, sul2, tetB, dfrA17. One of the E. coli isolates from swine with ST 410 was resistant to nine antibiotics and carried more than 28 virulence factors, and this ST has been shown to belong to an international high-risk clone. Our data suggests that ESBL producing E. coli are widely distributed in different animal sources, but swine and dairy cattle may be their main reservoir.

Succession patterns of the bacterial community in poultry litter after bird removal and sodium bisulfate application.

Sulfate-based acid amendments are used for treating litter between broiler chicken flocks and during grow-out for in-house ammonia abatement. These amendments reduce litter pH and inhibit ammonia volatilization by converting ammonia to nonvolatile ammonium. Research on the effects of acid amendments on litter microbiota is limited and usually done in microcosms, which do not replicate natural environments. In this study, we determined the changes in bacterial populations present in litter during downtime (the period after a flock was removed and before new broiler chicks were placed) and 24 h before and after the application of a sodium bisulfate (NaHSO4 )-based amendment. We used DNA sequencing technologies to characterize the litter microbiota, elucidating microbial shifts in litter samples with respect to downtime, litter depth, and NaHSO4 application. During downtime (∼18 d), the litter microbiota was dominated by Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Sodium bisulfate affected the microbiota in the top layer (3 cm) of reused litter topdressed with fresh pine shavings and resulted in an increase in Escherichia spp. and Faecalibacterium spp. and a decrease in members of the phylum Acidobacteria. Furthermore, culturable Escherichia coli decreased by 1.5 log units during downtime, but an increase was observed for topdressed litter after NaHSO4 was applied. Although the effect of acidifiers on ammonia reduction, bird performance, and litter performance are well documented, their effect on litter bacteria is not well understood. Our results suggest that acidifiers may perturb litter bacteria when topdressed with fresh pine shavings and that further research is required.

A Machine Vision-Based Method Optimized for Restoring Broiler Chicken Images Occluded by Feeding and Drinking Equipment.

The presence equipment (e.g., water pipes, feed buckets, and other presence equipment, etc.) in the poultry house can occlude the areas of broiler chickens taken via top view. This can affect the analysis of chicken behaviors through a vision-based machine learning imaging method. In our previous study, we developed a machine vision-based method for monitoring the broiler chicken floor distribution, and here we processed and restored the areas of broiler chickens which were occluded by presence equipment. To verify the performance of the developed restoration method, a top-view video of broiler chickens was recorded in two research broiler houses (240 birds equally raised in 12 pens per house). First, a target detection algorithm was used to initially detect the target areas in each image, and then Hough transform and color features were used to remove the occlusion equipment in the detection result further. In poultry images, the broiler chicken occluded by equipment has either two areas (TA) or one area (OA). To reconstruct the occluded area of broiler chickens, the linear restoration method and the elliptical fitting restoration method were developed and tested. Three evaluation indices of the overlap rate (OR), false-positive rate (FPR), and false-negative rate (FNR) were used to evaluate the restoration method. From images collected on d2, d9, d16, and d23, about 100-sample images were selected for testing the proposed method. And then, around 80 high-quality broiler areas detected were further evaluated for occlusion restoration. According to the results, the average value of OR, FPR, and FNR for TA was 0.8150, 0.0032, and 0.1850, respectively. For OA, the average values of OR, FPR, and FNR were 0.8788, 0.2227, and 0.1212, respectively. The study provides a new method for restoring occluded chicken areas that can hamper the success of vision-based machine predictions.