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).

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.

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.

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.

Reused poultry litter microbiome with competitive exclusion potential against Salmonella Heidelberg.

The success of poultry litter reuse in U.S. poultry production can be attributed to the efficient treatment methods used by producers during downtimes (the time lapse between consecutive flocks, during which the broiler house is empty). During this period, reused litter may be decaked, tilled/windrowed, or treated with acid-based amendments to reduce ammonia and bacteria levels. Competitive exclusion, pH, and temperature are proposed factors that influence the level of pathogens and the overall litter microbiome during downtimes. We previously reported on the bacterial genetic factors associated with the fitness of two strains of Salmonella enterica serovar Heidelberg (SH) incubated for 14 d in reused litter. Here, we investigated the physicochemical parameters and the microbiome of the litter correlating with SH abundance during this period. We used 16S ribosomal RNA gene sequencing to determine the litter microbiome and whole genome sequencing to characterize bacteria with competitive exclusion potential against SH. The ß diversity of the litter microbiome was significantly affected by the duration of incubation, microcosm, and microcosm plus Heidelberg strain combinations. In addition, ß diversity was significantly affected by litter parameters, including NH4 , pH, moisture, water activity, and aluminum. The major phyla observed in the reused litter throughout the 14-d incubation experiment were Firmicutes and Actinobacteria, although their abundance differed by microcosm and time. Amplicon-specific variants homologous to the members of the genera Nocardiopsis and Lentibacillus and the family Bacillaceae_2 were found to significantly correlate with the abundance of Salmonella. A consortium of Bacillus subtilis strains isolated from the litter microcosms reduced the growth of SH in vitro.

A Machine Vision-Based Method for Monitoring Broiler Chicken Floor Distribution.

The proper spatial distribution of chickens is an indication of a healthy flock. Routine inspections of broiler chicken floor distribution are done manually in commercial grow-out houses every day, which is labor intensive and time consuming. This task requires an efficient and automatic system that can monitor the chicken's floor distributions. In the current study, a machine vision-based method was developed and tested in an experimental broiler house. For the new method to recognize bird distribution in the images, the pen floor was virtually defined/divided into drinking, feeding, and rest/exercise zones. As broiler chickens grew, the images collected each day were analyzed separately to avoid biases caused by changes of body weight/size over time. About 7000 chicken areas/profiles were extracted from images collected from 18 to 35 days of age to build a BP neural network model for floor distribution analysis, and another 200 images were used to validate the model. The results showed that the identification accuracies of bird distribution in the drinking and feeding zones were 0.9419 and 0.9544, respectively. The correlation coefficient (R), mean square error (MSE), and mean absolute error (MAE) of the BP model were 0.996, 0.038, and 0.178, respectively, in our analysis of broiler distribution. Missed detections were mainly caused by interference with the equipment (e.g., the feeder hanging chain and water line); studies are ongoing to address these issues. This study provides the basis for devising a real-time evaluation tool to detect broiler chicken floor distribution and behavior in commercial facilities.

Horizontal Gene Transfer and Acquired Antibiotic Resistance in Salmonella enterica Serovar Heidelberg following In Vitro Incubation in Broiler Ceca.

The chicken gastrointestinal tract harbors microorganisms that play a role in the health and disease status of the host. The cecum is the part of the gut that carries the highest microbial densities, has the longest residence time of digesta, and is a vital site for urea recycling and water regulation. Therefore, the cecum provides a rich environment for bacteria to horizontally transfer genes between one another via mobile genetic elements such as plasmids and bacteriophages. In this study, we used broiler chicken cecum as a model to investigate antibiotic resistance genes that can be transferred in vitro from cecal flora to Salmonella enterica serovar Heidelberg. We used whole-genome sequencing and resistome enrichment to decipher the interactions between S Heidelberg, the gut microbiome, and acquired antibiotic resistance. After 48 h of incubation of ceca under microaerophilic conditions, we recovered one S Heidelberg isolate with an acquired IncK2 plasmid (88 kb) carrying an extended-spectrum-ß-lactamase gene (blaCMY-2). In vitro, this plasmid was transferable between Escherichia coli and S Heidelberg strains but transfer was unsuccessful between S Heidelberg strains. An in-depth genetic characterization of transferred plasmids suggests that they share significant homology with P1-like phages. This study contributes to our understanding of horizontal gene transfer between an important foodborne pathogen and the chicken gut microbiome.IMPORTANCES. Heidelberg is a clinically important serovar, linked to foodborne illness and among the top 5 serovars isolated from poultry in the United States and Canada. Acquisition of new genetic material from the microbial flora in the gastrointestinal tract of food animals, including broilers, may contribute to increased fitness of pathogens like S. Heidelberg and may increase their level of antibiotic tolerance. Therefore, it is critical to gain a better understanding of the interactions that occur between important pathogens and the commensals present in the animal gut and other agroecosystems. In this report, we show that the native flora in broiler ceca were capable of transferring mobile genetic elements carrying the AmpC ß-lactamase (blaCMY-2) gene to an important foodborne pathogen, S Heidelberg. The potential role for bacteriophage transduction is also discussed.