Chicken cecal microbial functional capacity and resistome differ by age and barn 1 disinfection practice

Yi Fan, Tingting Ju, Tulika Bhardwaj, Douglas R. Korver, Benjamin P. Willing. bioRxiv 2024.05.23.595585; doi: https://doi.org/10.1101/2024.05.23.595585

About

Livestock farming accounts for over 50% of antibiotic usage globally. Compared with other livestock species, chickens were reported to have the highest density of antibiotic resistance genes (ARGs) due to the high stocking density and short production cycle. Currently, biocidal agents such as benzalkonium chloride (BAC), hydrogen peroxide, glutaraldehyde, ethanol, and sodium hypochlorite have been widely applied in agriculture for disinfection purposes. While chemical disinfectants can inhibit antibiotic-resistant bacteria and destruct ARGs through oxidation, they may also induce bacterial adaptation, potentially through promoting antibiotic resistance through co-selection. With the controversial effect of chemical disinfectants on ARGs, there is limited information available regarding the effects of chemical disinfectant-treated rearing environments on the resistome in the gut of animals. In this sense, barn cleaning practices, which involve chemical disinfectants, may impact the presence and persistence of ARGs throughout production cycles. Consequently, from the perspective of food safety and environmental sustainability, it is important to explore the influence of chemical disinfectants usage in barn cleaning. In broiler chicken production, barn sanitation has been used to prevent disease transmission between flocks. Both full sanitation with chemical disinfectants (FD) and water-wash (WW) method are widely employed in the Canadian poultry production system with FD being required on a yearly basis. In this study, we sought to gain greater insight into the effects of barn sanitation and age on the functionality of the gut microbiome, specifically in terms of microbial metabolic capacity and the profile of antibiotic resistance genes.

Approach

The animal study was conducted on commercial broiler chicken farms in Alberta, Canada. During each production cycle, samples were collected from barns that had undergone two consecutive rounds of repeated cleaning treatments, including both full disinfection (FD) and water-wash (WW). For FD treatment, manure and litter were completely removed from the barn after chickens were depopulated. Subsequently, chemical disinfection was performed using foam containing 7% sodium hydroxide, 7% 2-(2-2-butoxyethoxy) ethanol, 6% sodium laureth sulfate, 5% sodium N-lauroyl sarcosinate, and 5% tetrasodium ethylenediaminetetraacetic acid on all surfaces within the facilities, followed by high- and low-pressure water rinse with water temperature set at 35℃. After the facilities were air-dried, foam containing 10% glutaraldehyde, 10% benzalkonium chloride, and 5% formic acid was applied to surfaces of the facilities for 60 mins followed by high-pressure water rinse, overnight air-dry, and fresh litter placement. For WW treatment, manure and used litter were removed, followed by low-pressure water rinse with the water temperature set at 35℃ for all facility surfaces, air-dry overnight, and fresh litter placement. A total of 14 production flocks were studies with 7 flocks to each treatment (FD or WW). The study included a total of 140 chickens with 35 chickens sampled from each treatment at each age (day 7 or day 30). In each production flock, Ross 308 broilers were used. The flock size was approximately 14,000 birds, with a final stocking density of 30 kg/m2. All chickens were fed an antibiotic free diet ad libitum and sent for processing at 32-35 days of age when the average target live weight of 1.8 kg was reached. At D7 and D30 of age, five broilers per flock were randomly selected from different areas within each barn and euthanized using cervical dislocation. Approximately 300 mg of cecal contents were collected using sterile technique, placed on dry ice until being transported to the lab, and stored at -80 ℃ for shotgun metagenomic sequencing. Antibiotic resistance-encoding genes were annotated against the Comprehensive Antibiotic Resistance Database via Resistance Gene Identifier.

Analysis of Results

In this study, shotgun metagenomic sequencing was used to provide a comprehensive profile of the cecal microbial community to further assess the effects of barn cleaning methods. At the phylum level, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria made up the majority cecal microbial communities both at day 7 and 30. However, there were shifts in the relative abundance of these phyla from D7-to D30-microbiota (D7 vs. D30: Firmicutes, 62.14% vs. 24.59%; Bacteroidetes, 25.57% vs. 53.27%; Proteobacteria, 8.21% vs. 15.59%; Actinobacteria, 0.76% vs. 5.92%, other phyla, 3.32% vs. 0.63%). PERMONOVA analyses based on Bray-Curtis distance dissimilarity matrix revealed that barn cleaning methods had limited impact on the D7 microbial community structure and a modest impact on D30 microbiota (R2 = 0.02, adonis P < 0.01). In addition, the Shannon index indicated that barn cleaning methods had minimal impact on alpha diversity at D7 (P = 0.96) and D30 (P = 0.25); however, differences were observed between D7 and D30 microbial communities as reflected by increased Shannon diversity with age (P < 0.05). At day 30, Ruminococcus torques, Barnesiella viscericola, Helicobacter pullorum, Faecalibacterium prausnitzii were more abundant in the ceca of the chickens from the WW treatment group, whereas Megamonas funiformis was more abundant in the chicken cecal microbiota of FD group. Compared with the modest impact on cecal microbial functional capacities by barn cleaning methods, age was shown as a strong factor affecting cecal microbial functionalities. The chicken cecal resistome was affected by the barn sanitation practices and sampling timepoints. At D30, the impacts of cleaning methods on the chicken gut microbiome were subtle, indicating that the barn cleaning practices had greater impacts in younger chickens. Some persistent ARG gene families (e.g. erm gene family) were enriched in the WW-derived chicken cecal microbiome at day 7.

Application

This is the first study to report the impact of disinfectants in broiler production on microbial functional capacity and resistome. We showed that the barn chemical disinfection may alter the chicken gut microbiota’s composition, leading to decreased microbial functional capacity for amino acid and SCFA metabolism. While our initial expectation was that disinfection might enhance the selection of antibiotic resistance genes (ARG), our results indicate that disinfection is linked to a reduced abundance of ARG. This finding suggests that the impact of disinfection on ARG transmission between flocks merits further evaluation as a potential strategy for controlling ARG spread.

Abstract

Chemical disinfectants and water-wash methods are widely employed in sanitizing broiler chicken barns. Previous studies showed that chemical disinfectants affect environmental microbial composition and antibiotic resistance genes (ARGs). However, little is known regarding how barn disinfection treatments impact the chicken gut resistome and microbial functionality. The current study compared the effects of chemical disinfection and water-wash method on the gut microbiome and resistome of commercial broilers using a crossover experimental design after 2 production cycles at 7 barns. Shotgun metagenomic sequencing performed on cecal contents collected at day 7 and 30 also allowed evaluation of age-associated characteristics of microbiome. Age of the chickens had the largest effects on the resistome, with younger birds having increased relative abundance of total ARGs (P<0.05) and differences in resistance mechanism, however, functional and resistome differences were also identified by barn sanitation practice. At day 7, chickens in chemically-disinfected barns had decreased functional capacity related to amino acid synthesis compared to the water-wash group. Additionally, genes related to stringent response were enriched in chickens raised under chemically-disinfected condition (FDR-P<0.05), suggesting selection for stress resistance. Consistently, lower abundance of genetic pathways encoding amino acid biosynthesis associated with cecal Helicobacter pullorum was observed in the disinfection group at day 30 compared to the water-wash group, with the same pattern in short-chain fatty acid biosynthesis (FDR-P<0.05). Overall, while the use of disinfectants in barn sanitation slightly affected the relative abundance of some ARGs in the gut, age had a dominant effect on the microbial functionality and resistome.

KEYWORDS: disinfectants, water-wash, sanitizing, broiler, barns