Antibiotic Resistance Genes of Class 1 Integrons in Chicken Microbiomes Modulated by Prebiotics

Class 1 integrons are genetic elements of bacteria that contribute to the abundance of antibiotic resistance. Poultry products are thought to be one of the most important reservoirs for transmission of antimicrobial resistance bacteria (AMR) via foodborne zoonotic pathogens reducing the effectiveness of antimicrobial treatments. Prebiotics have attracted attention as an aid to reduce pathogen loads and support the intestinal health of poultry. However, whether prebiotics directly reduce the expansion of bacterial populations carrying antibiotic resistance genes (ARG) or reduce their transmission from livestock, is not known. This thesis investigates the potential role of prebiotic galactooligosaccharides (GOS) in the mitigation of ARG prevalence in the microbiomes of broiler chickens reared under biosecure or commercial conditions. This study identified and estimated the prevalence of three conserved genes present in class 1 integrons (intI1, sul1 and qacEΔ1) from broiler chicken caecal contents reared in biosecure and commercial condition. The prevalence of gene cassettes (GCs) featuring ARGs were examined from birds fed either standard control or an isocaloric diet supplemented with GOS. Six GC types were identified by sequencing long-range PCR products: GCs-A (aadA2, linF), GCs-B-1 (dfrA1, ORF1, aadA24), GCs-B-2 (dfrA1, aadA1), GCs-C-1 (aadA1), GCs-C-2 (aadA1), and GCs-F (aadA9). The predominant GC in biosecure birds was GC-B2, whilst GC-A was more prevalent in commercial birds. Phenotypic antimicrobial resistance of the caecal microbiota was estimated by evaluation of trimethoprim coliform resistant populations and parallel determination of integrase gene copy number for birds fed either control or GOS diets. Alpha and beta diversities of the caecal bacterial communities were also determined using a 16S rRNA sequencing approach. Differences in the caecal communities were calculated using AMOVA and differentially abundant Operational Taxonomic Units (OTUs) identified by LEfSe (Linear discriminant analysis effect size). Feeding prebiotic GOS to 22 days of age mitigated the expansion of antibiotics resistance populations observed in the control microbiome composition for broiler chickens reared in a biosecure environment. Colonization by zoonotic Salmonella Enteritidis demonstrated that both Salmonella and GOS feed influence the structure of the gut microbiome. GOS treatment altered the proportions of specific OTUs in infected bird compared to non-infected. These important changes resulted in a faster clearance of Salmonella infection in GOS-fed birds compared to control fed birds, which was associated with a significant increase on Negativicutes at the expense of Clostridiales. It is hypothesized that this change restricts the abundance of Proteobacteria carrying antimicrobial resistance due to the depletion of oxygen. Thus, GOS feed modulates the broiler microbiome, which can have a positive impact on the safety of poultry products by reducing the incidence of foodborne pathogens, mitigating the antibiotic resistance load, and improving overall public and animal health

Antibiotic Resistance Genes of Class 1 Integrons in Chicken Microbiomes Modulated by Prebiotics

Class 1 integrons are genetic elements of bacteria that contribute to the abundance of antibiotic resistance. Poultry products are thought to be one of the most important reservoirs for transmission of antimicrobial resistance bacteria (AMR) via foodborne zoonotic pathogens reducing the effectiveness of antimicrobial treatments. Prebiotics have attracted attention as an aid to reduce pathogen loads and support the intestinal health of poultry. However, whether prebiotics directly reduce the expansion of bacterial populations carrying antibiotic resistance genes (ARG) or reduce their transmission from livestock, is not known. This thesis investigates the potential role of prebiotic galactooligosaccharides (GOS) in the mitigation of ARG prevalence in the microbiomes of broiler chickens reared under biosecure or commercial conditions. This study identified and estimated the prevalence of three conserved genes present in class 1 integrons (intI1, sul1 and qacEΔ1) from broiler chicken caecal contents reared in biosecure and commercial condition. The prevalence of gene cassettes (GCs) featuring ARGs were examined from birds fed either standard control or an isocaloric diet supplemented with GOS. Six GC types were identified by sequencing long-range PCR products: GCs-A (aadA2, linF), GCs-B-1 (dfrA1, ORF1, aadA24), GCs-B-2 (dfrA1, aadA1), GCs-C-1 (aadA1), GCs-C-2 (aadA1), and GCs-F (aadA9). The predominant GC in biosecure birds was GC-B2, whilst GC-A was more prevalent in commercial birds. Phenotypic antimicrobial resistance of the caecal microbiota was estimated by evaluation of trimethoprim coliform resistant populations and parallel determination of integrase gene copy number for birds fed either control or GOS diets. Alpha and beta diversities of the caecal bacterial communities were also determined using a 16S rRNA sequencing approach. Differences in the caecal communities were calculated using AMOVA and differentially abundant Operational Taxonomic Units (OTUs) identified by LEfSe (Linear discriminant analysis effect size). Feeding prebiotic GOS to 22 days of age mitigated the expansion of antibiotics resistance populations observed in the control microbiome composition for broiler chickens reared in a biosecure environment. Colonization by zoonotic Salmonella Enteritidis demonstrated that both Salmonella and GOS feed influence the structure of the gut microbiome. GOS treatment altered the proportions of specific OTUs in infected bird compared to non-infected. These important changes resulted in a faster clearance of Salmonella infection in GOS-fed birds compared to control fed birds, which was associated with a significant increase on Negativicutes at the expense of Clostridiales. It is hypothesized that this change restricts the abundance of Proteobacteria carrying antimicrobial resistance due to the depletion of oxygen. Thus, GOS feed modulates the broiler microbiome, which can have a positive impact on the safety of poultry products by reducing the incidence of foodborne pathogens, mitigating the antibiotic resistance load, and improving overall public and animal health

Prebiotic galactooligosaccharide feed modifies the chicken gut microbiota to efficiently clear Salmonella

Chicken meat is contaminated with Salmonella from the gut of infected chickens during slaughter. Eradication of Salmonella from broiler chickens through hygiene measures and/or vaccination is not cost-effective; complementary approaches are required. A mature gut microbiota obstructs Salmonella infection in chickens, and deliberate fortification of colonization resistance through prebiotic feed formulations would benefit public health and poultry production. Prebiotic galactooligosaccharides hastens Salmonella clearance from the gut of infected chickens. To better understand the role of galactooligosaccharides in colonization resistance, broiler chickens were raised on a wheat-soybean meal-based feed, with or without galactooligosaccharides for the first 24 days of life. Chickens were orally challenged with Salmonella enterica serovar Enteritidis at 20 days and the effect of supplementary galactooligosaccharides characterized by profiling Salmonella colonization, gut microbiota, innate immune response, and cecal short-chain fatty acid concentrations. Exposure to dietary galactooligosaccharides shortened the time to clear S. Enteritidis from the ceca. Differential abundance analysis of the cecal microbiota associated Salmonella challenge with a bacterial taxon belonging to the Acidaminococcaceae family (P < 0.005). Increased cecal concentrations of the short-chain fatty acids propionate and valerate were measured in Salmonella-challenged chickens sustained on either control or galactooligosaccharide-supplemented feed relative to mock-challenged controls; but far greater concentrations were detected in chickens fed a galactooligosaccharide-supplemented diet in early life. The abundance of the Acidaminococcaceae taxon exhibited a positive correlation with the cecal concentrations of propionate (ρ = 0.724, P = 0.008) and valerate (ρ = 0.71, P = 0.013). The absence of cecal pro-inflammatory transcriptional responses suggest that the rapid Salmonella clearance observed for the galactooligosaccharide-supplemented diet was not linked to innate immune function. IMPORTANCE: Work presented here identifies bacterial taxa responsible for colonization resistance to Salmonella in broiler chickens. Deliberate cultivation of these taxa with prebiotic galactooligosaccharide has potential as a straight-forward, safe, and cost-effective intervention against Salmonella. We hypothesize that catabolism of galactooligosaccharide and its breakdown products by indigenous microorganisms colonizing the chicken gut produce excess levels of propionate. In the absence of gross inflammation, propionate is inimical to Salmonella and hastens intestinal clearance