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 (SH), a model food pathogen, to test if chicks raised antibiotic-free harbor transferable AR. We challenged chicks with an antibiotic susceptible SH strain using various routes of inoculation and determined if SH isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole genome sequencing (WGS) to show that chicks grown without antibiotics harbored antimicrobial resistant SH population 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 SH infection perturbed the microbiota of broiler chicks and used metagenomics and WGS to confirm commensal Escherichia coli population as the main reservoir of IncI1 plasmid acquired by SH. The carriage of this IncI1 plasmid posed no fitness cost to SH but increased its fitness when exposed to acidic pH in vitro. These results suggest that HGT of plasmids carrying AR shaped the evolution of SH and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella.

#PBS -S /bin/bash #PBS -N #PBS -q batch #PBS -l nodes=2:ppn=4 #PBS -l walltime= #PBS -l mem=40gb #PBS -M #PBS -m ae cd $PBS_O_WORKDIR module load GATK/4.1.2.0-GCCcore-8.2.0-Java-1.8 module load SAMtools/1.10-GCC-8.2.0-2.31.1 module load BWA/0.7.17-foss-2016b bwa index SH-2813-ancestor.fasta samtools faidx SH-2813-ancestor.fasta bwa mem -k 24 -T 0 -B 100 -O 100 -E 100 -t 8 -R "@RG\tID:IVISII\tSM:SH-isolate-A\tPL:Illumina_Miseq\tLB:PE" SH-2813-ancestor.fasta SH-isolate-A_S11_L001_R1_001.fastq.gz SH-isolate-A-R2_001.fastq.gz > SH-isolate-A.sam samtools view -bS SH-isolate-A.sam > SH-isolate-A.bam samtools sort SH-isolate-A.bam -o SH-isolate-A_sorted.bam samtools rmdup SH-isolate-A_sorted.bam SH-isolate-A_rmdup.bam samtools index -b SH-isolate-A_rmdup.bam > SH-isolate-A gatk HaplotypeCaller -R CP016573.fasta -I SH-isolate-A_rmdup.bam -ploidy 1 -O SH-isolate-A.vcf Funding provided by: University of Georgia Research FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100012483Award Number: 58-6040-6-030Funding provided by: USDA-ARS*Crossref Funder Registry ID: Award Number: 6040‐32000‐010‐00‐DFunding provided by: USDA-ARSCrossref Funder Registry ID: Award Number: 6040‐32000‐010‐00‐D

Single nucleotide polymorphisms (SNPs) and indels present in sequenced isolates were determined by aligning raw sequence reads to the chromosome of S. Heidelberg ancestor strain (SH-2813-ancestor) using Burrows-Wheeler Aligner (BWA). SAM file sorting and removal of PCR duplicates were done using SAMtools. Genome Analysis ToolKit with a minimum mapping quality of 30 and a minimum base quality of 30 was used for SNP identification.