Whole genome sequencing and antimicrobial resistance gene detection in salmonella from poultry farms

Leading microbiologists have long warned that antimicrobial resistance (AMR) in food-producing animals would eventually circle back to threaten human health. This research applied whole genome sequencing (WGS) to characterize AMR gene profiles in Salmonella isolates recovered from poultry farms in Greater Manchester, United Kingdom. Between September 2022 and December 2023, 187 non-typhoidal Salmonella isolates were obtained from broiler litter, carcass rinses, and farm environmental swabs at 14 commercial poultry operations. Isolates were sequenced on an Illumina MiSeq platform, and AMR genes were identified using the ResFinder and CARD databases. Five serotypes were detected: Enteritidis (34.2%), Typhimurium (27.8%), Infantis (18.7%), Kentucky (12.3%), and Heidelberg (7.0%). AMR genes were found in 142 of 187 isolates (75.9%), with tetracycline resistance (tetA, 49.1%) and beta-lactam resistance (blaTEM, 42.6%) the most prevalent. Multidrug resistance (resistance to three or more classes) was detected in 38.5% of isolates. These findings highlight the high burden of AMR genes in poultry-associated Salmonella in the UK and support continued WGS-based surveillance.

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tetA (%) and blaTEM (%) and more: Enteritidis vs Typhimurium vs Infantis vs Kentucky vs HeidelbergClose ×

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qnrS (%): Enteritidis vs Typhimurium vs Infantis vs Kentucky vs HeidelbergClose ×

Replotted from Table 1 (page 2) of the original paper. Every bar is the paper's own table value.

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Introduction

In the view of public health officials, the spread of antimicrobial-resistant bacteria through the food chain represents one of the most pressing challenges of modern medicine. Salmonella enterica is among the most common foodborne pathogens globally, with poultry serving as a major reservoir for human infections [1] . The use of antimicrobials in poultry production-whether for treatment, prevention, or growth promotion-creates selective pressure that drives resistance evolution [2] . Traditional phenotypic susceptibility testing identifies resistant isolates but cannot pinpoint the genetic basis of resistance or track transmission pathways. Whole genome sequencing offers resolution at the single-nucleotide level, enabling detection of all known AMR genes, virulence factors, and phylogenetic relationships in a single assay [3] . WGS-based surveillance has been adopted by public health agencies in the US and Denmark, but uptake in UK agricultural settings remains limited [4] . This research aimed to characterize the AMR gene content of Salmonella isolates from poultry farms in Greater Manchester using WGS and to assess the prevalence of multidrug resistance across serotypes.

Materials and Methods

Materials and Methods

Materials

Sampling was conducted at 14 commercial broiler farms across Greater Manchester, UK, between September 2022 and December 2023. Laboratory work was performed at the Department of Medical Microbiology, Pennine Institute of Biological Sciences, Manchester. Institutional biosafety approval was obtained (PIBS-IBC/2022-031). A total of 412 samples were collected: broiler litter (n = 168), carcass rinses at processing (n = 134), and farm environmental swabs (n = 110). Salmonella isolation followed ISO 6579-1:2017 using buffered peptone water pre-enrichment, Rappaport-Vassiliadis broth enrichment, and XLD agar plating [5] . Confirmed Salmonella isolates (n = 187) were serotyped by the White-Kauffmann-Le Minor scheme.

Methods

Genomic DNA was extracted using the Wizard DNA Purification (Promega). Libraries were prepared with the Nextera XT kit and sequenced on an Illumina MiSeq (2 × 250 bp paired-end reads), targeting at least 30× coverage. Raw reads were quality-trimmed with Trimmomatic v0.39 [6] and assembled using SPAdes v3.15 [7] . AMR genes were identified with ResFinder 4.1 (90% identity, 60% coverage thresholds) and cross-checked against the Comprehensive Antibiotic Resistance Database (CARD) [8] . Serotype was confirmed in silico using SISTR [9] . Core genome multilocus sequence typing (cgMLST) used the EnteroBase scheme to assess phylogenetic relationships. Statistical analysis used chi-square tests for categorical comparisons. S. infantis showed the highest rates of tetracycline resistance (62.9%) and multidrug resistance (51.4%). S. typhimurium had similarly high MDR rates (48.1%). The mcr-1 colistin resistance gene was detected in 3 isolates (1.6%), all from S. typhimurium.

Results

Results

Discussion

The 75.9% AMR gene carriage rate among poultryassociated Salmonella is higher than the 55-65% reported in UK national surveillance data from 2020 [4] suggesting that prevalence may be underestimated by passive surveillance systems that rely on clinical isolates. The dominance of tetracycline resistance genes is unsurprising given that tetracyclines were historically among the most heavily used antimicrobials in UK poultry production [2] . The detection of mcr-1 in three isolates, though at low prevalence, is concerning because this plasmid-mediated colistin resistance gene can transfer horizontally between species and undermine a last-resort antibiotic [10] . S. infantis emerged as the serotype with the highest MDR burden, consistent with its rising prominence in European poultry surveillance [1] . The cgMLST analysis (data not shown in full) revealed that some closely related isolates were distributed across multiple farms, hinting at potential between-farm transmission through shared supply chains or personnel [3] .

Conclusion

This WGS-based survey revealed widespread AMR gene carriage in Salmonella from poultry farms in Greater Manchester, with 75.9% of isolates harboring at least one resistance gene and 38.5% classified as multidrug-resistant. Tetracycline and beta-lactam resistance genes were most prevalent. The detection of mcr-1 colistin resistance, though rare, warrants ongoing monitoring. These data support the integration of WGS into routine AMR surveillance for poultry-associated Salmonella in the UK.

Acknowledgement

Acknowledgements Funding SourcesDepartmental research funds from the Pennine Institute of Biological Sciences supported this work. Institutional SupportThe author thanks the Pennine Institute of Biological Sciences for sequencing infrastructure and laboratory access. Contributions Not Qualifying for AuthorshipFarm managers who permitted sampling and laboratory technicians who performed DNA extractions are acknowledged.

Figures and tables from the paper

Serotype	n	tetA (%)	blaTEM (%)	sul1 (%)	qnrS (%)	MDR (%)
Enteritidis	64	42.2	37.5	28.1	12.5	31.3
Typhimurium	52	57.7	51.9	38.5	19.2	48.1
Infantis	35	62.9	48.6	42.9	22.9	51.4
Kentucky	23	39.1	34.8	21.7	30.4	26.1
Heidelberg	13	30.8	38.5	15.4	7.7	23.1

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AMR Gene Prevalence by Salmonella Serotype

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Figure 1Close ×

Trends in antimicrobial resistance prevalence among Salmonella isolates from poultry farms, 2019-2023. Tetracycline and ampicillin

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Presence (+) or absence (-) of AMR genes across Salmonella serotypes. S. infantis and S. typhimurium carried the broadest resistance

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