skip to main content


Title: Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar Heidelberg
ABSTRACT 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.  more » « less
Award ID(s):
1450032
NSF-PAR ID:
10347179
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more » ; ; ; ; ; ; ; « less
Editor(s):
Garrido, Daniel
Date Published:
Journal Name:
mSystems
Volume:
6
Issue:
4
ISSN:
2379-5077
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    Salmonella enterica is one of the most common bacterial foodborne pathogens in the United States, causing illnesses that range from self-limiting gastroenteritis to more severe, life threatening invasive disease. Many Salmonella strains contain plasmids that carry virulence, antimicrobial resistance, and/or transfer genes which allow them to adapt to diverse environments, and these can include incompatibility group (Inc) FIB plasmids. This study was undertaken to evaluate the genomic and phenotypic characteristics of IncFIB-positive Salmonella enterica serovar Typhimurium isolates from food animal sources, to identify their plasmid content, assess antimicrobial resistance and virulence properties, and compare their genotypic isolates with more recently isolated S. Typhimurium isolates from food animal sources. Methods: We identified 71 S. Typhimurium isolates that carried IncFIB plasmids. These isolates were subjected to whole genome sequencing and evaluated for bacteriocin production, antimicrobial susceptibility, the ability to transfer resistance plasmids, and a subset was evaluated for their ability to invade and persist in intestinal human epithelial cells. Results: Approximately 30% of isolates (n = 21) displayed bacteriocin inhibition of Escherichia coli strain J53. Bioinformatic analyses using PlasmidFinder software confirmed that all isolates contained IncFIB plasmids along with multiple other plasmid replicon types. Comparative analyses showed that all strains carried multiple antimicrobial resistance genes and virulence factors including iron acquisition genes, such as iucABCD (75%), iutA (94%), sitABCD (76%) and sitAB (100%). In 17 cases (71%), IncFIB plasmids, along with other plasmid replicon types, were able to conjugally transfer antimicrobial resistance and virulence genes to the susceptible recipient strain. For ten strains, persistence cell counts (27%) were noted to be significantly higher than invasion bacterial cell counts. When the genome sequences of the study isolates collected from 1998–2003 were compared to those published from subsequent years (2005–2018), overlapping genotypes were found, indicating the perseverance of IncFIB positive strains in food animal populations. This study confirms that IncFIB plasmids can play a potential role in disseminating antimicrobial resistance and virulence genes amongst bacteria from several food animal species. 
    more » « less
  2. Abstract

    Modern large‐scale agricultural practices that incorporate high density farming with subtherapeutic antibiotic dosing are considered a major contributor to the rise of antibiotic‐resistant bacterial infections of humans with species ofSalmonellabeing a leading agriculture‐based bacterial infection. Microcin J25, a potent and highly stable antimicrobial peptide active against Enterobacteriaceae, is a candidate antimicrobial against multipleSalmonellaspecies. Emerging evidence supports the hypothesis that the composition of the microbiota of the gastrointestinal tract prevents a variety of diseases by preventing infectious agents from proliferating. Reducing clearance of off‐target bacteria may decrease susceptibility to secondary infection. Of the Enterobacteriaceae susceptible to microcin J25,Escherichia coliare the most abundant within the human gut. To explore the modulation of specificity, a collection of 207 mutants encompassing 12 positions in both the ring and loop of microcin J25 was built and tested for activity againstSalmonellaandE. colistrains. As has been found previously, mutational tolerance of ring residues was lower than loop residues, with 22% and 51% of mutations, respectively, retaining activity toward at least one target within the target organism test panel. The multitarget screening elucidated increased mutational tolerance at position G2, G3, and G14 than previously identified in panels composed of single targets. Multiple mutations conferred differential response between the different targets. Examination of specificity differences between mutants found that 30% showed significant improvements to specificity toward any of the targets. Generation and testing of a combinatorial library designed from the point‐mutant study revealed that microcin J25I13Treduces off‐target activity toward commensal human‐derivedE. coliisolates by 81% relative toSalmonella entericaserovar Enteritidis. These in vitro specificity improvements are likely to improve in vivo treatment efficacy by reducing clearance of commensal bacteria in the gastrointestinal tract of hosts.

     
    more » « less
  3. Abstract

    The acquisition of antimicrobial resistance (AR) genes has rendered important pathogens nearly or fully unresponsive to antibiotics. It has been suggested that pathogens acquire AR traits from the gut microbiota, which collectively serve as a global reservoir for AR genes conferring resistance to all classes of antibiotics. However, only a subset of AR genes confers resistance to clinically relevant antibiotics, and, although these AR gene profiles are well-characterized for common pathogens, less is known about their taxonomic associations and transfer potential within diverse members of the gut microbiota. We examined a collection of 14,850 human metagenomes and 1666 environmental metagenomes from 33 countries, in addition to nearly 600,000 isolate genomes, to gain insight into the global prevalence and taxonomic range of clinically relevant AR genes. We find that several of the most concerning AR genes, such as those encoding the cephalosporinaseCTX-Mand carbapenemasesKPC,IMP,NDM, andVIM, remain taxonomically restricted to Proteobacteria. EvencfiA, the most common carbapenemase gene within the human gut microbiome, remains tightly restricted toBacteroides, despite being found on a mobilizable plasmid. We confirmed these findings in gut microbiome samples from India, Honduras, Pakistan, and Vietnam, using a high-sensitivity single-cell fusion PCR approach. Focusing on a set of genes encoding carbapenemases and cephalosporinases, thus far restricted toBacteroidesspecies, we find that few mutations are required for efficacy in a different phylum, raising the question of why these genes have not spread more widely. Overall, these data suggest that globally prevalent, clinically relevant AR genes have not yet established themselves across diverse commensal gut microbiota.

     
    more » « less
  4. Staphylococcus aureus are human facultative pathogenic bacteria and can be found as contaminants in the environment. The aim of our study was to determine whether methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolated from coastal beach and river waters, anchialine pools, sand, and wastewater on the island of Hawaiʻi, Hawaiʻi, are a potential health risk. Samples were collected from three regions on Hawaiʻi Island from July to December 2020 during the COVID-19 pandemic and were characterized using whole-genome sequencing (WGS). From WGS data, multilocus sequence typing (MLST), SCCmec type, antimicrobial resistance genes, virulence factors, and plasmids were identified. Of the 361 samples, 98.1% were positive for Staphylococcus spp. and 7.2% were S. aureus positive (n = 26); nine MRSA and 27 MSSA strains were characterized; multiple isolates were chosen from the same sample in two sand and seven coastal beach water samples. The nine MRSA isolates were multi-drug resistant (6–9 genes) sequence type (ST) 8, clonal complex (CC) 8, SCCmec type IVa (USA300 clone), and were clonally related (0–16 SNP differences), and carried 16–19 virulence factors. The 27 MSSA isolates were grouped into eight CCs and 12 STs. Seventy-eight percent of the MSSA isolates carried 1–5 different antibiotic resistance genes and carried 5–19 virulence factors. We found S. aureus in coastal beach and river waters, anchialine pools, and sand at locations with limited human activity on the island of Hawaiʻi. This may be a public health hazard. 
    more » « less
  5. Abstract

    Plasmids are autonomous genetic elements that can be exchanged between microorganisms via horizontal gene transfer (HGT). Despite the central role they play in antibiotic resistance and modern biotechnology, our understanding of plasmids’ natural ecology is limited. Recent experiments have shown that plasmids can spread even when they are a burden to the cell, suggesting that natural plasmids may exist as parasites. Here, we use mathematical modeling to explore the ecology of such parasitic plasmids. We first develop models of single plasmids and find that a plasmid’s population dynamics and optimal infection strategy are strongly determined by the plasmid’s HGT mechanism. We then analyze models of co-infecting plasmids and show that parasitic plasmids are prone to a “tragedy of the commons” in which runaway plasmid invasion severely reduces host fitness. We propose that this tragedy of the commons is averted by selection between competing populations and demonstrate this effect in a metapopulation model. We derive predicted distributions of unique plasmid types in genomes—comparison to the distribution of plasmids in a collection of 17,725 genomes supports a model of parasitic plasmids with positive plasmid–plasmid interactions that ameliorate plasmid fitness costs or promote the invasion of new plasmids.

     
    more » « less