Dissemination of Cephalosporin Resistance Genes between Escherichia coli Strains from Farm Animals and Humans by Specific Plasmid Lineages

Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.

[1]  Fernando de la Cruz,et al.  Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences , 2014, PLoS genetics.

[2]  F. Baquero,et al.  Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. , 2008, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[3]  Alessandra Carattoli,et al.  Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. , 2010, The Journal of antimicrobial chemotherapy.

[4]  H. Goossens,et al.  Antimicrobial Drug Use and Resistance in Europe , 2008, Emerging infectious diseases.

[5]  Julian Parkhill,et al.  Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak. , 2012, The New England journal of medicine.

[6]  Lars Bolund,et al.  State of the art de novo assembly of human genomes from massively parallel sequencing data , 2010, Human Genomics.

[7]  Fernando de la Cruz,et al.  Mobility of Plasmids , 2010, Microbiology and Molecular Biology Reviews.

[8]  C. Salgado,et al.  Attributable Hospital Cost and Length of Stay Associated with Health Care-Associated Infections Caused by Antibiotic-Resistant Gram-Negative Bacteria , 2009, Antimicrobial Agents and Chemotherapy.

[9]  C. Jernberg,et al.  Characterization of plasmid-mediated AmpC-producing E. coli from Swedish broilers and association with human clinical isolates. , 2013, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[10]  D. Paterson,et al.  Extended-spectrum and CMY-type beta-lactamase-producing Escherichia coli in clinical samples and retail meat from Pittsburgh, USA and Seville, Spain. , 2010, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[11]  F. Mégraud,et al.  CTX-M-producing Escherichia coli in a maternity ward: a likely community importation and evidence of mother-to-neonate transmission. , 2010, The Journal of antimicrobial chemotherapy.

[12]  M. Nei,et al.  MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. , 2011, Molecular biology and evolution.

[13]  Daniel Falush,et al.  Sex and virulence in Escherichia coli: an evolutionary perspective , 2006, Molecular microbiology.

[14]  B. Duim,et al.  Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis. , 2010, The Journal of antimicrobial chemotherapy.

[15]  Hajo Grundmann,et al.  Mortality and Hospital Stay Associated with Resistant Staphylococcus aureus and Escherichia coli Bacteremia: Estimating the Burden of Antibiotic Resistance in Europe , 2011, PLoS medicine.

[16]  Fredj Tekaia,et al.  Genome Trees from Conservation Profiles , 2005, PLoS Comput. Biol..

[17]  Michael Y. Galperin,et al.  The COG database: new developments in phylogenetic classification of proteins from complete genomes , 2001, Nucleic Acids Res..

[18]  Alexandros Stamatakis,et al.  RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models , 2006, Bioinform..

[19]  C. Schapendonk,et al.  Identical plasmid AmpC beta-lactamase genes and plasmid types in E. coli isolates from patients and poultry meat in the Netherlands. , 2013, International journal of food microbiology.

[20]  N. Woodford,et al.  Complete Nucleotide Sequences of Plasmids pEK204, pEK499, and pEK516, Encoding CTX-M Enzymes in Three Major Escherichia coli Lineages from the United Kingdom, All Belonging to the International O25:H4-ST131 Clone , 2009, Antimicrobial Agents and Chemotherapy.

[21]  A. Carattoli,et al.  Identification of plasmids by PCR-based replicon typing. , 2005, Journal of microbiological methods.

[22]  F. Baquero,et al.  Dramatic Increase in Prevalence of Fecal Carriage of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae during Nonoutbreak Situations in Spain , 2004, Journal of Clinical Microbiology.

[23]  P. Savelkoul,et al.  Extended-spectrum β-lactamase-producing Escherichia coli from retail chicken meat and humans: comparison of strains, plasmids, resistance genes, and virulence factors. , 2013, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[24]  Eric S. Lander,et al.  Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011 , 2012, Proceedings of the National Academy of Sciences.

[25]  Y. Carmeli,et al.  Transfer of Carbapenem-Resistant Plasmid from Klebsiella pneumoniae ST258 to Escherichia coli in Patient , 2010, Emerging infectious diseases.

[26]  H. Sørum,et al.  Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments , 1994, Applied and environmental microbiology.

[27]  Akiko Ishiwa,et al.  PilV adhesins of plasmid R64 thin pili specifically bind to the lipopolysaccharides of recipient cells. , 2004, Journal of molecular biology.

[28]  C. Dierikx,et al.  Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry. , 2010, Veterinary microbiology.

[29]  Martin C. J. Maiden,et al.  BIGSdb: Scalable analysis of bacterial genome variation at the population level , 2010, BMC Bioinformatics.

[30]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[31]  D. Mackay,et al.  Sales of veterinary antibacterial agents in nine European countries during 2005-09: trends and patterns. , 2012, The Journal of antimicrobial chemotherapy.

[32]  J. Corander,et al.  The Evolutionary Path to Extraintestinal Pathogenic, Drug-Resistant Escherichia coli Is Marked by Drastic Reduction in Detectable Recombination within the Core Genome , 2013, Genome biology and evolution.

[33]  Adam Godzik,et al.  Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences , 2006, Bioinform..

[34]  Stefan Niemann,et al.  Whole Genome Sequencing versus Traditional Genotyping for Investigation of a Mycobacterium tuberculosis Outbreak: A Longitudinal Molecular Epidemiological Study , 2013, PLoS medicine.

[35]  J. Corander,et al.  Recent Recombination Events in the Core Genome Are Associated with Adaptive Evolution in Enterococcus faecium , 2013, Genome biology and evolution.

[36]  C. William Keevil,et al.  Horizontal Transfer of Antibiotic Resistance Genes on Abiotic Touch Surfaces: Implications for Public Health , 2012, mBio.

[37]  A. Carattoli,et al.  Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum beta-lactamases in Escherichia coli and Salmonella of human and animal origin. , 2008, The Journal of antimicrobial chemotherapy.

[38]  L. Peixe,et al.  Antibiotic resistance integrons and extended-spectrum {beta}-lactamases among Enterobacteriaceae isolates recovered from chickens and swine in Portugal. , 2008, The Journal of antimicrobial chemotherapy.

[39]  B. Liu,et al.  Rates of Mutation and Host Transmission for an Escherichia coli Clone over 3 Years , 2011, PloS one.

[40]  P. Savelkoul,et al.  Extended-Spectrum β-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands , 2011, Emerging infectious diseases.

[41]  M. Bonten,et al.  Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. , 2011, Clinical Microbiology and Infection.

[42]  J. Dewulf,et al.  Diversity of Extended-Spectrum β-Lactamases and Class C β-Lactamases among Cloacal Escherichia coli Isolates in Belgian Broiler Farms , 2008, Antimicrobial Agents and Chemotherapy.

[43]  Harry L. T. Mobley,et al.  Pathogenic Escherichia coli , 2004, Nature Reviews Microbiology.

[44]  Z. Iqbal,et al.  Rapid Whole-Genome Sequencing for Surveillance of Salmonella enterica Serovar Enteritidis , 2014, Emerging infectious diseases.

[45]  Rick L. Stevens,et al.  The RAST Server: Rapid Annotations using Subsystems Technology , 2008, BMC Genomics.

[46]  J. A. van der Goot,et al.  Extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli in Dutch broilers and broiler farmers. , 2013, The Journal of antimicrobial chemotherapy.

[47]  F. Aarestrup,et al.  Selection and Persistence of CTX-M-Producing Escherichia coli in the Intestinal Flora of Pigs Treated with Amoxicillin, Ceftiofur, or Cefquinome , 2008, Antimicrobial Agents and Chemotherapy.

[48]  Fernando de la Cruz,et al.  The diversity of conjugative relaxases and its application in plasmid classification. , 2009, FEMS microbiology reviews.

[49]  Adam M. Phillippy,et al.  Hawkeye and AMOS: visualizing and assessing the quality of genome assemblies , 2013, Briefings Bioinform..

[50]  C. Stoeckert,et al.  OrthoMCL: identification of ortholog groups for eukaryotic genomes. , 2003, Genome research.

[51]  Toni Gabaldón,et al.  trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses , 2009, Bioinform..

[52]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[53]  S. McEwen,et al.  Human Deaths and Third-Generation Cephalosporin use in Poultry, Europe , 2013, Emerging infectious diseases.

[54]  Ole Lund,et al.  Multilocus Sequence Typing of Total-Genome-Sequenced Bacteria , 2012, Journal of Clinical Microbiology.

[55]  P. Hawkey,et al.  The changing epidemiology of resistance. , 2009, The Journal of antimicrobial chemotherapy.

[56]  S. Salzberg,et al.  Versatile and open software for comparing large genomes , 2004, Genome Biology.

[57]  M. Mulvey,et al.  Characterization of plasmids encoding CMY-2 AmpC beta-lactamases from Escherichia coli in Canadian intensive care units. , 2009, Diagnostic microbiology and infectious disease.

[58]  A. Friedrich,et al.  Overview of molecular typing methods for outbreak detection and epidemiological surveillance. , 2013, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.