Genetic Determinants of Resistance to Extended-Spectrum Cephalosporin and Fluoroquinolone in Escherichia coli Isolated from Diseased Pigs in the United States

Abstract

Fluoroquinolones and cephalosporins are critically important antimicrobial classes for both human and veterinary medicine. We previously found a drastic increase in enrofloxacin resistance in clinical Escherichia coli isolates collected from diseased pigs from the United States over 10 years (2006 to 2016). However, the genetic determinants responsible for this increase have yet to be determined. The aim of the present study was to identify and characterize the genetic basis of resistance against fluoroquinolones (enrofloxacin) and extended-spectrum cephalosporins (ceftiofur) in swine E. coli isolates using whole-genome sequencing (WGS). bla_CMY-2 (carried by IncA/C2, IncI1, and IncI2 plasmids), bla_CTX-M (carried by IncF, IncHI2, and IncN plasmids), and bla_SHV-12 (carried by IncHI2 plasmids) genes were present in 87 (82.1%), 19 (17.9%), and 3 (2.83%) of the 106 ceftiofur-resistant isolates, respectively. Of the 110 enrofloxacin-resistant isolates, 90 (81.8%) had chromosomal mutations in gyrA, gyrB, parA, and parC genes. Plasmid-mediated quinolone resistance genes [qnrB77, qnrB2, qnrS1, qnrS2, and aac-(6)-lb'-cr] borne on ColE, IncQ2, IncN, IncF, and IncHI2 plasmids were present in 24 (21.8%) of the enrofloxacin-resistant isolates. Virulent IncF plasmids present in swine E. coli isolates were highly similar to epidemic plasmids identified globally. High-risk E. coli clones, such as ST744, ST457, ST131, ST69, ST10, ST73, ST410, ST12, ST127, ST167, ST58, ST88, ST617, ST23, etc., were also found in the U.S. swine population. Additionally, the colistin resistance gene (mcr-9) was present in several isolates. This study adds valuable information regarding resistance to critical antimicrobials with implications for both animal and human health.IMPORTANCE Understanding the genetic mechanisms conferring resistance is critical to design informed control and preventive measures, particularly when involving critically important antimicrobial classes such as extended-spectrum cephalosporins and fluoroquinolones. The genetic determinants of extended-spectrum cephalosporin and fluoroquinolone resistance were highly diverse, with multiple plasmids, insertion sequences, and genes playing key roles in mediating resistance in swine Escherichia coli Plasmids assembled in this study are known to be disseminated globally in both human and animal populations and environmental samples, and E. coli in pigs might be part of a global reservoir of key antimicrobial resistance (AMR) elements. Virulent plasmids found in this study have been shown to confer fitness advantages to pathogenic E. coli strains. The presence of international, high-risk zoonotic clones provides worrisome evidence that resistance in swine isolates may have indirect public health implications, and the swine population as a reservoir for these high-risk clones should be continuously monitored.

Keywords: ESBLs; PMQR; PacBio; USA; WGS; antimicrobial resistance; cephalosporin; epidemic plasmids; fluoroquinolone; high-risk clones; long-read sequencing; plasmids; swine.

FIG 1

Maximum likelihood tree constructed using the core gene alignment of Escherichia coli isolates collected from diseased pigs at UMN-VDL between 2014 and 2015. This unrooted tree was created by mapping raw reads on E. coli K-12 substrain MG1655 (accession NZ_AJGD00000000.1), followed by extraction of SNPs and phylogenetic tree construction (GTR plus gamma substitution model, 1,000 bootstrap replicates) using RAxML version 8.0. Raw reads mapped onto 84.1% to 94.9% of the reference sequence E. coli K-12 substrain MG1655. Phylogenetic tree was constructed using 100,569 recombinant-free sites. Ceftiofur and enrofloxacin MIC values (in μg/ml) are labeled in red and blue to denote resistant and susceptible isolates, respectively. Heat map shows presence of chromosomal mutations in quinolone resistance-determining regions (QRDRs) (green squares), plasmid-mediated quinolone resistance (PMQR) genes (blue stars), ESBL/pAmpC genes (red circles), and virulotypes (APEC, ExPEC, UPEC, ETEC, and STEC) (purple triangles). Median pairwise SNP distances (MPDs) were estimated using ST-specific references. Colored clusters represent groups of isolates with an SNP distance to the next closest isolate of less than 100. Unknown, plasmids/chromosomes carrying these genes were not identified for these isolates.

FIG 2

Circular maps representing comparisons of bla_CTX-M-14 (p77)- and bla_CTX-M-55 (p65)-carrying plasmids available at GenBank and plasmids assembled in this study. The innermost rings (not colored black) represent the top plasmids with high nucleotide identity and coverage with respect to reference plasmids (p77 and p65). The legends at the top left present plasmid name, country, animal species/human, and year of isolation, where available. Areas of the plasmids carrying AMR genes are presented in the outermost rings. AMR genes, genes associated with mobile elements, and virulence genes are colored and labeled in red, blue, and green, respectively. Truncated genes are presented with Δ as a prefix.

FIG 3

Circular map representing comparison of bla_CTX-M-27 (p37 and p62)-carrying plasmids available at GenBank and plasmids assembled in this study. The innermost rings (not colored black) represent the top plasmids with high nucleotide identity and coverage with respect to reference plasmid (p37). The legend at the top left presents plasmid name, country, animal species/human, and year of isolation, where available. Area of the plasmid carrying AMR genes is presented in the outermost ring. AMR genes, genes associated with mobile elements, and virulence genes are colored and labeled in red, blue, and green, respectively.

FIG 4

Circular maps representing comparisons of bla_CTX-M-15 (p1, p2, and p4)-carrying plasmids available at GenBank and plasmids assembled in this study. The innermost rings (not colored black) represent the top plasmids with high nucleotide identity and coverage with respect to reference plasmids (p1). There were no plasmids similar to p4. The legends at the top left present plasmid name, country, animal species/human, and year of isolation, where available. Areas of the plasmids carrying AMR genes are presented in outermost rings. AMR genes, genes associated with mobile elements, and virulence genes are colored and labeled in red, blue, and green, respectively. Truncated genes are presented with Δ as a prefix.

FIG 5

Circular maps representing comparisons of bla_SHV-12 (p33 and p39)-carrying plasmids available at GenBank and plasmids assembled in this study. The innermost rings (not colored black) represent the top plasmids with high nucleotide identity and coverage with respect to reference plasmids (p33 and p39). The legends at the top left present plasmid name, country, animal species/human, and year of isolation, where available. Areas of the plasmids carrying AMR genes are presented in the outermost rings. AMR genes, genes associated with mobile elements, and virulence genes are colored and labeled in red, blue, and green, respectively.

FIG 6

Circular maps representing comparisons of qnrB77 (p23)- and bla_CMY-2 (pCMY)-carrying plasmids available at GenBank and plasmids assembled in this study. The innermost rings (not colored black) represent the top plasmids with high nucleotide identity and coverage with respect to reference plasmids (pCMY). There were no plasmids similar to p23. The legends at the top left present plasmid name, country, animal species/human, and year of isolation, where available. Areas of the plasmids carrying AMR genes are presented in the outermost rings. AMR genes and genes associated with mobile elements are colored and labeled in red and blue, respectively. Truncated genes are presented with Δ as a prefix.

FIG 7

Maximum likelihood tree constructed using the core gene alignment of selected Escherichia coli isolates collected in this study and isolates available at Enterobase. E. coli isolates from Enterobase were selected by identifying those that were within 20 allelic differences (same HC20) of the isolates assembled in our study. This unrooted tree was created by mapping raw reads on E. coli K-12 substrain MG1655 (accession NZ_AJGD00000000.1), followed by extraction of SNPs and phylogenetic tree construction (GTR plus gamma substitution model, 1,000 bootstrap replicates) using RAxML version 8.0. Raw reads mapped onto 84.2% to 94.2% of the reference sequence E. coli K-12 substrain MG1655. Phylogenetic tree was constructed using 43,503 recombinant-free sites. Heat map shows presence of chromosomal mutations in quinolone resistance-determining regions (QRDRs) (green squares), plasmid-mediated quinolone resistance (PMQR) genes (blue stars), ESBL/pAmpC genes (red circles), and virulotypes (APEC, ExPEC, UPEC, and ETEC) (purple triangles). Median pairwise SNP distances (MPDs) were estimated using ST-specific references. Colored clusters represent groups of isolates with an SNP distance to the next closest isolate of less than 100. Unknown, plasmids/chromosomes carrying these genes were not identified for these isolates.