Whole-genome sequencing of gentamicin-resistant Campylobacter coli isolated from U.S. retail meats reveals novel plasmid-mediated aminoglycoside resistance genes

Abstract

Aminoglycoside resistance in Campylobacter has been routinely monitored in the United States in clinical isolates since 1996 and in retail meats since 2002. Gentamicin resistance first appeared in a single human isolate of Campylobacter coli in 2000 and in a single chicken meat isolate in 2007, after which it increased rapidly to account for 11.3% of human isolates and 12.5% of retail isolates in 2010. Pulsed-field gel electrophoresis analysis indicated that gentamicin-resistant C. coli isolates from retail meat were clonal. We sequenced the genomes of two strains of this clone using a next-generation sequencing technique in order to investigate the genetic basis for the resistance. The gaps of one strain were closed using optical mapping and Sanger sequencing, and this is the first completed genome of C. coli. The two genomes are highly similar to each other. A self-transmissible plasmid carrying multiple antibiotic resistance genes was revealed within both genomes, carrying genes encoding resistance to gentamicin, kanamycin, streptomycin, streptothricin, and tetracycline. Bioinformatics analysis and experimental results showed that gentamicin resistance was due to a phosphotransferase gene, aph(2")-Ig, not described previously. The phylogenetic relationship of this newly emerged clone to other Campylobacter spp. was determined by whole-genome single nucleotide polymorphisms (SNPs), which showed that it clustered with the other poultry isolates and was separated from isolates from livestock.

Fig 1

Chromosome features of Campylobacter coli strain N29710. Tracks are numbered from outside in, and the color code is given in the top of the figure. Track 1, size. Tracks 2 and 3, coding sequence (CDS), rRNA, and tRNA on forward and reverse strands, respectively. Three copies of rRNA operons are also marked on the map. Track 4, SNP between N29710 and N29716. Track 5, GC content. Track 6, GC skew [(G − C)/(G+C)]. The circular map was drawn using dnaplotter (22).

Fig 2

Phylogenetic tree generated from whole-genome SNPs. WGS data for C. coli and the complete genome sequence of C. jejuni were downloaded from GenBank (see Materials and Methods) and compared to those for C. coli N29710. H, human isolate; P, poultry isolate; B, bovine isolate; S, swine isolate. Human isolates are shaded blue, poultry isolates are shaded yellow, and swine and bovine isolates are shaded pink. The two strains sequenced in this study are marked with red stars. All bootstrap values are above 99% and are not shown.

Fig 3

Features of multidrug resistance plasmid pN29710-1 and comparison of antimicrobial resistance gene clusters in pN29710-1, SX81, and pCG8245. Tracks are numbered from outside in. Track 1, antimicrobial resistance island in SX81 (6). Track 2, antimicrobial resistance gene cluster in pCG8245 (4). Track 3, size of pN29710-1. Track 4, pN29710-1. Track 5, pTet (25), with large open area corresponding to insertion site of ARG clusters of pCG8245 and pN29710-1. Track 6, GC content of pN29710-1. Regions of GC content above average for the entire plasmid are drawn outside the ring and in red, while regions of GC content below average are inside the ring and in yellow. Gentamicin resistance genes are colored green, other antimicrobial resistance genes are colored magenta, and homologous regions in the antibiotic gene clusters are shaded orange.