Comparative Genomic Analysis of Third-Generation-Cephalosporin-Resistant Escherichia coli Harboring the <i>bla</i> _CMY-2 -Positive IncI1 Group, IncB/O/K/Z, and IncC Plasmids Isolated from Healthy Broilers in Japan

Abstract

The off-label use of third-generation cephalosporins (3GCs) during <i>in ovo</i> vaccination or vaccination of newly hatched chicks has been a common practice worldwide. CMY-2-producing <i>Escherichia coli</i> strains have been disseminated in broiler chicken production. The objective of this study was to determine the epidemiological linkage of <i>bla</i>_CMY-2-positive plasmids among broilers both within and outside Japan, because the grandparent stock and parent stock were imported into Japan. We examined the whole-genome sequences of 132 3GC-resistant <i>E. coli</i> isolates collected from healthy broilers during 2002 to 2014. The predominant 3GC resistance gene was <i>bla</i>_CMY-2, which was detected in the plasmids of 87 (65.9%) isolates. The main plasmid replicon types were IncI1-Iγ (<i>n</i> = 21; 24.1%), IncI (<i>n</i> = 12; 13.8%), IncB/O/K/Z (<i>n</i> = 28; 32.2%), and IncC (<i>n</i> = 22; 25.3%). Those plasmids were subjected to gene clustering, network analyses, and plasmid multilocus sequence typing (pMLST). The chromosomal DNA of isolates was subjected to MLST and single-nucleotide variant (SNV)-based phylogenetic analysis. MLST and SNV-based phylogenetic analysis revealed high diversity of <i>E. coli</i> isolates. The sequence type 429 (ST429) cluster harboring <i>bla</i>_CMY-2-positive IncB/O/K/Z was closely related to isolates from broilers in Germany harboring <i>bla</i>_CMY-2-positive IncB/O/K/Z. pST55-IncI, pST12-IncI1-Iγ, and pST3-IncC were prevalent in western Japan. pST12-IncI1-Iγ and pST3-IncC were closely related to plasmids detected in <i>E. coli</i> isolates from chickens in North America, whereas 26 IncB/O/K/Z types were related to those in Europe. These data will be useful to reveal the whole picture of transmission of CMY-2-producing bacteria inside and outside Japan.