Comparative Genomic Analysis of Third Generation Cephalosporin-Resistant Escherichia coli Harboring blaCMY-2-Positive IncI1 group, IncB/O/K/Z, and IncC Plasmids Isolated from Healthy Broilers in Japan.

: The off-label use of third generation cephalosporin (3GC) during in ovo vaccination or vaccination of newly hatched chicks, was a common practice worldwide. CMY-2-producing Escherichia coli have been disseminated among broiler production. The objectives of this study were to determine the epidemiological linkage of blaCMY-2-positive plasmids among broilers both within and outside Japan because grandparent stock and parent stock were imported in Japan. We examined the whole genome sequences of 132 3GC-resistant E. coli isolates collected from healthy broilers during 2002-2014. The predominant 3GC-resistance gene was blaCMY-2, which was detected in the plasmids of 87 (65.9%) isolates. The main plasmid replicon types were IncI1-Iγ (n=21; 24.1%), IncI (n=12; 13.8%), IncB/O/K/Z (n=28; 32.2%), and IncC (n=22; 25.3%). Those plasmids were subjected to gene clustering and network analyses and plasmid multi-locus 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 E. coli isolates. ST429 harboring blaCMY-2-positive IncB/O/K/Z was closely related to isolates from broiler in Germany harboring blaCMY-2-positive IncB/O/K/Z. pST55-IncI and pST12-IncI1-Iγ and pST3-IncC were prevalent in western Japan. pST12-IncI1-Iγ and pST3-IncC were closely related to those detected in E. coli isolates from chicken in American continent, whereas 26 IncB/O/K/Z were related to those in Europe. These data will be useful to reveal the whole picture of transmission of CMY-2-producing bacteria in and out of Japan.