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This study was undertaken to discern the differences of the multi-locus sequence typing (MLST), O serogroups, and virulence factors among 34 CTX-M-1 Escherichia coli, 49 CTX-M-9 strains and 23 non-CTX-M isolates from chickens in Henan province, China. The MLST scheme yielded 34 sequence types, in which ST155 and ST359 were frequent (17% and 15%, respectively) and associated with zoonotic disease. The irp-2 (20% versus 2%, P = 0.0001), traT (85.3% versus 56.5%, P = 0.019), and sfaS (70.6% versus 0, P = 0.021) were significantly more prevalent in CTX-M-1 E. coli than in non-CTX-M producers. Also, CTX-M-9 isolates carried more irp-2 (17% versus 2%, P = 0.023), iroN (71.4% versus 39.1%, P = 0.019), and iss (79.6% versus 39.1%, P = 0.002) genes. In conclusion, although the 106 isolates encompassed a great genetic diversity, the CTX-M isolates harbored more virulence factor genes than non-CTX-M producers.