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Objectives: The objectives of this study were to determine CTX-M-producing Escherichia coli ST131 strain prevalence in stool specimens from healthy subjects in central China and to molecularly characterize clonal groups. Methods: From November 2013 to January 2014, stool specimens from healthy individuals in Hunan Province were screened for ESBL-producing E. coli using chromogenic medium and CTX-M genotypes and phylogenetic groups were determined. ST131 clonal groups were detected by PCR and characterized for antibiotic resistance, fimH, gyrA and parC alleles, plasmid-mediated quinolone resistance determinants, virulence genotypes and PFGE patterns. Results: Among 563 subjects, 287 (51.0%) exhibited the presence of faecal ESBL-producing E. coli, all of which produced CTX-M enzymes. The most common CTX-M genotypes were CTX-M-14 (48.4%), CTX-M-15 (27.5%) and CTX-M-27 (15.0%). Of the 287 CTX-M-producing isolates, 32 (11.1%) belonged to the ST131 clone. O16-ST131 isolates were dominant (75%) and contained the fimH41 allele. The remaining eight (25%) ST131 isolates were of the O25b subgroup and contained fimH30 or fimH41. Ciprofloxacin resistance was found in 100% of the O25b-ST131 isolates, whereas only 8% of the O16-ST131 isolates were resistant. All of the O25b-ST131 isolates except one showed gyrA1AB and parC1aAB mutations; most of the O16-ST131 isolates had gyrA1A and parC1b mutations. The virulence genotypes of O16-ST131 resembled those of the O25b-ST131 isolates. The 32 ST131 isolates formed one large group at the 64% similarity level. They comprised 15 PFGE groups (defined at ≥85% similarity). Conclusions: O16-ST131 isolates have emerged as the predominant type of ST131 isolate in faecal CTX-M-producing E. coli in healthy individuals in China. [ABSTRACT FROM AUTHOR]