We investigated the impact of the hatchery practice of administering third-generation cephalosporin (3GC) on the selection and persistence of 3GC-resistant Escherichia coli in poultry. We studied 15 3GC-treated (TB) and 15 non-3GC-treated (NTB) broiler flocks and 12 3GC-treated (TL) and 10 non-3GC-treated (NTL) future layer flocks. Fecal samples from each flock were sampled before arrival on the farm (day 0), on day 2, on day 7, and then twice more. E. coli isolates were isolated on MacConkey agar without antibiotics and screened for 3GC resistance, and any 3GC-resistant E. coli isolates were further analyzed. 3GC-resistant E. coli isolates were found in all 3GC-treated flocks on at least one sampling date. The percentages of 3GC-resistant E. coli isolates were significantly higher in TB (41.5%) than in NTB (19.5%) flocks and in TL (49.5%) than in NTL (24.5%) flocks. In the day 2 samples, more than 80% of the E. coli strains isolated were 3GC resistant. 3GC-resistant E. coli strains were still detected at the end of the follow-up period in 6 out of 27 3GC-treated and 5 out of 25 non-3GC-treated flocks. Many 3GC-resistant E. coli strains were resistant to tetracycline, and there were significant differences in the percentages of resistance to sulfamethoxazoletrimethoprim, streptomycin, or gentamicin between treated and nontreated flocks. bla CTX-M and bla CMY-2 were the most frequently detected genes. These results clearly demonstrated that 3GC-resistant strains are introduced early in flocks and that the use of 3GC in hatcheries promotes the selection of 3GC-resistant E. coli. Measures must be implemented to avoid the spread and selection of 3GC-resistant strains.T hird-generation cephalosporins (3GC) have been classified as "critically important antimicrobials" in human medicine by the World Health Organization. In many European countries, there has been a dramatic increase in the prevalence of 3GC-resistant Escherichia coli in broilers and broiler meat (1-3), and these isolates are considered of public health concern.Several studies have suggested a possible link between use of 3GC in hatcheries and the increase in 3GC resistance in E. coli. The presence of these resistant isolates in the broiler production pyramid has been recently demonstrated (2, 4). 3GC antimicrobials are sometimes used to control the early mortality rate associated with E. coli. It is automatically administered in ovo to broilers (5) or by subcutaneous injection to 1-day-old future layers (6), together with Marek's disease vaccination. It is strongly suspected that use at hatcheries is responsible for the increase in 3GC resistance in poultry production (3,7,8). However, to our knowledge, no direct evidence of this potential cause-effect relationship has been published.The purpose of this study was to investigate the impact of hatchery use of 3GC on the selection and persistence of 3GC-resistant E. coli in poultry intestinal flora throughout the lifetime of the birds, by comparing flocks of broilers and future laying hens (layers) that ha...