The aim of the present study was to determine whether oral targeted recombinant beta-lactamase (TRBL) administration could overcome the development of ampicillin-induced resistance in the gut microbiota. Eighteen laboratory beagles with permanent jejunal fistula were randomised to receive ampicillin + placebo, ampicillin + TRBL or placebo. A total of 982 coliform isolates, collected from jejunal and faecal samples before, during and after the treatment were tested against nine antimicrobials. The proportion of ampicillin resistance (multi-resistance) among coliform isolates increased from 20 to 36% in the ampicillin + placebo group but far less, 20-36%, in the ampicillin + TRBL group. These results indicate that TRBL may prevent the emergence of beta-lactam-associated resistance in coliforms in the gut.