ABSENCE OF PERSISTENCE AND TRANSFER OF GENETIC MATERIAL BY RECOMBINANT ESCHERICHIA-COLI IN CONVENTIONAL, ANTIBIOTIC-TREATED MICE

Strain BST-1 is a derivative of Escherichia coli K-12 that carries a p lasmid designated pURA-4 and is the expression system used by The Upjo hn Company in the production of recombinant bovine somatotropin (rbSt) . This plasmid also encodes an ampicillin resistance gene. The plasmid less carrier strain, BST-1C, contains a gene for tetracycline resistan ce which is provided by the chromosomal insertion of the transposon Tn 10. Therefore, BST-1 is resistant to ampicillin and tetracycline, whil e BST-1C is resistant only to tetracycline. The Food and Drug Administ ration requested that we conduct an environmental assessment study to monitor the 'persistence of the recombinant live K-12 E. coli organism compared to the host E. coli organism'. In addition, we were requeste d to monitor 'the potential transfer of genetic material from (our) re combinant organism to the indigenous microflora' of the mouse gastroin testinal (GI) tract. The differences in persistence were determined by monitoring shedding of BST-1 and BST-1C in the feces of conventionall y reared, outbred mice inoculated with either of the two strains. Even with antibiotic selective pressure applied (tetracycline in the water ), BST-1 did not persist as well as the non-plasmid carrying parental stain, BST-1C. In the gene transfer experiments, transfer of pURA-4 wa s monitored by the appearance of the ampicillin resistance marker and/ or by hybridization assays for the rbSt gene in indigenous, mouse-colo nizing E. coli strains which had been made streptomycin resistant. At the limit of detection, no transfer of pURA-4 was detected either in v itro or in vivo. These data support an interpretation that BST-1 does not present an environmental hazard as measured by colonization/persis tence in the gut of convention ally reared mammals.