4 
would, thus, never reach high erwugh numbers in the huran intestine for 
plasmid tr 2 insfer to be detected if it occurred at low probability. The 
Ad hoc Working Gtoup suggested instead that the plasmids should be evaluated 
'Sor mobilizability using as host an ^ ooli strain that reeidily colonizes 
the hunan intestine. 
Dr. Levine said the risk assessment studies he would describe used ^ ooli 
strain HS-4 feeding studies to evaluate plasnid mobilizability. He said 
the study was to determine; (a) how well ^ ooli HS-4 colonized the hunan 
intestine; (b) viiether indigenous colifotms would continue to co-habitate 
in the colon with HS-4; (c) v4iether and with vhat frequency a mobilizable 
plaanid would be transferred by triple crosses in vivo from HS-4 into 
indigenous colifotms; (d) Whether and with whatTrequency a poorly mobili- 
zable plasmid would be transferred by triple crosses in vivo fron HS-4 
into indigenous colifotms; and (e) vihether and with vfiat frequency a poorly 
mobilizable plasmid would be transferred vivo, in the presence of a 
highly conjugative plasmid, fron HS-4 into indigenous colifotms. 
Dr. Levine said the experiments show that: (a) ^ ooli HS-4 very effectively 
colonizes the human intestine; (b) indigenous colifotms continue to cohabit 
the colon with HS-4 in most individuals; (c) mobilizable plasnids are 
transferred by triple crosses jn vivo from HS-4 into indigenous coliforms; 
(d) a poorly mobilizable plasmid is not detectably transferred by triple 
crosses in vivo from HS-4 into indigenous colifotms; and (e) a poorly 
mobilizaEIe plasmid transfers in the presence of a highly conjugative 
plasmid in vivo from HS-4 into indigenous coliforms. 
Cr. Levine anphasized that experiment (d) described above examining vhether 
a poorly mobilizable plasmid can be transferred ^ vivo by triple cross 
is the criticad risk assessrent study as it most resenbles the potential 
laboratory accident. He pointed out, ho%«*ever, that very large numbers of 
organisms, in sodiun bicarbonate to neutradize stomach acid, were fed to 
volunteers taking the antibiotic, tetracycline. The use of antacids and 
antibiotics is forbidden in a recombinant D^A laboratory, and the numbers 
of organisTts administered in the study are ixirealistically large in terms 
of vhat might occur in a laboratory accident. Even inder these unrealistic 
conditions vhich enhance the possibility of transmission, there was no 
demonstrable transfer of the poorly mobilizable plasmid. 
Dr. Levine said Dr. Stiart Levy of Tufts Lhiversity had also performed 
feeding studies. Dr. Levine said Dr. Levy fed volunteers a debilitated E. 
ooli K-12 strain containing a poorly mobilizable plasmid and two derepressed 
conjugative plasnids. The strain does not colonize, and no transfer of 
the plasmid to indigenous coliforms was observed. Dr. Levy's study, 
however, is more realistic in that it tests for plasmid transfer with a 
host-vector ^tem actually used in recombinant ENA research. Dr. Martin 
asked if conparable studies had been performed in mic£. Dr. Levine said 
that E. ooli is nc3t a major flora in the mouse and doesn't reach anyvhere 
near tRe ocxjoentration per gram of feceil material that <xie gets in nan; 
Ct. Levine felt that human feeding studies are the cnritical experiments. 
( 333 ] 
