/ nferliou* Disease I icu f of Recombinanl DSA 
621 
possible to convert the kl2 strain into an epi- 
demic pathogen. 
Whether the organism could Ire concerted to 
a pathogen even in terms of an individual labora- 
tory worker is still moot. It is recognized that a 
large inoculum would l>c required to induce in- 
testinal colonization or infection. Most ol the in- 
fectious diseases caused b\ E. coli arc associated 
with colonization ol Ixxlv surfaces and mucous 
membranes, particularly in the intestine. The 
lartors influencing colonization and those re- 
sponsiblc for disease production are controlled 
by a number of genetic elements. Even the sur- 
face antigens have to have certain antigenic struc- 
tures iu order to render the strain pathogenic. 
The other virulence factors constitute a diverse 
list controlled by chromosomal and extrachromo- 
somal genes. Hence, it is difficult to conceive that 
the multiple deficiencies of E. coll k 12 could be 
corrected by a random, or even designed, inser- 
tion of foreign DNA. Pike has recently analyzed 
3,921 cases of laboratory-associated infections 
[17]. Only two instances were caused by E. coli. 
In the 30 years that E. coli K12 has been used in 
genetics research, there have been no reported 
cases of laboratory acquired infections due to this 
organism Monitoring the fecal flora of laboratorv 
workers over a two-year period in a P-1 type of 
facility failed to reveal the K 12 organisms (marked 
bv nalidixic acid resistance) or the self transmis- 
sible plasmids used in the experiments [48] Never- 
thelcss. the issue of pathogenicity and coloniza- 
tion in an individual subject requires further 
investigation. 
Transmission of DNA from £. co/i K12 to 
Other Organisms 
A potential risk in recombinant DNA research 
is the transmission of the cloned DNA insert 
from the E. coli kl2 vector to other organ- 
isms within the intestinal flora. This concern has 
prompted a scries of investigations into the trans- 
missihility of plasmid DNA in £. coli K12. 
Smith fed 10" E. coli kl2 organisms to a normal 
volunteer [II]. He used several strains which 
contained self-transmissible plasmids of the F, 1. 
or A2 tr.mslei groups. These strains could trans- 
fer in vitro tire tetracycline resistance plasmids 
to an E. coli k!2 recipient and to resident E. 
coli from the normal flora of the volunteer. 
When the strains were fed to the volunteer, how- 
ever. they were eliminated from the feces within 
four days, and there was no evidence of in vivo 
plasmid transfer to resident strains or to suscep- 
tible k!2 and H123 E. coli strains fed in the 
same ingested sample. This experiment was re- 
peated, and again there was failure to transfer 
in vivo the tetracycline resistance plasmid. 
Anderson fed large numbers of £. coli kl2 
organisms which contained a nonconjugative plas- 
mid to eight volunteers [49]. In no instance was 
plasmid transfer to normal flora demonstrated in 
vivo. However, in vitro studies showed that £. 
coli strains from the normal flora of three of 
eight subjects carried transfer plasmids which 
could mobilize one of the nontransmissible plas- 
mids. but not the other. Anderson suggested that 
“transfer would therefore l>c possible if a suit- 
able ronjugative plasmid entered a strain carry- 
ing a nonautotransferring hybrid plasmid." 
Curtiss described in vitro transfer experiments 
in which he measured the mobilization of a se- 
ries of recently dcvelo]>ed. nonconjugative plas- 
mids under optimal laboratory conditions [46], 
He estimated that the maximal probability for 
transmission of such plasmid vectors from an £. 
coli kl2 host is 10 16 per surviving bacteria per 
day in the intestinal tract of warm-blooded ani- 
mals. He emphasized that the chance of transfer 
is even less since other factors, not taken into 
account, would reduce transfer in the intestinal 
tract. The in vivo deterrents include the fol- 
lowing (actors. (/) Diminished bacterial meta- 
Irolic activiiv leads to decreased conjugation. In 
the test tulre, the generation time of £. coli is 
20—10 min. but it is 4-6 hr in the intestinal tract. 
(2) Conjugation is inhibited by fatty acids, bile, 
and other constituents of the gut. (4) Conjuga- 
tion is inefficient at the pH and Eh (oxidation- 
reduction |jotential) of the intestine. 
It was the consensus of the participants that 
the transmissibility studies, while given some com- 
fort bv their negative findings, are not sufficient 
in number or in scojxr to exclude the potential 
risk in this area. In addition, several discussants 
raised the possibility of transfer of genetic mater- 
ial to indigenous flora and to somatic cells of the 
host by unknown mechanisms. Within the com- 
plex milieu of the intestinal tract, it is possible 
[ 159 ] 
