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Federal Register / Vol. 44, No. 232 / Friday, November 30, 1979 / Notices 
strain will either itself become established in 
the gut or lead to the persistence of an R- 
plasmid in another E. coli line following 
plasmid transfer. 
As such, it is therefore a composite 
measure of two separate processes: the 
probability of infection by the organisms 
under the conditions in which the laboratory 
work is carried out; and the ability of the 
organism, or its plasmid, to survive as a 
component of the faecal flora of the worker 
concerned. Although it could be argued that 
such a study is difficult to interpret because 
of its complexity, nevertheless the overall 
probability with which the process occurs is 
important in assessing the hazards of 
laboratory work with E. coli strains carrying 
R-plasmids. The results reported here suggest 
that E. coli K-12 lines, whether or not R- 
plasmids are carried, are unlikely to establish 
themselves as major components of the 
bacterial faecal flora of the workers handling 
the plasmids; and such a view is certainly 
consistent with the behaviour of E. coli K-12 
strains carrying R-plasmids when deliberate 
attempts are made to establish them in the 
human gut by feeding them to volunteers 
(Anderson, 1975; Williams-Smith, 1975). 
In the context of genetic manipulation 
experiments, these results suggest that some 
measure of safety attaches to the use of E. 
coli K-12 strains even in comparison to what 
might be expected for more commonly 
encountered smooth strains of E. coli. The 
margin of safety is increased very 
substantially when disabled versions of E. 
coli K-12, and/or plasmids that are not self- 
transmissible are used for in vitro 
recombination experiments. 
An update of these data, sent to the 
RAC and contained in “Background 
Documents on E. coli K-12/P1 
Recommendation,” ijs the report 
“Summary of Conclusions from Feeding 
and Monitoring Experiments” by M. 
Richmond, as presented to the Risk- 
Assessment Subcommittee of COGENE 
on March 30, 1979. He notes, “Over a 
period of 26 months in Bristol, 12 months 
in London and 12 months in Seattle 
involving 64 subjects, there was no 
evidence that laboratory workers, or 
members of their families, acquired 
either bacterial strains or plasmids that 
were employed in the laboratory. This 
was true even in a few individuals 
working in the laboratory who received 
short courses of antibiotic therapy or, in 
one case, an individual who was on 
prophylactic antibiotic therapy.” 
Will E. coli K-12 Carrying Recombinant 
DNA Survive? 
The Environmental Impact 
Assessment of July 28, 1978, said: 
There are various indications that both 
host bacteria and plasmid or virus vectors 
containing inserted foreign DNA are less 
likely to survive and multiply than are the 
original organisms, except for the very 
unusual instances where the foreign DNA 
supplies some function, such as antibiotic 
resistance, that favors the organism in a 
particular, non-natural environment. Natural 
selection results in the survival of only well- 
balanced and efficient organisms; unneeded 
genetic material tends to be lost. Essential 
functions are carefully controlled and are 
switched on and off as needed. 
The activity of a particular gene product 
depends upon, and in turn influences, many 
other functions of a cell. Such uncontrolled, 
nonessential properties as might be 
introduced by foreign genes would probably 
not result in any advantage to the survival 
and multiplication of an otherwise well- 
balanced organism. Rather, the new 
properties might be expected to confer some 
relative disability. It is unlikely that 
elimination of a gene product by insertion of 
a foreign DNA sequence would be 
advantageous. More likely than not, any new 
properties derived from insertion of foreign 
DNA would confer some relative disability 
on the recipient organism. Therefore, it is 
probable that bacterial cells, plasmids, or 
viruses containing inserted foreign DNA 
would multiply more slowly in nature than 
the same cells or vectors without foreign 
DNA; and in a natural competitive 
environment, those organisms containing 
recombinant DNA would generally be 
expected to disappear. 
New data relevant to this issue, sent 
to the RAC and contained in the 
“Background Documents on E. coli K- 
12/Pl Recommendation,” were supplied 
by Dr. Donald Brown of the Carnegie 
Institution of Washington. E. coli K-12 
containing no recombinant DNA were 
mixed with E. coli K-12 containing a 
number of different inserts (DNA from 
Xenopus laevis, Drosophila 
melanogaster, or Bombyx mori). In each 
case the E. coli K-12 containing no 
recombinant DNA outgrew the E. coli 
K-12 containing recombinant DNA. 
Recent data sent to the RAC and 
contained in the “Background 
Documents on E. coli K-12/P1 
Recommendation” supplied by Dr. Paul 
Burnett of Lilly Research Laboratories 
on May 8, 1979, indicated that when E. 
coli K-12 carrying recombinant plasmids 
containing insulin gene sequences were 
fed to conventional rats or mice, “the 
strain and plasmid are lost very 
rapidly.” 
Will Recombinant DNA Be Transmitted 
from E. coli K-12 to Other Organisms? 
As described in the Environmental 
Impact Assessment (Federal Register 
July 28, 1978): 
While it would appear impossible to render 
E. coli K-12 pathogenic by the introduction of 
foreign DNA, there is still to be considered 
whether the inserted fragment could be 
transmitted to another bacterium with which 
the K-12 comes in contact, including other 
strains of E. coli. Such a transmission might 
convert the recipient into a pathogen or 
render a pathogen more viable. The case of 
plasmid vectors is considered first. 
Plasmids are intracellular particles 
composed of DNA and not dependent on 
chromosomes for their replication. Hence, 
they can be used as vectors, or vehicles for 
transporting foreign DNA into the bacterial 
host, where they multiply and propagate the 
genes they bear. Certain plasmids (called 
"conjugative") are inherently able to migrate 
from one bacterial cell to another. These are 
prohibited for nearly all recombinant DNA 
experiments. Only plasmids not capable or 
barely capable of spontaneous intercellular 
migration ("nonconjugative") may be used. 
As summarized by Dr. Sherwood 
Gorbach (Journal of Infectious Diseases 
137, 615, 1978): 
Smith (1978) fed 10 9 £. coli K-12 organisms 
to a normal volunteer. He used several 
strains which contained self-transmissible 
plasmids of the F. I. or A2 transfer groups. 
These strains could transfer in vitro the 
tetracycline resistance plasmids to an E. coli 
K-12 recipient and to resident E. coli from the 
normal flora of the volunteer. When the 
strains were fed to the volunteer, however, 
they were eliminated from the feces within 
four days, and there was no evidence of in 
vivo plasmid transfer to resident strains or to 
susceptible K-12 and H123 E. coli strains fed 
in the same ingested sample. This experiment 
was repeated, and again there was failure to 
transfer in vivo the tetracycline resistance 
plasmid. 
Anderson (1978) fed large number of E. coli 
K-12 organisms which contained a 
nonconjugative plasmid to eight volunteers. 
In no instance was plasmid transfer to 
normal flora demonstrated in vivo. However, 
in vitro studies showed that E. coli strains 
from the normal flora of three of eight 
subjects carried transfer plasmids whicn 
could mobilize one of the nontransmissible 
plasmids, but not the other. Anderson 
suggested that "transfer would therefore be 
possible if a suitable conjugative plasmid 
entered a strain carrying a 
nonautotransferring hybrid plasmid." 
Curtiss (1978) described in vitro transfer 
experiments in which he measured the 
mobilization of a series of recently 
developed, nonconjugative plasmids under 
optimal laboratory conditions. He estimated 
that the maximal probability for transmission 
of such plasmid vectors from an E. coli K-12 
host is 10* 16 per surviving bacteria per day in 
the intestinal tract of warm-blooded animals. 
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 
included the following factors. (1) Diminished 
bacterial metabolic activity leads to 
decreased conjugation. In the test tube, the 
generation time of E. coli is 20-40 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. (3) 
Conjugation is inefficient at the pH and Eh 
(oxidation-reduction potential) of the 
intestine. 
A study by Dr. Dean Hamer (Science 
196, 220, 1977) looking at the ability of 
conjugative sex factors to mobilize 
nonconjugative plasmids with or 
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