Federal Register / Vol. 44, No. 232 / Friday, November 30, 1979 / Notices 
69239 
A critical constellation of virulence factors 
required by a microorganism in order to 
produce disease: (1) survival in the 
environment so that it can spread from 
animal to animal. (2) some mechanism for 
penetrating the skin or a mucosal surface 
such as the bowel, genitourinary tract, or 
oropharynx, (3) multiplication within the 
host, (4) systemic spread within the host. (5) 
resistance to host defense mechanisms, and 
(6) production of toxin or some other 
mechanism to damage the host to cause those 
symptoms associated with 'disease.' Freter 
has emphasized that the absence of any one 
of these characteristics will break the chain 
of events, rendering the microorganism 
avirulenl . ... £. coli K-12 is intrinsically 
impaired in most, if not all. of these 
properties. 
Can E. coli K-12 Be Made Pathogenic 
By The Insertion Of Recombinant DNA? 
As discussed in the Environmental 
Impact Assessment which appeared in 
the Federal Register on July 28, 1978: 
Seeking a consensus on the matter of risk 
assessment in recombinant DNA research, 
with particular reference to the use of E. coli. 
the National Institutes of Health sponsored a 
workshop in Falmouth, Mass., on June 20-21, 
1977. In attendance were approximately 50 
invited participants and observers, from the 
United States and abroad, including experts 
on all aspects of infectious disease. The 
following excerpt from a letter by the 
workshop chairman. Sherwood L Corbach. to 
Donald S. Fredrickson summarizes the 
principal conclusion: . . . “The participants 
arrived at unanimous agreement that E. coli 
K-12 cannot be converted into an epidemic 
pathogen by laboratory manipulations with 
DNA inserts. On the basis of extensive 
studies already completed, it appears that E. 
coli K-12 does not implant in the intestinal 
tract of man. There is no evidence that non- 
transmissible pjasmids can be spread from E. 
coli K-12 to other host bacteria within the 
gut. Finally, extensive studies in the 
laboratory to induce virulence in E. coli K-12 
by insertion of known plasmids and 
chromosomal segments coding for virulence 
factors, using standard bacterial genetic 
techniques, have proven unsuccessful in 
producing a fully pathogenic strain. As a 
result of these discussions, it was believed 
that the proposed hazards concerning £. coli 
K-12 as an epidemic pathogen have been 
overstated. Such concerns are not compatible 
with the extensive scientific evidence that 
has already been accumulated, all of which 
provides assurance that £ coli K-12 is 
inherently enfeebled and not capable of 
pathogenic transformation by DNA 
insertions." 
Does The Introduction of Eukaryotic 
Shotgun DNA Into E. coli Alter Its 
Pathogenicity? 
Numerous "shotgun" experiments 
have been performed, inserting pieces of 
eukaryotic DNA into E. coli K-12. In 
very few instances hatfe the resulting 
recombinant-DNA-bearing bacteria 
been specifically tested for 
pathogenicity. A paper sent to the RAC, 
and contained in "Background 
Documents on E. coli K-12/P1 
Recommendation," reports on such a 
study by Drs. Hardy Chan, David 
Botstein, Wallace Rowe, and Malcolm 
Martin. 
Shotgun DNA from Saccharomyces 
cerevisiae ligated into the plasmid pMB9 
was inserted into E. coli K-12. Weanling 
mice were injected intracerebrally and 
intraperitoneally with the recombinant- 
DNA-containing E. coli K-12. Mice were 
sacrificed, the brains cultured in broth, 
and the broth culture injected into 
another group of mice, for five serial 
passages. The results showed that 
“Shotgun cloning of the majority of the 
genome of Saccharomyces into E. coli 
12-K did not yield any clone with 
increased virulence for mice or with 
increased ability to adapt to mouse 
virulence as compared with K-12 
carrying a non-recombinant plasmid." 
Does E. coli K-12 Implant in the 
Intestinal Tract of Laboratory Animals? 
As summarized by Dr. Sherwood 
Gorbach [Journal of Infectious Disease 
137. 615, 1978): 
A number of investigators have tried in 
vain to implant £. coli K-12 in the intestinal 
tract of laboratory animals. Negative results 
have been noted in mice. rats, chickens, pigs, 
and calves. £. coli K-12 has been able to 
colonize the stomach of starved sheep. 
Dr. Rolf Freter [Journal of Infectious 
Diseases 137, 624, 1978) reported that 
chi-1666 (an E. coli K-12 strain) 
"persisted beyond seven days in only 
one of a total of 144 conventional mice 
fed this bacterium." However, when the 
E. coli K-12 were first implanted in 
"germ-free" mice which were 
"conventionalized" later, "the strains 
established themselves in the 
indigenous flora for the length of the 
experiments (up to 85 days) at 
population levels normally assumed by 
E. coli in the conventional mouse." 
Does E. coli K-12 Implant in the Human 
Intestine? 
As noted in the Environmental Impact 
Statement on the 1976 Guidelines: 
Experiments have shown that even after 
normal humans have ingested up to 
10.000.000,000 K-12 cells, only transient 
multiplications of the bacteria in the 
intestines can be observed, and that after a 
time no K-12 can be detected in the feces. 
Thus, K-12 does not establish itself as a 
permanent resident of normal human beings. 
It might be pointed out that animals, 
including humans, ingest large numbers of 
bacteria of many species daily. Most of these 
do not take up long-term residency. For 
example, a normal portion of yogurt may 
contain ten billion cells of the bacteria 
Lactobacillus vulgaris; in spite of daily 
consumption, the Lactobacillus quickly 
disappears from the human bowel. 
As summarized by Dr. Sherwood 
Gorbach [Journal of Infectious Disease 
137. 615, 1978): 
Since colonization of the intestine is felt to 
be an initial event in many pathologic states 
involving £. coli, it is natural that this feature 
has been the subject of several 
investigations. It is fair to state that there 
have been no instances in which the ingested 
strain of £. coli K-12 has been implanted in 
the human intestine. Smith fed eight different 
£. coli K-12 strains, containing various 
transmissible plasmids, to a volunteer at high 
doses (10 s ). Some strains could not be 
isolated at all from the feces, while others 
persisted in progressively reduced counts for 
a period of up to four days. The experiment 
was subsequently repeated, and there was 
again no persistence beyond four days. To 
increase the likelihood of implanting £. coli 
K-12 in the gut. Smith then used a K-12 strain 
that had inserted in it the colicin V (ColV) 
plasmid of a wild-type £. coli (ColV promotes 
the survival of wild-type £. coli in the 
intestinal tract.) When a volunteer consumed 
the K-12 strains with and without the ColV 
plasmid, the strains were eliminated from the 
feces in an equal time frame, none persisting 
more than four days. 
Anderson attempted similar implantations 
with £. coli K-12 strains in eight volunteers 
who received dose& of up to 10'° organisms. 
The maximal period of fecal excretion of the 
test strains was six days, with a mean of 
three days. These studies were repeated, 
again using eight volunteers, and the same 
findings were observed. Gorbach reported at 
this meeting unsuccessful attempts at 
implanting £. coli K-12 in two patients with 
defective bacterial clearing mechanisms in 
the small bowel. In one case, the patient has 
a stricture in the ileum, and the other patient 
had severe diarrhea due to cholera. The K-12 
strain was eliminated from the small bowel 
and faeces within 24 hours in both patients. 
Does E. coli K-12 Infect Laboratory 
Personnel? 
A study of non-recombinant DNA 
workers in a laboratory studying E. coli 
K-12 and transmission-proficient R- 
plasmids (V. Petrocheilou and M. H. 
Richmond, Gene 2, 323, 1977) concluded 
that: 
Faeces of laboratory workers who handled 
nalidixic acid-resistant Eschericjiia coli K-12 
and R plasmids with multiple drug resistance 
markers were monitored every 2 or 3 days for 
over a 2-year period. Neither the K-12 
bacteria nor any of these plasmids were ever 
found in the faeces. Since these R-plasmids 
are transmission-proficient and the work was 
carried out without any special precautions, 
one may conclude that there is not likely to 
be any practical risk for transmission of 
recombinant DNA when cloned in currently 
used transmission-deficient plasmids of £. 
coli K-12. ... It is important to realize that 
studies carried out in this way give an overall 
view of the probability that a laboratory 
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