Federal Register / Vol. 46. No. Ill / Wednesday. June 10. 1981 / Notices 
30775 
was found to transfer to coliforms at a 
frequency no higher than 8 x 10"*. This 
value is 10'* lower than seen when the 
plasmid is tested under the best 
laboratory conditions. Thus pBR322. 
which is transferable by F*like R 
plasmids at 10'* —10" 'would be 
expected to be transferred at an 
extremely low frequency; probably 
below experiemntally detectable levels. 
The same is presumable true of potential 
mobilization by the second conjugative 
plasmid, although the data for their 
mobilization of pBR322 are not readily 
available. We would estimate that 
pBR322 would be transferred at a 
frequency at least 10* —10* fold less 
than the conjugatable plasmid, i.e.. 10'* 
- 10 ' 
These studies were supported by 
Contract NOl-AI-72529. Tufts 
University, Stuart Levy, principal 
Investigator. 
b. Progress on the evaluation of the 
transmission of vectors from E. coU K- 
12 to other bacteria In the 
gastrointestinal tract of animals and 
human beings has been made via three 
approaches. 
(1) Data reported in section a. by two 
of the contractors relates directly to this 
question. 
(2) A meeting was convened on 
August 30. 1979 at the NIH with the 
purpose of considering Falmouth 
Workshop Protocols I and II (jour. 
Infect. Dis. 157, 704-706. 1978) and some 
related Issues. Dr. Stanley Falkow 
served as Chairman and sixteen 
participants and NIH staff attended. 
Protocol I addressed the colonization 
and transmission of plasmids from £ 
coU K-12 in the gastrointestinal tract of 
humans to other bacterial strains in the 
intestinal flora. The Working Croup 
unanimously recommended that the 
NIAID not initiate new studies to pursue 
the investigations as written in Protocol 
I. This judgment was based on a review 
of data that existed at the time of the 
Falmouth Workshop, a consideration of 
some newer published data and the 
results of contracts that NIAID was 
supporting. As written by the Falmouth 
Workshop Participants, the experiments 
were to be based on £. coH K-12 and 
this was judged to not be a fruitful 
experimantal model. It was a clear 
consensus of the Croup that, based on 
the available data, it can be predicted 
that only negative results will be 
obtained and that limited resources 
could be better expended in other 
pursuits. 
Protocol II was designed to study the 
transmission of plasmids from £ coH K- 
12. including Chi 1778. into the normal 
intestinal flora utilizing a germ-free 
mouse model. 
The Working Croup unanimously 
supported the view that the NIAID 
should not initiate new studies for the 
Protocol as it was written, but to rely on 
the contracts to supply some additional 
data. This judgment was based on the 
same reasoning and data base 
considered for Protocol I. 
During discussion of the issues cited 
above it was obvious that the Working 
Croup felt that a more benendal use of 
monies would be to support the training 
of workers in good microbiological 
laboratory practices and to support 
research aimed at gaining a better basic 
scientific understanding of bacterial 
colonization and plasmid mobilization. 
Toward this last goal the Working 
Croup strongly recommended that the 
NIAID support studies that could obtain 
quantitative data, expand scientific 
knowledge in an important area and 
which may prove useful at some future 
date for risk assessment. The Croup felt 
that such studies should be performed 
directly in humans and employ wild 
type £ coU (not K-12). Strain HS 
containing pBR324 was suggested as a 
good initial combination, and the study 
should be developed to assay for both 
survival and transfer to the indigenous 
flora. [These studies are now in their 
early stages and being performed by an 
NIAID contractor at the University of 
Maryland Medical School.) 
The full transcript of this meeting and 
a verbal report was made to RAC at 
their March 1960 meeting and the 
recommendations were approved by 
that Committee. 
(3) NIAID identified two relevant 
grant applications, and the National 
Advisory Allergy and Infectious 
Diseases Council supported selective 
payment of these projects for inclusion 
into the risk assessment program. 
One grantee (Dr. Rolf Freter. 
University of Michigan. A1 15279) will 
focus on the mechanisms that control 
human and animal gut flora. Of the four 
stated proposed aims of the research 
plan three relate to issues of importance 
to the NIH Recombinant DNA Risk 
Assessment Program. They are: (1) 
characterize and extend the application 
of anaerobic continuous flow cultures. 
(2) analyze the efficiency of plasmid and 
bacteriophage transfer, and (3) 
determine whether human microflora 
can be maintained in gnotobiotic mice 
and in anaerobic continuous flow 
cultures. The issue of mobilization of 
vector plasmids to the indigenous flora 
has always been a concern when 
considering the use of £ coli K-12 based 
host-vector systems. Most recently 
concern has been expressed over the 
potential for exchange of plasmids in the 
intestinal tract between the 
Enterobacteriaceae and the anaerobic 
flora, principally members of the genus 
Bacteroides. This project has the 
capacity for filling a significant void in 
experimental data. 
A second grantee (Dr. Paul Cohen. 
University of Rhode Island. AI 16370) 
will be exploring a related issue which 
focuses on the molecular mechanisms of 
£ co// colonization of the intestine, 
specifically on the relative importance 
of plasmid or chromosomal 
determinants of colonization. 
At this point in time the majority of 
experiments using recombinant DNA 
technology employ host-vector systems 
based on £ coli K-12 and its plasmids 
or bacteriophages. Prominent among the 
scenarios raised early in the debate over 
use of this technology was the possible 
colonization of the intestinal tract by 
host-vector systems followed by various 
consequences due to the elaboration of 
a product which would cause harm to 
the individual by either direct or indirect 
mechanisms. There now are a 
considerable number of studies 
describing the survival of various types 
of £ coli in the intestinal tracts of man 
and mice and they demonstrate a 
tremendous disparity in the survivability 
and colonization potential of such 
strains. A complete understanding of 
those factors that control survival and 
colonization may permit the 
development of both safer and more 
useful £ coH hosts in the future as well 
as perhaps provide data suggesting 
adjustments in the physical containment 
requirements of the NIH Guidelines 
governing use of this technology. 
c. Experiments testing £ coli K-12 
host-vector systems carrying 
recombinant DNA for virulence have 
been done by NIH scientists (Dr. 
Malcolm Martin and colleagues). The 
studies were designed to determine the 
pathogenicity and stability of shotgun 
clones qf Saccharomyces DNA when 
used with both plasmid and 
bacteriophage vectors. In this 
experimental model, which utilized 
mice, there was no evidence that the 
presence of segments of the entire yeast 
genome altered the inherently low 
pathogenicity of £. coli K-12 in any way. 
d. A Workshop was convened to 
consider two areas [within the NIH 
Program] recommended by the Risk 
Assessment Subcommittee of RAC. This 
Workshop was designed to define the 
scientific issues and assess the potential 
risks of: (a) Possible direct adverse 
effects of hormone-producing strains of 
£. coH K-12 and (b) The possible 
occurrence of autoantibodies or 
autoreactive cells due to the production 
of eukaryotic polypeptides (including 
[ 137 ] 
