Federal Register / Vol. 47, No. 235 / Tuesday. December 7, 1982 / Notices 
55107 
two to three; times more mucouse gel 
than either F-10 col' or E. coli J5-3. 
Most of the difference in mucous gel 
binding abilities of the throe strains was 
accounted for by the relatively greater 
ability of F-18 lipopolysaccharide (l.l’S) 
to bind a specific 26.000 dealton protein. 
The investigators conclude that these 
results show that the two strains with 
altered I. PS (i.e.. F-18 col" and J5-3) are 
poor colonizers relative to F-18, 
suggesting that LPS may play an 
important role in the colonization of the 
mouse colon by E. coli. 
3. Adhesion of E. coli Strains to 
Mouse Mucous Gel. The investigators 
have developed an adhesion assay in 
which E. coli strains were tested for 
their abilities to adhere to both mouse 
colonic mucous gel and mouse small 
intestine mucous gel. Nonradioactive 
mucous gel was allowed to bind 
overnight at 4° to polystyrene tissue 
culture wells. The next day the bound 
mucous gel was exposed to 
radioactively labeled E. coli (1 X 
lO'^cpm per bacterium) at 370°, and 
following a one hour incubation period, 
unbound E. coli were removed and the 
number of bacteria adhering to the 
mucous gel was determined. Thus far, 
they have tested adherence of several E. 
coli strains both to large and small 
intestine mucous gel and, as a control, to 
bovine serum albumin. E. coli strains 
which have been shown to be 
enteropathogenic for suckling mice (i.e., 
K88 + ) adhere to small and large 
intestine mucous gel 20 fold better than 
their nonenteropathogenic counterparts 
(i.e., normal fecal strains) and 
approximately 50 fold better than to 
bovine serum albumin. 
Enteropathogenic strains of E. coli that 
do not infect mice (i.e., K99°, 987 — , 
CFA/I") did not adhere to mouse 
mucous gel above the level of normal 
human fecal strains. Finally, neither 
normal rabbit serum, anti-K99 serum, 
nor anti-987 serum blocked K88 + strains 
adhering to mucous gel; but anti-K88 
serum blocked adherence completely. 
The investigators conclude that these 
results suggest that mucous gel may be 
important in the onset of infection by 
enteropathogenic E. coli strains. 
A report by this investigator on the 
effect of plasmid gene expression on the 
colonizing ability of E. coli HS in mice 
has recently appeared (Recombinant 
DNA Technical Bulletin, 5, 1-4, (1982). 
During the coming year, Dr. Cohen’s 
laboratory will be evaluating the role of 
E. coli lipopolysaccharide and intestinal 
mucous gel in the colonization process 
of normal fecal and enteropathogenic E. 
coli strains. 
U. Mechanisms That Control Human 
and Animal Gat Flora 
The National Advisory Allergy and 
Infectious Diseases Council had 
recommended for selective payment a 
project that will focus on the 
mechanisms that control human and 
animal gut flora. This grant (AI 17154) 
was awarded to the University of 
Michigan on behalf of Dr. Rolf Freter. 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 
continous flow (CF) cultures. The issue 
of mobilization of vector plasmids to the 
indigenous flora has always been a 
concern when considering the use of E. 
coli K-12 based host-vector systems. 
Concern has been expressed over the 
potential for exchange of plasmids 
between the Enterobacteriaceae and the 
anaerobic flora, principally members of 
the genus Bacteroids. 
Dr. Freter’s group continues to place 
emphasis on studies of plasmid transfer 
in the mouse intestine and in continuous 
flow cultures of natural or synthetic 
mouse large intestinal flora. 
Mathematical models have been further 
developed. The main aim still is to 
determine “fertility,” i.e., the Intrinsic 
ability of various host-plasmid 
combinations to transfer the plasmid 
under various environmental conditions 
likely to occur in the gut. To this has 
been added an extensive evaluation of 
other factors which are important in 
plasmid transfer. For this reason, the 
models also evaluate the effects of 
plasmid segregation, reduction of the 
growth rates of plasmid-bearing 
bacterial hosts, repression of transfer 
functions, competition for nutrients and 
the effect of bacterial attachment to the 
wall of the gut or culture vessel. The 
investigators have gained confidence in 
the validity of these mathematical 
models as they were able to reproduce a 
number of known phenomena such as 
the repression of fertility of the Rl 
plasmid, as well as known differences in 
the transmission and mobilization of the 
plasmids studied. 
Dr. Freter draws the following 
conclusions from the data: (a) fertility of 
plasmid-bearing E. coli in the normal 
intestine was not impaired. The 
observed low rates of plasmid transfer 
in the normal gut can be explained on 
quantitative grounds alone and do not 
require the postulation of hypothetical 
inhibitory mechanisms: (b) conditions 
for long-term spread and maintenance 
throughout human or animal populations 
of a diversity of conjugative and non- 
conjugativc plasmids may be optimal 
among E. coli strains of low fertility, as 
are found among wild type strains: (c) E. 
coli strains carrying plasmid pBR322 
plus Rldrdl9 were impaired in their 
ability to transfer Rldrdl9, but strains 
carrying pBR322 were significantly 
better recipients of Rldrdl9 than 
plasmid-free recipient E. coli : (d) long- 
term coexistence of plasmid-bearing and 
plasmid-free E. coli. which occured in 
spite of undiminished fertility, appeared 
to be due to a detrimental effect of the 
plasmid on the growth rate of its host 
bacterium, rather than due to high rates 
of plasmid segregation. 
The investigators conclude that the 
long-term interactions observed were 
often the consequences of minor 
differences in parameters such as 
growth rates, fertility, rates of 
segregation, etc., which were decisive 
determinants of the ultimate fates of the 
plasmids and their hosts, but which 
were too small to be detected except by 
precise mathematical analysis of long- 
term experiments. 
Mathematical reconstructions by their 
current mathematical model of plasmid 
transfer in the human gut on the basis of 
experimental data published by earlier 
investigators, are consistent with the 
conclusion that the quantitative aspects 
of fertility and plasmid transfer in the 
human gut are similar to those in mice 
and CF cultures. It appears that, even in 
the absence of selection, plasmid 
transfer occurs consistently in the 
human gut, but that the resulting 
transconjugant E. coli populations are 
too small to be detected regularly with 
the culture methods employed by other 
investigators. 
Three papers reporting on these 
studies have been submitted to a 
scientific journal for publication. Work 
in Dr. Freter’s laboratory is continuing 
on a new mathematical model of 
plasmid transfer among E. coli 
embedded in mucous gel. 
E. Intestinal Absorption of Peptide 
Hormones. 
In April 1980, NIAID convened a 
Workshop on Recombinant DNA Risk 
Assessment. 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 E. coli 
K-12, and (b) the possible occurrence of 
autoantibodies or autoreactive cells due 
to the production of eukaryotic 
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