30774 
Federal Register / Vol. 46, No. Ill / Wednesday, June 10, 1981 / Notices 
The data on biological survival 
characteristics are applicable to 
evaluation of potential dosage to 
workers or to the environments both 
from accidents creating massive release 
and from the continuing releases 
incidental to the labortory procedures. 
Evaluation of the effect of survival of 
the agents in the course of routine work 
should assist in prudent and cost- 
effective application of physical and 
biological containment. Further, it can 
lend emphasis to the areas in which 
training in “good practice” can be most 
productive. 
This work was supported by 
Interagency Agreement YOI-Al-70003, 
Naval Biosciences Labpratory, Robert J. 
Heckley, principal investigator. 
(2) The Survival of EKl and EK2 
Systems in Sewage Treatment Plant 
Models. 
In an early series of studies utilizing 
bench scale models of wastewater 
treatment facilities, £. coli DPSOsupF, E. 
coli Chi 1776 and phage Charon 4A 
concentrations were shown to be 
reduced by at least two orders of 
magnitude by a conventional treatment 
chain (including primary settling 
followed by activated sludge treatmeht 
and anaerobic digestion of all sludges 
generated]. This work was described in 
brief form in the Recombinant DNA 
Technical Bulletin o/July 1979. 
Similar results are now available for 
the survival of E. coli Chi 2656 (which 
carries plasmid pBR322) and for E. coli 
GF 2174 (carrying plasmid pBR325). 
Evidence also was sought in these 
studies for transfer of the plasmids to 
indigenous coliforms and, within the 
sensitivity of the methods used, such 
fransfer was not demonstrated. 
Experiments favoring plasmid transfer 
in raw wastewater and in primary 
biological sludges also failed to 
demonstrate such mobilization. 
Likewise, E. coli 2e0lC carrying a 
lambda prophage (Xc/857/i/n5 plac5) 
was removed effectively by wastewater 
treatment; no free lambda phage was 
observed at any point in the treatment 
train. 
Studies have been completed 
measuring the survival of an indigenous 
E. coli and E. coli K-12 in these 
treatment plant models as well. The 
results of experiments utilizing such 
genetically tagged organisms confirm an 
earlier report that conventional 
domestic wastewater treatment 
processes will result in at least two logic 
reductions in concentration of 
indigenous EKl and EK2 organisms. 
These results are similar to those 
obtained with the indigenous 
wastewater microflora. Further 
reduction of these host-vector 
populations will depend on appropriate 
sludge treatment and effluent 
disinfection. 
This work was supported by Contract 
NOl-AI-82566, University of Texas, San 
Antonio, Bernard Sagik, principal 
investigator. 
(3] The contractor who was 
performing tests in mice and in cultures 
made significant progress during the 
intervening year. The most notable 
result was the establishment of a 
mathematical model to study the 
exchange of plasmids between normal 
hosts and indigenous bacteria. 
Plasmid transfer in continuous flow 
(CF) cultures of defined or natural 
intestinal flora occurred with similar 
efficiency as in pure cultures in vitro. 
One can conclude that the capacity of E. 
coli K-12 strains to function as plasmid 
donors or recipients was not impaired 
by the presence of an indigenous 
microflora. E. coli Chi 1776 donated 
plasmid Rldrdl9 with somewhat lower 
efficiency than a standard E. coli K-12 
donor. 
Plasmid transfer in mice harboring a 
defined intestinal microflora appears to 
have the same degree of efficiency as in 
the CF cultures, because the rate 
constants calculated on the basis of the 
equations are similar to those obtained 
in CF cultures. However, the validity of 
this mathematical treatment for mice is 
not.completely certain, because the 
mathematical model is based on the 
assumption that the bacteria are freely 
suspended, which is not likely to be the 
case in the animal. Nevertheless, the 
data suggest that plasmid transfer 
efficiency in the gut should not differ 
profoundly from that in CF cultures. 
These conclusions are based on 
similarities of transfer rate constants as 
measures of donor and recipient 
capacity. 
The actual rates of transfer are, of 
course, critically dependent on the 
concentrations of donors and recipients. 
The E. coli populations in normal mice 
(and in people) are so low (approx. 10® 
per ml gut content], that little plasmid 
transfer occmred in the mice after the 
initial 20 hours of the experiment, i.e., 
after the large inoculum of donors had 
passed through the animals. This was 
true in spite of the fact that the resident 
E. coli were artificially implanted, highly 
efficient recipients for the highly 
efficient conjugative plasmid. It is not 
very common to find E. coli of such high 
recipient capacity in natural gut flora. 
Despite the fact that the capacities of 
the E. coli strains to donate or receive 
the plasmid were not seriously impaired 
in the gut, the quantitative parameters in 
the normal gut are such that little actual 
transfer occurred once the large initial 
inoculum of donors had been eliminated. 
From these data one may tentatively 
conclude that the probability for 
triparental transfer of a non-conjugative 
plasmid in the normal gut would be 
exceedingly small. The only 
circumstances in which one could 
imagine a realistic possibility for 
triparental transfer to occur, would be at 
times when abnormally high E. coli 
populations are present in the gut, as 
may be the case during fasting and other 
types of stress, in diarrhea, or as a 
consequence of antibiotic therapy. 
These studies were supported by 
Contract NOl-AI-623518, University of 
Michigan, Rolf Freter, principal 
investigator. 
(4] The major effort of the contractor 
who was testing in both mice and 
humans in the interim has been to 
initiate triparental mating studies using 
Chi 1666 (an EKl host], the plasmid 
pBR322, and two additional mobilizing 
plasmids. 
The studies are designed to determine 
if the presence of a mobilizing plasmid 
in a bacterial host cell can cause the 
transfer of the non-conjugative pBR322 
to the indigenous microflora of either 
mice or humans. 
Two studies have been done in mice. 
In both cases germ-free micfrwere 
colonized with human E. coli to provide 
an array of potential recipients and then 
fed the Chi 1666 containing the three * 
plasmids. In the first study, 5x10 * 
organisms were fed once and fecal : 
samples from the mice were pooled to 
facilitate assay; Chi 1666 could only be 
isolated during the first day of the study. ! 
In the second mouse study, the Chi 1666 || 
containing the plasmids were fed daily , 
for 4 days to the mice and their drinking 
water contained tetracycline. In this , 
latter group the Chi 1666 survived at | 
levels of lOVgram or less for the 4 days. 1 
In neither case, however, was the 1 1 
pBR322 mobilized to the indigenous 
flora. 
Finally, Chi 1666 containing pBR322 
and the two mobilizing plasmids was 
fed to 4 human subjects. More than 70 , 
fecal samples were collected during the 
study and a large number of cultures 
and subcultures (2500] performed. The 
Chi 1666 survived from 3V2 to 7 days andi 
that interval corresponds well with 
previous data from studies in which Chi Li 
1666 was fed to humans with and 
without pBR322. While the data for y 
transfer of pBR322 await DNA:DNA 
colony hybridization and further |f|j 
confirmation, from the data already ] 
available on the two transferable lij 
plasmids, we can make certain itj 
estimates. Highly transferable pSL222-4 ; 
