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Federal Register / Vol. 44, No. 232 / Friday, November 30, 1979 / Notices. 
Il-B-l-a-(12). The laboratory shall be kept 
neat and clean. 
Two requirements of Pi are most 
important. First, all biological wastes 
are to be decontaminated. E. coli are 
very sensitive to chemical disinfection. 
( Disinfection , Sterilization and 
Preservation, Seymour S. Black, editor, 
second edition, Lea and Febiger, 1977.) 
Second, mouth pipetting is prohibited. In 
the 1976 NIH Guidelines, mouth- 
pipetting was permitted at the PI level. 
Mouth-pipetting was prohibited at the 
Pi level in the Guidelines promulgated 
in December 1978. The "Decision 
Document” accompanying proposed 
revised guidelines in the Federal 
Register on July 28, 1978, said: 
A number of commentators have urged that 
mouth pipetting be prohibited at the PI level 
of physical containment. This is strongly 
endorsed by NIH safety experts, who point 
out that this is an important safety feature, 
and that efficient new mechanical pipetting 
aids should not greatly hamper researchers. 
Also, the EMBO Standing Advisory 
Committee on Recombinant DNA Research 
"believes that mouth pipetting should be 
prohibited in P2-P4 laboratories.” In addition, 
the Working Group of American virologists 
which met on April 6-7, 1978, to review the 
report of the U.S.-EMBO Workshop To 
Assess Risks for Recoipbinant Experiments 
Involving the Genomes of Animal, Plant, and 
Insect Viruses wrote the following in their 
report: "In its deliberations, the Working 
Group was impressed with the safeguards 
afforded by a ban on mouth pipetting for 
recombinant DA experiments involving E. 
coli K-12 host-vectors. The group felt that the 
only plausible way E. coli K-12 could gain 
entry into laboratory workers was by oral 
ingestion. The analysis contained in the U.S.- 
EMBO Report was predicated on the remote 
possibility that E. coli K-12, containing 
eukaryotic viral DNA, would be swallowed 
and the viral DNA insert would be delivered 
to a tissue in the body which ordinarily 
would be inaccessible to the virus. A 
prohibition of mouth pipetting would clearly 
prevent this sequence of events from even 
beginning. The Working Group therefore, 
recommended that no mouth pipetting be 
allowed at any level of physical containment 
(including PI) when working with E. coli K- 
12 .” 
The Environmental Impact 
Assessment accompanying proposed 
revised guidelines in the Federal 
Register on July 28, 1978, said: 
A major change * * * is the banning of 
mouth pipetting at the Pi level, meaning that 
mouth pipetting is now banned for all 
experiments covered by the guidelines. Since 
the only plausible way E. coli K-12 could 
gain entry into laboratory workers is by oral 
ingestion, this ban greatly reduces the 
possibility that any organisms containing 
recombinant DNA will escape and thus 
minimizes the risk of environmental impact. 
Pl-type laboratories are used around 
the world for diagnostic microbiological 
purposes, which involve handling many 
common pathogens. There is no 
evidence that the public has ever been 
endangered by the diagnostic and 
research efforts of the many private, 
city, county, and State microbiological 
laboratories associated with hospitals 
and public health departments. While 
accidental infections in laboratory 
workers have occurred, no epidemic or 
hazard to the public health has ever 
been shown to have arisen from work 
conducted in accordance with the basic 
safe practices required in a Pl-type 
laboratory. 
The main additional safety features of 
P2 and P3 as compared with Pi are the 
use of a biological safety cabinet, 
controlled air flow, and access control. 
These features are of primary 
importance in dealing with organisms 
transmitted by the respiratory route. For 
enteric organisms, such as E. coli, the 
main route of transmission is oral. Thus 
the ban on mouth pipetting, the 
requirement for decontamination, and 
observance of basic personal hygiene 
and sanitation practice, as required at 
the Pi physical containment level, are 
the essential safety features. The 
additional features of P2 and P3 provide 
additional safeguards against inhalation 
exposure, which is not a route of 
infection for E. coli. 
What is The Probability of E. coli 
Causing An Epidemic By Person-lo- 
Person Spread? 
There are E. coli strains (other than 
E. coli K-12) which can cause disease in 
man. However, even for these, epidemic 
spread by person-to-person contact is 
extremely unlikely in adults. As 
summarized by Dr. Sherwood Gorbach 
(Journal of Infectious Diseases 137, 615, 
1978): 
The organisms are transmitted by the fecal- 
oral route. Person-to-person spread is rather 
unlikely, and for this reason secondary 
transmission of E. coli from the index case to 
another person is rarely observed. These 
epidemiologic characteristics are based on 
the requirement for a large oral inoculum of 
E. coli to initiate disease, an inoculum 
estimated to be at least 10 6 -10 10 organisms. 
Under natural circumstances, only highly 
contaminated sources such as food and water 
can serve as vehicles pf transmission. 
As discussed in an April 12, 1977, 
letter from Dr. Roy Curtiss (reprinted in 
the Environmental Impact Statement on 
the 1976 Guidelines): 
In terms of communicability of E. coli K-12, 
we know that enteric diseases caused by 
enteropathogenic E. coli and various strains 
of Shigella, Salmonella and Vibrio are 
transmitted by contaminated food and water 
and that manifestation of disease symptoms 
requires consumption of approximately one 
million bacteria. Such enteric diseases are 
seldom spread by aerosols ... In terms of 
the more usual means for spread of enteric 
pathogens, it is evident that enteric diseases 
are very well controlled in the United States 
by sanitary engineering. 
Certain strains of E. coli, such as 
those which produce enterotoxins, are 
responsible for a portion of acute 
diarrheal diseases of childhood and 
have been related to hospital nursery 
outbreaks of diarrheal disease. Data 
collected thus far suggest that E. coli 
diarrheal disease is relatively 
uncommon in the United States, but is 
more common in the developing world. 
Dr. Eugene Gangarosa, writing in the 
Journal of Infectious Diseases 137, 634, 
1978, concluded as follows: 
Current evidence suggests that 
diarrheagenic E. coli are not important 
causes of disease in the sanitized urban 
centers of the United States at this time. 
However, enterotoxigenic E. coli are a 
leading cause of diarrhea among travelers 
who visit developing countries. The failure of 
diarrheagenic E. coli pathogens to gain a 
foothold in this country, despite problems 
with enteropathogenic E. coli in nurseries 
during the 1940’s and 1950’s and the more 
recent multiple introductions of 
enterotoxigenic E. coli by travelers returning 
from developing areas of the world, 
demonstrates the epidemiologic impotence of 
diarrheagenic E. coli in the relatively 
sanitized environment of the United States. 
Nondiarrheagenic E. coli seem to be major 
pathogens in community-acquired and 
nosocomial infections in extraintestinal sites. 
In my judgment, the potential for an 
epidemic caused by the genetically 
manipulated K-12 strain of E. coli does not 
seem remotely possible. Even if disease could 
occur, it seems most unlikely that 
transmission from the laboratory to the 
community would take place, because of the 
reasons cited previously. 
Is E. coli K-12 Pathogenic? 
The “E. coli K-12 /PI 
Recommendation” mandates the use of 
E. coli K-12 hosts. Although there are 
pathogenic strains of E. coli, the 
enfeebled laboratory strain E. coli K-12 
is not pathogenic. As noted in the 
Environmental Impact Assessment 
published in the Federal Register on July 
28, 1978: 
The laboratory variants of K-12 permitted 
in recombinant DNA experiments have never 
been reported cause disease, even in 
laboratory workers. K-12 has been grown in 
large quantities up to hundreds of liters 
containing as many as a trillion bacteria. 
These cultures have been produced in 
countless laboratories the world over, and 
under containment conditions lower than the 
minimal ones in the NIH guidelines. 
As summarized by Dr. Sherwood 
Gorbach (Journal of Infectious Diseases 
137, 615, 1978): 
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