Federal Register / Vol. 46, No. 233 / Friday. December 4, 1981 / Notices 
59393 
pertinent to discuss "risk” relevant to 
working with E. coli K-12. 
A. History and Description ofD. coli K- 
12 
E. coli K-12 was first isolated from the 
intestine of a patient convalescing from 
diphtheria in Stanford University 
Hospital in the 1920’s. Thereafter it was 
stored in the laboratory as a stock 
strain. Presumably, at the time of initial 
isolation it was a smooth strain adapted 
to colonization of the human colon. By 
the term smooth is meant the fact that 
the strain produces a complete 
lipopolysaccharide O antigen on its 
surface with many polymeric 
polysaccharide side chains. Over the 
course of years of laboratory passage E. 
coli K-12 has become irreversibly rough. 
By dint of its rough state, E. coli K-12 
has lost a major prerequisite required 
for successful colonization of man and 
animals. 
1. E. coli as a Pathogen. It is well- 
recognized that within the enormous 
species, E. coli, are strains that are true 
pathogens for man and animals. 
Depending on strain and host, certain E. 
coli can cause diarrheal illiness, 
dysentery, urinary tract infection or 
meningitis. While this is true, it is now 
known that such pathogenicity is not a 
general or even a potential feature of all 
E. coli. Rather, a constellation of 
specific virulence factors must be 
present in certain E. coli for them to be 
able to cause disease in man (or 
animals) and such virulence factors tend 
to be stable in only a relatively small 
number of strains or serotypes. 
For example: 
A. E. coli meningitis — Trauma or 
neurosurgical intervention which 
breaches the integrity of the meningeal 
coverings of the central nervous system 
can result in direct contamination and 
nosocomial infection with many gram- 
negative or gram-positive organisms. In 
the absence of the above, for all intdnts 
and purposes, primary E. coli meningitis 
is limited to infants less than three 
months of age; in fact most cases occur 
in neonates within 28 days of birth. In 
the early 1970’s it was discovered that 
the E. coli that cause meningitis in 
infants have in common a surface 
capsular polysaccharide, Kl, which 
confers virulence [1,2). By mid-infancy 
most individuals are already resistant to 
Kl encapsulated E. coli. 
b. Diarrheal Illness — Certain E. coli 
can cause watery diarrhea. These 
strains, which include enterotoxigenic 
and enteropathogenic E. coli, possess a 
constellation of virulence properties 
which in tandem result in the ability to 
cause diarrhea. For example, 
enterotoxigenic E. coli that elaborate 
both heatlabile and heatstable 
enterotoxins usually also possess 
fimbrial colonization factors (adhesion 
pili) and such strains are usually limited 
to perhaps a dozen of the 164 E. coli O 
serogroups. Studies in pigs involving 
attempts to "create” a diarrheal 
pathogen by insertion of genes for 
adhesion pili (K88 antigen fimbriae) and 
enterotoxin into a K-12 strain were 
unsuccessful, (5) showing that the 
constellation of virulence properties of 
true porcine pathogens is more complex. 
c. Dysentery — Certain strains of E. 
coli exist that are capable of causing a 
clinical syndrome identical to bacillary 
dysentery. Such strains are referred to 
as enteroinvasive and are limited to a 
very few E. coli O serogroups. 
Biochemically and serologically these 
strains bear many relationships to 
shigella. 
d. Urinary Tract Infections — It is now 
recognized that the E. coli strains that 
are associated with upper urinary tract 
infections possess mannose resistant 
hemagglutinin, and fimbrial colonization 
factors that allow adhesion to epithelial 
cells of the urinary tract and prevent 
dislodgement by urine flow. 
Furthermore, it is currently recognized 
that urinary tract epithelial cells of 
certain individuals are more receptive 
than others to adhesion by E. coli 
strains possessing the fimbrial 
colonization factors. 
2. Inoculum Size and Mode of 
Infection — It has been shown with 
volunteer studies that rather high 
inocula (10® — 10‘”) and neutraliation of 
gastric acid (for example with NaHCOs) 
are required to ensure colonization by 
enterotoxigenic coli. Volunteers with 
E. coli diarrheal disease did not transmit 
the infection by direct contact to 
uninoculated control volunteers with 
whom they were living in close quarters 
[ 4 ]. 
Volunteers challenged with 
enteropathogenic E. coli excreted large 
numbers of E. coli in thier stools and 
were colonized in their proximal small 
intestine but were not colonized in the 
throat (5). 
The thrust of all these observations is 
that: E. coli enteropathogens are 
transmitted via large inocual contained 
within contaminated food and water 
vehicles. They are not spread by 
airborne or direct contact routes. In 
order to colonize the human gut, even 
with a known enteric pathogen, a large 
inoculum is required and modifications 
must be made (e.g., ingestion of 
NaHCOs) to ensure survival of the E. 
coli through the gastric acid barrier. 
Thus, based on current knowledge and 
data it is extremely unlikely that 
individuals working in a recombinant 
DNA research laboratory could become 
colonized in their intestine by droplet, 
aerosol or contact transmission. 
3. Colonizability of Human Intestine 
by E. coli K-12: Summary of Feeding 
Studies — Four groups have carried out 
studies in which high inocula (10* — 10'® 
organisms) of E. coli K-12 strains were 
fed to Volunteers (d-S). The common 
observation from these studies is that E. 
coli K-12 is unable to colonize the 
human intestine. While counts of 10* — 
10’ K-12 organisms per gram of stool 
could be cultured on the first day 
postinfection, levels dropped to 10’ or 
10* by day 2 and K-12 was usually no 
longer detectable by day 5. In contrast, 
ingestion of 10'® organisms of a smooth 
non-pathogenic normal intestinal flora 
strain [E. coli HS) resulted in prolonged 
excretion of the organism (weeks) in 
high titer (10® — 10*/gram stool), 
demonstrating clear-cut colonization 
[ 8 , 10 ). 
There is only one report of apparent in 
vivo transmission of a conjugative 
plasmid from K-12 to normal host flora 
(7). In this instance, one day post- 
ingestion small niunbers (10 '/gram 
stool) of resident coliforms were found 
to be carring the K-12 plasmid. The 
putative recombinants were detectable 
for only one day (7). 
Feeding studies in volunteers have 
also been carried out with E. coli xl778, 
a highly defective, fastidious variant of 
K-12 that hac been certified as host 
organism in EK2 levels of biological 
containment (S). xl776 was not detected 
in stools of volunteers following 
ingestion. When volunteers were fed 
xl776 containing plasmid pBR322, low 
levels of excreation, (lO'-lO^am for 
two days) were observed. 
In summary, the above-mentioned 
feeding studies demonstrate that even 
after direct ingestion of large inocula, E. 
coli K-12 does not readily colonize the 
human intestine. 
4. Active Bacteriologic Surveillance 
for K-12 Colonization — During a two 
year period fecal cultures were obtained 
every two to three days from laboratory 
workers handling nalidixic acid- 
resistant E. coli K-12 containing 
transmission-proficient R plasmids. 
These workers practiced no special 
precautions other than good 
microbiologic technique. Neither E. coli 
K-12 nor the R factors utilized in the 
laboratory were recovered from any 
stool culture at any time. These data 
demonstrate under “field conditions” 
that colonization of E. coli K-12 or 
acquisition by resident intestinal 
coliform flora of R factors used in the 
laboratory do not occur. 
S 
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