7 
Holmes , and appropriate language was added to Appendix F of the Guidelines • 
The language stipulated that if the investigators wish to proceed with the 
experiments in the NTH P4 facility, a prior review would be conducted to 
advise NIH whether the proposal had sufficient scientific merit to justify 
the use of the NIH P4 facility. A subsequent review by the Bacteriology 
and fycology Study Section of the NIH indicated that the proposal had 
sufficient scientific merit. 
In a second submission dated December 8, 1983, Drs. O'Brien and Holmes 
requested reconsideration at this RAC meeting of containment levels in 
view of information which has recently become available. They requested 
approval at the P2 level of physical containment for the following reasons: 
(1) Epidemiology studies have been performed on over 150 Eh coli 
strains isolated from human and animal stools. These studies 
have shown that the majority (80%) of the strains made detectable 
levels of Shiga-like toxin. Moreover, four of four substrains of 
the well-characterized bacterium Eh_ coli K-12 were shown to make 
low levels of the toxin. Thus, dealing of the Shiga-like toxin 
gene from clinical isolates of Eh_ coli into laboratory strains of 
E. coli K-12 will not involve the introduction of a "foreign" 
toxin into the organism. 
(2) Production of low levels of Shiga-like toxin was observed in 2 of 
15 normal human gut flora Eh_ coli strains from asymptomatic infants. 
( 3 ) Strains of Vibrio cholerae and Vibrio parahaemolyticus were tested 
and shewn to produce Shiga-like toxin. Thus, the gene(s) for Shiga- 
like toxin are present in naturally occurring isolates of the family 
Vlbrionaceae and are not restricted to the Enterobacteriaceae . In 
volunteer studies, seme of the strains of cholerae that produce 
Shiga-like toxin did not cause disease. Therefore, the ability to 
produce Shiga-like toxin is not equivalent with virulence in humans 
challenged by the oral route. 
(4) Phages from two clinic ad isolates of Eh_ coli have been shown to 
control high-level production of Shiga-like toxin in Eh coli K-12 
host strains by phage conversion. Thus, either the structural 
gene(s) for the Shiga-like toxin or regulatory genes that control 
high-level production of the toxin are present on wild- type phages 
fran clinical isolates of Eh_ coli . In this sense, "cloning" of 
genes that affect production of Shiga-like toxin onto phage gencmes 
has already occurred in nature. 
The investigators argued that based on the occurrence of the gene(s) for 
Shiga-like toxin in severed different bacterial genera, the a virulence of 
seme bacterial strains that produce Shiga— like toxin in human subjects 
challenged orally, and occurrence of converting phages in clinical isolates 
of E. ooli controlling high-level production of Shiga-like toxin, there 
is little justification to require the maximum possible level of physical 
[252] 
