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INTRODUCTION TO REVISED APPLICATION: 
The attached proposal is a revision of application 0 1 R29 
AI26669-01 originally submitted October 1987, Portions of the 
text which have undergone significant revision are indicated by 
a line in the right margin. Specific revisions in the application 
include : 
Clinical significance of vancomycin resistance : Criticisms 
raised in the Summary Statement included the lack of evidence of 
pathogenicity of Leuconostoc and the rarity of vancomycin resistance 
among Gram positive bacteria. At the time of the original submission, 
only one unpublished report described the isolation of Leuconostoc 
from blood cultures. Five new reports published within the past year 
describe vancomycin resistant (MIC > 256 ug/ml) Leuconostoc isolates 
from clinically significant specimens, including blood and cerebrospinal 
fluid, suggesting that with growing awareness and proper identification, 
these organisms are being increasingly recognized as pathogens. The 
association between treatment with vancomycin and colonization or infection 
with Leuconostoc species has also been documented in the literature. 
Additionally, reports have appeared of other vancomycin resistant 
Gram positive bacteria, including S^. f aecium and f aecalis , 
indicating that vancomycin resistance among Gram positive bacteria 
is emerging as a more widespread clinical problem. Furthermore, 
a group of French investigators has transferred vancomycin resistance 
from resistant enterococci into j>. sanguis via conjugation, 
demonstrating that interspecies transfer of vancomycin resistance 
may occur among Streptococcaceae . 
Mechanisms of resistance : Further experiments, as delineated 
in the Preliminary Studies section, have excluded increased 
surface hyarophobicity and increased pool levels of peptidoglvcan 
precursors as mechanisms of resistance, narrowing the focus of 
proposed experiments. Three highly susceptible isolates (MIC N < 2.5 
ug/ml) are now included for comparative studies. 
Genetics of vancomycin resistance : The entire genetics section 
of the original application has been revised. Experiments to attempt 
transfer into unencapsulated S. pneumoniae have been deleted. The 
amended proposal provides preliminary data from this laboratory 
suggesting that vancomycin resistance is chromosomally meidated in 
Leuconostoc. New experiments propose to clone the genetic determinants 
of vancomycin resistance in S^. sanguis , and to examine their 
potential relationship to vancomycin resistance geses of enterococci 
by DNA-DNA hybridization. 
Recombinant DNA Research, Volume 13 
[ 417 ] 
