Sandra Handwerger, M.D. 
PRINCIPAL INVESTIGATOR'PROGRAM DIRECTOR 
Preliminary experiments have excluded antibiotic inactivation, 
increased surface hydrophobicity , replacement of D-amino acids 
by L-amino acids in cell wall peptides, and increased pool 
levels of peptidoglycan precursors as mechanisms of resistance, 
and suggested that resistance is chromosomal ly mediated. 
Specifically, the aims of this proposal are to: 
1. distinguish among the remaining possible mechanisms of 
resistance to vancomycin: 
a. bacterial cell surface alterations causing decreased 
antibiotic penetration 
b. hyperproduction of individual peptidoglycan precursor 
moieties 
c. alteration in the structure of peptidoglycan preventing 
antibiotic binding 
2. characterize the genetic determinants of vancomycin 
resistance in Leuconostoc, and determine the potential for 
transfer of vancomycin resistance to other Gram positive 
bacteria 
B. BACKGROUND AND SIGNIFICANCE 
Vancomycin, a cell wall active antibiotic, was isolated in 
1955 from Streptomyces orientalis found in soil samples from a 
Borneo jungle (12,19). Shortly after it became commercially 
available, reports of toxicity appeared, and within a few years 
vancomycin was largely displaced by the newly developed 
semisynthetic penicillins. However, renewed interest in 
vancomycin has arisen over the last several years. The 
widespread emergence of methicillin resistance among 
Staphylococcus aureus and the more frequent occurrence of 
nosocomial infections with organisms such as Staphylococcus 
epidermidis and Corynebacteria have necessitated increased used 
of vancomycin. Recent preparations of vancomycin which are 
highly purified have shown less toxicity, and its use has been 
expanded to include first-line therapy for many nosocomial 
infections and surgical prophylaxis (19,28,68). The recent 
observation of vancomycin resistance in Gram positive bacteria 
(6,7,8,11,30,57,59,66) raises grave concerns. Were significant 
vancomycin resistance to emerge among Gram positive pathogens, 
no effective therapy would be available for many 
life-threatening infections. 
Vancomycin is a glycopeptide antibiotic with molecular 
weight of 1448. The structure is that of a heptapeptide with 
two ether bonds and a carbon-carbon bond joining the 
substituents to form three large rings. A disaccharide 
(composed of glucose and vancosamine) is bound to the peptide 
backbone but is not part of the tricyclic structure (52). The 
primary mechanism of action of vancomycin is inhibition of 
peptidoglycan synthesis, which appears to occur at the cell 
wall-membrane interface. Vancomycin binds strongly to peptides 
terminating in D-alanyl-D-alanine (45) ; three dimensional models 
of the antibiotic reveal a cleft in the molecule which is 
probably the site of binding to this acyl-dipeptide (60) . 
However, the D-alanyl-D-alanine terminus is common to both the 
polymerized cell wall and the precursors of peptidoglycan 
Recombinant DNA Research, Volume 13 
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