PRINCIPAL INVESTIGATOR' PROGRAM DIRECTOR: R-.nR-- M fl 
DESC =, = ~iCN State the aoplication s prcac. long-term oOiectives anc scecilic aims, making reference tc "~e neaith relateaness of the o. r o;ect. 
Desc ;e concisely the exoerimentai cesign and methocs ‘or achieving these goais. Avoid summaries c‘ cast accomolisnments and the use 
or tre • - st cerscn. This acstract is meant to serve as a succinct anc accurate cescnption of the proposed work wnen separated from the 
application. DO NOT EXCEED THE SPACE PROVIDED. 
Antibiotic resistance among Gram positive bacteria has been increasing over the 
past decade. Recently elucidated mechanisms of resistance have included 
alterations in essential bacterial structures, e.g. .alterations in the enzymes 
require'd for peptidoglycan assembly, which are responsible for intrinsic beta- ; 
lactam resistance. Vancomycin, an antibinr.lc with a unique mechanism of action, 
has been widely used for treatment of infections due to Gram positive bacteria 
resistant to other antibiotics. Vancomycin's site of antibacterial activity, 
the D-alanyl-D-alanine terminus of peptidoglycan precursors, is common to 
all bacteria; development of resistance to vancomycin has therefore been 
considered highly unlikely. Nonetheless, within the past several years, a 
number of highly vancomycin resistant Gram positive bacteria have been isolated 
, from clinically significant specimens. The aim of the proposed project is to 
elucidate the mechanism of vancomycin resistance and characterize the 
genetic determinants encoding such resistance. Leuconostoc, members of 
the Streptococcaceae which frequently demonstrate high level resistance to 
vancomycin, will serve as a model of resistance in Gram positive bacteria. 
Preliminary studies in this laboratory have excluded antibiotic destruction, 
altered surface hydrophobicity , and hyperproduction of peptidoglycan precursors 
as mechanisms of resistance in Leuconostoc, and point to chromosomal mediation 
of the trait. The proposed project aims' to distinguish among mechanisms of 
resistance by ;mparing relevant cell surface structures in resistant and 
susceptible ; lins of Leuconostoc, specifically by: measurement of vancomycin 
binding to ce walls and cell wall stem peptides, characterization of stem 
peptides and UDP-linked peptidoglycan precursors, and identification of 
ceil surface barriers to drug penetration. The genetic determinants of vancomycin 
resistance will be cloned in S . sanguis , and the potential for transfer to 
streptococci by transformation or conjugation will be tested. 
Elucidation of the mechanism of vancomycin resistance may yield new insights 
into the strategies involved in resistance, and suggest alternate targets of action. 
KEY RERSONNEL ENGAGED ON PROJECT 
NAME. OEGREEfSL SSN 
I POSITION TITLE AND ROLE IN PROJECT DEPARTMENT AND ORGANIZATION 
Sandra Handverger, M.D. 
SS # 121-44-0-18 
Patricia May Donath, 3.S. 
SS :/ 114-20-8499 
Principal Investigator 
Research Assistant 
Division of Infectious 
Diseases, Beth Israel 
Medical Center 
Recombinant DNA Research, Volume 13 
[415] 
