PRINCIPAL INVEST1GAT0R/PR0GRAM DIRECTOR 
Sandra Handwerger, M.D. 
A. SPECIFIC AIMS 
Antibiotic resistance among Gram positive bacteria has been 
increasing over the last decade. Recently elucidated mechanisms 
of resistance in Gram positive organisms have included 
remarkably fundamental alterations in bacterial structure. For 
example, alterations in the enzymes required for the final steps 
in peptidoglycan assembly, the penicillin binding proteins, have 
been identified as the cause of intrinsic beta lactam 
resistance. The emergence of antibiotic resistance due to 
alterations in such structures essential to bacterial growth had 
been previously considered extremely improbable. Nonetheless, 
it appears that with sufficient selective pressure, increasingly 
sophisticated mechanisms of resistance will arise among human 
pathogens . 
Vancomycin, a cell wall inhibitor with activity against 
virtually all Gram positive organisms, appears to act by binding 
to the D-alanyl-D-alanine terminus of peptidoglycan components. 
Its unique mechanism of action permits the use of vancomycin for 
infections due to Gram positive bacteria resistant to all beta 
lactam antibiotics. Thus vancomycin has increasingly been used 
as the antibiotic of last resort, for treatment of infections 
due to resistant staphylococci and streptococci. In view of the 
ubiquitious nature of the D-alanyl-D-alanine target among 
bacteria, acquisition of resistance to vancomycin has been 
considered to be highly unlikely. 
However, within the past several years, reports of 
bacteremia, meningitis, and peritonitis due to vancomycin 
resistant Gram positive bacteria have appeared from the United 
States, Europe and Africa (6,7,8,11,30,57,59,66). Although 
vancomycin resistant organisms reported to date represent a 
number of species, including Streptococcus faecium , S. faecalis , 
and Staphylococcus hemolyticus , the most commonly reported 
isolates are Leuconostoc species (6,7,8,11,30,57). These 
organisms, members of the Streptococcaceae which normally reside 
in soil and vegetable matter, have only recently have been 
described as human pathogens . Leuconostoc cause infections most 
frequently in the hospitalized or immunosuppressed patient, 
although cases of serious infections in otherwise healthy 
persons have been reported. These organisms may demonstrate a 
remarkable degree of resistance to vancomycin (minimum 
inhibitory concentration [MIC] > 256 ug/ml) (30,39), and appear 
particularly to colonize the gastrointestinal tract of patients 
receiving vancomycin therapy (2,30) . Thus they are of particular 
interest both in their intrinsic pathogenicity and in the 
possibility that they may confer resistance upon other 
gastrointestinal flora such as enterococci. Elucidation of the 
mechanism of resistance in these strains may yield new insights 
into the strategies involved in antibiotic resistance and its 
transfer among species and genera. Furthermore, identification 
of mechanisms of resistance due to alterations in peptidoglycan 
synthesis may suggest alternate targets of antibiotic action. 
The aims of the proposed investigation are to identify the 
mechanism of high-level vancomycin resistance in Leuconostoc, 
and to characterize the genetic determinants of such resistance. 
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Recombinant DNA Research, Volume 13 
