PRINCIPAL INVESTIGATOR/PROGRAM DIRECTOR: 
Sandra Handwerger, M.D. 
traits tested. Repeated "curing" of these partially cured 
isolates is presently underway. 
For transformation experiments, DNA was prepared by the 
method of Marmur (43) , except that Leuconostoc species were 
lysed by exposure to mutanolysin (50 U/ml) and lysozyme (1 
mg/ml) prior to addition of SDS. S. sanguis V288 was grown to 
competence in brain heart infusion (BHI) with 10% fetal calf 
serum, and frozen with 10% glycerol at -70°C (53) . For 
transformation, aliquots of competent cells were thawed and 
incubated with 20 ug/ml DNA for 15 minutes, treated with DNase, 
and plated in BHI agar with 5% sheep blood over a base layer of 
BHI agar. After 90 minutes incubation to allow expression, an 
over layer of antibiotic containing BHI agar was added. When the 
donor DNA was L. mesenteroides 688St, a spontaneous 
streptomycin resistant mutant of 688, transformants growing on 
200 ug/ml ^treptomycin were obtained at a frequency of 6 x 10 2 
per 7 x 10 competent cells; this was comparable to the 
frequency obtained using the spontaneous resistant S. pneumoniae 
R6St as a donor (6 x 10 Z per 8 x 10 7 competent cells). However, 
when strain VR or VR1 was used as DNA donor, and transformants 
selected with vancomycin agar (20 ug/ml) , no vancomycin 
resistant transformants were obtained. Lack of transformants may 
be related DNA preparation technique resulting in shear of large 
fragments, or lack of homologous regions allowing recombination. 
Alternatively, S. sanguis may express vancomycin resistance 
inducibly rather than constituitively , as has been shown when 
the DNA donor is S. faecalis . Therefore, experiments are 
presently underway to test for inducible resistance: 
transformants are screened by 18 hour incubation with sub-MIC 
concentrations of vancomycin (agar overlayer of 0.5 ug/ml) 
followed by a second overlayer containing a higher concentration 
of vancomycin (20 ug/ml) . 
D. EXPERIMENTAL. DESIGN AND METHODS 
The intent of the proposed project is two fold; these 
avenues of investigation will be pursued concomitantly. The 
first aim is to determine the mechanism of resistance to 
vancomycin. The second aim is to characterize the genetic 
determinants of resistance, and further to develop a pair of 
isogenic strains for more detailed mechanistic studies. 
I. Mechanism of Resistance: 
Preliminary experiments have excluded antibiotic 
destruction or surface adsorption, increased surface 
hydrophobicity , replacement of D-amino acids by L-amino acids in 
cell wall peptides, and increased pool levels of N-Ac-hexosamine 
containing precursors as mechanisms of resistance in the 
clinical isolates of Leuconostoc. The experiments described 
below aim to distinguish between: a) bacterial cell surface 
alterations causing decreased penetration of the antibiotic, b) 
altered production of target sites, i.e. specific peptidoglycan 
precursors moietis, and c) alteration of peptidoglycan structure 
preventing binding to the target of antibiotic action. 
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Recombinant DNA Research, Volume 13 
