PRINCIPAL INVESTIGATOFVPROGRAM DIRECTOR. 
Sandra Handwerger, M.D. 
D-amino acids by L-amino acids, alterations in surface 
hydrophobicity and significantly increased pools of cell wall 
precursors. Thus the proposed project will aim to examine 
structural alterations resulting in a barrier to antibiotic 
penetration or decreased binding of vancomycin to peptidoglycan 
precursors . 
II. Genetics of vancomycin resistance: 
Little is known about the genetics of Leuconostoc in 
general, and the site of genetic information encoding vancomycin 
resistance is unknown. Data from enterococci indicate that 
vancomycin resistance may be plasmid mediated, but also occurs 
in plasmid-free strains, suggesting potential transfer via 
transposon. Most Leuconostoc strains carry plasmids, but no 
association with antibiotic resistance phenotypes have been 
established (49,54). 
Similarly, preliminary experiments in this laboratory 
showed both susceptible and resistant strains to carry a number 
of plasmids, without apparent correlation to susceptibility 
pattern. A rapid plasmid DNA procedure was used which is a 
modification of several published procedures (4,48). Cultures (4 
ml) were grown to late log phase in MRS medium, and the pH 
adjusted to 7.0 with NaOH. Cells were harvested by 
centrifugation, washed and resuspended in 1 ml 0.05 M tris HC1, 
0.01 M EDTA (TE buffer), pH 8.0. Cells were lysed by incubation 
with lysozyme (1 mg/ml) and mutanolysin (50 U/ml) for 6 minutes 
at 37°C. TE buffer, pH 12.3, with 4% SDS was added, the mixture 
incubated for 10 minutes, and neutralized with 30 ul of 2.0 M 
tris pH 7.0. Chromosomal DNA was precipitated by addition of 240 
ul 5.0 M NaCl and chilling on ice. Debris was removed by 
centrifugation (10,000 x g) . Plasmid DNA was precipitated with 
cold ethanol, extracted with phenol, and resuspended in 0.05 M 
tris, 0.005 EDTA, 0.05 M NaCl, pH 8.0. The DNA content of each 
preparation was estimated by spotting aliquots onto ethidium 
bromide-agarose plates and comparing to standards of salmon 
sperm DNA under UV illumination. Approximately 0.2 ug DNA of 
each preparation was used in agarose gel electrophoresis (0.6 to 
0.8%, 5 V/in) . Results are shown in table 2. Both susceptible 
and resistant strains tested carried plasmids of similar 
molecular sizes, and two vancomycin resistant strains appeared 
to harbor no plasmids. These preliminary findings suggest that 
the genetic determinants of vancomycin resistance, at least in 
some strains of Leuconostoc, reside on the chromosome. 
Strains VR1 and VR were "cured" by incubation with one-half 
MIC of novobiocin or rifampin for 18 hours, followed by plating 
on tryptose soy agar (TSA) . Colonies were then replica plated 
onto agar containing vancomycin (20 ug/ml) , lactose indicator 
agar, and sucrose agar. No colonies were identified that had 
lost vancomycin resistance, lactose fermentation, or dextran 
production. Screening of novobiocin treated colonies by agarose 
gel electrophoresis revealed no loss of plasmids. Screening of 
rifampin treated colonies revealed single plasmid loss in 
approximately 10% of colonies, without loss of the phenotypic 
Recombinant DNA Research, Volume 13 
[427] 
