PRINCIPAL INVESTIGATORPRCGRAM DIRECTOR Sandra Handwerger, M.D. 
property was also apparent after treatment with suprainhibi tory 
concentrations of vancomycin; no decrease in viability occurred 
after 24 hours of incubation with two times the MIC of 
vancomycin. 
a) Antibiotic inactivation: To determine whether vancomycin 
resistance was due to either destruction or surface adsorption 
of the antibiotic, strain VR was incubated with either 32 or 500 
ug/ml of vancomycin for 18 hours at 37° C in TSB or c + y. After 
centrifugation, the supernatant fluid was passed through a 
sterile filter and used in two fold dilutions to determine the 
concentration of the antibiotic using a bioassay (MIC 
determination) with susceptible strains of S. pneumoniae and S . 
sanguis . Vancomycin incubated in sterile medium for 18 hours at 
37° C was used as a control. No change in the titer of 
vancomycin was observed after incubation of the antibiotic for 
2, 4 or 18 hours with strain VR prior to filtration and dilution 
for use in the bioassay. 
In order to test a possible removal of vancomycin through 
weak or strong adsorption to the surface of the VR cells, the 
vancomycin-containing media was either "gently" separated from 
the bacteria by careful sterile-filtration or exposed to shear 
by vortexing with glass beads and centrifuged prior to 
sterile-filtration. Results of these experiments are summarized 
in table 1. Neither procedure caused a significant decrease in 
the concentration of vancomycin as determined by the bioassay. 
In order to exclude -more complex mechanisms of drug 
"inactivation," strain VR was cocultivated with a vancomycin 
sensitive pneumococcal strain equipped with a streptomycin 
resistance marker (R6St, vancomycin MIC 0.25 ug/ml). VR was 
incubated with R6St in tubes containing twofold dilutions of 
vancomycin (ranging from 0.1 to 32 ug/ml) for 6 hours. Aliquots 
from each tube were plated in c+y agar and c+y agar containing 
100 ug/ml streptomycin to allow selective scoring of survivors. 
The presence of VR cells did not affect the bactericidal effect 
of vancomycin on the pneumococcal indicator strain; no colonies 
of R6St survived 6 hours incubation with 0.25 ug/ml or more of 
vancomycin. 
In a similar experiment, the rate of killing of R6St by 
vancomycin (2.5 ug/ml) in the presence and absence of strain VR 
was determined by plating aliquots of cultures every 2 hours. 
There was no difference in the number of R6St colonies surviving 
exposure to vancomycin. 
Thus, inactivation of the antibiotic in the broadest sense 
of the word seems to be excluded as the mechanism of vancomycin 
resistance . 
b) Increased production of antibiotic targets (effect of 
precursor production) : To test the possibility that strain VR 
may overproduce pentapeptide containing peptidoglycan 
precursors, 3 antibiotics capable of blocking earlier steps in 
cell wall synthesis were used in combination with sub-MIC 
concentrations of vancomycin. VR was incubated in MRS medium 
with sub-MIC concentrations of bacitracin (which prevents 
regeneration of the unaecaprenyl carrier) , D-cycloserine (which 
inhibits production of D-alanvl-D-alanine) , or LY146032 (a 
peptoliae antibiotic which inhibits production of UDP-linked 
[424] 
Recombinant DNA Research, Volume 13 
