PRINCIPAL INVESTIGATOR/PROGRAM DIRECTOR 
Sandra Handwerger, M.D. 
a) Decreased antibiotic penetration : 
The first potential mechanism of resistance to be studied 
is a barrier to penetration of the antibiotic. This hypothetical 
barrier might be present at the level of the cell wall, 
hindering antibiotic penetration through the wall, or at the 
membrane, potentially protecting sites of beta-lactam action 
(penicillin binding proteins) as well as the membrane-bound cell 
wall precursors. Elucidation of the nature and location of such 
structures might also aid in pinpointing the site of 
vancomycin's wall-inhibitory action. 
Vancomycin binding to whole cells and purified cell walls; 
To initially assess the role of antibiotic penetration in 
resistance, binding of vancomycin to purified cell walls of a 
susceptible strain and a resistant strain will be compared to 
that of whole cells. While development of an isogenic pair is 
underway, strains 688 (MIC = 1.25 ug/ml) and VR1 (MIC > 512 
ug/ml) will be used for comparison in initial experiments. 
These strains are identical in their fermentation reactions, and 
show identical protein patterns in whole cell lysates separated 
by SDS-polyacry lamide gel electrophoresis. 
Purified cell walls will be prepared as described above, 
omitting the LiCl and EDTA steps. h propriony lvancomycin will 
be prepared by the method of Fong et al (14) . Aliquots of mid 
log phase cells will be exposed to various sub-inhibitory 
concentrations of labelled vancomycin. At various time points, 
cells will be harvested, washed, and aliquots removed for 
determination of binding by scintillation counting. Binding to 
equivalent aliquots of cell wall preparations (as determined by 
dry weight of cells) will be measured by the same method. 
The results of these initial experiments will aid in 
distinguishing among the broad types of antibiotic resistance 
and serve to direct the course of subsequent investigations. 
Although any binding which occurs may be non-specific, the lack 
of detectable binding to both walls and cells would suggest an 
alteration in target structure or a structural barrier at the 
level of the cell wall. Increased binding to isolated cell walls 
as compared to whole cells would suggest the possibility of a 
permeability barrier which is removed by cell wall purification 
(e.g., membrane-bound, or a wall-bound structure lost during the 
cell wall preparation procedure) . 
Teichoic acids: To assess the possibility that teichoic 
acids represent a wall bound barrier to antibiotic penetration, 
cell walls will be prepared with both sodium hydroxide and 
trichloroacetic acid extraction to remove teichoic acids, and 
vancomycin binding to these preparations assayed as described 
above. In the event of a significant difference in binding 
between cell walls prepared to retain or to remove teichoic 
acids, the teichoic acids of sensitive and resistant strains 
will be compared with regard to structure and sites of 
attachment to the glycan moiety of the cell wall. Previous cell 
wall analysis suggests that some L. mesenteroides contain a 
glycerol teichoic acid with rhamnose and glucose substituents 
(27) . 
Recombinant DNA Research, Volume 13 
[429] 
