PRINCIPAL INVESTIGATOR/PROGRAM DIRECTOR. 
Sandra Handwerger, M.D. 
centrifugation, resuspended in 100 mM MgSO and incubated with 
10 ug/ml DNase and 50 ug/ml RNase for 2 hours. CaCl 2 (10 mM) and 
trypsin (100 ug/ml) were added, and the mixture incubated 
overnight. The mixture was boiled in 1% SDS for 30 minutes, 
washed extensively, boiled in 8M LiCl for 15 minutes, washed and 
boiled in 100 mM EDTA for 15 minutes. After washing, the 
preparation was lyophilized prior to weighing. 
Lyophilized cell wall preparations were hydrolysed in 6N 
HC1 for 16 hours at 110 C. Samples were analyzed on a Durrum 
D-500 amino acid analyzer with detection by the ninhydrin 
reaction. Acid was removed from an aliquot of hydrolysed wall by 
evaporation and the preparation resuspended in 10 mM 
triethylamine buffer and incubated with D-amino acid oxidase (20 
ug/ml) overnight at 37 C. Treated and untreated aliquots were 
then subjected to derivatization with phenylisothiocyanate and 
amino acid analysis by high performance liquid chromatography. 
Amino acid analysis by both methods revealed a molar ratio 
of alanineiglutamic acid : lysine : serine of 3 . 1 : 1 . 3 : 1 : 0 . 5 . After 
treatment with D-amino acid oxidase, the ratio of alanine to 
lysine was decreased to 2:1. These results are consistent with 
the peptidoglycan structure A3«( of Schleifer and Kandler (58) , 
with interpeptide crosslinks of L-alanyl-L-alanyl-serine. Thus 
vancomycin resistance in strain VR is not due to a change in 
composition of stem peptides or replacement of 
D-alanyl-D-alanine with L-amino acids. 
d) Alteration in antibiotic penetration : Decreased 
penetration of the antibiotic would appear to be an unlikely 
mechanism of resistance in Gram positive bacteria, which lack 
the outer membrane present in Gram negative organisms. Previous 
studies using electron microscopy suggest the surface structure 
of Leuconostoc is similar to that of other Streptococcaceae (5) . 
Some isolates of Leuconostoc produce large dextran capsules 
(diameter up to 6 urn) which conceivably might hinder antibiotic 
penetration in .a fashion analogous to the alginate layer of some 
Pseudomonas species (62) . Preliminary experiments showed that 
vancomycin resistant isolates retained their high level 
resistance after growth for 20 generations in sucrose free 
medium, or in the presence of dextranase (10 U/ml), excluding 
hindrance of vancomycin penetration by dextran. 
Preliminary experiments also revealed an unusual property of 
Leuconostoc in their selective susceptibility to beta lactam 
antibiotics. Ten beta lactam antibiotics selected for their 
differences in degree of hydrophobici ty were tested against 
three vancomycin resistant strains of Leuconstoc (VR, VR1 , VR2) . 
The log of the MIC showed a strong inverse correlation with the 
log of P , the partition coefficient of octanol :water for each 
antibiotic tested (linear regressions, r = -.85 to -.89, p < 
.001), suggesting a potential barrier to hydrophilic antibiotics 
(69) . However, comparison of the susceptible strains 688 and 39 
revealed a similar correlation. Furthermore, direct measurement 
of cell surface hydrophobicity by hexadecane : water partitioning 
(56) revealed no significant differences between vancomycin 
susceptible and resistant strains. 
In summary, several mechanisms of vancomycin resistance 
have been excluded: destruction of antibiotic, replacement of 
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Recombinant DNA Research, Volume 13 
