PRINCIPAL INVESTIGATOR'PROGRAM DIRECTOR. 
Sandra Handwerger, M.D. 
In isolates obtained from clinical sources, as well as 
laboratory mutants and genetic transformants, low and 
intermediate penicillin resistance was shown to be correlated 
with stepwise decreases in the affinity of the penicillin 
binding proteins (PBPs) for penicillin (24; Appendix B) . In very 
highly resistant clinical isolates from South Africa, resistance 
was associated with an alteration in molecular size of the 
largest PBP as well as diminished affinity for penicillin and 
decreased PBP production (25; Appendix C) . Examination of highly 
resistant pneumococci from other locales showed slight 
variations in PBPs, but in all the highest molecular weight PBP 
was replaced by an immunological ly related protein with 
diminished affinity for penicillin (20; Appendix D) . 
Preliminary studies of vancomycin resistance in this 
laboratory have been aimed at distinguishing among the four 
general types of resistance: a) antibiotic inactivation, b) 
increased production of antibiotic targets, c) structural 
alteration of antibiotic target, and d) decreased penetration 
of the antibiotic. Initial experiments have used five strains 
which were isolated from blood cultures of hospitalized 
patients: VR and CH ( Leuconostoc paramesenteroides ) , and VR1, 
VR2 , and MM ( L.mesenteroides ) (30); and six from agricultural 
sources: 33313 ( L. paramesenteroides ) , and 14AM, 688, 39, 8293, 
and Va ( L. mesenteroides ) . All strains were identified on the 
basis of gas production from glucose and typical fermentation 
and hydrolysis reactions (16,30). 
MICs of leuconostoc for vancomycin were determined by 
two-fold dilutions of antibiotic in in four media: c+y, a 
semisynthetic casein hydrolysate medium containing 0.1% yeast, 
MRS medium (42), tryptic soy broth (TSB) and Mueller Hinton 
broth. The MIC for vancomycin of the five clinical isolates 
tested ( L. mesenteroides strains VR1 , VR2 , MM, and L. 
paramesenteroides strains VR and CH) was 512 ug/ml in, the four 
media tested, with initial inocula of either 10 4 or 10 b cfu/ml. 
Assays performed at 25 c C, 30°C, 37°C, and 42°C, or at pH ranging 
from 4.65 to 8.0, showed no difference in MIC. Strain VR has 
maintained its high level resistance after three years of frozen 
storage and after growth for over 50 generations in 
antibiotic-free media. The MIC of strain VR for ristocetin and 
each of the semisynthetic vancomycin analogues A47934, A41030A, 
A35512B, N-demethyl vancomycin , aglycovancomycin (provided by 
Robert Hamill, Ph.D., Eli Lilly and Co.), and for actaplanin G, 
tested in c+y media, was greater than 100 ug/ml. The susceptible 
isolates of Leuconostoc obtained from industrial sources, 
strains 688, 14AM, and 39, had MIC's for vancomycin of < 2.5 
ug/ml; the resistant isolates 33313, 8293, and Va showed MIC's 
of > 512 ug/ml. 
Preliminary experiments also revealed a marked degree of 
"tolerance" (26; Appendix E) , i.e., resistance to the lytic and 
killing effects of cell wall active antibiotics, for both 
vancomycin resistant and susceptible strains. Cultures of 
strains VR, VR1 or 688 to which penicillin was added at five 
times the MIC showed inhibition of growth, but no decrease in 
optical density was observed, and killing was minimal (one log 
decrease in viability after 24 hours of incubation) . This 
Recombinant DNA Research, Volume 13 
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