I III; M.W ENGLAND JorKNAI. Of MEDICINE. 
July 21, I'.ia8 
s l re p ti >t; t.i ii i i ! i antibiotics was due to the production of 
<i ribosoim inclhyla.se. 
Selection of Mutants Resistant to LY 145032 
Single sli p mutants of strains BM4147 and 
IIM 1152 were obtained, at a IVc(jueney of 10 -8 , on 
plates containing 8 and 2 mg of LY1 16032 per liter, 
respectively. Two clones, BM H 18 and BM 1153 ( Ta- 
ble 1), were studied further. The minimal inhibitory 
concentrations of LY140032 for the two mutants were 
16 and 4 mg per liter, respectively, whereas those of 
vancomycin and teicoplanin ('Table 2) remained un- 
changed. Strain BM4153-1 (Table 1), which had lost 
plasmid pll’817 upon curing, remained resistant to 
LYM6032 but became susceptible to glycopcptidcs 
(Table 2). The growth characteristics of the mutants 
and of the parental strains were compared by nephe- 
CN 
p" 
i 
<N 
N 
m 
«n 
O 
Kt 
?— 
r— 
F- 
9460 - 
6660 - 
4500 - 
2850 - 
1980 - 
11 60 - 
800 - 
Figure 1. Analysis of Plasmid DNA by Agarose-Gel 
Electrophoresis. 
Plasmid DNA (2 to 3 /ig) was digested with Hitid\\\. Fragments 
obtained by digestion of bacteriophage AC1857 DNA with Hind\\\ 
(A a ) and Psfl (A b ) served as internal standards. 34 The sizes of 
certain A DNA fragments are indicated in base pairs at the left. 
The smaller fragments are not visible. 
Figure 2. Analysis of Plasmid DNA by Agarose-Gel Electrophore- 
sis (Left) and by Hybridization (Right). 
Plasmid DNA was digested with Hind III. Fragments obtained by 
digestion of Acl857 DNA with PstA were used as standards for 
molecular size. The resulting fragments were fractionated by 
agarose-gel electrophoresis, transferred to a nitrocellulose 
sheet, 29 and hybridized to in vitro 32 P-labeled 27 plP816 DNA. 
lometry and were found to be indistinguishable (data 
not shown). In three independent experiments, we 
were unable to obtain spontaneous LY 146032-resist- 
ant or vancomycin-resistant mutants of Staphylococcus 
aureus RX450, E.faecalis JH2-2, or E.faecium BM4147- 
1, BM4152-1, BM4105, or BM4106. 
Discussion 
We have demonstrated that high-level resistance to 
vancomycin and teicoplanin in E. faecium strains 
BM4147 and BM4152 is plasmid mediated. Loss or 
acquisition of resistance to glycopeptide antibiotics 
correlated with the absence or presence of plasmid 
DNA. The two clinical isolates differed in their carbo- 
hydrate-fermentation patterns, their antibiotic-resist- 
ance phenotypes (which were very broad), and their 
plasmid content (Table 1). The resistance plasmids, 
p I P3 1 6 and pIP817, were distinct although related. 
They differed in size (34 and 38 kb, respectively), in 
restriction-endonuclease-gcnerated patterns (Tig. 1), 
and in the resistance phenotype they conferred (Ta- 
ble 1). However, hybridization experiments revealed 
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Recombinant DNA Research, Volume 13 
