V..I 3 1'» No. 3 
MEDICAL INTELLIGENCE — I.ECI.LRCQ F.T AL. 
Table 2. Minimal Inhibitory Concentrations (MIC) of Glycnpop- 
tidos and a UpopepUcic against Bacterial Strains. 
Vv-'T/MION 
Ti K«*rt^vsiN 
LY1WI32 
E. [deciiun IIM4I47 
1024 
MIC (••> g per liter) 
512 
2 
E fueiiut’i nMII47-| 
4 
1 
2 
F furcium BM4I48 
1024 
512 
16 
E fiiecium BM4I52 
512 
64 
0.50 
E fuicinm BM4I52-1 
0.30 
0.25 
0.50 
F. /uMiim UM4I53 
512 
64 
4 
E finium IJM415J.1 
0.50 
0.25 
4 
5. sangtiu Oiallts 
0.50 
0.12 
0.50 
S wigtiiJ BM4I63 
0.50 
0.12 
0.50 
5 BM4163* 
8 
4 
U.50 
5 BM4I64 
2 
2 
0.50 
5 BM4I64* 
64 
8 
0.50 
• \f:t* inCwO. 1 »■:!) 0 !0 rrg of v».-..o.-r}«in u« lo.i pl.inin pci liter. 
rr Tm. Resistance to aminoglycosides was due to syn- 
thesis of a 6'-amiiiogl\ coside acctyltransfcrase (data 
not show n). F. fc.'cium BM4152 was also resistant to 
high levels of stnplorm cin, through the production of 
a (j-amiimglycosidc nucleotidyltransferase, and to ma- 
eirilidc, liiKusamidc, and streptograrnin B-lype antibi- 
otics constitutive!) - . 
In curing experiments with novobiocin, resistance 
to ghcopeptidcs was lost at a low rate in strain 
BNIIl'17 (approximately 5 percent of 1000 colonics 
tested). In contrast, resistance to glycopcptides, 
together with resistance to m acrolide-lincosamide- 
streplogramin and streptomycin, was lost spontane- 
ously at a high rate in strain BM4152 (approxi- 
mately 60 percent of 200 colonies tested). Two 
susceptible clones, BM4 147-1 and BM41 52- 1 (derived 
from BM1147 and B.M4I32, respectively; Table 1) 
were studied further. The minimal inhibitory concen- 
trations of various antibiotics for the parental 
and the cured derivative strains arc shown in Tables 2 
and 3. 
Plasmid DNA was isolated from strains BM4147, 
BM H52, and BM4I52-1 and anal) zed by agarose-gel 
electrophoresis. Strain BM4147 carried a single 34-kb 
plasmid, pIP3l6, which was absent in BM4147-1. 
Strain BM4I52 contained two plasmids, p I P8 1 7 and 
pi P8 18 (data not show n). Plasmid pi P8 1 7 (38 kb) was 
absent in BM 1152-1, whereas p 1 P3 1 8 (approximately 
100 kb) was present in both strains. Plasmids plP816 
and p I P8 1 7 were not transferable by conjugation to 
F.. /moil is JH2-2 or BMII10. Plasmid DNA purified 
from the wild-type strains B.M4147 and BM4152 
was used to transform Streptococcus sanguis strain 
Challis, and clones were selected on plates containing 
vancomycin, er\ thrornycin, or streptomycin. Analysis 
of transformants on vancomycin revealed a 100 
percent Colransfer of resistance to teicoplanin for 
both donor strains. Selection for the transfer of 
vancomycin, macrolide-Iincos.imide-streptogramin, 
or streptomycin resistance from BM4152 revealed co- 
transfer of all three resistance phenotypes. Two 
clones, BM 1163 and BM416I (Tabic 1), were studied 
further. Clycoprptidc resistance in the transformants 
appeared to be inducible by suhinliibitory concen- 
trations of vancomycin or teicoplanin (Table 2). 
The minimal inhibitory concentrations of glycopcp- 
tides, after 10 generations in antibiotic-free medium, 
were identical to those before induction (data not 
show n). 
The plasmid DNA from strains BM4147, BM4152, 
BM 11 52-1, BM 1163, and BM4164 was purified by 
ultracentrifugation and analyzed by agarose-gel elec- 
trophoresis before (data not shown) and after di- 
gestion with Hindi \\ endonuclease (Fig. 1). Strain 
BM4163 was resistant to vancomycin after acquisition 
of pIP816, whereas strain BM4164 was resistant to 
gl scope j) tide, macrolidc-lincosamidc-streptogramin 
antibiotics, and streptomycin after acquisition of 
pi P8I 7. The patterns generated by Hindlll (Fig. 1), 
/feoRI, and DamW I (data not shown) for the two plas- 
mids weic distinct, although certain fragments had 
similar electrophoretic properties. Plasmid p!P816 
had a size of 34 kb and 10 Hindi 1 1 -generated frag- 
ments, whereas p I P8 1 7 had a size of 38 kb and 12 
fragments. 
Analysis of Plasmid DNA by Hybridization 
Plasmid DNA from BM4152 and BM4152-1 was 
hybridized to p I P8 1 6 (Fig. 2) and to aadF or ermAM 
probes (data not shown). The homologous reaction 
was used as an internal standard. All the Hindlll frag- 
ments, except that of 4.5 kb of p I P8 1 7, hybridized to 
p I P8 1 6. A minority of fragments of the cry ptic plas- 
mid p I P8 1 8 also shared homology with p I P8 1 6. 
Probes intragenic to aadF and ermAM hybridized to 
p I P8 1 7 DNA, thereby confirming that the resistance 
of BM4152 to streptomycin was due to synthesis of a 
6-aminoglycosidc nucleotidyltransferase and indicat- 
ing that the resistance to macrolidc-lincosamide- 
Table 3. Minimal Inhibitory Concentrations (MIC) of Various Anti- 
biotics against E faecium Strains. 
Antibiotic 
Strain 
BM4I47 
BM1I47-1 
BM41J2 
BVU152-1 
MIC (mg per tiler) 
Penicillin G 
61 
64 
16 
16 
Ainpicillin 
16 
16 
8 
i 
Piperacillin 
64 
61 
16 
16 
Imipenem 
32 
32 
16 
16 
Streptomycin 
128 
128 
2048 
128 
Kanamycin 
512 
512 
512 
512 
Tobramycin 
256 
256 
1024 
1024 
AmiVjcin 
64 
64 
128 
128 
Gentamicin 
8 
8 
16 
16 
Sisomicin 
64 
64 
64 
64 
Netilmicin 
64 
64 
128 
123 
Erythromycin 
2 
2 
>4096 
4 
Li neomycin 
16 
16 
2048 
16 
Tetracycline 
64 
64 
64 
64 
Minocycline 
32 
32 
32 
32 
Recombinant DNA Research, Volume 13 
[411] 
