Current Drug Targets, 2006, 7, 000-000 1

1389-4501/06 $50.00+.00 2006 Bentham Science Publishers Ltd.

The Mechanism of Plasmid Curing in Bacteria
Gabriella Spengler
1,
*
, Annamria Molnr
1
, Zsuzsanna Schelz
1
, Leonard Amaral
2
, Derek Sharples
3
,
J oseph Molnr
1
1
Institute of Medical Microbiology and Immunobiology, Albert Szent-Gyrgyi Medical Centre, University of Szeged,
Szeged, Hungary;
2
Unit of Mycobacteriology, UPMM, Institute of Hygiene and Tropical Medicine, Universidade Nova
de Lisboa, Lisbon, Portugal and
3
School of Pharmacy and Pharmaceutical Sciences, University of Manchester,
Manchester, UK
Abstract: Bacterial plasmids have a major impact on metabolic function. Lactose fermentation of E. coli or hemolysin B
transporter expressed by the plasmids that carry these respective genes could be readily obviated by heterocyclic com-
pounds that readily bind to plasmid DNA. These compounds could also reverse the resistance to antibiotics of E. coli ,
Enterobacter, Proteus, Staphylococcus and Yersinia strains by eliminating plasmids. However, the frequency and extent
of this effect was significantly less than might have been expected based on a complex interaction with plasmid DNA. The
effects of heterocyclic compounds on the plasmids responsible for the virulence of Yersinia and A. tumefaciens, or on
nodulation, nitrogen fixation of Rhizobia accounted for the elimination of 0.1 to 1.0 % of plasmids present in the popula-
tions studied. Bacterial plasmids can be eliminated from bacterial species grown as pure or mixed bacterial cultures in the
presence of sub-inhibitory concentrations of non-mutagenic heterocyclic compounds.
The antiplasmid action of the compounds depends on the chemical structure of amphiphillic compounds having a planar
ring system with substitution in the L-molecular region. A symmetrical -electron conjugation at the highest occupied
molecular orbitals favours the antiplasmid effect.
The antiplasmid effect of heterocyclic compounds is expressed differentially in accordance with the structural form of the
DNA to which they bind. In this manner extrachromosomal plasmid DNA that exists in a superhelical state binds more
compound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries the
plasmid. It can also be noted that these compounds are not mutagenic and their antiplasmid effects correlate with the en-
ergy of HOMO-orbitals.
Plasmid elimination is considered also to take place in ecosystems containing numerous bacterial species. This opens up a
new perspective in rational drug design against bacterial plasmids. The inhibition of conjugational transfer of antibiotic
resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of
plasmid replication at various stages, as shown in the rolling circle model (replication, partition, conjugal transfer) may
also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and
antibiotic resistance.
The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find a
correlation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmid
elimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducing
mutations.
INTRODUCTION
This report reviews the mechanisms by which the an-
tiplasmid activity of heterocyclic compounds is expressed in
plasmid carrying bacteria and the reversal of drug resistance
by these bacteria by elimination of plasmids containing anti-
biotic resistant genes and the relationship of these mecha-
nisms to the inhibition of antibiotic efflux produced by these
same compounds. The complete inhibition of plasmid en-
coded hemolysin B transporter activity and poor elimination
of plasmid DNA encouraged the study of the role of the
*Address correspondence to this author at the Institute of Medical Microbi-
ology and Immunobiology, Albert Szent-Gyrgyi Medical Centre, Univer-
sity of Szeged, Szeged, Hungary; E-mail: spenglerg@freemail.hu
membrane efflux pumps in bacteria and the recognition of
the importance of ABC transporters in general.
The study of the potential release and acquisition of re-
sistance genes presents an interesting research challenge.
The phenomenon has resulted in part from the misuse of
antibiotics and from horizontal gene transfer in the ecosys-
tem. This horizontal gene transfer contributes to evolutionary
processes in a wide variety of organisms. In this process the
auto-transmissible plasmids have a key role in the expansion
of gene pools involved in gene transfer between various or-
ganisms [1]. Other types of gene transfer can occur at the
intracellular level and between bacterial chromosomes,
plasmids and transposons. The third level of antibiotic resis-
tance transmission occurs between microorganisms and ani-
mals, humans included.
2 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
RELATIONSHIP BETWEEN THE CHEMICAL
STRUCTURE AND THE ANTIBACTERIAL EFFECT
Heterocyclic compounds such as phenothiazines have
been shown to possess activity against a large number of
bacterial species [2-5]. E. coli however is relatively resistant
to phenothiazines having MIC values that range from 100 to
150 g/ml [6, 7]
Despite this innate resistance, phenothiazines are quite
effective in the elimination of plasmids from this bacterium
[8]. The use of E. coli in studies that attempt to relate the
structure of the heterocyclic phenothiazine to the degree of
antimicrobial activity and to its antiplasmid activity may
therefore provide essential insights that may in turn lead to
the rational synthesis of new antimicrobial agents. This has
been achieved by studies that test both the antibacterial and
antiplasmid activities of heterocyclic compounds grouped
into two major categories, namely, substituted phenothiazi-
nes and non-phenothiazine heterocyclic compounds.
The activities of substituted phenothiazine compounds
against the plasmid model strain of E. coli K12LE140 are
presented in Table 1. The results for non-phenothiazine het-
erocyclic compounds in Table 2. As shown in Table 1 all of
the compounds listed possess activity against E. coli
K12LE140. However, the phenothiazine derivatives of
Group A are in general, more effective than those of Groups
B, D, E and F. Group A consists of chloro substituted phe-
nothiazines all of which have been derived from the parent
chlorpromazine.
The antibacterial activity of promazine, which has equal
neuroleptic activity to chlorpromazine but with milder side
effects, is similar to that of chlorpromazine as has 2-chloro-
10-(2-dimethylaminoethyl)phenothiazine. The antibacterial
activity significantly increases in the derivatives, des-dime-
thylchlorpromazine (MIC 0.73g/ml) and des-methylchlor-
promazine (MIC of 0.47g/ml), primary and secondary
amines being more effective as antibacterial agents. This
finding demonstrates the importance of amine order in the
antibacterial effect of phenothiazines. In contrast to this, the
derivative 2-chloro-7-hydroxyphenothiazine is far less active
(MIC 16g/ml). The differential antibacterial activity of
these derivatives suggests that substitution in the ring system
and the conformation of the side chain can modify the an-
timicrobial effect.
The antibacterial activity of chlorpromazine can be sig-
nificantly decreased by oxidation at the S-5 atom as shown
by Table 1 Group B. Position 5 seems to be an important site
on the ring in determining antibacterial activity (CPZ-5-
oxide MIC 24.3g/ml) whereas oxidation at the phenothiaz-
ine ring nitrogen has no effect on the antibacterial activity
(CPZ-N-oxide MIC 2.1g/ml). The irrelevance of the phe-
nothiazine ring nitrogen is also evidenced by the decreased
effect noted with CPZ-5, N-dioxide (MIC 27.0g/ml), an
effect that is essentially the result of oxidation at the S-5
position. Trihydroxylation of carbons 3, 7, and 8 reduces the
antibacterial activity of the CPZ derivative whereas single or
dihydroxylation at these carbon atoms does not significantly
affect the activity. One can therefore conclude that reduction
of CPZ antibacterial activity is focused on the Sulfur atom at
position 5 and on substitution at Carbons 3, 7 and 8, 6, 9.
The antibacterial activity of thioridazine, a derivative of
CPZ that is substituted by sulfur at carbon 2, is equal to that
of CPZ [9-11]. Phenothiazine enantiomers show similar ac-
tivities to CPZ (Table 1 Group D). The phenothiazine dyes
methylene blue and toluidine blue have MICs that are con-
siderably lower than those shown by CPZ or thioridazine.
The importance of substitution at C-2 is thus exemplified by
the presence of chloride and sulfur, respectively. The anti-
bacterial activity of non-phenothiazine tricyclic compounds
is presented in Table 2. Other than the demonstration that
many tricyclic compounds do indeed have antibacterial ac-
tivity, and that this activity can be modified by selective sub-
stitution at important sites of the rings, it can be noted that
the tricyclic psychopharmacons imipramine and desipramine
that have non-planar tricyclic skeletons have similar anti-
bacterial activities and become relatively inactive on satura-
tion of the ring system (Table 2 Group A). It can be con-
cluded that one of the aromatic rings is necessary for the
antibacterial activity although which of the three rings is
primarily responsible is yet to be determined.
ANTIPLASMID EFFECTS IN VITRO
Bacteria have developed the ability to resist the toxic
action of antibiotics by a variety of mechanisms such as:
enzymatic inactivation, as is frequently the case with resis-
tance to -lactams and aminoglycosides [12-15], alteration
of the permeability of the cell envelope to given antibiotics
[16-20] and modification of the relevant antibiotic target
[21]. To date all bacteria studied have been shown to have
efflux pumps that are capable of extruding antibiotics when
the concentration of the antibiotic is of itself, not sufficiently
toxic to the organism [22].
Phenothiazines, which have been shown to have antimi-
crobial activity against bacteria (see Table 1), have also been
shown to inhibit the efflux pumps of bacteria whenever this
has been studied [20, 23-27]. These phenothiazines have also
been shown to cause the elimination of plasmids from bacte-
ria [28-30, 33]. A relationship between antimicrobial activ-
ity, elimination of bacterial plasmids and the inhibition of
efflux pumps that extrude antibiotics is strongly suggested
by a number of studies that have investigated agents that
exhibit all three effects [34-38].
The relationship between agents that have antibacterial
and antiplasmid activities and also act as inhibitors of bacte-
rial efflux pumps has been further studied. In addition,
agents such as promethazine and acridine orange, which also
have these activities, have also been shown to reverse antibi-
otic resistance in various bacterial species, when applied as
controls [39-41]. Such reversal of resistance is associated
with the ability of the agent to inhibit the efflux pump. This
confers on the bacteria the ability to grow in concentrations
of an antibiotic that would normally be toxic to the bacteria.
The compounds listed in Tables 1 and 2, and whose mo-
lecular structures are described by Fig. (2), have been inves-
tigated for their ability to eliminate bacterial plasmids. It is
important to note that plasmid elimination in some com-
pounds only occurs at sub-inhibitory concentrations of the
respective agent. The use of sub-inhibitory concentrations
allows the bacterium to grow. This is a necessary pre-
condition for the demonstration of plasmid elimination as
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 3
Table 1. The MIC Values and the Plasmid Curing Effect of Phenothiazine Tricyclic Compounds on E. coli K12LE140 Strain
Compounds
MIC values
M(x10
-4
)
Plasmid curing % at
subinhibitory concentration
A) Phenothiazine derivatives
Chlorpromazine (CPZ) 2.0 29.0
Promazine 3.09 26.0
2-Chloro-10-(2-dimethylaminoaethyl)phenothiazine 3.1 90.0
Desdimethylchlorpromazine 0.73 8.0
Desmethylchlorpromazine 0.47 10.0
Chlorpromazine-methylammonium-iodide 4.7 38.0
2-chloro-7-hydroxyphenothiazine 16.0 0.1
B) Hydroxy-, silcoxy- and oxo-chlorpromazine (CPZ) derivatives
7-hydroxyCPZ 2.42 2.0
3,7-dihydroxyCPZ 5.7 0.8
7,8-dihydroxyCPZ 3.6 0.3
7,8-dioxoCPZ 3.87 0.1
6,9-dihydroxyCPZ 5.6 0.01>
6,9-dioxoCPZ 6.4 0.01>
8-hydroxyCPZ 0.9 1.0
3,7,8-trihydroxyCPZ 22.5 0.01>
CPZ-5-oxide 24.3 0.01>
CPZ-5,N-dioxide 27.0 0.01>
CPZ-N-oxide 2.1 2.0
7-(dimethyl-tert-butyl-siloxy)CPZ 2.2 10.0
7,8-bis-( dimethyl-tert-butyl-siloxyCPZ 3.5 0.01>
6,9-bis-( dimethyl-tert-butyl-siloxy)CPZ 3.5 0.01>
C) Thioalkyl-substituted phenothiazines
Thioridazine 1.9 34.0
Thiethylperazine 2.5 31.0
D) Phenothiazine enantiomers
Levomepromazine 3.5 20.0
Dextromepromazine 3.5 18.0
E) Phenothiazines with different side chains
diethazine 4.3 18.0
promethazine 3.0 25.0
thiasinamide 8.0 0.01>
Bis-(2-chloro-10--propyl-phenothiazine-,-glutamylamid 0.9 2.0
F) Dyes
Methylene blue 16.3 0.01>
Toluidine blue 17.0 0.01>
4 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
Table 2. The MIC Values and the Plasmid Curing Effect of Non-Phenothiazine Tricyclic Compounds on E. Coli K12LE140
Compounds
MIC values
M(x10
-4
)
Plasmid curing % at
subinhibitory concentration
A) Hydroxy- and siloxy derivatives of dibenzoazepines (DMI)
desipramine (DMI) 3.7 23.0
DMIH (partially saturated DMI) 4.3 16.0
DMIH2 (saturated DMI) 30.0 0.5
Imipramine 5.6 40.0
Imipramine (saturated) 23.9 0.01>
Imipramine-methylammoniumiodide 10.0 7.0
2-hydroxyimipramine 5.4 4.0
3-chloro-8-hydroxyimipramine 1.8 3.9
3-chloro-8-dimethyl-tert-butyl-siloxyimipramine 3.5 6.0
B) Dibenzocycloheptane derivatives
Protriptyline 2.5 22.0
Amitriptyline 5.1 50.0
Amitriptyline-methylammoniumiodide 7.1 0.5
C) Anthracene derivatives
1-dimethylamino-3-(1-anthryl)-3-propanol 7.1 5.0
1-dimethylamino-3-(2-anthryl)-3-propanol 10.7 0.2
1-dimethylamino-3-(9-anthryl)-3-propanol 32.2 24.0
Maprotiline 1.7 90.0
4,4-bis-dimethylaminodiphenylmethane 30.0 0.01>
D) Thioxanthene derivatives
Chlorprothixene 1.7 32.0
Cis-clopenthixol 2.3 26.0
Trans-clopenthixol 1.2 33.0
E) Cannabis and phenanthryl derivatives

8
-tetrahydrocannabinol 3.1 0.01>

9
-tetrahydrocannabinol 3.3 0.01>
Cannabinol 3.2 0.01>
Cannabidiol 9.6 0.3
Cannabidiolic acid 13.9 2.5
Tetrahydrocannabidiolic acid 14.0 30.0
Morphine 20.0 0.01>
1-dimethylamino-3-(9-phenanthryl)-3-propanol 4.1 40.0
F) Fluorescent Dyes
Ethidiumbromide 3.8 0.1
Acridine orange 4.1 20.0
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 5
Table 3. The effect of inoculum size (number of generations during the growth of bacteria in the presence of compounds) on the
plasmid elimination of imipramine (4.68x10
-4
M) on E. coli K12 Flac strain
Inoculum/mL at the beginning
of the experiments
Number of colony formers x10
7
Plasmidless (lac
-
) colonies % Number of colonies tested
2x10
2
3.0 12.0 1050
2x10
3
8.0 57.0 840
2x10
4
12.0 6.0 520
2x10
5
16.0 1.0 642
2x10
6
20.0 0.1 811
2x10
8
22.0 0.1 876
plasmid replication is dependent upon bacterial replication
and the number of subsequent generations (Table 3).
After incubation of the bacterium carrying the lac plas-
mid with sub-inhibitory concentrations of the agents, ali-
quots of the culture are streaked onto drug-free medium
containing eosin-methylene blue (EMB). The EMB reagent
aids in the identification of colonies that either contain or are
deficient in the lac plasmid, showing deep violet (lac
+
) or
pink (lac
-
), respectively. The percentage of plasmid elimina-
tion (curing) is determined by the number of pink colonies
divided by the total number of colonies (pink and deep vio-
let) present on the surface of the agar plate. The majority of
the substituted phenothiazines of Groups A and C have high
plasmid curing activities below the MIC at sub-inhibitory
concentrations. The most active agent of these groups is 2-
chloro-10-(2-dimethylaminoaethyl)-phenothiazine which has
a plasmid curing activity of 90% and an MIC of 3.1g/ml.
The important contribution of electronic configuration to
plasmid curing activity is further shown by compounds such
as thioridazine, thiethylperazine and promazine derivatives,
all of which have symmetrical -electron distributions in the
L-molecular region (Fig. (1), Fig. (2) structures 22-27). An-
tidepressants possessing a non planar tricyclic structure with
a secondary amine side chain are more effective in eliminat-
ing plasmid than are compounds with tertiary amine side
chains, whereas the plasmid eliminating potency of quater-
nary amines is much weaker probably because they are un-
able to cross the bacterial cell membrane. The 7-hydroxy, 7,
8-dihydroxy and 7, 8-dioxo substituted CPZ derivatives also
appear to have lower plasmid curing effects than CPZ. Nei-
ther the 6, 9-dihydroxy, 6, 9-dioxo, 5-oxide nor the 7, 8- and
6, 9-siloxy derivatives exert any significant antiplasmid ac-
tivity. These derivatives are considerably more polar than
chlorpromazine as indicated by the comparative ClogP
values (ClogP: CPZ =+5.50, 7-OHCPZ =+4.58, 7, 8-
DiOHCPZ =2.06, 3, 7, 8-TriOHCPZ =0.77). This would
imply that cell penetration is an important factor to be con-
sidered in assessing plasmid curing activity.
It can be concluded from these results that substitution in
the aromatic rings might prevent an interaction between the
-electrons of the phenothiazine ring and target molecules
essential to plasmid replication. Structure 29 for example has
some marginal plasmid curing activity that might be related
to the additional electron cloud provided by the second
chlorpromazine ring. The plasmid curing activity of non-
phenothiazine tricyclic compounds is similar to that of the
substituted phenothiazines. The MIC values and antiplasmid
effects do not directly correlate but some of the compounds
were effective in sub-inhibitory concentrations. A large
number of compounds only have antibacterial activity with-
out any antiplasmid activity however some degree of anti-
bacterial activity is essential for antiplasmid activity. The
cannabinol derivatives are rather interesting with respect to
plasmid curing activity which is present in tetrahydocan-
nabidiolic acid, and to a lesser extent in cannabidiolic acid
and absent in cannabinol, tetrahydrocannabinols and can-
nabidiol.
Fig. (1). Electronic Structure of the phenothiazine skeleton.
The substituted secondary amines, desipramine and pro-
triptyline, eliminated F lac plasmid at lower concentrations
than did the substituted tertiary amines alimemazine and
noxiptyline (Table 4). The majority of phenothiazines elimi-
nated plasmid with a frequency similar to the antidepressants
(Table 4). Compounds such as chlorpromazine and imi-
pramine may become attached through their cationic amine
group to phospholipids in the cell membrane, while the hy-
drophobic part of the molecule intercalates between the fatty
acid chains of the membrane. This would indicate that the
plasmid eliminating ability of a given compound may be a
function of the membrane-lipid: water distribution coeffi-
cient. This can be substantiated by the fact that the distribu-
tion coefficient for chlorpromazine is 1700 and for imi-
pramine is 2395. Conversely the distribution coefficient for
lidocaine is only 17.
6 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
Phenothiazine derivative
1. 2-chloro-7-hydroxyphenothiazine

S
N
H
HO
Cl
16. CPZ-5-oxide

S
N
N
H3C CH3
Cl
O
2. 2-chloro-10-(2-dimethylaminoaethyl)-phenothiazine

H3C
S
N
Cl
N
CH3
17. CPZ-5N-oxide

S
N
N
+
Cl
O
CH3
O
3. Promazine
S
N
N
H
3
C CH
3
18. CPZ-N-oxide

S
N
N
+
CH3
O
4. Desdimethyl-CPZ
S
N
NH
2
Cl
19. 7-(dimethyl-tertbutyl-siloxy)CPZ

S
N
N
+
Cl
CH3
O
O
Si
CH
3
H
3
C
CH
3
H3C
H
3
C
5. Desmethyl-CPZ
S
N
NH Cl
H
3
C
20. 7,8-bis-(dimethyl-tert-butyl-siloxy)CPZ
S
N
N
H
3
C
Cl
O
Si
CH
3
H
3
C
CH
3
H
3
C
H3C
O
Si
H
3
C
CH
3
CH
3
H
3
C
H
3
C
CH
3
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 7
(Fig. 2) contd.
Phenothiazine derivative
6. Chlorpromazine (CPZ)

S
N
N Cl
H3C CH3
21. 6,9-bis-(dimethyl-tert-butyl-siloxy)CPZ

S
N
N
H
3
C
Cl
CH
3
O
O
Si
Si H
3
C
CH
3
CH
3
CH
3
CH
3
H
3
C CH
3
CH
3
CH
3
CH
3
7. CPZ methylammoniumiodide

S
N
N
+
Cl
H
3
C CH
3
H
3
C
I
-
22. Thioridazine

S
N
N
N
CH
3
S
CH3
8. 7-hydroxyCPZ

S
N
N Cl
H3C CH3
OH
23. Thiethylperazine

S
N
S
CH
3
N
CH
3
9. 3,7-hydroxyCPZ

S
N
N Cl
H3C CH3
OH
OH
24. Levomepromazine

S
N
O
CH
3
N
CH3
CH
3
H
3
C
8 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
(Fig. 2) contd.
Phenothiazine derivative
10. 7,8-hydroxyCPZ

S
N
N Cl
H3C CH3
OH
HO
25. Dextromepromazine
S
N
O
CH
3
N
CH
3
H
3
C
H3C
11. 7,8-dioxoCPZ

S
N
N Cl
H3C CH3
O
O
26. Diethazine

S
N
N
H
3
C
H3C
12. 6,9-dihydroxyCPZ

S
N
N
H3C CH3
OH
HO
27. Promethazine

S
N
N
CH
3
H
3
C
CH3
13. 6,9-dioxoCPZ

S
N
N
H3C CH3
O
O
Cl
28. Tiazinamium

S
N
N
+
CH
3
H
3
C
H3C
CH3
14. 8-hydroxyCPZ

S
N
N
H3C CH3
Cl
HO
29. Bis-(2-chloro-10--propyl)-phenothiazine-,-glutamylamide

S
N
NH
O
H
2
N
Cl
HN
O
N
S Cl
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 9
(Fig. 2) contd.
Phenothiazine derivative
15. 3,7,8-trihydroxyCPZ

S
N
N
H
3
C CH
3
Cl
HO
OH
OH
30. Methylene blue
S
+
N
N
H
3
C
CH3
N
CH
3
CH3
31. Toluidine blue
S
N
N
H
3
C
CH
3
NH2
Cl
CH
3
Non-phenothiazine derivatives
32. Desipramine (DMI)
N
N
H
CH3
46. 1-dimethylamino-3-(9-anthryl)-3-propanol
N
CH
3
CH
3
HO
33. DMIH
N
N
H
CH3
47. Maprotiline
N
CH3 H3C
34. DMIH2
N
N
H
CH3
48. 4,4-dimethylaminodiphenylmethane
N N
H3C
CH
3
CH3
CH
3
10 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
(Fig. 2) contd.
Non-phenothiazine derivatives
35. Imipramine
N
N
CH3
CH
3
49. Chlorprothixene
S
N
H
3
C
CH
3
Cl
36. Imipramine (saturated)
N
N
CH3
CH
3
50. Cis-chlopenthixol
S
N
N
OH
37. Imipramine methylammoniumiodide
N
N
+
CH
3
H
3
C
CH3
I
-
51. Trans-chlopenthixol
S
N
N
OH
38. 2-hydroxyimipramine
N
N
CH
3
CH
3
OH
52.
8
-tetrahydrocannabinol
O
CH3
CH
3
CH
3
H3C OH
39. 3-chloro-8-hydroxyimipramine
N
N
CH
3
CH
3
Cl
OH
53.
9
-tetrahydrocannabinol
O
CH3
CH
3
CH
3
H3C OH
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 11
(Fig. 2) contd.
Non-phenothiazine derivatives
40. 3-chloro-8-dimethyl-tertbutyl-siloxy-imipramine
N
N
CH
3
CH
3
Cl
O
Si
CH
3
H
3
C
H
3
C
CH
3
H3C
54. Cannabinol
O
CH3
CH
3
CH
3
H3C OH
41. Protriptyline
N
H
CH3
55. Cannabidiol
OH
H
2
C CH
3
HO
CH
3
CH
3
42. Amitriptyline
N
CH3
CH
3
56. Cannabidiolic acid
OH
H
2
C CH
3
CH
3
CH3
O
OH
HO
43. Amitriptyline-methylammoniumiodide
N
+
CH
3
H
3
C
CH3
I
-
57. Morphine
O
N
CH
3
44. 1-dimethylamino-3(1-anthryl)-3-propanol
HO
N
CH
3
CH
3
58. 1-dimethylamino-3(9-phenanthryl)-3-propanol
N
CH
3
CH
3
OH
12 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
(Fig. 2) contd.
Non-phenothiazine derivatives
45. 1-dimethylamino-3(2-anthryl)-3-propanole
OH
N CH
3
H
3
C
59. Ethidiumbromide
CH
3
NH
2
H
2
N
60. Acridine orange
CH
3
N N N
H
3
C
CH
3
CH
3
Fig. (2). Molecular structures of agents examined for antimicrobial and plasmid curing activity.
Table 4. Plasmid Elimination Action of Various Psychotherapeutics
Compounds
Concentration
g/mL
Plasmid elimination
percent
Colonies
Examined
MIC
g/mL
Desipramine 50 23,4 3600 70
Trimipramine 180 25,8 5700 200
Protriptyline 50 22,9 4400 80
Noxiptyline 260 41,0 3300 300
Promazine 60 40,0 6950 90
Trimeprazine 130 22,0 4700 150
Trifluopromazine 60 25,0 3500 80
Chlorprothixine 40 87,0 3450 50
Profenamine 220 54,0 7100 230
Thiazinamium 190 0,0 5000 230
Chlorpromazine methoioiodide 160 24,0 2900 180
Toluidine blue 240 0,0 3500 280
Lidocaine 2950 0,3 4360 3500
Procaine 18000 0,0 4020 >20.000
The plasmid curing activity of promethazine and tri-
fluoperazine was investigated by replica plating [32] on
plasmid-mediated doxycycline resistant bacterial strains iso-
lated from clinical specimens. The frequency of plasmid
elimination was low for the majority of the tested strains,
despite the formation of complexes between antiplasmid
compounds (promethazine, 9-aminoacridine) and plasmid
DNA isolated from the plasmid containing clinical strains. It
may be suggested that inefficient penetration of the an-
tiplasmid compounds was responsible for the weak plasmid
curing effect in these clinical isolates. It is probable that the
cell wall/cell membrane served as a barrier resulting in weak
plasmid elimination [20]. Indirect evidence also supports
this, since the co-administration of verapamil (Ca
2+
-anta-
gonist) and trifluoperazine (intercalating plasmid curing
compound) resulted in a remarkable increase in plasmid
elimination [unpublished results].
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 13
The inhibition of plasmid replication resulted from a sin-
gle nick, outside of the replication origo of the superhelical
structure. The process leads to further relaxation of plasmid
DNA. Intercalation of the compounds was proved by an in-
crease in the melting point of the DNA and by circular di-
chroism (Fig. (3) [14]). When the native plasmid DNA and
its promethazine complex were analysed by agarose-gel
electrophoresis, the superhelical form was missing from the
promethazine treated plasmid DNA. The open circular and
linear forms of plasmid DNA were present in the pro-
methazine treated samples in increased proportions [42-44]
(Fig. (4)).
Fig. (3). Intercalation of the antiplasmid compound into the plasmid
DNS. (In: Waring, M.J . (1966) Symp. Soc. Gen. Microb. 16, 235-
265).
Plasmid replication can also be inhibited by blocking the
activity of the enzyme DNA gyrase. This time the introduc-
tion of superhelical turns into plasmid DNA is prevented.
The activity of DNA gyrase isolated from M. luteus was in-
hibited in the presence of promethazine and imipramine [45],
both compounds having plasmid curing effects. Apart from
general intercalation into dsDNA, a specific binding site for
phenothiazine intercalation was found in the GC-rich region.
[46, 47]. This means that the expression of the GC rich pro-
moter of the MDR-1 gene responsible for drug resistance of
cancer cells can be blocked simultaneously.
All of the tested plasmids were curable from E. coli
strains, except the E1 colicin [43, 45]. The simultaneous
elimination of Flac and pBR322 plasmid from E. coli also
showed relatively high frequency. It turns out that the curing
efficiency of phenothiazines depends also on the host cells,
since the same plasmid was curable with different frequen-
cies from various E. coli strains [44, 48]. The Ca
++
binding
protein encoding the plasmid responsible for the virulence of
Y. enterocolitica and Y. pseudotuberculosis isolates was
cured with rather low frequency [49].
The antiplasmid compounds were also able to inhibit
plasmid transfer [8, 50]. In these experiments E. coli r144drd
Kmr strain with depressed pilus synthesis was used as a do-
nor, and a chromosomally sodium azide resistant E. coli
strain was used as a recipient. A dose dependent inhibition of
conjugal plasmid transfer was observed [42] where both
transconjugal DNA synthesis and mating pair formations
were inhibited. The inactivation of sex pili was shown by
Fig. (4). The effect of the binding of ethidium bromide to SV40
DNA I and II. The upper part of the diagram presents three stages
in the reversible binding of dye to SV40 DNA I.
a) represents the dye-free molecule with 14 superhelical turns; the
addition of 420 molecules of ethidium bromide completely unwinds
the superhelical turns to form the relaxed molecule (b). The addi-
tion of a further 720 dye molecules leads to the formation of a posi-
tive superhelical molecule, shown in (c). The lower part of the dia-
gram shows three stages in the reversible binding of dye to SV40
DNA II, which remains relaxed throughout. (e) represents the re-
laxed, nicked molecule with the same number of dye molecules
bound as to the relaxed, intact molecules in (b). The arrows joining
(b) and (e) indicate that the nick may be introduced or repaired
without change of the 420 molecules of ethidium bromide bound.
The nicked molecule is nearly saturated, as shown in (f), with 1860
molecules of ethidium bromide bound. Introduction of a single-
strand scisson into I in the presence of a high dye concentration
results an irreversible unwinding of the superhelix. (In: Waring, M
(1970) J. Mol. Biol. 51, 247-279).
the inhibition of absorption of pilus specific phage [39, 40]
(Tables 5, 6).
ANTIPLASMID EFFECTS IN VIVO
The adhesion of urinary pathogen E. coli strains was in-
hibited in HEP2 tissue culture cells by inhibition of the adhe-
sive type I and p-fimbriae [51]. Based on these results the
synergistic effect of promethazine with gentamycin was
studied in children with frequently recurring pyelonephritis
[52] and in adults [53, 54]. In these experiments 10 children
14 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
Table 5. The Effect of Substituted Phenothiazines on R-Plasmid Transfer in E. coli Strains
No. of cells/mL
Compounds
Transconjugant x 10
4
Donor x 10
8
Recipient x 10
8
A) Phenothiazine derivatives
2-chloro-7-hydroxyphenothiazine 0.2 1.6 2.6
2-chloro-10-(2-dimethylaminoaethyl)phenothiazine 0.12 1.4 1.8
Promazine 0.30 1.0 1.9
DesdimethylCPZ 0.1 1.1 1.9
DesmethylCPZ 0.15 0.9 1.6
Chlorpromazine (CPZ) 0.2 0.8 1.4
CPZ-methylammoniumIodide 0.4 1.9 2.1
B) Hydroxy-, siloxy- and oxo-CPZ derivatives
7-hydorxyCPZ 0.15 0.85 1.2
3,7-dihydroxyCPZ 0.08 0.9 0.5
7,8-dihydroxyCPZ 0.07 0.8 0.77
7,8-dioxoCPZ 0.05 0.5 0.9
6,9-dihydroxyCPZ 0.07 1.5 0.9
6,9-dioxoCPZ 0.45 1.5 2.2
8-hydroxyCPZ 0.08 1.2 2.1
3,7,8-trihydroxyCPZ 1.9 1.7 1.8
CPZ-5-oxide 1.5 2.6 1.0
CPZ-5, N-dioxide 2.0 1.6 1.4
CPZ-N-oxide 0.05 1.3 1.1
7-(dimethyl-tertbutyl-siloxy)-CPZ 3.1 2.4 2.2
7,8-bis-(dimethyl-tertbutyl-siloxy)CPZ 3.5 2.8 1.9
6,9-bis-(dimethyl-tertbutyl-siloxy)CPZ 2.3 2.6 1.5
C) Thioalkyl-substituated phenothiazines
Thioridazine 0.8 0.8 2.2
Thiethylperazine 0.63 0.6 1.1
D) Phenothiazine enantiomers
Levomepromazine 0.09 1.2 2.2
Dextromepromazine 0.09 1.4 2.4
E) Phenothiazines with different side chains
Diethazine 0.25 1.9 2.1
Promethazine 0.18 1.3 1.6
Thiazinamine 0.95 1.5 2.1
Bis-(2-chlor-10--propyl)phenothiazine-,-glutamyl-amide 1.8 1.6 1.8
F) Dyes
Methylene blue 1.2 1.4 2.4
Tholuidine blue 1.9 1.7 2.9
Control 3.4 2.1 2.8
Concentration of thecompounds: 50% MIC. Thesamples wereincubated for 120 min. at 37C. Thedata aretheresults of two parallel experiments.
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 15
Table 6. The Effect of Non-Phenothiazine Tricyclic Compounds on R-Plasmid Transfer of E. coli Strains
No. of cells/mL
Compounds
Transconjugant x10
4
Donor x10
8
Recipient x10
8
A) Hydroxy- and siloxy derivatives of dibenzoazepines
Dezipramine (DMI) 0.02 0.75 1.9
DMIH (partially saturated DMI) 0.04 1.3 1.6
DMIH2 (saturated DMI) 2.5 1.8 2.1
Imipramine 0.23 0.8 1.4
Imipramine (saturated) 3.0 1.7 2.3
Imipramine methylammoniumiodide 0.7 1.5 2.4
2-hydroxyimipramine 0.1 1.0 1.7
3-chloro-8-hydroxyimipramine 0.05 1.05 1.5
3-chloro-8-dimethyl-tertbutyl-siloxyimipramine 0.38 1.5 2.4
B) Dibenzocycloheptane derivatives
Protriptyline 0.34 1.3 1.15
Amitriptyline 0.23 0.9 0.87
Amitriptyline methylammoniumiodide 0.7 1.4 2.3
C) Anthracene derivatives
1-dimethylamino-3-(1-anthryl)-3-propanol 0.3 0.4 0.9
1-dimethylamino-3-(2-anthryl)-3-propanol 0.06 0.9 0.65
1-dimethylamino-3-(9-anthryl)-3-propanol 0.08 0.8 1.7
Maprotiline 0.16 1.5 1.9
4,4-bis-dimethylaminodiphenylmethane 2.5 2.0 2.3
D) Thioxanthene derivatives
Chlorprothixene 0.6 1.3 1.8
Cis-clopenthixol 0.4 1.1 1.5
Trans-clopenthixol 0.1 0.9 1.5
E) Cannabis and phenanthryl derivatives

8
-tetrahydrocannabinol 3.0 1.5 1.9

9
-tetrahydrocannabinol 2.2 2.2 2.5
Cannabinol 1.7 1.9 2.0
Cannabidiol 1.5 1.8 1.5
Cannabidiolic acid 2.1 1.9 2.0
Morphine 3.0 1.9 1.8
1-dimethylamino-3-(9-phenanthryl)-3-propanol 1.2 2.0 1.9
F) Dyes
Ethidiumbromide 0.34 1.2 1.9
Acridine orange 0.12 0.6 0.9
Control 3.1 1.9 2.1
Concentration of thecompounds: 50% MIC. Thesamples wereincubated for 120 min. at 37C. Thedata aretheresults of two parallel experiments.
16 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
were given a combination of gentamycin and promethazine
for 7 days (Group l.). In the second group 11 children re-
ceived gentamycin alone and in the 3
rd
group 19 children
were on long-term oral antibiotic prophylaxis with episodes
of intensive treatment. Over a three year follow up period,
the number of pyelonephritis recurrences were significantly
lower in Group1 than in Groups 2 and 3. Plasmid elimination
was also studied by in vivo or ex vivo experiments, when the
plasmid profile of freshly isolated colonies from the urine of
promethazine and imipramine treated adult patients were
compared. A heterogenicity of antibiotic sensitivities and the
plasmid profile of bacteria isolated from promethazine or
imipramine treated patients was found after only five days
treatment with tricyclic psychopharmacons. These changes
were found in only a few percent of the E. coli colonies iso-
lated from the urine of treated patients, therefore it has to be
considered that plasmid elimination has no clinical impor-
tance on its own [43]. Among 50 treated patients, 12 had
significant bacteriuria, however 21 of the control 50 patients
also had significant bacteriuria. The resistance patterns of ten
different isolates from the urine of maprotiline treated pa-
tients changed after 5 days of treatment, when carbenicillin
and cefuroxime resistance was eliminated from three isolates
[52]. A similar in vivo curing was also found by Mehtar [55].
The in vitro effective concentrations of plasmid curing com-
pounds were found to be higher than the achievable serum
concentrations in patients, consequently the frequency of in
vivo plasmid curing was low. Synergism between antibiotics
and antiplasmid effect can be produced by complex mecha-
nisms.
STRUCTURE ACTIVITY RELATIONSHIPS
It has been proposed that the conjugated -electron sys-
tem of the tricyclic skeleton has a special importance where
the conjugation of the -electrons of the two aromatic rings
is symmetrical to the L-molecular region (Fig. (1)). It is rea-
sonable to conclude that the antibacterial and antiplasmid
effect noted with active phenothiazines or tricyclic non-
phenothiazines is dependent on the available -electrons and
their distribution.
Thus knowing the mechanism of plasmid curing, an ideal
plasmid curing or resistance modifier compound can be pro-
posed. The correlation between antiplasmid effects and
chemical structure of the curing compounds was studied. In
quantitative structure activity relationship (QSAR) studies it
was shown that the HOMO orbital energy, the symmetry of
the electron distribution to the L-molecular region and the
superdelocalizability of the electron system of tricyclic
skeleton on atoms 10, 12 and 13 were responsible for effec-
tive binding through stacking interaction to the biological
target eg. DNA or DNA gyrase. Based on the results of the
QSAR correlation [56-61] novel substituted anthranyl- com-
pounds were synthesized and found to possess remarkable
plasmid curing effects [62-64]. The antiplasmid action of
tricyclics on the other hand was found to be rather specific
and depended upon the chemical structures of the com-
pounds [14, 65].
Drug treatment of bacterial cells resulted in the inhibition
of plasmid replication and finally in the formation of plas-
mid-free cells. In order to analyze the mechanism of action at
the molecular level the effects of these drugs at several
points in the course of transformation, in plasmid DNA rep-
lication and the topological state of plasmid DNA was ex-
amined (Fig. (5)).
Fig. (5). Electrophoretic analysis of antiplasmid effects of pro-
methazine.
Samples:
1. E. coli HB101/pBR322 culture was treated with 250 g/mL pro-
methazine; 2. E. coli HB101/pBR322 culture was treated with 1000
g/mL promethazine; 3. E. coli HB101/pBR322 culture was treated
with 2000 g/mL promethazine; 4. E. coli HB101/pBR322 cells
were lysed with lysosyme and 250 g/mL promethazine was added
to the lysed cells; 5. E. coli HB101/pBR322 cells were lysed with
lysosyme and 1000 g/mL promethazine was added to the lysed
cells; 6. E. coli HB101/pBR322 cells were lysed with lysosyme and
2000 g/mL promethazine was added to the lysed cells; 7. pBR322
plasmid DNA; 8. pBR322 DNA was treated with Hind III at 37C
for 2 hours, which made the DNA linear; 9. pBR322 plasmid DNA.
a: linear; b: relaxed circular; c: covalently closed circular. (In: Mol-
nr J , Fldek S, Nakamura MJ , Rausch H, Domonkos K, Szab M.
(1992) APMIS Suppl. 30/100, 24-31).
Two possible target sites were identified in plasmid DNA
replication. One of them involved membrane binding sites,
the other one is in DNA replication. The other effect ob-
served in vivo and in vitro was the influence on the topologi-
cal state of plasmid DNA (Fig. 4). The presence of tricyclic
drugs promoted the relaxation of plasmid DNA by interfer-
ing with the supercoiling activity of DNA gyrase causing a
cessation in plasmid replication.
Therefore in order to improve drug design, some quan-
tum chemical parameters, including: electron superdelo-
calizibility, HOMO, LUMO orbital energies, size of the Van
der Waals surface in a watery environment were calculated
by several computer programs (MM2, CNDO) for phenothi-
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 17
azines, acridines and naphthyridines. Based on these results
new anthracene derivatives were synthesized [66-70]. In this
way the antiplasmid and carcinogenic molecular orbitals
were clearly differentiated [71, 72]. Based on the QSAR
studies, new compounds with well-defined and predictable
electronic structures in the molecular orbitals [31] were pre-
pared. The compounds possessed antiplasmid activity but
displayed no mutagenic or carcinogenic effects [59, 60]. The
binding affinity changes caused by promethazine and imi-
pramine indicated that the resulting plasmid elimination was
connected at least partly to membrane proteins and plasmid
DNA complexes (Fig. (5)).
Drugs may affect the binding affinity of replicating plas-
mid DNA to membrane proteins producing more stable
complexes that negatively interfere with the processing of
the replication fork and the expression of plasmid encoded
genes. This was shown for the F lac plasmid.
EVALUATION OF PLASMID CURING
Although bacterial resistance is different from eukaryotic
resistance in many respects there are common sensitive
points, such as transporter protein mediated efflux pump
systems. In this respect the mechanism of resistance in bacte-
ria, protozoa and tumour cells is similar and therefore it may
be possible to overcome it in a similar way.
In preliminary in vivo experiments it was shown that the
efficiency of plasmid curing was rather low and apparently
of little practical importance. However considering the exis-
tence of efflux pump inhibitors it was decided to check the
results of the interaction of plasmid curing compounds and
antibiotics in vitro. Among various plasmids, the hemolysin
and tetracyline transporter encoding plasmid was eliminated
from the bacteria. Detailed analysis of the plasmidless bacte-
ria showed that the MIC value for tetracyline was reduced in
the plasmidless bacterial cells. In addition the antibacterial
effect of tetracycline is synergized by plasmid curing com-
pounds. This was independent from the elimination of plas-
mid DNA [52]. This resistance modifying effect of selected
phenothiazines and structurally related compounds was
similar for all individual cells of the culture, showing that the
inhibition of drug efflux was able to increase the intracellular
concentration of chemotherapeutics in bacteria and cancer
cells. Plasmid elimination was shown to exist in microbial
eco-systems, however a number of clinical isolates were
resistant to many antibiotics and simultaneously had very
low sensitivity to high concentrations of the plasmid curing
compounds. Using mixed cultures, including plasmid bear-
ing bacterial cells (E. coli K12 LE 140), the conditions of a
polimicrobial flora were simulated. Plasmid elimination was
studied under various conditions, including co-inhabiting
bacteria, at various temperatures and in the presence of the
plasmid curing promethazine. It was established that plasmid
elimination from E. coli K12 LE 140 promoted by sub-
inhibitory concentrations of promethazine was significantly
exalted by elevation of temperature either in the monoculture
or when incubated together with either B. cereus or S. epi-
dermidis. The efficiency of plasmid elimination of phenothi-
azine was markedly enhanced by the presence of a second
species of bacteria [73].
Doxycycline resistant bacterial isolates from clinical
specimens were found to have a cell membrane that was im-
permeable to resistance modifying antiplasmid drugs. The
frequency of the elimination of tetracycline resistance was
low for the majority of strains investigated, despite the fact
that it has been shown that complexes are formed between
antiplasmid compounds and plasmid DNA. The results sug-
gest that the curing effects were dependent on the perme-
ability barrier of the clinical isolates studied. Promethazine
and trifluoperazine as well 9-aminoacridine have pronounced
plasmid curing activity in E. coli K12 LE 140 and these ef-
fects were substantially enhanced by administering the pro-
ton pump inhibitor trifluoromethyl ketone at concentrations
ranging from 0.05 to 1.0 mg/L. Thus it can be shown that
various type of resistance modifying drugs, such as calcium-
channel blockers or proton pump inhibitors, can enhance the
activity of plasmid curing drugs. The results suggest that
inhibitors of membrane ABC transporters and proton pumps
may be combined to produce plasmid curing in some antibi-
otic-resistant bacterial strains [20].
There is evidence for that the activity of plasmid medi-
ated haemolysin transporter in bacteria [74] and other bacte-
rial transport proteins have a close homology to mammalian
multidrug resistance transporters, the so called efflux pumps
[75]. This multidrug resistance mechanism was modified in
both bacteria and in cancer cells by antiplasmid compounds
[76]. The intracellular accumulation of antibiotics or che-
motherapeutics increases as a consequence of decreased an-
tibiotic efflux in both bacterial and tumour cell systems. The
inhibition of the efflux pump is the same for all individual
members of the population of bacterial and cancer cells,
however the antiplasmid effects occurred in only a small
fraction of the growing bacterial cell populations [76] but the
inhibition of exporter proteins can also be exploited to in-
crease plasmid curing.
IMPORTANCE OF PLASMIDS AND ABC TRANS-
PORTERS
Membrane transporters can be encoded by genes local-
ised on the chromosomes and on plasmids such as haemo-
lysin and tetracycline transporters [77, 78]. The multidrug
membrane transporters are classified into two main groups
such as ABC transporters and proton pump systems based on
energetic requirements and the second class is sub-divided
into a large number of subclasses.
Some transporters such as the tetracycline efflux protein
mediate the extrusion of the particular antibiotic. This active
efflux is important in ensuring a significant level of resis-
tance to tetracycline or other antibiotics. In contrast the tet
transporters are multidrug-transporters, which confer resis-
tance against a wide variety of structurally unrelated com-
pounds [79-82]. The mdr transporters can be inhibited a wide
variety of compounds such as uncouplers and calcium an-
tagonists .The proton motive force utilizing efflux pumps is
sensitive to compounds that dissipate the proton gradient in
the membrane. These pumps mediate the efflux of xenobiot-
ics in a coupled exchange with protons.
The majority of multidrug transporters use ATP as the
energy to pump the antibiotics out of the cells, while the sec-
ond largest groups of transporters utilize the transmembrane-
18 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
proton gradient to drive the antibiotics or other xenobiotics
out of the cells. Several subclasses belong into this group
[83-85, 89, 90]. Experiments suggested that drug resistance
by bacteria and cancer cells can be achieved in various ways,
however the inhibition of efflux pump systems is the most
promising mechanism in this respect because the intracellu-
lar concentration of antibiotics is enhanced in all individual
cells of the population simultaneously and at much lower
concentration of resistance modifier compounds than is
needed for plasmid elimination. This resistance modifying
effect is apparently independent from the antibacterial or
cytotoxic effects.
CONCLUSIONS
The aim of the study was to clarify the mechanisms of
action of the drugs on plasmid replication and to improve the
curing activity of existing compounds by computer aided
drug design in order to obtain effective plasmid curing com-
pounds. The ordered replication, partition and segregation of
plasmid DNA is essential, if they are not to be lost from their
host cells [42, 86].
From the mechanisms described above it is evident that
the spread of antibiotic resistance can be reduced at different
levels in a rather complex approach. Apparently one of the
simplest ways to reduce antibiotic resistance is to halt plas-
mid replication, partition and transfer by plasmid elimina-
tion. Consequently the co-administration of antibiotics and
resistance modifiers can be recommended in serious poly or
multidrug resistant infections as well as in multidrug resis-
tant cancer. The inhibition of the efflux-pump systems in the
membrane of bacteria or in cancer cells occurs simultane-
ously in all the individual cells of the resistant population,
therefore blockade of the efflux pumps as targets would ap-
pear to be an effective approach to combinational chemo-
therapy. The comparison of minimal inhibitory concentra-
tions of drug candidates measured on the haemolysin trans-
porter plasmid containing E. coli cells and on its plasmidless
derivative can be exploited for pre-screening particular re-
sistance modifiers (Fig. (6)) [23]. In addition, since plasmid
curing compounds destabilize the maintenance of extra
chromosomal elements in a fraction of bacterial population
Fig. (6). Schematic structure of the HlyA translocon complex.
(http://bmec1.igmors.u-psud.fr).
without mutagenic effect, the results can be exploited in or-
der to isolate plasmid free bacteria for biotechnology without
any risk of mutations.
The main goal of this study is to investigate ways of
combating antibiotic resistance by the selective inhibition of
plasmid replication and transfer and by blocking the bacterial
efflux pumps. The results of the model experiments on the
synergistic interactions between antibiotics and resistance
modifiers found in vitro can be exploited in the rational drug
design of drugs to counteract antibiotic resistant pathogens.
ACKNOWLEDGEMENTS
These studies were supported by the Szeged Foundation
for Cancer Research and Cooperation in Science and Tech-
nology, COST Action B16 "Reversal of Antibiotic Resis-
tance" at the European Commission.
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