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Gabriella Spengler1,*, Annamária Molnár1, Zsuzsanna Schelz1, Leonard Amaral2, Derek Sharples3,
Joseph Molnár1
1
Institute of Medical Microbiology and Immunobiology, Albert Szent-Györgyi Medical Centre, University of Szeged,
Szeged, Hungary; 2Unit of Mycobacteriology, UPMM, Institute of Hygiene and Tropical Medicine, Universidade Nova
de Lisboa, Lisbon, Portugal and 3School 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 π-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.
Table 1. The MIC Values and the Plasmid Curing Effect of Phenothiazine Tricyclic Compounds on E. coli K12LE140 Strain
A) Phenothiazine derivatives
C) Thioalkyl-substituted phenothiazines
D) Phenothiazine enantiomers
F) Dyes
Table 2. The MIC Values and the Plasmid Curing Effect of Non-Phenothiazine Tricyclic Compounds on E. Coli K12LE140
B) Dibenzocycloheptane derivatives
C) Anthracene derivatives
D) Thioxanthene derivatives
∆ -tetrahydrocannabinol
9
3.3 0.01>
F) Fluorescent Dyes
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 F’lac strain
plasmid replication is dependent upon bacterial replication chlorpromazine ring. The plasmid curing activity of non-
and the number of subsequent generations (Table 3). phenothiazine tricyclic compounds is similar to that of the
substituted phenothiazines. The MIC values and antiplasmid
After incubation of the bacterium carrying the lac plas-
effects do not directly correlate but some of the compounds
mid with sub-inhibitory concentrations of the agents, ali-
were effective in sub-inhibitory concentrations. A large
quots of the culture are streaked onto drug-free medium
containing eosin-methylene blue (EMB). The EMB reagent number of compounds only have antibacterial activity with-
out any antiplasmid activity however some degree of anti-
aids in the identification of colonies that either contain or are
bacterial activity is essential for antiplasmid activity. The
deficient in the lac plasmid, showing deep violet (lac+) or
cannabinol derivatives are rather interesting with respect to
pink (lac -), respectively. The percentage of plasmid elimina-
plasmid curing activity which is present in tetrahydocan-
tion (curing) is determined by the number of pink colonies
nabidiolic acid, and to a lesser extent in cannabidiolic acid
divided by the total number of colonies (pink and deep vio-
and absent in cannabinol, tetrahydrocannabinols and can-
let) present on the surface of the agar plate. The majority of
nabidiol.
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 3.1µg/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 π-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- Fig. (1). Electronic Structure of the phenothiazine skeleton.
able to cross the bacterial cell membrane. The 7-hydroxy, 7,
8-dihydroxy and 7, 8-dioxo substituted CPZ derivatives also The substituted secondary amines, desipramine and pro-
appear to have lower plasmid curing effects than CPZ. Nei- triptyline, eliminated F’ lac plasmid at lower concentrations
ther the 6, 9-dihydroxy, 6, 9-dioxo, 5-oxide nor the 7, 8- and than did the substituted tertiary amines alimemazine and
6, 9-siloxy derivatives exert any significant antiplasmid ac- noxiptyline (Table 4). The majority of phenothiazines elimi-
tivity. These derivatives are considerably more polar than nated plasmid with a frequency similar to the antidepressants
chlorpromazine as indicated by the comparative ClogP (Table 4). Compounds such as chlorpromazine and imi-
values (ClogP: CPZ = +5.50, 7-OHCPZ = + 4.58, 7, 8- pramine may become attached through their cationic amine
DiOHCPZ = 2.06, 3, 7, 8-TriOHCPZ = 0.77). This would group to phospholipids in the cell membrane, while the hy-
imply that cell penetration is an important factor to be con- drophobic part of the molecule intercalates between the fatty
sidered in assessing plasmid curing activity. acid chains of the membrane. This would indicate that the
It can be concluded from these results that substitution in plasmid eliminating ability of a given compound may be a
the aromatic rings might prevent an interaction between the function of the membrane-lipid: water distribution coeffi-
π-electrons of the phenothiazine ring and target molecules cient. This can be substantiated by the fact that the distribu-
essential to plasmid replication. Structure 29 for example has tion coefficient for chlorpromazine is 1700 and for imi-
some marginal plasmid curing activity that might be related pramine is 2395. Conversely the distribution coefficient for
to the additional π electron cloud provided by the second lidocaine is only 17.
6 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
Phenothiazine derivative
1. 2-chloro-7-hydroxyphenothiazine 16. CPZ-5-oxide
HO S
O
S
N Cl
H N
N Cl
H3C CH3
H3C
N N+ Cl
O
CH3
CH3
S
S
N
N
N+
N O
H3C CH3 CH3
S
N
NH2 Cl
N+ Cl
O
CH3
N Cl
H3C CH3
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 7
(Fig. 2) contd….
Phenothiazine derivative
CH3 CH3
H3C Si CH3
S CH3
O
N
CH3 O S
H3C Si CH3 N
N Cl
H3C CH3 CH3 CH3
N Cl
H3C CH3
S S
N N
N S
N+ Cl
H3C CH3
H3C CH3
I- N
CH3
OH
S
S N
CH3
N
N
S
N Cl CH3
H3C CH3
OH
S
S N
N
CH3
OH N O
N Cl H3C CH3 CH3
H3C CH3
8 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
(Fig. 2) contd….
Phenothiazine derivative
10. 7,8-hydroxyCPZ 25. Dextromepromazine
OH
HO
S
S N
N
H3C
N O
N Cl H3C CH3 CH3
H3C CH3
N
S
N
N
H3C
N Cl H3C
H3C CH3
HO S
N
N
CH3
H3C N
N CH3
H3C CH3
CH3
H3C
N+
N Cl CH3
H3C CH3 H3C
S N
N
Cl
NH
N Cl O
H3C CH3 O
H2N
HN
Cl S
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 9
(Fig. 2) contd….
Phenothiazine derivative
OH N
HO
H3C CH3
N S+ N
S CH3 CH3
N
OH
N Cl
H3C CH3
N CH3
H3C
N S NH2
CH3 Cl
Non-phenothiazine derivatives
32. Desipramine (DMI) 46. 1-dimethylamino-3-(9-anthryl)-3-propanol
CH3
N
H
N
HO
CH3
N
CH3
CH3
N
H
N
N
H3C CH3
CH3
N
H H3C CH3
N N N
CH3 CH3
10 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.
(Fig. 2) contd….
Non-phenothiazine derivatives
35. Imipramine 49. Chlorprothixene
CH3
N
N CH3
S
N CH3
H3C
Cl
CH3
N
N CH3
S
OH
CH3 OH
N+
CH3
N H3C N
I-
CH3 CH3
CH3
N O
N CH3
OH
H3C OH CH3
Cl CH3
CH3 CH3
N O
N CH3
H3C OH CH3
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 54. Cannabinol
Cl CH3
CH3 CH3
N O
N CH3
H3C OH CH3
H3C CH3
O
H3C Si
H3C CH3
CH3
CH3
N
H OH
H2C CH3
HO
CH3
CH3
CH3
N
OH O
CH3
OH
H2C CH3
HO
CH3
CH3
N+ O
CH3
H3C -
I
N
CH3
CH3
CH3
N
N CH3
CH3 OH
HO
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 59. Ethidium bromide
H3C
N CH3 CH3
OH
H2N NH2
CH3 CH3
N N N
H3C CH3
Fig. (2). Molecular structures of agents examined for antimicrobial and plasmid curing activity.
The plasmid curing activity of promethazine and tri- tiplasmid compounds was responsible for the weak plasmid
fluoperazine was investigated by replica plating [32] on curing effect in these clinical isolates. It is probable that the
plasmid-mediated doxycycline resistant bacterial strains iso- cell wall/cell membrane served as a barrier resulting in weak
lated from clinical specimens. The frequency of plasmid plasmid elimination [20]. Indirect evidence also supports
elimination was low for the majority of the tested strains, this, since the co-administration of verapamil (Ca2+-anta-
despite the formation of complexes between antiplasmid gonist) and trifluoperazine (intercalating plasmid curing
compounds (promethazine, 9-aminoacridine) and plasmid compound) resulted in a remarkable increase in plasmid
DNA isolated from the plasmid containing clinical strains. It elimination [unpublished results].
may be suggested that inefficient penetration of the an-
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- transconjugal DNA synthesis and mating pair formations
gle nick, outside of the replication origo of the superhelical were inhibited. The inactivation of sex pili was shown by
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)).
No. of cells/mL
Compounds
Transconjugant x 104 Donor x 108 Recipient x 108
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-methylammonium Iodide 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-γ-propyl)phenothiazine-α,γ-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 the compounds: 50% MIC. The samples were incubated for 120 min. at 37°C. The data are the results 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 x104 Donor x108 Recipient x108
B) Dibenzocycloheptane derivatives
C) Anthracene derivatives
D) Thioxanthene derivatives
∆ -tetrahydrocannabinol
9
2.2 2.2 2.5
F) Dyes
were given a combination of gentamycin and promethazine the molecular level the effects of these drugs at several
for 7 days (Group l.). In the second group 11 children re- points in the course of transformation, in plasmid DNA rep-
ceived gentamycin alone and in the 3rd group 19 children lication and the topological state of plasmid DNA was ex-
were on long-term oral antibiotic prophylaxis with episodes amined (Fig. (5)).
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. Fig. (5). Electrophoretic analysis of antiplasmid effects of pro-
methazine.
STRUCTURE ACTIVITY RELATIONSHIPS
Samples:
It has been proposed that the conjugated π-electron sys-
1. E. coli HB101/pBR322 culture was treated with 250 µg/mL pro-
tem of the tricyclic skeleton has a special importance where
methazine; 2. E. coli HB101/pBR322 culture was treated with 1000
the conjugation of the π-electrons of the two aromatic rings
µg/mL promethazine; 3. E. coli HB101/pBR322 culture was treated
is symmetrical to the L-molecular region (Fig. (1)). It is rea-
with 2000 µg/mL promethazine; 4. E. coli HB101/pBR322 cells
sonable to conclude that the antibacterial and antiplasmid
were lysed with lysosyme and 250 µg/mL promethazine was added
effect noted with active phenothiazines or tricyclic non-
to the lysed cells; 5. E. coli HB101/pBR322 cells were lysed with
phenothiazines is dependent on the available π-electrons and
lysosyme and 1000 µg/mL promethazine was added to the lysed
their distribution.
cells; 6. E. coli HB101/pBR322 cells were lysed with lysosyme and
Thus knowing the mechanism of plasmid curing, an ideal 2000 µg/mL promethazine was added to the lysed cells; 7. pBR322
plasmid curing or resistance modifier compound can be pro- plasmid DNA; 8. pBR322 DNA was treated with Hind III at 37°C
posed. The correlation between antiplasmid effects and for 2 hours, which made the DNA linear; 9. pBR322 plasmid DNA.
chemical structure of the curing compounds was studied. In
a: linear; b: relaxed circular; c: covalently closed circular. (In: Mol-
quantitative structure activity relationship (QSAR) studies it
nár J, Földeák S, Nakamura MJ, Rausch H, Domonkos K, Szabó M.
was shown that the HOMO orbital energy, the symmetry of
(1992) APMIS Suppl. 30/100, 24-31).
the π electron distribution to the L-molecular region and the
superdelocalizability of the π electron system of tricyclic Two possible target sites were identified in plasmid DNA
skeleton on atoms 10, 12 and 13 were responsible for effec- replication. One of them involved membrane binding sites,
tive binding through stacking interaction to the biological the other one is in DNA replication. The other effect ob-
target eg. DNA or DNA gyrase. Based on the results of the served in vivo and in vitro was the influence on the topologi-
QSAR correlation [56-61] novel substituted anthranyl- com- cal state of plasmid DNA (Fig. 4). The presence of tricyclic
pounds were synthesized and found to possess remarkable drugs promoted the relaxation of plasmid DNA by interfer-
plasmid curing effects [62-64]. The antiplasmid action of ing with the supercoiling activity of DNA gyrase causing a
tricyclics on the other hand was found to be rather specific cessation in plasmid replication.
and depended upon the chemical structures of the com-
Therefore in order to improve drug design, some quan-
pounds [14, 65].
tum chemical parameters, including: π electron superdelo-
Drug treatment of bacterial cells resulted in the inhibition calizibility, HOMO, LUMO orbital energies, size of the Van
of plasmid replication and finally in the formation of plas- der Waals’ surface in a watery environment were calculated
mid-free cells. In order to analyze the mechanism of action at 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 Doxycycline resistant bacterial isolates from clinical
new anthracene derivatives were synthesized [66-70]. In this specimens were found to have a cell membrane that was im-
way the antiplasmid and carcinogenic molecular orbitals permeable to resistance modifying antiplasmid drugs. The
were clearly differentiated [71, 72]. Based on the QSAR frequency of the elimination of tetracycline resistance was
studies, new compounds with well-defined and predictable low for the majority of strains investigated, despite the fact
electronic structures in the molecular orbitals [31] were pre- that it has been shown that complexes are formed between
pared. The compounds possessed antiplasmid activity but antiplasmid compounds and plasmid DNA. The results sug-
displayed no mutagenic or carcinogenic effects [59, 60]. The gest that the curing effects were dependent on the perme-
binding affinity changes caused by promethazine and imi- ability barrier of the clinical isolates studied. Promethazine
pramine indicated that the resulting plasmid elimination was and trifluoperazine as well 9-aminoacridine have pronounced
connected at least partly to membrane proteins and plasmid plasmid curing activity in E. coli K12 LE 140 and these ef-
DNA complexes (Fig. (5)). fects were substantially enhanced by administering the pro-
ton pump inhibitor trifluoromethyl ketone at concentrations
Drugs may affect the binding affinity of replicating plas-
ranging from 0.05 to 1.0 mg/L. Thus it can be shown that
mid DNA to membrane proteins producing more stable
complexes that negatively interfere with the processing of various type of resistance modifying drugs, such as calcium-
the replication fork and the expression of plasmid encoded channel blockers or proton pump inhibitors, can enhance the
genes. This was shown for the F’ lac plasmid. activity of plasmid curing drugs. The results suggest that
inhibitors of membrane ABC transporters and proton pumps
EVALUATION OF PLASMID CURING may be combined to produce plasmid curing in some antibi-
otic-resistant bacterial strains [20].
Although bacterial resistance is different from eukaryotic
There is evidence for that the activity of plasmid medi-
resistance in many respects there are common sensitive
ated haemolysin transporter in bacteria [74] and other bacte-
points, such as transporter protein mediated efflux pump
rial transport proteins have a close homology to mammalian
systems. In this respect the mechanism of resistance in bacte-
multidrug resistance transporters, the so called efflux pumps
ria, protozoa and tumour cells is similar and therefore it may [75]. This multidrug resistance mechanism was modified in
be possible to overcome it in a similar way.
both bacteria and in cancer cells by antiplasmid compounds
In preliminary in vivo experiments it was shown that the [76]. The intracellular accumulation of antibiotics or che-
efficiency of plasmid curing was rather low and apparently motherapeutics increases as a consequence of decreased an-
of little practical importance. However considering the exis- tibiotic efflux in both bacterial and tumour cell systems. The
tence of efflux pump inhibitors it was decided to check the inhibition of the efflux pump is the same for all individual
results of the interaction of plasmid curing compounds and members of the population of bacterial and cancer cells,
antibiotics in vitro. Among various plasmids, the hemolysin however the antiplasmid effects occurred in only a small
and tetracyline transporter encoding plasmid was eliminated fraction of the growing bacterial cell populations [76] but the
from the bacteria. Detailed analysis of the plasmidless bacte- inhibition of exporter proteins can also be exploited to in-
ria showed that the MIC value for tetracyline was reduced in crease plasmid curing.
the plasmidless bacterial cells. In addition the antibacterial
effect of tetracycline is synergized by plasmid curing com- IMPORTANCE OF PLASMIDS AND ABC TRANS-
pounds. This was independent from the elimination of plas- PORTERS
mid DNA [52]. This resistance modifying effect of selected
Membrane transporters can be encoded by genes local-
phenothiazines and structurally related compounds was
similar for all individual cells of the culture, showing that the ised on the chromosomes and on plasmids such as haemo-
inhibition of drug efflux was able to increase the intracellular lysin and tetracycline transporters [77, 78]. The multidrug
membrane transporters are classified into two main groups
concentration of chemotherapeutics in bacteria and cancer
such as ABC transporters and proton pump systems based on
cells. Plasmid elimination was shown to exist in microbial
energetic requirements and the second class is sub-divided
eco-systems, however a number of clinical isolates were
into a large number of subclasses.
resistant to many antibiotics and simultaneously had very
low sensitivity to high concentrations of the plasmid curing Some transporters such as the tetracycline efflux protein
compounds. Using mixed cultures, including plasmid bear- mediate the extrusion of the particular antibiotic. This active
ing bacterial cells (E. coli K12 LE 140), the conditions of a efflux is important in ensuring a significant level of resis-
polimicrobial flora were simulated. Plasmid elimination was tance to tetracycline or other antibiotics. In contrast the tet
studied under various conditions, including co-inhabiting transporters are multidrug-transporters, which confer resis-
bacteria, at various temperatures and in the presence of the tance against a wide variety of structurally unrelated com-
plasmid curing promethazine. It was established that plasmid pounds [79-82]. The mdr transporters can be inhibited a wide
elimination from E. coli K12 LE 140 promoted by sub- variety of compounds such as uncouplers and calcium an-
inhibitory concentrations of promethazine was significantly tagonists .The proton motive force utilizing efflux pumps is
exalted by elevation of temperature either in the monoculture sensitive to compounds that dissipate the proton gradient in
or when incubated together with either B. cereus or S. epi- the membrane. These pumps mediate the efflux of xenobiot-
dermidis. The efficiency of plasmid elimination of phenothi- ics in a coupled exchange with protons.
azine was markedly enhanced by the presence of a second
The majority of multidrug transporters use ATP as the
species of bacteria [73].
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 without mutagenic effect, the results can be exploited in or-
out of the cells. Several subclasses belong into this group der to isolate plasmid free bacteria for biotechnology without
[83-85, 89, 90]. Experiments suggested that drug resistance any risk of mutations.
by bacteria and cancer cells can be achieved in various ways,
The main goal of this study is to investigate ways of
however the inhibition of efflux pump systems is the most
combating antibiotic resistance by the selective inhibition of
promising mechanism in this respect because the intracellu- plasmid replication and transfer and by blocking the bacterial
lar concentration of antibiotics is enhanced in all individual efflux pumps. The results of the model experiments on the
cells of the population simultaneously and at much lower synergistic interactions between antibiotics and resistance
concentration of resistance modifier compounds than is modifiers found in vitro can be exploited in the rational drug
needed for plasmid elimination. This resistance modifying
design of drugs to counteract antibiotic resistant pathogens.
effect is apparently independent from the antibacterial or
cytotoxic effects.
ACKNOWLEDGEMENTS
CONCLUSIONS These studies were supported by the Szeged Foundation
for Cancer Research and Cooperation in Science and Tech-
The aim of the study was to clarify the mechanisms of
nology, COST Action B16 "Reversal of Antibiotic Resis-
action of the drugs on plasmid replication and to improve the
tance" at the European Commission.
curing activity of existing compounds by computer aided
drug design in order to obtain effective plasmid curing com-
REFERENCES
pounds. The ordered replication, partition and segregation of
plasmid DNA is essential, if they are not to be lost from their [1] Carlos, F., Cuevas, A. and Chicure, M.E. (1992) Cell, 70, 189-199.
host cells [42, 86]. [2] Amaral, L., Viveiros, M. and Molnar, J. (2004) In Vivo, (In Press).
[3] Ordway, D., Viveiros, M., Leandro, C. and Amaral, L. (2003)
From the mechanisms described above it is evident that Antimicrob. Agents Chemother., 47, 917-922.
the spread of antibiotic resistance can be reduced at different [4] Amaral, L., Kristiansen, J.E., Thomsen, V.F. and Markowich, B.
(2000) Int. J. Antimicrob. Agents, 14, 225-229.
levels in a rather complex approach. Apparently one of the [5] Amaral, L., Kristiansen, J. E., Abebe, L.S. and Millet, W. (1996) J.
simplest ways to reduce antibiotic resistance is to halt plas- Antimicrob. Chemother., 38, 1049-1053.
mid replication, partition and transfer by plasmid elimina- [6] Amaral, L., Kristiansen, J.E. and Lorian, V. (1992) J. Antimicrob.
tion. Consequently the co-administration of antibiotics and Chemother., 30, 556-558.
[7] Amaral, L. and Lorian, V. (1991) Antimicrobial Agents Che-
resistance modifiers can be recommended in serious poly or mother., 35, 1923-1924.
multidrug resistant infections as well as in multidrug resis- [8] Csiszár, K. and Molnár, J. (1992) Anticancer Res., 12, 2267-2272.
tant cancer. The inhibition of the efflux-pump systems in the [9] Martins, M., Bleiss, W., Marko, A., Ordway, D., Viveiros, M.,
membrane of bacteria or in cancer cells occurs simultane- Leandro, C., Pacheco, T., Molnar, J., Kristiansen, J.E. and Amaral,
ously in all the individual cells of the resistant population, L. (2004) In Vivo, (In Press)
[10] Kristiansen, M.K., Leandro, C., Ordway, D., Martins, M., Viveiros,
therefore blockade of the efflux pumps as targets would ap- M., Pacheco, T., Kristiansen, J.E. and Amaral, L. (2003) Int. J. An-
pear to be an effective approach to combinational chemo- timicrobial Agents, 22, 250-255.
therapy. The comparison of minimal inhibitory concentra- [11] Ordway, D., Viveiros, M., Leandro, C. and Amaral, L. (2002) J.
tions of drug candidates measured on the haemolysin trans- Infect. Chemother., 8, 227-231.
[12] Davies, J. (1994) Science, 264, 375-382.
porter plasmid containing E. coli cells and on its plasmidless [13] Shaw, K.J., Rather, P.N., Hare, R.S. and Miller, G.H. (1993) Mi-
derivative can be exploited for pre-screening particular re- crobiol. Rev., 57, 138-163.
sistance modifiers (Fig. (6)) [23]. In addition, since plasmid [14] Barabás, K. and Molnár, J. (1980) Acta Microbiol. Acad. Sci.
curing compounds destabilize the maintenance of extra Hung., 27, 55-61.
chromosomal elements in a fraction of bacterial population [15] Jacobs, M.R., Anon, J. and Appelbaum, P.C. (2004) Clin. Lab.
Med., 24, 419-453.
[16] Nikaido, H. (1994) Science, 264, 382-388.
[17] Hiroshi, N. (2001) Cell Develop. Biol., 12, 215-223.
[18] Li, X.Z., Nikaido, H. (2004) Drugs, 64, 159-204.
[19] Pages, J.M. (2004) Med. Sci., (Paris), 20, 346-351.
[20] Spengler, G., Miczak, A., Hajdu, E., Kawase, M., Amaral, L. and
Molnar, J. (2003) Int. J. Antimicrob. Agents, 22, 223-237.
[21] Epstein, B.J., Gums, J.G. and Drlica, K. (2004) Ann. Pharma-
cother., 38, 1675-1682.
[22] Levy, S. (1992) Antimicrob. Agents Chemother., 36, 695-703.
[23] Molnar, J., Hever, A., Fakla, I., Fischer, J., Ocsovski, I. and
Aszalos, A. (1997) Anticancer Res., 17, 481-486.
[24] Kaatz, G.W., Moudgal, V.V., Seo, S.M. and Kristiansen J.E. (2003)
Antimicrob. Agents Chemother., 47, 719-726.
[25] Kristiansen, M.M., Leandro, C., Ordway, D., Martins, M.,
Viveiros, M., Pacheco, T., Kristiansen, J.E. and Amaral, L. (2003)
Int. J. Antimicrob. Agents, 22, 250-253.
[26] Hendricks, O., Butterworth, T.S. and Kristiansen, J.E. (2003) Int. J.
Antimicrob. Agents, 22, 262-264.
[27] Viverios, M., Portugal, I., Bettencourt, R., Victor, T.C., Jordaan,
A.M., Leandro, C., Ordway, D. and Amaral, L. (2002) Antimicrob.
Agents. Chemother., 46, 2804-2810.
[28] Molnar, J., Mandi, Y. and Kiraly, J. (1976) Acta Microbiol. Acad.
Fig. (6). Schematic structure of the HlyA translocon complex. Sci. Hung., 23, 45-54.
(http://bmec1.igmors.u-psud.fr).
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 19
[29] Molnar, J., Schneider, B., Mandi, Y., Farkas, S. and Holland, I.B. [59] Molnár, J., Petöfi, Sz., Kurihara, T., Sakagami, H. and Motohashi,
(1980) Acta Microbiol. Acad. Sci. Hung., 27, 309-315. N. (1993) Anticancer Res., 13, 263-266.
[30] Molnar, J., Mandi, Y. and Foldeak, S. (1982) Acta Microbiol. [60] Molnár, J., Sakagami, H. and Motohashi, N. (1993) Anticancer
Acad. Sci, Hung., 29, 17-25. Res., 13, 1019-1026.
[31] Molnar, J., Galfi, M., Lozsa, A. and Nakamura, M.J. (1984) Res. [61] Molnár, J. (1990) in Electropharmacology, (Keyzer, Gutmann, and
Commun. Chem. Pathol. Pharmacol., 43, 235-249. Eckert eds.) CRC, pp. 205-219.
[32] Lederberg, J. and Lederberg, E.M. (1952) J. Bact., 63, 399-406. [62] Molnár, J., Földeák, S., Hegyes, P., Schneider, B. and Holland, I.B.
[33] Molnar, A., Amaral, L. and Molnar, J. (2003) Int. J. Antimicrob. (1979) Biochem. Pharmacol., 28, 261-265.
Agents, 22, 217-222. [63] Molnár, J. (1997) Acta Microbiol. Immunol. Hung., 44, 21-26.
[34] Kawase, M., Motohashi, N., Sakagami, H., Kanamoto, T., Naka- [64] Molnár, J. and Schneider, B. (1978) Acta Microbiol. Acad. Sci.
shima, H., Ferenczy, L., Wolfard, K., Miskolci, C. and Molnar, J. Hung., 25, 291-298.
(2001) Int. J. Antimicrob. Agents, 18, 161-165. [65] Farkas, S. and Molnár, J. (1979) Acta Microbiol. Acad. Sci. Hung.,
[35] Spengler, G., Molnar, A., Klausz, G., Mandi, Y., Kawase, M., 26, 351-361.
Motohashi, N. and Molnar, J. (2004) Int. J. Antimicrob. Agents, 23, [66] Motohashi, N., Sakagami, H., Kurihara, T., Csúri, K. and Molnár,
631-633. J. (1992) Anticancer Res., 12, 135-140.
[36] Molnar, A., Wolfard, K., Kawase, M., Motohashi, N. and Molnar, [67] Molnár, J. and Földeák, S. (1994) Gyógyszerészet., 38, 1-7.
J. (2004) In Vivo, 18, 505-507. [68] Molnár, J. (1999) Orvosi. Hetilap., 140, 2155-2160.
[37] Molnar, J., Molnar, A., Spengler, G. and Mandi, Y. (2004) Acta [69] Molnár, J., Béládi, I. and Holland, I.B. (1978) Genet. Res. Camb.,
Microbiol. Immunol. Hung., 51, 333-349. 31, 197-201.
[38] Spengler, G., Molnar, A., Klausz, G., Mandi, Y., Kawase, M., [70] Molnár, J., Csiszár, K., Nishioka, I. and Shoyama, Y. (1986) Acta
Motohashi, N. and Molnar, J. (2004) Acta Microbiol. Immunol. Microbiol. Hung., 33, 221-231.
Hung., 51, 351-358. [71] Tanaka, M., Wayda, K., Molnár, J., Párkányi, C., Aaron, J-J. and
[39] Mándi, Y., Molnár, J., Holland, I.B. and Béládi, I. (1976) Genet. Motohashi, N. (1997) Anticancer Res., 17, 839-842.
Res. Camb., 26, 109-111. [72] Motohashi, N., Kawase, M., Saito, S., Miskolci, C.S., Berek, L. and
[40] Molnár, J., Király, J. and Mándi, Y. (1975) Experientia, 31, 444- Molnár, J. (1999) Anticancer Res., 19, 5075-5078.
445. [73] Molnár, A., Amaral, L. and Molnár, J. (2003) Int. J. Antimicrob.
[41] Molnár, J., Batho, N., Csik, V., Chevalier, J. and Cremieux, A. Agents, 22, 217-222.
(1993) Acta Microbiologica Hung., 40, 91-99. [74] Gerlach, J.H., Endicott, J.A., Juranka, P.F., Henderson, G.,
[42] Molnár, J. (1988) Find. Exp. Clin. Pharmacol., 10, 467-474. Deuchars, K.I. and Ling, V. (1986) Nature, 324, 485-489.
[43] Molnár, J., Földeák, S., Nakamura, M.J., Rausch, H., Domonkos, [75] Gross, P., Croop, J. and Housman, D. (1986) Cell, 47, 371-390.
K. and Szabó, M. (1992) APMIS Suppl., 30, 24-31. [76] Molnar, J., Hevér., A Fakla, I., Fischer, J., Ocsovszki, I. and
[44] Molnár, J., Mándi, Y. and Földeák, S. (1982) Acta Microbiol. Aszaklos, A. (1997) Anticancer Res., 17, 481-486.
Acad. Sci. Hung., 29, 17-25. [77] Rosenberg, E.M., Ma, D. and Nikaido, H. (2000) J. Bacteriol., 182,
[45] Molnár, J., Bathó, N., Csík, V., Chevalier, J. and Cremieux, A. 754-1756.
(1995) Acta Microbiol. Immunol. Hung., 42, 277-285. [78] Baranova, N.N. and Neyfakh, A.A. (1997) Antimicrob. Agents
[46] Miskolci., Cs., Labádi, I., Kurihara, T., Motohashi, N. and Molnár, Chemother., 41, 1396-1398.
J. (2000) Int. J. Antimicrob. Agents, 14, 243-247. [79] Lewis, K., Hooper, D.C. and Ouellette, M. (1997) ASM News, 63,
[47] Kurihara, T., Motohashi, N., Kobayashi, H., Yamanaka, W., Do- 605-610.
hyashki., S-I. and Molnár, J. (1998) Anticancer Res., 18, 3493- [80] Nikaido, H. (1996) J. Bacteriol., 178, 5853-5859.
3498. [81] Paulsen, I.T., Brown, M.H. and Skurray, R.A. (1996) Microbiol.
[48] Szabó, M., Molnár, J., Bánfalvi, Zs. and Motohashi, N. (1992) Rev., 60, 575-608.
Anticancer Res., 12, 1667-1670. [82] van Veen, H.W., Callaghan, R., Soceneantu, L., Sardini, A.,
[49] Molnár, J. and Nakamura, M.J. (1988) Acta Microbiol. Hung., 35, Konigs, W.N. and Higgins, C.F. (1998) Nature, 391, 291-295.
309-314. [83] Paulsen, I.T., Skurray, R.A., Tam, R., Saier, M.H. Jr., Turner, R.J.,
[50] Mándi, Y. and Molnár, J. (1981) Acad. Sci. Hung., 28, 205-210. Weiner, J.H., Goldberg, E.B. and Grinius, L.L. (1996) Mol. Micro-
[51] Molnár, J., Mucsi, I. and Kása, P. (1983) Zbl. Bakt. Hyg. I. Abt. biol., 19, 1167-1175.
Orig. A., 254, 388-396. [84] Brown, M.H., Paulsen, I.T. and Skuarry, R.A. (1999) Mol. Micro-
[52] Molnár, J., Haszon, I., Bodrogi, T., Martonyi, E. and Turi, S. biol., 31, 394-395.
(1990) Int. J. Urol. Nephrol., 22, 405-411. [85] Saier, M.H. Jr., Tam, R., Reizer, A. and Reizer, J. (1994) Mol.
[53] Kásler, M., Molnár, J., Ágoston, E. and Poczik, M. (1982) Urol. Microbiol., 11, 841-847.
Nephrol. Szle., 9, 135-138. [86] Molnár, J., Domonkos, K., Mándi, Y., Földeák, S. and Holland,
[54] Kásler, M., Molnár, J. and Poczik, M. (1982) Urol. Nephrol. Szle ., I.B. (1980) in: Phenothiazines and Structurally related Drugs, Ba-
9, 130-133. sic and Clinical Studies; (Usdin, Eckert, and Forrest eds.) Elsevier
[55] Methar, S., Blakemore, P.D. and Ellis, K. (1987) Amer. J. Med., 82 North Holland Inc., pp. 115-116.
55-57. [87] Brennan, R.G. (2001) Sem. Cell Develop. Biol., 12, 201-204.
[56] Molnár, J. (1986) Doctoral Thesis Szeged. [88] Orlowski, S. and Garrigos, M. (1999) Anticancer Res., 19, 3109-
[57] Molnár, J., Gálfi, M., Lózsa, A. and Nakamura, M.J. (1984) Res. 3124.
Commun. Chem. Pathol. Pharmacol., 43, 235-249. [89] Putman, M., van Veen, H.W. and Konigs, W.N. (2000) Microbiol.
[58] Molnár, J., Földeák, S., Nakamura, M.J., Gaizer, F. and Gutmann, Mol. Biol. Rev., 64, 672-693.
F. (1991) Xenobiotica, 21, 309-319. [90] Alekshun, M.N. and Levy, S.B. (1997) Antimicrob. Agents Che-
mother., 41, 2067-2075.