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During the last decades, in consequence of human activities, a great quantity and
variety of industrial and agricultural pollution, sludge, biocid chemicals, among them
xenobiotics (artifical and naturally foreign chemicals, e.g. pesticides), communal
waste, detergents, and not used or expired medicine, etc. have reached the biosphere
(the ecosystems, the lower soil, the waters and the air).
The survey and evaluation of the influence of the pollution in the environment on
living organisms, including microorganisms should be done with an exhaustive
analyses of the interaction among them. Primarily, the microorganisms have a
reducant role in the biosphere because of the reduction, the transformation and the
detoxication of the pollutants in the soil and water are the result of the activities of
microorganisms.
The essential first step of the nitrogen cycle for living organisms is the biological
fixing of the atmospheric nitrogen. Esclusevily prokaryote, free living or
microsymbiotic organisms (bacteria) that synthetise nitrogenase enzyme systems are
able to do this. Many of the genes that are responsible for symbiosis, nodulation of the
root and fixing nitrogen are located on the plasmids (extra chromosomic DNA).
Organic and inorganic xenobiotics that reach the soil can inhibit the appearance and
prevailance of the characteristics coded on these plasmids.
In a permanently polluted environment, soil and water, only microbes that became
resistant to the given pollutant can survive. These overtake the place and to some
extent the ecological function of the extenct sensitive organisms.
An enzyme system coded on the plasmids is often responsible for the resistance of
the organisms. The resistance (R)-plasmids can code resistance to several antibiotics
and heavy metals simultaniously. Bacteria can transmit the resistance coded on
plasmids not only to successive cells but also to other bacteria living in their
surroundings.
1
The aim of the investigation is to study the influence of different xenobiotics, e.g.
pesticides, unused medicine (polycondensed heterocyclic compounds) and mineral
salts on Bradyhrizobium, Rhizobium, Sinorhizobium and Escherichia coli strains
in vitro.
2
MATERIAL AND METHOD
Experiments were studied the effects of semi conductive salt, of heavy metals salts:
Ag, Hg (II), Cd, Co, Cu, Fe (II), Mn (II), Pb, Zn, and Se (IV), dithiocarbamate
derivatives (cineb and mankoceb), fenothiazine compounds (e.g. promethazine),
dibenzoazepine derivatives (timipramine) and monocyclic compounds with
tercieramine side chain on the following strains: E. coli (6), Bradyrhizobium sp.
(Acacia) (1), B. japonicum (2), Rhizobium leguminosarum bv. phaseoli (2),
R. leguminosarum bv. trifolii (1), R. leguminosarum bv. viciae (2), and Sinorhizobium
meliloti (2) in vitro.
The minimal inhihitory and bactericid concentration (MIC and MBC) of mineral
salts and organic compounds with broth dilution method were determined. E. coli
strains was cultured in MTY (minimal tripton-yeast extract) broth for 24 hours at
37 °C in aerob thermostat without airing. Rhizobium and Sinorhizobium strains were
cultured for 48 hours, Bradyhizobium strains for 72 hours at 28°C in aerob thermostat
without airing in YMB (yeast extract-mannit) broth.
The MIC value of antibiotics was determined by the help of microdilutive method
(Eliopoulus and Moellering 1991), and a semiquantitive „E-test” (White et al. 1996).
The interaction of antibiotics (ampicillin and gentamicin) and the resistance
changing to promethazine was determined by Checkerboard titration and the Growth
Rate Constant (GRC) was determined by „Time-Killing” method (Eliopoulus and
Moellering 1991).
The R-plasmids on E. coli and R. leguminosarum bv. trifolii strains were eliminated
with broth delutive method in the presence of different concentrations of imipramin.
Dilution was made row from the broth cultures from which was dispersed 0,1 mL on
MTY and YMA agar plates.
3
Replicas were made on plate medium containing antibiotics (with the help of sterile
velvet disk). After incubation, compared with the colonies appered were on master
plates without antibiotics and the colonies that became sensitive were marked
(Lederberg and Lederberg 1952).
The loss of R-plasmid of Rhizobium and Sinorhizobium strains (in presence of
0,5 x MIC HgCl2 or CuCl2) after heat treatment (4 hours at 60 °C or 1 hour at 70 °C)
was demonstrated according to Lederberg and Lederberg (1952) method.
The plasmid elimination of F’ lac was studied by broth delutive method in the
presence of different concentrations of three cyclic compounds. Then the colonies
holding plasmid (dark purple) were separated from the colonies that lost their plasmid
(pink) on eozin-methylene-blue (EMB)-lactose agar.
The elimination of F’lac and R-plasmids was demonstrated with agarose gel
electrophoresis by Birbain and Doly’s (1979, cit. Czirók 1999) alkalinic method.
The inhibition of R-plasmids transfer among E. coli strains was established presence
of three cyclic compounds with Beverly et. al’s (1981) method.
4
- Four seedlings were sowed in each dish and repeated the experiment
5 times.
- Culture of R. leguminosarum bv. phaseoli was added three times in each
pot in 5 mL YMB culture fluid, 100 mL of seedling culture solution was
added per pot that contained trace elements.
- 200 mL of solution of the different concentrations of the compounds
(promethazine and trimipramine) were poured on the perlite, which used
for the preparation of the seedlings as well.
- The plants were watered daily or every second day as needed with
tapwater (about 200-250 mL/pot).
The results of the experiment were evaluated after 6 weeks. The nodules were counted
on the roots of each plant (the ones bigger than 5 mm, the ones between 3-5 mm and
the ones smaller than 3 mm), roots were weighed and then dried until constant weight.
The results were evaluated with statistical methods.
5
RESULTS
1. Antibacterial effects
The MIC and MBC values of mineral salts, pesticides, polycondensed heterocyclic
compounds and monocyclic compounds with tercieramine side chain were
determined. Ag and Hg salts proved to be the most toxic among the mineral salts. The
MIC values were between 2 and 20 µM, the MBC values were laid between 5 and
40 µM. The Bradyrhizobium, Rhizobium and Sinorhizobium strains were more
sensitive to Ag+ ions than E. coli strains. In case of Hg2+ ions the tendency was
reversed.
The MIC values against Cd2+, Co2+, Cu2+, Ni2+ and Zn2+ ions were laid between 80
and 2500 µM. The E. coli K12 R144 drd+ strain proved to be the most resistant. The
MIC values were against Fe2+, Pb2+, Mn2+ and Se4+ ions were laid beteween 1250 and
10000 µM.
The toxic range of mineral salts in case of Bradyrhizobium, Rhizobium,
Sinorhizobium and E. coli strains showed deviation.
The MIC values of pesticides were between 320 and 10000 µM. Mankoceb proved
to be the most toxic.
Also, it was determined that 10 µM Fe2+ + 1 µM Mo6+ and 50 µM MgCl2 reduce
the antibacterial effect of the used metals salts and pesticides.
The antibacterial effect of phenothiazine derivatives and other polycondensed
heterocyclic compounds on E. coli K12 LE140 strain was examined. The strain
(E. coli K12 LE 140) had a medium or greater level of resistance against these
compounds. The MIC values were laid between 190 and 1406 µM. Monocyclic
compounds (lidocaine and procaine) had no antibacterial effect. In the antibacterial
sensitivity tests, the facultative pathogen E. coli and the symbiont N2 fixing strains
often showed resistance against 6 or 8 kinds of antibiotics. This indicates that
symbiont N2 fixing strains had also met antibiotics producing fungi and bacteria and
in effect resistance had grown in them. On the other hand they could take resistance
coding genes with plasmid transfer as well. The most sensitive was R. leguminosarum
bv. phaseoli (Bab 5/3) strain which resisted only 4 antibiotics. The most resistant
6
strains were S. meliloti 11, E. coli K12 R144 drd+ and E. coli K12 RP4, which carried
resistance against 10 antibiotics.
The interaction between antibiotics and fenothiazine derivatives was established and
the combination of ampicillin and prometazine had a synergic antibacterial effect on
E. coli K12 LE 140 strain. On the other hand the combination of gentamicin and
promethazine had an additíve effect on E. coli K12 LE 140 and R. leguminosarum bv.
phaseoli 6 strains.
The elimination of F،lac plasmids on E. coli K12 LE 140 strain was examined in the
presence of fenothiazine, dibenzoazepine and dibenzocycloheptane derivatives,
monocyclic compounds with tercieramine side chain and pesticides. The pesticides,
the procaine and the toluidin blue did not show a plasmid elimining effect. The other
compounds eliminated F’lac plasmid in 0.01-52 %.
R-plasmid elimination on E. coli and R. leguminosarum bv. trifolii strains was
studied in the presence of imipramine. The imipramine eliminated R-plasmids in
0.15-3.5 % and F’lac plasmid in 38.5 % from the cells.
Heat shock (60-70°C) and heat shock with heavy metal salts (HgCl2, CuCl2) caused
plasmid elimination (3.2-10.3 %) in Rhizobium and Sinorhizobium strains, which
could be demonstrated with agarose gel electrophoresis as well.
Akridin-orange and methylene blue significantly increased the F’lac plasmid
eliminative effect of dezipramine, chlorprotixene and triflupromazine. But the
presence of 50 mM Mg2+ inhibited significantly the F’lac plasmid elimination from
E. coli cells.
7
4. The inhibition of nodulation ability of R. leguminosarum bv. phaseoli in vivo
The effect of promethazine and trimipramine on the formation of root nudules and
root hair of beans (Phaseolus vulgaris L.) was examined with the pretreatment of the
seedling in vivo.
It can be established on the basis of the results that both compounds, the
promethazine in the concentration of 40 µg/mL (140 µM) and the trimipramine in that
of 60 µg/mL (204 µM), significantly inhibited the formation of root hair and root
nodules.
The results prove that the seedling is more sensitive to the two compounds than the
microsymbiont strain whose multiplication was not inhibited by these concentrations.
8
NEW SCIENTIFIC RESULTS
9
7. The fenothiazines and similar componds with three rings did not have an
influence on the formation of conjugation pairing (in the used concentration) but they
inhibited the process of plasmid transfer among E. coli cells. Some compounds that
did not have a plasmid eliminating effect, e.g. tiazinamine, toluidin blue, and
lidocaine were effective in inhibiting plasmid transfer.
8. It found that the interaction of stress factors (physical and chemical) increases
plasmid elimination. Following heat shock (4 hours at 60 °C, or 1 hour at 70 °C) or
heat shock and the presence of a small concentration (0.5 x MIC) of heavy metal salts
(Hg, Cu), the surviving cells of Sinorhizobium and Rhizobium strains could lose
1-2 plasmids at the same time, among them the megaplasmids which are essential in
forming symbiosis with plants.
9. It has been established that bean (Phaseolus vulgaris L.) seedling is sensitive to
40 µg/mL (140 µM) promethazine and 60 µg/mL (204 µM) trimipramine
concentration, which do not yet influence the multuplication of R. leguminosarum bv.
phaseoli strains. The compounds with three rings can inhibit the development of the
seedling, the multiplication of plant cells by forming chemical bonds at certain points
of the seedling. In consequence of this, the weight of dry root hair and the number of
nodules bigger than 5 mm and ones between 3-5 mm have decreased significantly.
10
CONCLUSION AND RECOMMENDATIONS
11
SUMMARY
12
The gentamicin-prometazine combination showed on additíve antibacterial effect on
both strains.
It was examined F’lac and R-plasmid elimination on E. coli strains in the presence of
fenothiazine derivatives, dibenzoazepine, dibenzocycloheptane and monocyclic
compounds with tercieramine side chain. It was established that the same compounds
can eliminate R-plasmids in 0.2-3.5 % but F’lac plasmids in 10 times higher percent.
The kanamycin-resistance of R. leguminosarum bv. trifolii was eliminated by
imipramine in 0.15 %. In Rhizobium and Sinorhizobium strains, heat shock caused a
plasmid loss.
Heat shock (60-70°C) or heat shock together with heavy metals (0.5 x MIC Hg or
Cu) could cause plasmid loss (in 3.2-10.3 %) in E. coli and Rhizobium strains.The
change in the plasmid profile could be seen compared to control.
Akridin orange and methylene blue significantly increased the F’lac plasmid
eliminative effect of dezipramine, chlorprotixene, trimipramine and triflupromazine.
50 mM Mg2+ inhibited F’lac plasmid elimination.
Promethazine, triflupromazine, dezipramine, chlorprotixene, trimipramine and two
more compounds: toluidin blue and tiazinamine (which did not have a plasmid
eliminative effect) inhibited successfully conjugation plasmid transfer among E. coli
strains.
It was examined the effect of promethazine and trimipramine on the formation of
nodules and root hair of the bean (Phaseolus vulgaris L.) with the pretreatment of the
seedling in vivo. The results show that both compounds, promethazine in the
concentration of 40 µg/mL (140 µM) and trimipramine in 60 µg/mL (204 µM)
inhibited significantly the formation of root hair and nodules thus N2 fixing as well.
13
References
14
PUBLICATIONS RELATED TO THE THESIS
Dissertation
Publications
Molnár, J., Domonkos, K., Földeák, S., Farkas, S., Manian, A. (1979): Effect of
influence factors to plasmid elimination of psychopharmacons.
The celldivision pharmacology, 8/2, 291-307.
Farkas, S., Molnár, J. (1979): Elimination of F’lac plasmid by different psycho-
tropic drugs and some related compounds.
Acta Microbiol. Acad. Sci. Hung. 26, 351-361.
Molnár, J., Schneider, B., Mándi, Y., Farkas, S., Holland, I. B. (1980): New
mechanism of plasmid curing by psychotropic drugs.
Acta Microbiol. Acad. Sci. Hung. 27, 309-315.
Farkas, S. (1993): The F’lac plasmid elimination and inhibitory of R144plasmid
tansfer in E. coli strains.
DATE Scientific Publications, Ed.: Mucsi, I. and Szabó, J. pp. 146-157.
Gunics, Gy., Motohashi, N., Amaral, L., Farkas, S. and Molnár J. (2000):
Interaction between antibiotics and non-conventional antibiotics on bacteria.
Int. J. Antimicrob. Agents. 14, 239-242.
IF: 1,141
Gunics, Gy., Motohashi, N., Molnár, J., Farkas, S., Kawase, M., Saito, S.,
Shah, A. (2001): Enhanced antibacterial effect of erythromycin in the presence of
3,5-dibenzoil-1,4-dihydropyridines.
Anticancer Res. 21, 269-273.
IF: 1,416
15
Farkas, S., Bayoumi Hamuda, H. A. E. F., Kecskés, M. Jr., Gunics, Gy.,
Kecskés, M. (2002): In vitro antibacterial activity of single, double and triple
combinations of metal salts on Rhizobium and Escherichia strains.
In preparation for publication.
Gunics, Gy., Farkas, S., Motohashi, N., Shah, A., Harsukh, G., Kawase, M.,
Molnár, J. (2002): Interaction between 3,5-diacetyl-1,4-dihydropiridines and
Proceedings
16
promethazine.
Scient. of Szabolcs-Szatmár-Bereg Country of Sci. Acad. Hung.
Nyíregyháza, Vol. 2, pp. 353-362. 2002.
Abstracts
Gunics, Gy., Farkas, S., Motohashi, N., Shah., A., Kawase, M., S. Saito, S.,
Amaral, L., Molnár, J. (2000): Dihydropyridines as resistance modifiers
in vitro on E. coli strains.
Third Europen Congress of Chemotherapy, Madrid, Spain, 8-9 May.
J. Antimicrob. Chemother. Suppl. A. pp. 85.
IF: 2.563
Farkas, S., Gunics, Gy., Hegedűs, A., Kecskés, M. (2000): Susceptibility of
Rhizobium and E. coli strains to different antibiotics.
Acta Microb. Immun. Hung. 47, pp. 308.
Detail of Book
17
Publishing of Agricultural. Budapest, pp. 312-325.
Farkas, S., Péntek, M., Gunics, Gy., Molnár, J. (1988) : Antiplasmid effect of silil-
substitualted benzoic acid derivatives.
Congress of the Hungarian Society for Microbiology, Kaposvár, Aug. 25-27.
1988.
Farkas, S., Gunics, Gy., Molnár, J. (1989) : Investigation of the penicilline-
resistance and beta-hemolysis loss on Staphylococcus aureus strains.
Congress of the Hungarian Society for Chemotherapy, Hajdúszoboszló,
10-13, May. 1989.
Farkas, S., Gunics, Gy., Molnár, J. (1989): Alteration of attribution of
Staphylococcus aureus strains by plasmid elimination compounds.
Congress of the Hungarian Society for Microbiology, Eger, 24-26, Aug. 1989.
Farkas, S., Hegedüs, A., Várady, Gy., Kecskés, M. (1996): Effect of some
xenobiotics on Escherichia coli, Rhizobium meliloti and R. leguminosarum
strains to reproduction.
Congress of the Hungarian Society for Microbiology, Nyíregyháza,
21-23, Aug. 1996.
Farkas, S., Gunics, Gy., Hegedüs, A., Kecskés, M. (1996) : Antibacterial effect
plasmid elimination and inhibition of R144 plasmid transfer by triflu-
promazine and trimipramine in Escherichia coli and Rhizobium strains.
Szabolcs-Szatmár-Bereg Country Academic Committee IX. International
18
Gunics, Gy., Farkas, S., Molnár, J. (1997) : Elimination of antibiotic resistance
from Acinetobacter calcoaceticus strains in the presence promethazine,
imipramine and SDS.
Congress of the Hungarian Society for Microbiology, Szekszárd, 25-27, Aug.
19
10-12, Oct. 2001.
Farkas, S., Bayoumi Hamuda, H. A. E. F., Kecskés, M: (2001): Antibacterial and
plasmid elimination effect of heavy metal and heat stresson Rhizobium and
Sinorhizobium strains.
Congress of the Hungarian Society for Microbiology,
Balatonfüred, 10-12. Oct. 2001.
Farkas, S., Bayoumi Hamuda, H. E. A. F., Kecskés, M, Jr., Gunics, Gy.,
Kecskés, M. (2002): Effect of ampicillin and gentamicin on E. coli,
Bradyrhizobium, Rhizobium and Sinorhizobium strains modified by
Promethazine.
Scientific of Szabolcs-Szatmár-Bereg Country of Sci. Hung. Acad.
Nyíregyháza, 29. Sept. 2002.
Farkas, S., Bayoumi Hamuda, H. A. E. F., Kecskés. M. (2002): The inhibition of
nodulation ability of Rhizobium leguminosarum bv. phaseoli on root of
beans (Phaseolus vulgaris L.) in vivo.
Congress of the Hungarian Society for Microbiology,
Balatonfüred, 8-10. Oct. 2002.
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