Study The Effects of Ethidium Bromide, Sds and Elevated Temperature On Stability of Multiple Antibiotic Resistances Plasmids of

Iraqi J. Biotech.
9(4): 797-811 (2010)
Rabab O. Radi and Fryad H. Rahman
STUDY THE EFFECTS OF ETHIDIUM BROMIDE, SDS

AND ELEVATED TEMPERATURE ON STABILITY OF
MULTIPLE ANTIBIOTIC RESISTANCES PLASMIDS OF
Pseudomonas aeruginosa
Rabab O. Radi1*
Fryad H. Rahman2
1*
Biology Department, College of Sciences, Babylon University

Biology Department, College of Sciences, Sulaimaniya University
Received 15/9/2009
Accepted 2/3/2010
ABSTRACT
Three clinical isolates of Pseudomonas aeruginosa were isolated and identified
from wound, burns and ear infections. They had multiple resistance to
Chloramphenicol, Erythromycin, Lincomycin, Tetracycline, Trimethoprime,
Amikacin, Neomycin, Rifampicin, Streptomycin, Pencillins and Cefalosporins
antibiotics. All isolates produced pyocyanin and two of them produced
extracellular proteases. The plasmid profile of the isolates appeared the presence
two small plasmid and single mega-plasmid bands on agarose gel electrophoresis.
Spontaneous curing experiment showed all plasmids are stable within bacterial
cells. The isolates appeared partial elimination of multiple antibiotic resistances
after treated with 700g/ml ethidium bromide or 1% SDS that indicates they
harbor more than one small plasmid had the same molecular size. While elevated
temperature (46C) is more efficient to cure all plasmids than chemical agents
,therefore the isolates became sensitive to all tested antibiotic except lincomycin.
All the curing experiments appeared no effect on lincomycin resistance gene and
protease encoding genes or cured them from bacteria that indicates these genes
may be located on bacterial chromosomal DNA.
Key words: Pseudomonas aeruginosa, Plasmid profile, Antibiotic resistance, Curing plasmid
*To whom correspondence should be addressed (E-mail: [email protected])
797
PDF created with pdfFactory Pro trial version www.pdffactory.com
)Iraqi J. Biotech. 9(4): 797-811 (2010
SDS

2
2
2009/9/15
2010/3 /2
Spontaneously curing
)700/( SDS%1
.

.
.
798
Iraqi J. Biotech. 9(4): 797-811 (2010)
INTRODUCTION
Pseudomonas is a gram negative, straight or curved but not helical, aerobic, single
cell, non-spore forming, motile by polar(monotrichous)flagella, forming pili . It belongs
to bacterial family Pseudomonadaceae(1).
P. aeruginosa is notorious for its resistance to antibiotics and is, therefore, a
particularly dangerous and dreaded pathogen; there is much attention paid to the study
of antibiotic resistance in P. aeruginosa. It maintains antibiotic resistance plasmid (Rfactor). These plasmids are transmissible to sensitive bacteria and make them acquire
resistance to antibiotics, and have the ability to genetic recombination through
conjugation, transformation, and transduction. Moreover, there are other genes
belonging to antibiotics resistance located on chromosome(2). Multidrug active efflux
systems have recently been recognized in a number of bacteria as efficient mechanisms
of resistance in P. aeruginosa, by which antibiotics are expelled from the cells by
membrane transporter proteins, the so-called drug-efflux pumps(3).
The bacterial cells may loss their plasmids during cell division; these types of cell
were said to be cured. Curing may occur naturally through cell division or by treating
the cells with chemical and physical agents(4). Plasmid curing agents imply the possible
involvement of extrachromosomal elements in the biosynthesis of secondary
metabolites. The DNA interacting agent ethidium bromide can be used to eliminate
certain plasmids at high efficiency. The plasmids are cured during cell division if the
plasmid has no portioning system, called par function. The copy number of plasmid is 4
after cell division and 8 before cell division. If the plasmids are equal segregated into
the two daughter cell, each will get four plasmids, but the plasmid usually will not be
equally distributed; then one daughter will get more than the other, with certain
probability, one cell will get all of the plasmids and the other cell will be cured. For
cells with one copy number such as F-plasmid, so the probability 1/2 of cells would be
cured during each generation(4).
There are number of reports demonstrating the ability of various chemical and
physical agents to increase the rate of loss of plasmid DNA from bacteria such as
ethidium bromide inhibits RNA and DNA polymerase, and intercalate between basepairs of DNA. Stanier et al.(5) reported that the elimination of plasmids by dyes and
other agents reflects the ability of such agents to inhibit plasmid replication at
concentration that does not affect the chromosome. Pseudomonas putida MCM B-408
capable of utilizing caprolactam (monomer of nylon-6)as the sole source of carbon and
nitrogen was found to harbor a single32-Kb plasmid, acridine orange, ethidium
bromide, mitomycin and sodium dodesyle sulfate(SDS) failed to cure the plasmid and
the phenotype changed, while elevated temperature alone(40C) was found to be
ineffective in curing phenotype, whereas the plasmid was cured at a frequency of 2.63%
when acridine orange and elevated temperature (40C) were used together.
Ingram et al.(6) found that drug resistance of P. aeruginosa could be eliminated from
RP1-containig strains by treatment with SDS, and Pattnakik et al.(7) found that acridine
orange could not affect P. aeruginosa due to impermeability of cell wall, while
ethidium bromide and SDS affected on curing antibiotic resistance plasmid at a
concentration of 1-2% and 700-3000g/ml for SDS and ethidium bromide respectively.
799
Iraqi J. Biotech. 9(4): 797-811 (2010)
Same observations were obtained when Al-Amir(8) treated isolate of P. aeruginosa

with acridine orange up to 1000g/ml, no cured cells were obtained and the plasmid
profile of cured cells was the same as untreated samples, and concluded that acridine
orange had no effect on Pseudomonas isolates as curing agent, while ethidium bromide
was effective at 600, 700 and 1000g/ml for the same isolates of Pseudomonas.
From all the previous data, Pseudomonas isolates appeared different responses to
chemical and physical agents as plasmid curing agents, therefore the present research
study the effect of curing agents (some chemical agents and elevated temperature) on
plasmid elimination from Pseudomonas isolates, in the other hand it was aimed to
confirm the location of resistance genes and protease encoding gene on the plasmid or
chromosome in order to perform cloning to protease encoding gene in the next research.
MATERIALS AND METHODS

Bacterial strains
Pseudomonas aeruginosa isolated from different human infections were transferred
to the laboratory and activated using brain heart infusion broth. After activation
inoculated on the MacConKey agar, asingle colonies were selected, for more
purification, inoculated on the selective medium cetrimide agar, and oxidase test was
done, positive isolates, and microscopically Gram negative rod shape, identified
provisionally as Pseudomonas aeruginosa, subcultured on nutrient agar slants, after
incubation at 37C for 24 hr, stored at 4C, till other bacteriological tests were done (9).
The api 20E Micro tube system (BioMerieux SA, Lyon, France) was used. This system
is a standardized, miniaturized version of conventional procedures for the identification
of Enterobacteriaceae and other Gram negative bacteria.
Antibiotic susceptibility test
Antibiotic susceptibility test by disk-diffusion method was performed according
Bauer et al. method (1966) that described in Baron and Finegold(10) and the results was
compared with standard inhibition zone according Wikler et al.(11).
Antibiotic resistance test by pour method was used to screen the genetic transfer of
antibiotic resistances in studied isolates was preformed according to Sambrook et
al.(12) and Baron and Finegold(10).
Total DNA extraction by salting out method
Total DNA content of P. aeruginosa isolates was extracted according to salting out
method (13). Plasmid DNA content was extracted by using alkaline lyses(12).
Agarose Gel electrophoresistechnique
Agarose Gel was prepared according to the method of Sambrook et al.(12); agarose
gel was prepared by using 0.7% agarose gel. The electrophoresis were run at 6volt/cm
for3hrs. The gels were illuminated with ultraviolet transilluminator, and then
photographed by digital camera.
Spontaneous curing
This method for plasmid curing in P. aeruginosa was described by Meyer(14) as
follows The 10ml of nutrient broth was inoculated by a single colony of P. aeruginosa
isolate, then incubated with shaking 100rpm at 37C for 24hr. Several dilutions were
prepared, and 0.1ml. of last three dilutions were spread on to nutrient agar plates, after
that the plates were incubated at 37C for 24hr.
800
Iraqi J. Biotech. 9(4): 797-811 (2010)
A master plate containing 100 colonies was made, then these colonies transferred to
different antibiotic agar plates and the results were recorded.The curing frequency was
calculated according to the following equation :
Frequency of Curing = No. of Curing cells/No. of plated cells
Curing with ethidium bromide
This method was described by Darfeuille-Michanol et al.(15), elimination of
antibiotics resistance plasmid DNA from P. aeruginosa isolates was done by Ethidium
Bromide, as follow: Ten ml of nutrient broth containing 700g/ml Ethidium Bromide
was inoculated with 0.3ml of overnight culture of P. aeruginosa isolates, incubated at
37C for 24 48 and 72hrs. Serial dilution was performed up to 10-7 by 0.1ml of interval
incubated samples, and 0.1ml of last three dilutions were plated on nutrient agar plates,
then all plates were incubated at 37C for 24hr. Several colonies were transferred for
plasmid DNA extraction and electrophoresis achieved to observe the loss of the
plasmids by gel electrophoresis techniques.
Curing of plasmid by sodium dodecyl sulfate
Plasmid curing by SDS was done by the method described by Tomoeda et al.(16), as
follows; Test tube containing 5ml of nutrient broth was prepared with adding of
appropriate antibiotic at final concentration (50g/ml), then inoculated with single
colony of P. aeruginosa isolates, and incubated at 37C for 24 hrs. The dilution was
prepared up to 10-3 dilution by nutrient broth containing (0.05%, 0.1%, 0.25%, 0.5%,
1%, 2% and 2.5%) (W/V) (SDS), then third dilution incubated at 37C for 24 hrs., serial
dilutions were prepared up to10-6, then 0.1ml of last three dilutions were spread on
nutrient agar plates that contained appropriate antibiotics and incubated at 37C for 24
hr, until the result appeared.
Plasmid curing by physical agents ( elevated temperature)
A single colony of P. aeruginosa isolate was inoculated into 10ml of nutrient broth,
after incubation at 37C for 24 hrs, then 0.2ml.of bacterial culture was inoculated to
10ml. of fresh nutrient broth, and incubated at elevated temperature from 20to 46C for
24hr with shaking 100rpm, after incubation time several dilutions were performed up to
10-7 dilutions, then 0.1ml. of last three dilutions were spread on plates of nutrient agar
which contain different antibiotics and incubated at 37C for 24hr. After that, the results
were recorded by the loss of ability of the tested bacteria to survive on the medium
which contains the antibacterial agents (17).
Selection of the cured bacterial cells
In all treatment of curing agents, master plates were prepared containing100 bacterial
treated colonies. Replica plating technique was used, in order to determine the cured
cells on to the nutrient agar plates containing the antibiotics separately for each the
isolate, and untreated cells used as control. The results were recorded, then selected the
curried cells and stored for next tests, to be sure that the plasmid was cured through
comparing with the original strains and plasmid DNA extracted from cured cells for
Agarose gel electrophoresis study (12).
801
Iraqi J. Biotech. 9(4): 797-811 (2010)
RESULTS AND DISCUSSION

Isolation and identification of Pseudomonas aeruginosa
Three isolates of P. aeruginosa were isolated from different human infections (ear,
wounds, and burns), from Teaching, and Emergency Hospitals in Sulaimaniya City. All
bacterial isolates were characterized selectively using cetrimide medium, cultural and
morphological characteristics. The colonies of P. aeruginosa isolates were studied using
nutrient agar plates and MacConkey's agar plates. They had fried-egg appearance,
smooth with flat edge and an elevated appearance. All of these isolates produce
pyocyanin (blue green pigment), which is in accordance with that is mentioned by
Todar(1). P. aeruginosa does not ferment lactose and is differentiated from lactose
fermenting bacteria (Enterobacteriaceae). Culture is the specific test for diagnosis of P.
aeruginosa infection. The bacterial cells from smear preparation are gram negative, rodshaped, and occur as single, in pairs, or in short chains, they regards P. aeruginosa,
which in accordance with previous observation (1,18). The bacterial colonies are able to
grow at 41C but not at 4C. These criteria are used for the identification of P.
aeruginosa from other species; this is in agreement with(18), who found that P
aeruginosa have the ability to grow at 41C and produce pyocyanin after growing on
cetrimide medium. Two of P. aeruginosa isolates produced extracellular proteases.
Furthermore, biochemical tests were performed to support the results above, using api
20E test which is the rapid accurate technique for the identification of the family
Enterobacteriaceae and Gram negative bacilli(19).
All the isolates were oxidase positive, which is regarded an important characteristics
for these bacteria and the identification of P. aeruginosa strains is usually based on
clinical morphology, oxidase positively, the presence of characteristics pigments, and
growth at 42C as described by Jawetz et al.(18).
Antibiotic resistance of P. aeruginosa isolates
All isolates show resistance to ampicillin, amoxicillin, carbencillin, chloramphenicol,
cefotaxime, erythromycin, lincomycin, penicillin, tetracycline and trimethoprim), while
they show variable resistance to amikacin, , neomycin, rifampicin and streptomycin and
sensitive to ciprofloxacin and gentamycin Table(1).
802
Iraqi J. Biotech. 9(4): 797-811 (2010)
Table (1): The resistance of P. aeruginosa isolates to antibiotics

Antibiotic
sensitivity
tests
Ap
Ak
Ax
Car
Cip
Cm
CTX
Ery
Gm
Lin
N
Pi
Rif
Sm
Tc
Tri
Isolation
source
Isolate No of P. aeruginosa
3
+
+
+
+
+
+
+
+
+
+
+
+
+
Ear
19
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Wound
27
+
+
+
+
+
+
+
+
+
+
Burn
-The symbols (+): Resistance to Antibiotics, (-): Sensitive to Antibiotics, and (I): intermediate.
-Ap: ampicillin, Ak: amikacin, Ax: amoxicillin, Car:carbencillin, Cip: ciprofloxacin, Cm:
chloramphenicol, Ctx: cefotaxime, Ery:erythromycin, Gm:gentamycin,Lin:lincomycin,N:neomycin,
Pi: penicillin, Rif: rifampicin, Sm: streptomycin, Tc: tetracycline, and tri: trimethoprim.
Originally P.aeruginosa harbor R-plasmid encoding multiple antibiotics resistances

(2).The epidemiology of drug resistance in the Enterobacteriaceae, Gramnegative
bacilli and some of the Gram-positive cocci undergo a remarkable change in character
with the widespread occurrence of resistance transfer factors (RTF). RTF may transfer
to drug-sensitive strains by conjugation in much the same way and with much the same
type of kinetics as F transfer in E. coli. Furthermore, RTF can act as sex factors in
promoting conjugation and transfer of chromosome(20).
The fluorinated quinolones, in particular ciprofloxacin, are still active against P.
aeruginosa. Resistance may nevertheless, emerge during long term treatment of chronic
infections. Resistance to other antibiotic including cephalosporins and
antipseudomonal antibiotics may also occur in future(21). Given this drug-resistant
nature of P. aeruginosa, it is important from a public health viewpoint to know whether
RTF can either occur in this species or be transferred to it from the Enterobacteria.
The plasmid profile of P. aeruginosa isolates
Electrophoresis characterization of total DNA and plasmid DNA content of P.
aeruginosa isolates obtained from different human infections were extracted by salting
out and carried out for migration using 0.7% agarose gel, at 50volt for 3hrs. All isolates
revealed that the presence one single large plasmid and two small plasmid bands that
indicated by agarose gel electrophoresis Figure (1).
Gabisoniia etal.(22)elucidated that plasmid size bearing antibiotic resistance
characteristics in P. aeruginosa ranged between (20-100) mega Dalton. Nordmann(23)
found that the size of plasmid ranged between (1.9-45.0) MD also reported that the size
of plasmid in the bacteria ranged between (4-80) Kbp.
803
Iraqi J. Biotech. 9(4): 797-811 (2010)
Curing experiments among different P. aeruginosa isolates

Spontaneous curing
Spontaneous curing of the plasmid DNA contents of P. aeruginosa P3, P19, and P27
isolates were performed according to Meyer(15). No spontaneously losses of antibiotic
resistance genes were obtained for any of the tested isolates that may be indicates the
antibiotics resistance plasmids in these isolates are segregate regularly and are stable
within them cells whether antibiotics present or absent. Hardy(24) reported that the
plasmid appeared to have evolved particularly in a genius way of increasing its stability,
through decreasing cell division. Snyder and Champness(4) explained that since cells
are seldom cured of even low-copy number plasmids, some mechanism must ensure that
plasmids, especially those with low copy numbers, will be partitioned faithfully into the
daughter cells each time the cell divides.
Curing with chemical physical agents
Three plasmid curing agents, Sodium dodecyle sulphate (SDS), Ethidium bromide and
elevated temperature were used to cure plasmid DNA that confer the antibiotic
resistance in the P. aeruginosa isolates.
Curing by Ethidium bromide
Ethidium bromide was used as a curing agent according to the method of which
described by Shahid and Malik(21). The minimal inhibitory concentration of ethidium
bromide was determined for the bacterial isolates in trypticase soy broth (TSB) and the
highest concentration permitting growth was used for plasmid curing.
Table(2) showed the effect of EB at concentration of 700g/ml as a curing agent to
DNA plasmid of P. aeruginosa P3, P19, and P27 isolates at different incubation times
(24, and 48 hrs). The result shows that the ethidium bromide had no effect on curing of
plasmid DNA carrying the ampicillin, chloramphenicol, erythromycin, lincomycin,
streptomycin and trimethoprim resistance genes, for all tested isolates, for different
incubation periods. Whereas the other antibiotics resistance characteristics are missing
from the isolates. The P. aeruginosa P3 isolate was missing its resistance to amikacin
and carbencillin and the P. aeruginosa P19 and P27 were missing their resistances to
carbencillin and tetracycline after EB treatment for24hrs. or more. That may be
indicated to the resistances to either amikacin and carbencillin or carbencillin and
tetracycline are encoded by small plasmids in P. aeruginosa isolates as appeared on gel
electrophoresis.
804
Iraqi J. Biotech. 9(4): 797-811 (2010)
Table (2): Screening of cured P.aeruginosa isolates by 700g/ml of

Ethidium bromide in different incubationperiods.
Antibiotic
antibiotics Resistance of cured
Antibiotics resistance of P. isolates
aeruginosa
24 hours 48 hours
isolates
P3 P19 P27 P3 P19 P27 P3 P19 P27
Ap
Ak
Car
Cm
Ery
Lin
Sm
Tc
Tri
Proteases
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-The symbols (+): Resistance to Antibiotics & proteases producer, (-):Sensitive to Antibiotics and
non proteases producer. *Ap: ampicillin, Ak: amikacin, Car: carbencillin, Cm: chloramphenicol,
Ery: erythromycin, Gm: gentamycin, Lin: lincomycin, Sm: streptomycin, Tc: tetracycline, and tri:
trimethoprim.
Elsewhere, ethidium bromide at different concentrations was unable to affect the

protease production genes after the two incubation periods; from the results, again we
conclude that the proteases gene may be located on chromosomal DNA of studied P.
aeruginosa isolates, and our results re-confirmed (agree) with that obtained by Guzzo et
al.(25).
In general, EB affect the plasmid DNA encoding to amikacin, carbencillin, and
tetracycline resistances with various rates, the antibiotic resistance genes may be located
on low copy number plasmid; this agrees with Keyser et al(26) who reported that low
copy number plasmid was efficiently cured by EB. The agents causing complete
inhibition of plasmid replication like Acridine orange and Ethidium bromide, intercalate
between base pairs in DNA. Furthermore, they suggested that differences in DNA
polymerase and RNA polymerase sensitive are responsible for differences in EB
sensitivity to bacterial strains due to differences in the rate of agent's penetration in
different strain of Enterobacteriaceae.
Rubins and Rosenblum(27)speculated that further exposure to EB the rate of
elimination decreased and resistance to EB increased, and resistance levels tended to
increase slightly after 24hrs of growth in EB. This finding agrees with our obtained
results. The previous Table(2) showed the plasmids carrying antibiotic resistance genes
were not eliminated with EB such as ampicillin, chloramphenicol, erythromycin, and
trimethoprim resistances for all tested isolates, and amikacin, streptomycin, and
tetracycline resistances for some isolates. This could be due to high copy number of
these plasmids in these isolates; this agree with that documented by Pallida et al.(28)
who demonstrated that the percent of cured plasmid DNA is not more than 20% in
optimal conditions in P. aeruginosa.
805
Iraqi J. Biotech. 9(4): 797-811 (2010)
Curing by sodium dodecyle sulphate (SDS)

Curing experiments with different concentrations of SDS were performed on the
Pseudomonal isolates to determine changes in plasmid content associated with
antibiotic resistance pattern.
Table(3)showed the effect of 1%(w/v) SDS as curing agent on the plasmid DNA of P.
aeruginosa P3, P19, P27 isolates with three incubation times 24, 48, and 72hrs. The
results demonstrated that all isolates are maintains their resistances to lincomycin and
erythromycin after 1% SDS treatment for three incubation periods, thats indicated their
encoding genes of resistances my be located on the mega plasmid or chromosomal
DNA. The P. aeruginosa P3 isolate appeared losing or missing its resistances to
amikacin, carbencillin, chloramphenicol, streptomycin and tetracycline after 24hrs of
SDS treatment. That may be indicated that the resistance genes my be located on the
two small plasmids at the same molecular weight as shown in a plasmid profile on
agarose gel electrophoresis Figure(1). One of them encoding amikacin and carbencillin
resistances comparison with the previous results of curing by ethidium bromide and the
other plasmid encoding to chloramphenicol, streptomycin and tetracycline resistances.
The P. aeruginosa P19 isolate appeared missing its resistances to amikacin,
carbencillin and streptomycin after 24hrs of SDS treatment. Subsequently, after 48hrs.
it was missing its resistance to ampicillin. That may be indicated its resistance genes
my be located on three small plasmids that had the same molecular size which appeared
on agarose gel electrophoresis. One of them encoding amikacin, resistance, the second
plasmid encoding ampicillin resistance and the third plasmid encoding carbencillin and
streptomycin resistances comparison with the previous results of curing by ethidium
bromide.
The P. aeruginosa P27 isolate appeared missing its resistances to ampicillin, and
trimethoprim after 24hrs. of SDS treatment. Subsequently, it was missing its resistance
to chloramphenicol after 48hrs. finally it is missing tetracycline and carbencillin
resistances after 72hrs. of treatment. That may be indicated its resistance genes my be
located on three plasmids, two of them had the same molecular size and the other large
plasmid which appeared on agarose gel electrophoresis. The first one encoding
ampicillin and trimethoprim resistances, the second encoding chloramphenicol
resistance and the third plasmid encoding tetracycline and carbencillin resistances that
correspond the missing plasmid after ethidium bromide treatment. Elsewhere, the ability
to protease production remains active inP19 and P27 on skimmed milk agar pates after
SDS treatment for 24,48, and 72hrs.of incubation period, the results indicate that the
genes responsible for protease production may be located on the chromosomal DNA of
P. aeruginosa isolates, and this results agree with these obtained by Guzzo et al.(25).
806
Iraqi J. Biotech. 9(4): 797-811 (2010)
Table(3):Screening of cured P.aeruginosa isolates by antibiotic resistance after 1%SDS

treatment for different incubation times.
Antibiotic
Screening of cured isolates by antibiotic resistance
Antibioti
resistance of
24hrs
48hrs
72hrs
cs
P. aeruginosa
isolates
P3
P19
P27
P3
P19
P27
P3
Ap
Ak
P19
P27
P3
P19
P27
Car
Cm
Ery
Lin
Sm
Tc
Tri
Proteases
-The symbols (+): Resistance to Antibiotics & proteases producer, (-): Sensitive to Antibiotics
& non proteases producer.- Ap: ampicillin, Ak: amikacin, Car: carbencillin, Cm: chloramphenicol,
Ery: erythromycin, Lin: lincomycin, Sm: streptomycin, Tc: tetracycline, and tri: trimethoprim.
Large plasmid
Chromosome
Small plasmid
Figure(1): The plasmid profile of P. aeruginosa isolates and cured isolates after
treated with 1 % Sodium Dodecyle Sulfate (SDS) for 72 hrs.
The DNA plasmid extracted by alkaline lyses (Kado and Liu, 1981)
and migrated on agarose gel 0.7%, 50 volt, for 6hr.
Lane 1: DNA content of cured P. aeruginosa P27 isolate
Lane 2: DNA content of proteases producing P. aeruginosa P27 isolate
Lane3 : DNA content of cured P. aeruginosa P3 isolate
Lane 5: DNA content of non-proteases producing P. aeruginosa P3 isolate.
Lane 6: DNA content of proteases producing P. aeruginosa P19 isolate.
807
Iraqi J. Biotech. 9(4): 797-811 (2010)
The results revealed that the P. aeruginosa isolates respond in different rate to 1%
SDS, and this may be related to the permeability through outer membrane, and to the
location of antibiotic resistance genes which carried on different plasmids. Sonstein and
Baldwin(29) elucidate that the effectiveness of SDS may be related to plasmid copy
number, or amount of enzyme which inactivate antibiotics.
Agarose gel electrophoresis in Figure(1) of the curried isolates shows that loosing
small plasmid in P. aeruginosa P3 and P. aeruginosa P19 isolates after1% SDS
treatment. This result documented that two mega plasmids remain after curing in both
P. aeruginosa P27 and P19 isolates. Furthermore, two plasmids among three plasmid
DNA were cured after using 1% SDS in P. aeruginosa P27 isolates during all
incubation times. The cured plasmid may be the R-plasmid which harbors most of
antibiotics resistance genes.
Adachi et al.(30) founded that SDS was only effective in elimination of sex (F) and Rplasmids in E. coli in a high frequency at concentration higher than 1%, and reported
that the longer incubation times (24 to 72 hrs.), higher the frequency of sensitive.
Curing by elevated temperature.
Elevated temperature (46C) was used to cure the plasmid DNA that confer resistance
to antibiotics from P. aeruginosa P3, P19, and P27 isolates.
The results showed that two of treated isolates (P. aeruginosa P3 and P27) appear
sensitive to all antibiotics except lincomycin Table (4) that indicated the isolates were
missing their plasmids as revealed that DNA contents of their cured cells on gel
Electrophoresis (Figure2) whereas the lincomycin resistance are encoded by
chromosomal gene. While P. aeruginosa P19 isolate maintains its resistance to
lincomycin as well as chloramphenicol, and trimethoprim.
Table (4): The effect of elevated temperature (46C) on elimination the plasmids of P. aeruginosa
isolates.
Antibiotics
Antibiotic susceptibility test of P.

aeruginosa isolates
Screening of cured isolates by antibiotic

susceptibility test
Ap
Ak
Car
Cm
Ery
Lin
P3
+
+
+
+
+
+
P19
+
+
+
+
+
+
P27
+
+
+
+
+
P3
+
P19
+
+
P27
+
Sm
Tc
Tri
Proteases
+
+
+
-
+
+
+
+
+
+
+
+
+
* The symbols (+): Resistance to Antibiotics & proteases producer, (-): Sensitive to Antibiotics &
non proteases producer.
* Ap: ampicillin, Ak: amikacin, Car: carbencillin, Cm: chloramphenicol, Ery: erythromycin, Gm:
gentamycin, Lin: lincomycin, Sm: streptomycin, Tc: tetracycline, and Tri: trimethoprim.
808
Iraqi J. Biotech. 9(4): 797-811 (2010)
That indicated this isolate miss its small plasmid only as showed that the DNA content
of its cured cells on agarose gel electrophoresis (Fig. 2) and the other resistances that
not losing may be encoded by plasmid or chromosomal genes. Elsewhere, the
production of proteases by the P. aeruginosa P19 and P27 isolates remain active after
expositing to elevated temperature (46C), the results indicated that the genes of
proteases production may be located on chromosomal DNA in P. aeruginosa isolates;
this agrees with that observed by Michael et al.(25).
11 2 2 3
34
45
56
Large plasmid
Chromosomal DNA
Small plasmids
Figure(2): The plasmid profile of P. aeruginosa isolates and cured isolates after
treated with elevated temperature ( 46C)
The DNA plasmid extracted by alkaline lyses (Kado and Liu, 1981) and migrated
on agarose gel 0.7%, 50 volt, for 2hrs.
Lane 1: DNA content of non-proteases producing P. aeruginosa P3 isolate.
Lane 2: DNA content of proteases producing P. aeruginosa P19 isolate.
Lane3: DNA content of proteases producing P. aeruginosa P27 isolate
From the obtained results, a conclusion can be made that curing by elevated
temperature is the most efficient method among others. This may be due to the fact that
the enzymes which contribute in the DNA replication processes are more affected by
this high temperature. This inactivation of these enzymes may be due to the change in
the folding of polypeptide at this temperature, i.e. the enzymes are sensitive to elevated
temperature(17).
Furthermore, enzymatic activity declines above the specific temperature that is
characteristic of the heat stability of the particular enzyme(24). However, plasmids
appear to be dependent on host enzymes for their replication, therefore, most of the
proteins synthesized during changing (converting) of temperature might be utilized for
cell division, by that, chance of plasmid replication decreases then curing occurred. The
results obtained by elevated temperature indicate that the genes which are located on the
chromosomal DNA of all tested isolates for example (proteases gene and Lincomycin
resistance gene) were not affected by high temperature comparing with that encoded by
plasmids DNA.
809
Iraqi J. Biotech. 9(4): 797-811 (2010)
Previously few studies have been performed on the effect of temperature on the DNA
synthesis and plasmid curing. Al-Amir(8) documented that there is a clear effect of
elevated temperature on P. aeruginosa isolates plasmids, which agree with the results of
the present study. Kheder(17) found that the 46C affected on the antibiotic resistance
plasmids DNA for four tested isolates and curing was obtained among them except for
the genes responsible for Lincomycin, because they are encoded chromosomally.
REFERENCES
1- Todar, K.(2004). Pseudomonas aeruginosa. University of Wisconsin-Madison.
Todar's online text book of bacteriology.
2- Foster, T. J.(1983). Plasmid-determined resistance to antimicrobial drugs and toxic
metal ions in bacteria. Microbiol. Rev., 47: 361-409.
3- Lomovskaya, O.; Warren, M. S.; Lee, A.; Galazzo, J.; Fronko, R.; Lee, M.; Blais, J.;
Cho, D.; Chamberland, S.; Renau, T.; Leger, R.; Hesker, S.; Hoshino, K.; Ishido,
H. and Lee, V. J.(2001). Identification and Characterization of Inhibitors of
Multidrug Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents
for Combination Therapy. J. Antimicrob. Chemoth., 45(1): 105-116.
4- Snyder, L. and Champness, W.(1997). Molecular Genetics of Bacteria. American
Society for Microbiology. ASM press Washington D.C.
5- Stanier, R.Y.; Adelberg, E. A. and Ingraham, J.L.(1984). General Microbiology. 4th
ed. The MacMailian Press LTD, London and Basingstoke.
6- Ingram, L.; Syker, R.B.; Grinsted, J.; Saunders, J. R. and Richmond, M. H.(1972). A
transmissible resistance element from a strain of P. aeruginosa containing no
detectable extrachromosomal DNA. J.Gene. Microbiol., 72: 269-279.
7- Pattnakik, S.; Rath, C. and Subramyam, V.(1995). Characterization of resistance of
essential oils in a strain of Pseudomonas aeruginosa VR-6. J. Microbiol., 81
(326): 29-31.
8- Al-Amir, L. A. K.(1999). Molecular study of virulence factor in P. aeruginosa. Ph.D.
thesis. College of Science. University of Baghdad. Iraq.
9- Holt, J. G.; Krieg, N. R.; Sneath, P. H. A.; Staley, J. T. and Williams, S. T.(1994).
Bergeys manual of determinative bacteriology. 9th edition. Lippincott William &
Wilkins.
10- -Baron, E. J. and Finegold, S. M.(1990). Bailey and Scott,s diagnostic
microbiology. 8th ed. The C.V. Mosby company. Missouri, 171-186, 363-376387-395-396-397-398-406.
11- Wikler, M. A.; Cockeril, F. R.; Dudley, M. N..; Eliopoulos, G. M.; Hecht, D. W.;
Hindler, J. F.; Low, D. E.; Sheehan, D. J.; Tenover, F. C.; Turnidge, J. C.;
Weinstein, M. P.; Zimmer, B. L.; Ferraro, M. J. and Swenson, J. M.(2006).
Performance standards for antimicrobial disk susceptibility tests. Approved
standard 9ed. Vol.26 Clinical and Laboratory Standard Institute.
12- Sambrook, J.; Fritsch, E. F. and Maniatis, T.(1989). Molecular Cloning: a
laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press. Cold Spring
Harbor, NY.
13- Pospiech, A. and Neuman, A.(1995). Preperation and analysis of genomic and
plasmid DNA. In: Genomic DNA isolation (ed. T. Kieserv) Johnines Center,
USA.
810
Iraqi J. Biotech. 9(4): 797-811 (2010)
14- Meyer, R.(1974). Alternate forms of the resistance factors R1 in Proteus mirabilis.
J. Bacteriol., 118(3): 1010-1019.
15- Darfeuille-Michand, A.; Forestier, C.; Joly, B. and Cluzel, R.(1986). Identification
of a nonfimbrial adhesive factor of an Enterotoxigenic Escherichia coli strain. J.
Infect. Immun., 52(2): 468-475.
16- Tomoeda, M.; Inuzuka, M.; Anto, S.; Konishi, M.(1974). Curing action of sodium
dodecyl sulfate on a Proteus mirabilis R+ strain. J. Bacteriol., 120(3): 11581163.
17- Kheder, A. K.(2002). Studies on antibiotic resistance by plasmid of Pseudomonas
aeruginosa. Ph.D. thesis. College of Education . University of Salahaddin, Iraq.
18- Jawetz, E.; Melinick, J. L. and Adelberg, E. A.(1998). Medical microbiology. 21th
ed. Lange Medical Pubilcation, Colifornia.
19- Kurlandsky, L. E. and Fader, R. C.(2000). In vitro activity of aminocycline against
respiratory pathogens from patients with cystic fibrosis. J. Pediatr. Pulmonol.,
29: 210-212.
20- Small, P. M.; Shafer, R.W. and Hopewell, P. C.(1993). Exogenous re-infection with
multidrug resistant Mycobacterium tuberculosis in patients with advanced HIV
infection. N. Engl. J. Med., 328: 1137-1144.
21- Shahid, M. and Malik, M.(2004). Plasmid mediated Amikacin resistance in clinical
isolates of Pseudomonas aeruginosa. Indian J. Med. Microbiol., 22(3): 182-184.
22- Gabisoniia, T. G.; Calushka, F. P. and Chaishvili, T. G.(1992). Conjugative R
plasmids isolated from hospital strains of Pseudomonas aeruginosa. J. Antibio.
Chemoth., 37(12): 39-41.
23- Nikaido, H.(1994). Prevention of drug access to bacterial targets: Permeability
barriers and active efflux. J. Science, 264: p 382-388.
24- Hardy, K.(1986). Bacterial plasmid.2ndEdition. American Society for Microbiology.
1913 street N. W. Washington D. C.(20006) USA.
25- Guzzo, J.; Murgier, M.; Filloux, A. and Lazdunski, A.(1990). Cloning of the
Pseudomonas aeruginosa Alkaline Protease Gene and Secretion of the Protease
into the Medium by Escherichia coli. J. Bacteriol., 172(2): 942948.
26- Keyser, H. H.; Bohlool, B. B.; Hu, T.S.; and Weber, D.F.(1982). Fast growing
Rhizobia isolated from root nodules of soybean. Science, 215: 1631-1632.
27- Rubins, S. j. and Rosenblum, E. D.(1971). Effect of the recipient strain and
ultraviolet irradiation on transduction kinetics of the penicillinase plasmid of
Staphylococcus aureus. J. Bacteriol., 108: 1192-1199.
28- Padilla, C.; Salazar, M. and Faundez, O.(1992). Range of action and Genetic
Bacteriocine codification of Pseudomonas aeruginosa isolated from three
different ecological niches. J. App. Bacteriol., 73(6): 497-500.
29- Sonstein, S. A. and Baldwin, J. N.(1972). Loss of the penicillinase plasmid after
treatment of Staphylococcus aureus with sodium dodecyle sulfate. J. Bacteriol.,
109: 262-265.
30- Adachi, H.; Nakano, M.; Inuzuka, M. and Tomoeda, M.(1972). Specific role of sex
pili in the effective eliminatory action of sodium dodecyle sulfate on sex and drug
resistance factors in Escherichia coli. J. Bacteriol., 109(3): 1114-1124.
811

Study The Effects of Ethidium Bromide, Sds and Elevated Temperature On Stability of Multiple Antibiotic Resistances Plasmids of

Uploaded by

Copyright:

Available Formats

Study The Effects of Ethidium Bromide, Sds and Elevated Temperature On Stability of Multiple Antibiotic Resistances Plasmids of

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Study The Effects of Ethidium Bromide, Sds and Elevated Temperature On Stability of Multiple Antibiotic Resistances Plasmids of

Uploaded by

Copyright:

Available Formats

Iraqi J. Biotech.

9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman