Download Pseudomonas aeruginosa

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Plasmid mediated antibiotic resistance and pigment production in clinical
isolates of Pseudomonas aeruginosa.
Ahlam K. Al-Yasseen(PhD)*
Israa K. Al-Yasiri(Msc)**
Kifah J. Al-Yakobbi(Msc)***
Libna M. Abas(Bsc)
*Biological Department Education Collage for Girls Kufa University .
** Dentistry Collage Kufa University .
***Medicine Collage Kufa University.
Abstract:
Twelve’s multidrug resistant (MDR) isolates of Pseudomonas aeruginosa obtained from
hospitalized patients were selected for plasmid detection and conjugation experiments. These
isolates were also studied for plasmid mediated resistance and pigment production. All isolates
were found to harbor R plasmid, conjugation experiments showed that resistance to most
antibiotic and pigment production was plasmid mediated. It is suggest that plasmid should be
characterized in all MDR P. aeruginosa strains and nation wide antibiotic policy should be
made to minimize the emergence of drug resistance.
:‫الخالصــــة‬
‫تم عزل وتشخيص اثنا عشرة عزلة متعددة المقاومة للمضادات الحيوية من بكتيريا‬
‫ أجريت تجارب االقتران البكتيري لمعرفة دور البال زميدات‬. Pseudomonas aeruginosa ‫في انتقال صفة المقاومـة‬
‫ أبدت جميع العزالت امتالكها لبالزميدات المقاومة‬.‫للمضادات الحيوية وإنتاج الصبغة من الخاليا الواهبة إلى الخاليا المستلمة‬
‫ لذا ينبغي‬,ً‫وأظهرت نتائج االقتران البكتيري إن انتقال صفة المقاومة ألغلب المضادات الحيوية وإنتاج الصبغة كان بالزميديا‬
‫إجراء المزيد من الدراسات حول عزل وتشخيص البالزميدات في العزالت المتعددة المقاومة للمضادات الحيوية وإنتاج‬
.‫مضادات ذات فعالية أوسع لتقليل ظهور المقاومة للمضادات الحيوية‬
Introduction
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Pseudomonas aeruginosa is an important opportunistic pathogen that cause sever infection
such as septicemia in immunocompromised patient, cystic fibrosis and it is a common cause of
nosocomial infection(1).
Although antibiotics are though to be the most effective form of therapy against infections
caused by this microorganism , they are frequently ineffective due to its innate resistance to
various antimicrobial agents(2). Antibiotic resistance in bacteria has reach a near crisis point in
nosocomial health care, with many bacterial isolates now multiresistant as a result of the
acquition of additional DNA element. Resistance gene can occur on chromosomes, transferable
plasmid, transposons or jumping gene and specialized transposons called integrons that can
assemble multiple resistance genes in to cassette(3).
The relationship between antibiotic usage and emergence of resistance is complex and better
understood through mathematical modeling methods derived from population genetic , this
models show the frequency of antibiotic resistance affected by the incidence of treatment, the
probability of resistance given treatment, the duration of infections of patient, the degree of
reduction in competitive fitness of bacteria from treatment. Such models suggest that antibiotic
resistance emerges rapidly under antimicrobial selective pressure but degree of reduction in
resistance is proportional to the degree of reduction in drug consumption (4). The evolution of
multidrug resistant plasmid often involves a site specific integration of antibiotic- resistance
determinants(5).
Eelier studies have shown that genes for resistance markers do occur on plasmids and they can
be transferable(6-8), and most of them have demonstrated it by plasmid curing experiments
alone(9).
The pathogenicity of P. aeruginosa is largely caused by multiple bacterial virulence factors
and genetic flexibility enabling it to survive in varied environment. Some of these factors help
colonization, whereas other facilitate bacterial invasion. Most P. aeruginosa stains secrete
pyocyanin (N-methyl-1-hydroxyphenazine), the pigment that give blue-green color to the
bacterial colonies(10). High concentration of pyocyanin are detected in pulmonary secretion of
patient with cystic fibrosis where exerts a pro-inflammatory effect, disrupts the bronchial
epithelium and impairs ciliary function. Pyocynin also interferes with the antioxidant defense in
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the lung and facilitates oxidative damage to the lung epithelium through inhibition of catalase
activity(11).
In the present study we demonstrate the role of plasmid in distribution of multidrug-resistance,
pyocyanin production in P. aeruginosa.
Materials and methods:
 Bacteria:
P. aeruginosa isolates were obtained from urine, sputum, wound and burned patients and
were identified by conventional methods (12) on the basis of pigment production oxidase test,
glucose fermentation , hydrolysis of arginine and nitrate production.
 Antibiotic susceptibility test:
All isolates were analyzed for the presence of drug resistance by the method of Bauer et.al.(13),
on Mueller Hinton agar (HiMedia.) by using commercial available paper discs. The antibiotic
discs and their concentration used in this study are shown in Table 1. Escherichia coli MM294
rifr was used as quality control organism. Two multidrug resistance isolates were selected for
plasmid detection and conjugation experiments.

Plasmid profile:
Plasmid profile was carried out using the large scale alkaline lyses method as described by
Davis et.al.(14).plasmid samples were electrophoresed through 0.8% agarose (Sigma) in TBE
buffer at 100V, 60mA for 1hour by method of Portnoy et.al.(15). The gel was stained in 0.5µg/ml
of ethidium bromide solution.

Conjugation test:
Conjugation experiments were carried out according to Davis et.al.(14)by using P. aeruginosa
strain as donor and E. coli MM294 rifr which was sensitive to all the previously tested
antibiotics except rifampicin and non pigment production as recipient. The presence of plasmid
in transconjugant was checked through electrophoresis and the transconjugants were also tested
for pigment production and for each antibiotic resistance already recorded for the donor strains.
Table 1: Antibiotic susceptibility pattern of P. aeruginosa isolated from
patients by disc susceptibility testing .
hospitalized
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Antibiotic
β- Lactam antibiotic:
Ampiclox
Cephalothin
Oxacillin
Carbencillin
Clindamycin
Cephalosorine:
Cefotaxime
Ciprofloxacin
Cephalexin
Clarithromycin
Lincomycin
Aminoglycosides:
Amikacin
Neomycin
Kanamycin
Gentamicin
Sterptomycin
Quinolones:
Nalidixic acid
Others:
Chloramphenicol
TrimethoprimSulfamethoxazal
Rifampicin
Concentration
% of strains (n = 12 )
µg/disc Sensitive Intermediate Resistant
3
1
100
2
-
16.7
16.7
-
83.3
100
83.3
100
100
30
5
30
15
2
25
75
-
16.7
16.7
16.7
16.7
-
58.4
8.3
83.3
83.3
100
3
30
30
30
30
83.3
33.3
33.3
33.3
-
58.4
16.7
-
16.7
8.3
66.7
50.
100
30
33.3
-
66.7
5
33.3
16.7
25
66.7
58.3
5
-
8.3
91.7
Results:

Morphological and phenotypic characteristics:
Morphological and phenotypic characteristics were examined for each all of which were
consistent with the description of typical pseudomonad to MacFadden Manual for Systemic
Bacteriology(12), which describes the genus as being Gram-negative, non-spore forming, motile,
catalase and oxidase positive, straight or slightly curved rods. The strains gave positive results
for agrinine hydrolysis and the differ however in their respective pigment production,
characterized for P. aeruginosa by production of fluorescent pigment, particularly pyocyanin.
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
Antibiotic susceptibility tests and plasmid profile :
The results of antimicrobial susceptibility and pigment production of 12 isolates of P.
aeruginosa included in the present study are shown in Table 1. only two strains of multiple
antibiotic resistant P. aeruginosa isolates were examined for the presence of plasmid. These
Large plasmid bands
Chromosomal DNA
Small plasmid bands
Transconjugant
P. aeruginosa 30
Transconjugant
P. aeruginosa 50
E. coli MM294
isolates were found to harbor one to three plasmid bands (Fig 1).
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Fig
1: Agarose gel alectrophoresis of Pseudomonas aeruginosa original isolates and
transconjugants isolates.

Conjugation experiments:
Conjugation experiments were attempted on these isolates to determined changes in plasmid
content associated with antibiotic pattern and pigment production. Its was noted that (Table 2)
the transconjugants became resistance to most antibiotics used which confirm that all antibiotics
resistance determinants has been transferred form donor cell to the recipient cell. On the other
hand the genetic determinants of pigment production also has been transferred to the recipient
cell as shown in (Table 2) which revealed the ability of transconjugant isolates to produce
pyocyanin.
Agarose gel electrophoresis of the transconjugant showed the transferring of plasmid bands
form donor to the recipient cell (Fig 1).
Table 2: Antibiotic susceptibility pattern and pigment production of P. aeruginosa and
transconjugant strains.
Antibiotic
Pigment production
β- Lactam antibiotic:
Ampiclox
Cephalothin
50*
+
R
No. of isolates
50t**
3*
+
+
R
R
R
R
3t**
+
R
R
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Oxacillin
Carbencillin
Clindamycin
Cephalosorine:
Cefotaxime
Ciprofloxacin
Cephalexin
Clarithromycin
Lincomycin
Aminoglycosides:
Amikacin
Neomycin
Kanamycin
Gentamicin
Sterptomycin
Quinolones:
Nalidixic acid
Others:
Chloramphenicol
TrimethoprimSulfamethoxazal
Rifampicin
* Original stain of P. aeruginosa
I
R
R
R
R
R
R
R
R
R
R
R
I
S
R
R
R
25
S
R
R
R
I
S
R
R
R
R
S
R
R
R
S
S
S
S
R
S
S
S
S
R
S
R
R
I
R
S
R
R
R
R
S
S
R
R
S
I
S
R
R
R
R
R
R
R
R
R
** Trasconjugant strain.
Discussion:
P. aeruginosa is currently one of the most frequently nosocomial pathogen and the infections
due to this organism are often difficult to treat due to antibiotic resistance (16). The mechanisms
of resistance to antibiotics include reduced cell wall permeability, presence of lipopolysaccharid
in the outer membrane, production of chromosomal and plasmid mediated β- Lactamase ,
aminoglycoside -modifying enzymes and an active multidrug efflux mechanism(17).
In the present study a marked increase of resistant strains to various antimicrobial agents was
observed which corresponds with recent findings by others (18). Resistance to some antibiotics
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such as cefotaxim, neomycin and kanamycin showed increase in comparison with previous
studies(19), this might be due to the variation in the usage of antibiotics. The overall evolution of
antibiotic resistance can be attributed to various factors like spread of trasposons or R- plasmids
to various pathogens mainly because of the selective forces imposed by human due to the over
use of antibiotics. A review of the published research reveals over 40 known resistance gene to
contain the nucleotide sequences associated with the site specific recombination site, these
include gene encoding resistance to antiseptic, disinfectant and β - Lactamas (20).
The intermediate to low resistance that observed in our results may also be chromosomally
encoded or can possibly be on transposon, which might jump on to a plasmid that can be spread
to different species and genera. The relationship between certain plasmid and resistance to some
antibiotics has been reported previously (21).
Pigment production is contributory phenotypic characteristic in the classification of P.
aeruginosa . A results from selective plating identified isolates with the ability to produce
pyocyanin (blue-green) a redox-active metabolite, and this character can be transfer with
antibiotic resistance by conjugation. The pigment has been determined to display antibiotic,
antifungal and cytotoxic properties therefore contributes to the pathogenesis of P. aeruginosa as
a human pathogen and this is considered to be an infection associated virulence factor (22).
In conclusion we suggest that plasmids should be characterized in all MDR P. aeruginosa
strains and nation wide antibiotic policy should be made to minimize the emergence of drugresistance.
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