STUDY THE EFFECTS OF ETHIDIUM BROMIDE ...

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Sep 15, 2009 - temperature (46ºC) is more efficient to cure all plasmids than chemical agents. ,therefore the isolates became sensitive to all tested antibiotic ...
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*

2

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 700µg/ml ethidium bromide or 1% SDS that indicates they harbor more than one small plasmid had the same molecular size. While elevated temperature (46ºC) 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])

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‫)‪Iraqi J. Biotech. 9(4): 797-811 (2010‬‬

‫‪Rabab O. Radi and Fryad H. Rahman‬‬

‫ﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭﺍﺕ ﺒﺭﻭﻤﻴﺩ ﺍﻻﺜﻴﺩﻴﻭﻡ ﻭﻤﺎﺩﺓ ‪ SDS‬ﻭﺩﺭﺠﺔ ﺍﻟﺤﺭﺍﺭﺓ ﺍﻟﻤﺭﺘﻔﻌﺔ ﻓﻲ ﺜﺒﺎﺕ‬ ‫ﺒﻼﺯﻤﻴﺩﺍﺕ ﺍﻟﻤﻘﺎﻭﻤﺔ ﺍﻟﻤﺘﻌﺩﺩﺓ ﻟﻠﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ ﻟﺒﻜﺘﺭﻴﺎ‬ ‫‪Pseudomonas aeruginosa‬‬ ‫‪2‬‬

‫‪1‬‬

‫ﻓﺮﯾﺎد ﺣﻤﺪ رﺣﻤﻦ‬

‫رﺑﺎب ﻋﻤﺮان راﺿﻲ‬ ‫‪1‬ﻗﺴﻡ ﻋﻠﻭﻡ ﺍﻟﺤﻴﺎﺓ‪ ،‬ﻜﻠﻴﺔ ﺍﻟﻌﻠﻭﻡ‪ ،‬ﺠﺎﻤﻌﺔ ﺒﺎﺒل‬

‫‪2‬ﻗﺴﻡ ﻋﻠﻭﻡ ﺍﻟﺤﻴﺎﺓ‪ ،‬ﻜﻠﻴﺔ ﺍﻟﻌﻠﻭﻡ‪ ،‬ﺠﺎﻤﻌﺔ ﺍﻟﺴﻠﻴﻤﺎﻨﻴﺔ‬ ‫ﺍﻹﺴﺘﻼﻡ ‪2009/9/15‬‬

‫ﺍﻟﻘﺒﻭل‪2010/3 /2‬‬

‫ﺍﻟﺨﻼﺼﺔ‬ ‫ﻋﺯﻟﺕ ﻭﺸﺨﺼﺕ ﺜﻼﺙ ﺴﻼﻻﺕ ﺴﺭﻴﺭﻴﺔ ﻤﻥ ﺒﻜﺘﺭﻴﺎ ‪ Pseudomonas aeruginosa‬ﻤﻥ ﺃﺨﻤـﺎﺝ ﺍﻟﺠـﺭﻭﺡ‬

‫ﻭﺍﻟﺤﺭﻭﻕ ﻭﺍﻷﺫﻥ‪ .‬ﺇﻤﺘﻠﻜﺕ ﺍﻟﻌﺯﻻﺕ ﺍﻟﻤﻘﺎﻭﻤﺔ ﺍﻟﻤﺘﻌﺩﺩﺓ ﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﻜﻠﻭﺭﺍﻤﻔﻴﻨﻜﻭل ﻭﺍﻻﺭﺜﺭﻭﻤﺎﻴﺴﻴﻥ ﻭﺍﻟﻠﻨﻜﻭﻤﺎﻴﺴﻴﻥ‬ ‫ﻭﺍﻟﺘﺘﺭﺍﺴﺎﻴﻜﻠﻴﻥ ﻭﺍﻟﺘﺭﺍﻤﺜﺒﺭﻡ ﻭﺍﻷﻤﻴﻜﺎﺴـﻴﻥ ﻭﺍﻟﻨﻴﻭﻤﺎﻴـﺴﻴﻥ ﻭﺍﻟﺭﻴﻔﺎﻤﺒـﺴﻴﻥ ﻭﺍﻟﺴﺘﺭﺒﺘﻭﻤﺎﻴـﺴﻴﻥ ﻭﺍﻟﺒﻨـﺴﻠﻴﻨﺎﺕ‬

‫ﻭﺍﻟﺴﻴﻔﺎﻟﻭﺴﺒﻭﺭﻴﻨﺎﺕ‪ .‬ﻜﺎﻨﺕ ﻜﺎﻓﺔ ﺍﻟﻌﺯﻻﺕ ﻤﻨﺘﺠﺔ ﻟﺼﺒﻐﺔ ﺍﻟﺒﺎﻴﻭﺴﻴﺎﻨﻴﻥ ﻭ ﻋـﺯﻟﺘﻴﻥ ﻤﻨﻬـﺎ ﻤﻨﺘﺠـﺔ ﻟﻠﺒﺭﻭﺘﻴﻴـﺯﺍﺕ‬

‫ﺍﻟﺨﺎﺭﺠﻴﺔ‪ .‬ﺃﻅﻬﺭﺕ ﻨﺘﺎﺌﺞ ﺍﻟﺘﺭﺤﻴل ﺍﻟﻜﻬﺭﺒﺎﺌﻲ ﻟﻠﻨﺴﻕ ﺍﻟﺒﻼﺯﻤﻴﺩﻱ ﻓﻲ ﻫﻼﻡ ﺍﻷﻜﺎﺭﻭﺯ ﺒﺄﻥ ﺍﻟﻌﺯﻻﺕ ﺘﺸﺘﺭﻙ ﺒﻭﺠـﻭﺩ‬

‫ﺤﺯﻤﺘﻴﻥ ﺒﻼﺯﻤﻴﺩﻴﺔ ﺼﻐﻴﺭﺓ ﺍﻟﺤﺠﻡ ﻭﺤﺯﻤﺔ ﺒﻼﺯﻤﻴﺩﻴﺔ ﻤﻔﺭﺩﺓ ﻜﺒﻴﺭﺓ ﺍﻟﺤﺠﻡ‪ .‬ﺃﻅﻬﺭﺕ ﻨﺘـﺎﺌﺞ ﺍﻟﺘﺤﻴﻴـﺩ ﺍﻟﺘﻠﻘـﺎﺌﻲ‬

‫‪ Spontaneously curing‬ﻋﺩﻡ ﻓﻘﺩﺍﻥ ﺍﻟﺒﻼﺯﻤﻴﺩﺍﺕ ﻓﻲ ﺃﻱ ﻤﻥ ﺍﻟﻌﺯﻻﺕ ﺍﻟﺜﻼﺜﺔ ﺍﻟﻤﺩﺭﻭﺴﺔ ﻤﻤﺎ ﻴﺸﻴﺭ ﺇﻟﻰ ﺃﻨﻬـﺎ‬

‫ﺜﺎﺒﺘﺔ ﻭﻤﺴﺘﻘﺭﺓ ﻓﻲ ﺨﻼﻴﺎ ﺍﻟﺒﻜﺘﺭﻴﺎ‪ .‬ﺃﻅﻬﺭﺕ ﺍﻟﻌﺯﻻﺕ ﻓﻘﺩﺍﻨﻬﺎ ﺍﻟﺠﺯﺌﻲ ﻟﻠﻤﻘﺎﻭﻤﺔ ﺍﻟﻤﺘﻌﺩﺩﺓ ﻟﻠﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ ﺒﻌـﺩ‬

‫ﻤﻌﺎﻤﻠﺘﻬﺎ ﺒﺒﺭﻭﻤﻴﺩ ﺍﻻﺜﻴﺩﻴﻭﻡ )‪700‬ﻤﺎﻴﻜﻭﻏﺭﺍﻡ‪/‬ﻤﻠﻴﻠﺘﺭ( ﺃﻭ‪ ،SDS%1‬ﻤﻤﺎ ﻴﺸﻴﺭ ﺇﻟﻰ ﺃﻥ ﺍﻟﻌﺯﻻﺕ ﻗﺩ ﺘﺤﺘﻭﻱ ﻋﻠـﻰ‬

‫ﺃﻜﺜﺭ ﻤﻥ ﺒﻼﺯﻤﻴﺩ ﺼﻐﻴﺭ ﻟﻬﺎ ﺫﺍﺕ ﺍﻟﺤﺠﻡ ﺍﻟﺠﺯﻴﺌﻲ‪ .‬ﺒﻴﻨﻤﺎ ﻜﺎﻨﺕ ﺩﺭﺠﺔ ﺍﻟﺤﺭﺍﺭﺓ ﺍﻟﻤﺭﺘﻔﻌﺔ ﺃﻜﺜﺭ ﻜﻔﺎﺀﺓ ﻓﻲ ﺘﺤﻴﻴـﺩ‬ ‫ﻜﺎﻓﺔ ﺍﻟﺒﻼﺯﻤﻴﺩﺍﺕ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻟﻤﻭﺍﺩ ﺍﻟﻜﻴﻤﻴﺎﺌﻴﺔ ﻟﺫﻟﻙ ﺃﺼﺒﺤﺕ ﻜﺎﻓﺔ ﺍﻟﻌﺯﻻﺕ ﺤﺴﺎﺴﺔ ﻟﻠﻤﻀﺎﺩﺍﺕ ﺍﻟﻤﺩﺭﻭﺴﺔ ﺒﺈﺴﺘﺜﻨﺎﺀ‬ ‫ﺍﻟﻠﻨﻜﻭﻤﻴﺴﻴﻥ‪ .‬ﺃﻅﻬﺭﺕ ﺘﺠﺎﺭﺏ ﺍﻟﺘﺤﻴﻴﺩ ﺒﺄﻨﻬﺎ ﻋﺩﻴﻤﺔ ﺍﻟﺘﺄﺜﻴﺭ ﻓﻲ ﺠﻴﻥ ﻤﻘﺎﻭﻤﺔ ﺍﻟﻠﻨﻜﻭﻤﻴﺴﻴﻥ ﻭﺍﻟﺠﻴﻨﺎﺕ ﺍﻟﻤﺸﻔﺭﺓ ﻹﻨﺘﺎﺝ‬ ‫ﺍﻟﺒﺭﻭﺘﻴﺯﺍﺕ ﻭﺘﺤﻴﻴﺩﻫﺎ ﻤﻥ ﺍﻟﻌﺯﻻﺕ ﺍﻟﺜﻼﺙ ﻤﻤﺎ ﻗﺩ ﻴﺸﻴﺭ ﺇﻟﻰ ﺃﻥ ﻫﺫﻩ ﺍﻟﺠﻴﻨﺎﺕ ﻤﺤﻤﻭﻟﺔ ﻋﻠﻰ ﻜﺭﻭﻤﻭﺴﻭﻡ ﺍﻟﺒﻜﺘﺭﻴﺎ‪.‬‬

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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(40°C) 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 (40°C) 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 700-3000µg/ml for SDS and ethidium bromide respectively.

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

Same observations were obtained when Al-Amir(8) treated isolate of P. aeruginosa with acridine orange up to 1000µg/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 1000µg/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 37°C for 24 hr, stored at 4°C, 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 37°C 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 37°C for 24hr.

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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 700µg/ml Ethidium Bromide was inoculated with 0.3ml of overnight culture of P. aeruginosa isolates, incubated at 37°C 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 37°C 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 (50µg/ml), then inoculated with single colony of P. aeruginosa isolates, and incubated at 37°C 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 37°C 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 37°C 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 37°C for 24 hrs, then 0.2ml.of bacterial culture was inoculated to 10ml. of fresh nutrient broth, and incubated at elevated temperature from 20to 46°C 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 37°C 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).

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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 41°C but not at 4°C. 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 41°C 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 42°C 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).

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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, Gram–negative 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 cephalosporin’s 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.

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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 700µg/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.

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

Table (2): Screening of cured P.aeruginosa isolates by 700µg/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.

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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, that’s 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).

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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.

1

2

3

4

5

6

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 4: DNA content of cured P. aeruginosa P19 isolate Lane 5: DNA content of non-proteases producing P. aeruginosa P3 isolate. Lane 6: DNA content of proteases producing P. aeruginosa P19 isolate.

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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 (46°C) 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 (46°C) 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.

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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 (46°C), 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

6

Large plasmid Chromosomal DNA

Small plasmids

Figure(2): The plasmid profile of P. aeruginosa isolates and cured isolates after treated with elevated temperature ( 46°C) 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 Lane 4: DNA content of cured P. aeruginosa P3 isolate Lane 5: DNA content of cured P. aeruginosa P19 isolate Lane 6: DNA content of cured 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.

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Iraqi J. Biotech. 9(4): 797-811 (2010)

Rabab O. Radi and Fryad H. Rahman

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 46°C 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.

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Rabab O. Radi and Fryad H. Rahman

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): 942–948. 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.

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