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1 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 2 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 3 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 4 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. 5 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). 6 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 7 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 8 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. References: 1- Filhol L., Levi J., Bento C and Rozov T. (1999). PCR identification of Pseudomonas aeruginosa and direct detection in clinical samples from cystic fibrosis patients. J. Med. Microbiol. 48: 357-361. 2- Matsumoto T., Furuya N., Tateda K., Ohno A and Yamaguchik. (1999). Effect of passive immunotherapy on murine gut-derived sepsis caused by Pseudomonas aeruginosa.J. Med. Microbiol. 48: 765-770. 9 3- Shlaes DM., Gerding DN, John JF Jr., Duncan RA and Craig WA. (1997). Society for healthcare epidemiology of America and infections disease society of America joint committee on the prevention of antimicrobial resistance in hospitals. Infect. Contral Hosp Epidemiol. 18: 275-291. 4- Austin Dj., Kristinsson KG., and Anderson RM .(1999). The relationship between the volume of antimicrobial consumption in human communities and the frequency of resistance . proc. Natl Acad Sci. USA. 96:1152-1156. 5- Spencer DH., Kas A., Smith EE., Smis EH and Olson MV. (2003). Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa. J. Bacteriol. 185: 13161325. 6- Youssef MT., Malkawi HI, Shurman AA., and Andremont Ao. (1999). Molecular typing of multi-resistant Klebsiella pneumonia isolated from northern Jordan. J. Trop Pediatr. 45:271-277. 7- Khaled AE., Samir A and Ali ZE.(2000). Antibiotic resistance of bacteria isolated from urine specimens in Amman Jordan .J. Med. 34(2): 117-122. 8- Shahid M., Malik A., and Sheeba, M. (2003). Multidrug resistant Pseudomonas aeruginosa strain harboring R-plasmids and AmpC β-Lactamases isolated from hospitalized burn patients in a tertiary care hospital of North India. FEMS Microbiol. Lett. 228(2): 181-186. 9- Shahid M and Malik A.(2004). Plasmid mediated Amikacin resistance in clinical isolates of Pseudomonas aeruginosa. India J Med. Microbiol. 22(3): 182-184. 10- Ruxana T., Timothy S, John W and Aice S. (2005). Pathogen. Host interactions in Pseudomonas aeruginosa pneumonia. American J. Resp. Cri. Car. Med. 171: 1209-1223. 11- O'Malley YQ., Reszka KJ., Rasmussen GT and Britigan B.E. (2003). The pseudomonas secretary product pyocyanin inhibits catalas activity in human lung epithelial cells. Am. J. physiol. Lung cell Mol physiol. 285:L1077-L1086. 12- McFadden. JF. (2000). Biochemical test for identification of medical bacteria. 3 ed ed. Lippicott Williams and wilkins A Walters company. Philadelphia. Baltimore, New York. London. Hong Kong. Sydney. Tokyo. 13- Bauer AW, Kirby WMM., Sherris JC and Turk M. (1966). Antibiotic susceptibility testing by a standardized single disc method . Am. J. clin. Pathol. 45:493-496. 10 14- Davis LG., Dibner MD and battey JF (1986). Basic methods in molecular biology. Elsevier science publishing Co. Inc. New York .99. 15- Portnoy DA., Moseley SL., and Fatkow S. (1981). Characterization of plasmids and plasmid associated determinants of Yersinia enterocolitica pathogenesis. Infect immunol. 31(2): 775-782. 16- Emori TG and Gaynes RP. (1993). An overview of nosocomial infections. Including the role of the microbiology laboratory . Clin. Microbiol Rev. 6(4): 428-442. 17- Prince A. (1986). Antibiotic resistance of Pseudomonas species. J. pediatr. 108:830-834. 18- Akiyoshi. TK., Intentsu O, Toyoko I and Sachiko G. (2005). An epidemiological study of the susceptibility and frequency of multiple- drug resistant strains of Pseudomonas aeruginosa. J. Infect. Chemother. 11(2): 64. 19- Nikibin VS., Abdi. A., Feizabadi MM and Gharavi S. (2007). Pulsed field gel electrophoresis and plasmid profile of Pseudomonas aeruginosa at two hospitals in Tehran , Iran. Indian J med. Res. 126: 146-151. 20- Shahcheraghi F., Feizabadi MM. Yamin V and Abedian Z. (2003). Server determination, drug resistance patterns and plasmid profiles of Pseudomonas aeruginosa isolated from burn patients at two hospitals of Tehran (Iran). Burns. 29: 547-551. 21- Ferguson D., Cahillo J., and Quilty B. (2007). Phenotypic, molecular and antibiotic resistance profiling of nosocomial Pseudomonas aeruginosa strains isolated from two Irish hospitals. 22- Lee B., Haagensen JAJ., Ciofu O, Andersen JB and Molins. (2005). Heterogencity of biofilms formed by non-mucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J. Clin. Microbiol. 43(10): 5247-5255. 11 12 13