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 ~  Abstract
 ~  Materials and Me...
 ~  Curing experiments
 ~  Transformation e...
 ~  Results
 ~  Discussion
 ~  References

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Year : 2004  |  Volume : 22  |  Issue : 3  |  Page : 182-184
 

Plasmid mediated amikacin resistance in clinical isolates of pseudomonas aeruginosa


Department of Microbiology, JN Medical College, Aligarh Muslim University, Aligarh - 202 002, India

Correspondence Address:
Department of Microbiology, JN Medical College, Aligarh Muslim University, Aligarh - 202 002, India

 ~ Abstract 

Ten multidrug resistant (MDR) isolates of Pseudomonas aeruginosa, obtained from hospitalized burn patients, were selected for plasmid detection, curing and transformation experiments. These isolates were also studied for plasmid mediated resistance. All the isolates were found to harbour R plasmid. Curing and transformation experiments showed that resistance to amikacin was plasmid mediated. -lactamase production was also tested. It is suggested that plasmids should be characterised in all MDR P. aeruginosa strains and a nation wide antibiotic policy should be made to minimise the emergence of drug resistance.

How to cite this article:
Shahid M, Malik A. Plasmid mediated amikacin resistance in clinical isolates of pseudomonas aeruginosa . Indian J Med Microbiol 2004;22:182-4


How to cite this URL:
Shahid M, Malik A. Plasmid mediated amikacin resistance in clinical isolates of pseudomonas aeruginosa . Indian J Med Microbiol [serial online] 2004 [cited 2019 Dec 9];22:182-4. Available from: http://www.ijmm.org/text.asp?2004/22/3/182/11215


In the past few decades Pseudomonas aeruginosa has been increasingly recognised as a pathogen in a variety of serious infections in hospitalized patients especially with impaired immune defences.[1] This organism is an important opportunistic pathogen with innate resistance to many antibiotics. Despite innate resistance, additional acquired resistance due to plasmids is also found in P.aeruginosa. Plasmid mediated resistance to various antimicrobial drugs have been demonstrated by various workers[2],[3] and most of them have demonstrated it by plasmid curing experiments alone. In the present study, we have isolated a plasmid responsible for multidrug resistance in P. aeruginosa isolates from burn patients and an attempt was made to demonstrate the drug resistance by plasmid curing experiments as well as by transformation experiments.

 ~ Materials and Methods Top

The present study was carried out between July 2002 and December 2002. P.aeruginosa isolates were obtained from the pus culture of hospitalized burn patients and were analysed for the presence of drug resistance by the method of Bauer et al[4] on Mueller Hinton agar (HiMedia) by using commercially available paper discs (HiMedia, India). The antibiotic discs and their concentrations used in the present study are shown in [Figure - 1].
Ten multidrug resistant isolates, which were also resistant to amikacin, were selected for plasmid detection, curing and transformation experiments and also for b-lactamase detection. Plasmid isolation was carried out using the large scale alkaline lysis method as described by Davis et al.[5]
Plasmid samples (25 L) were electrophoresed through 0.8% agarose (type 1; Sigma) in TBE buffer at 150V, 60mA for 5.5 hours by method of Portnoy et al.[6] The gel was stained for 2 hours in 0.5 g/L of ethidium bromide solution and destained in water.

 ~ Curing experiments Top

Ethidium bromide (Sibisco Research Lab. Ltd. India) was used as curing agent. The method described by Guerry and Colwell[7] as adopted in our previous experiments[8] was used. 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. Subcultures were initially done on trypticase soy agar and the colonies were tested for antibiotic sensitivity.

 ~ Transformation experiments Top

Transformation experiments were carried out according to Davis et al[9] as described by us elsewhere[8] by using P. aeruginosa strains as donor and  Escherichia More Details coli strain HB101, which was sensitive to all the previously tested drugs, as recipient. The presence of plasmid in transformants was checked through electrophoresis and the transformants were also tested for each drug resistance already recorded for the donor strain.
Beta lactamase production
The iodometric method of Miles et al[10] was used for the detection of b-lactamase production.

 ~ Results Top

The results of the antimicrobial susceptibility of the P. aeruginosa isolates included in the present study are shown in [Figure - 1]. All 10 MDR P. aeruginosa isolates were examined for the presence of plasmid. All the P. aeruginosa isolates were found to harbour a single and similar plasmid [Figure:2].
Curing and transformation experiments were attempted on these isolates to determine changes in plasmid content associated with antibiotic resistance pattern. After curing experiments the loss of antibiotic resistance was concomitant with the loss of plasmid content [Figure:2]. It was noted that the isolates became sensitive to amikacin while they remained resistant to carbenicillin and clindamycin. Similarly, the transformants (E. coli HB 101) became resistant to amikacin after the transformation experiments while E. coli HB 101 was sensitive to all the drugs prior to the experiments. Agarose gel electrophoresis of the tranformants showed the presence of transforming plasmid [Figure:2]. Out of 10 P. aeruginosa isolates tested, 4 (40%) showed the presence of b-lactamase by the iodometric method.

 ~ Discussion Top

P. aeruginosa is currently one of the most frequent nosocomial pathogen and the infections due to this organism are often difficult to treat due to antibiotic resistance.[11] The mechanisms of resistance to antibiotics include reduced cell wall permeability, production of chromosomal and plasmid mediated b-lactamases,[12] aminoglycoside-modifying enzymes[13] and an active multidrug efflux mechanism.[14]
An alarming increase in resistance of Pseudomonas spp. to various antimicrobial agents has been reported by many workers [15] but studies demonstrating the relation of plasmid and drug resistance in clinical isolates of P. aeruginosa by curing and transformation experiments is scanty in our country. In the present study we have isolated a plasmid from MDR P. aeruginosa isolates, which was found responsible for amikacin resistance. An important striking feature found in this study was increased resistance to amikacin and tobramycin whereas the strains were sensitive to gentamicin and netilmicin (80% and 70% respectively). Various workers from our country have reported the opposite i.e., increased sensitivity of P. aeruginosa strains to amikacin and resistance to gentamicin.[16],[17] This might be due to the variations in the usage of antibiotics in different geographical areas.
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 antibiotics including cephalosporins and anti-pseudomonal antibiotics may also occur in future. Therefore, to combat this problem, efforts should be made to isolate and characterise plasmids responsible for resistance in MDR P. aeruginosa strains from all over the country and a nation wide antibiotic policy should be defined after evaluating the effectiveness of the regime so that misuse of antibiotics is minimised and also the emergence of multidrug resistant organism can be restricted. This is a preliminary study on plasmid mediated amikacin resistance in P. aeruginosa isolates, however, there is a need for a large scale study to find out the plasmid mediated drug resistance in P. aeruginosa along with isolation and characterisation of the plasmid (s). 

 ~ References Top

1.Neu HC. The role of Pseudomonas aeruginosa in infections. J Antimicrob Chemother 1983;11:Suppl B 1-13.  Back to cited text no. 1    
2.Karunasagar I, Nagesha CN, Karunasagar I. Screening of Vibrio parahemolyticus isolates for plasmids. Indian J Med Res 1987;86:711-715.  Back to cited text no. 2    
3.Kawakami Y, Mikoshiba F, Nagasaki S, Matsumoto H, Tazaki T. Prevalence of Pseudomonas aeruginosa strains possessing R factors in a hospital. J Antibiot 1972;25(10):607-609.  Back to cited text no. 3    
4.Bauer AW, Kirby WMM, Sherris JC, Turk M. Antibiotic susceptibility testing by a standardised single disc method. Am J Clin Pathol 1966;45:493-496.  Back to cited text no. 4    
5.Davis LG, Dibner MD, Battey JF. Large scale Alkaline lysis method of Plasmid purification. In: Basic methods in molecular biology. (Elsevier Science Publishing Co. Inc., New York) 1986a:99.  Back to cited text no. 5    
6.Portnoy DA, Moseley SL, Fatkow S. Characterisation of plasmids and plasmid associated determinants of Yersinia enterocolitica pathogenesis. Infect Immunol 1981;31(2):775-782.  Back to cited text no. 6    
7.Guerry P, Colwell RR. Isolation of cryptic plasmid deoxyribonucleic acid from Kanagawa positive strains of Vibrio parahemolyticus. Infect Immunol 1977;16(1):328-334.  Back to cited text no. 7    
8.Shahid M, Malik A, Sheeba. Multidrug resistant Pseudomonas aeruginosa strains harbouring R-Plasmids and AmpC ?-lactamases isolated from hospitalised burn patients in a tertiary care hospital of North India. FEMS Microbiol Lett 2003;228(2):181-186.  Back to cited text no. 8    
9.Davis LG, Dibner MD, Battey JF. Basic methods in molecular biology (Elsevier Science Publishing Co. Inc., New York) 1986b:90-92.  Back to cited text no. 9    
10.Miles RS, Amyes SGB. Laboratory control of antimicrobial therapy. In: Mackie and Mc Cartney Practical Medical Microbiology, 14th ed. Collee JG, Fraser AG, Marmion BP, Simmons A, Eds. (Churchill Livingstone, New York) 1996: 167.  Back to cited text no. 10    
11.Emori TG, Gaynes RP. An overview of nosocomial infections, including the role of the microbiology laboratory. Clin Microbiol Rev 1993;6(4):428-442.  Back to cited text no. 11    
12.Livermore DM. Role of Beta-lactamase and impermeability in the resistance of Pseudomonas aeruginosa. Antibiot Chemother 1989;42:257-263.  Back to cited text no. 12    
13.Prince A. Antibiotic resistance of Pseudomonas species. J Pediatr 1986;108:830-834.  Back to cited text no. 13    
14.Li X-Z, Livermore DM, Nikaido H. Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol and norfloxacin. Antimicrob Agents Chemother 1994;38(8):1732-1741.  Back to cited text no. 14    
15.Paul K, De PK, Bhattacharya S. Comparative efficacy of fluoroquinolones, aminoglycosides ureidopenicillins and newer cephalosporins against Pseudomonas spp. Indian J Med Res 1992;95:136-138.  Back to cited text no. 15    
16.Nagoba BS, Deshmukh SR, Gude UG, Gomashe AV, Wadher BJ. In vitro susceptibility of Pseudomonas aeruginosa to different antibiotics. Indian J Med Microbiol 1997;15(4):185-186.  Back to cited text no. 16    
17.Veenu, Sikka R, Arora DR. Isolation and susceptibility pattern of nonfermenting gram- negative bacilli from clinical samples. Indian J Med Microbiol 1998;17(1):14-18.  Back to cited text no. 17    
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