PLASMID PROFILE AND CURING, BIOFILM DETECTION AMONG ESBL PRODUCING ISOLATES OF ACINETOBACTER SPECIES
Objective: The aim of the present study is to evaluate the plasmid profile and curing in extended spectrum beta-lactamases (ESBL) producers and know the association of biofilm formation among ESBLs.
Methods: Standard disk diffusion method (Kirby Bauerâ€™s method) was performed to know the sensitivity and resistance patterns for commonly used antibiotics and clinical laboratory standard institute (CLSI) recommended confirmatory phenotypic method was used to confirming ESBL producers. 56 ESBL producers were subjected to plasmid profiling and curing. The tube (qualitative) and microtitre (quantitative) methods were performed to detect the biofilm formation.
Results: In all ESBL producers, a single plasmid was found with approximately 21, 226 bp. After plasmid curing, the resistance patterns against the third generation cephalosporin group of antibiotics were transformed into susceptible for all bacterial isolates. In tube method, 11 (19.64%) isolates were strong adherent, 08 (14.28%) were either weakly adherent or non-biofilm producers. In the microtitre plate method, 09 (16.07%) were considered as positive, and 47 (83.92%) were considered as negative for biofilm formation.
Conclusion: We found a single resistant plasmid in all isolates and after curing of plasmids, the transformation of resistance to susceptibility against third generation drugs were observed. So, we concluded that ESBL mediated antimicrobial resistance mechanisms in Acinetobacter spp. is maybe plasmid-mediated in the tested isolates. However, there was no association found between biofilm producers and ESBLs, this baseline information will be helpful to study more in this area.
Keywords: Acinetobacter spp, Biofilm detection, ESBL, Plasmid profile and curing
2. Payne DJ, Cramp R, Winstanley DJ, Knowles DJ. Comparative activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases. Antimicrob Agents Chemother 1994;38:767-72.
3. Lipps G. Plasmids, Current Research, and Future Trends. Horizon Scientific Press UK; 1008. p. 263.
4. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167-93.
5. Saitou K, Furuhata K, Kawakami Y, Fukuyama M. Biofilm formation abilities and disinfectant resistance of Pseudomonas aeruginosa isolated from cockroaches captured in hospitals. Biocontrol Sci 2009;14:65-8.
6. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standard disk diffusion method. Am J Clin Pathol 1966;45:493-6.
7. Clinical Laboratory Standards Institute: Performance standards for antimicrobial susceptibility testing. Twenty-second informational supplement. Wayne, PA, USA: CLSI; 2012. p. M100-S22.
8. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor; 1989.
9. Deshpande NM, Dhakephalkar PK, Kanekar PP. Plasmid mediated-dimethoate degradation in Pseudomonas aeruginosa MCMB-427. Lett Appl Microbiol 2001;33:275-9.
10. Christensen GD, Simpson WA, Younger JA, Baddour LM, Barrett FF, Melton DM, et al. Adherence of coagulase negative Staphylococci to plastic tissue culture: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 1985;22:996-1006.
11. Chaudhury A, Nagaraja M, Kumar AG. Potential of biofilm formation by Staphylococci correlation with methicillin susceptibility. Indian J Med Microbiol 2009;27:377-8.
12. Ndugulile F, Jureen R, Harthug S, Urassa W, Langeland N. Extended-spectrum beta-lactamases among Gram-negative bacteria of nosocomial origin from an intensive care unit of a tertiary health facility in Tanzania. BMC Infect Dis 2005;5:86.
13. Kumar E, Usha K, Chaudhury A, Ramana BV, Sai Gopal DVR. Detection of AmpC Î²-lactamases production in Acinetobacter species by an inhibitor (disk) based & modified three-dimensional (enzyme extraction) methods. Indian J Med Res 2014;140:688-90.
14. Tullu MS, Deshmukh CT, Baveja SM. Bacterial profile and antimicrobial susceptibility pattern in catheter-related nosocomial infections. J Postgrad Med 1998;44:7-13.
15. Kumar E, Usha K, Ramana BV, Chaudhury A, Gopal DVR. Molecular characterization of plasmid-mediated blactx-M15 extended spectrum Î² lactamase (esbls) in Acinetobacter spp. Isolates from intensive care unit patients, at a tertiary care hospital, South India. Indian J Med Microbiol 2015;33:601-2.
16. Shafaq Aiyaz Hassan, Syed Asfar Jamal, Mustafa Kamal. The occurrence of multidrug resistant and ESBL producing E. coli causing urinary tract infections. J Basic Appl Sci 2011;7:39-43.
17. Iroha IR, Oji AE, Afiukwa TN, Nwuzo AC, Ejikeugwu PC. Extended spectrum betaâ€“lactamase (ESBL) mediated resistance to antibiotics among Klebsiella Pneumoniae in enugu metropolis. Maced J Med Sci 2009;2:196-9.
18. Winokur PL, Canton R, Casellas JM, Legakis N. Variations in the prevalence of strains expressing an extended-spectrum beta-lactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific region. Clin Infect Dis 2001;2(Suppl 2):94-103.
19. Iraj Alipourfard, Nilufar Yeasmin Nili. Antibiogram of extended spectrum beta-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae isolated from hospital samples. Bangladesh J Med Microbiol 2010;4:32-6.
20. Hirakata Y, Matsuda J, Miyazaki Y, Kamihira S, Kawakami S, Miyazawa Y, et al. Regional variation in the prevalence of extended-spectrum beta-lactamase producing clinical isolates in the Asia-pacific region (SENTRY 1998-2002). Diagn Microbiol Infect Dis 2005;52:323-9.
21. Jabeen K, Zafar A, Hassan R. Frequency and sensitivity pattern of extended spectrum beta-lactamase producing isolates in a tertiary care hospital laboratory of Pakistan. J Pak Med Assoc 2005;55:436-9.
22. Sader HS, Hsiung A, Fritsche TR, Jones RN. Comparative activities of cefepime and piperacillin/tazobactam tested against a global collection of Escherichia coli and Klebsiella spp. with an ESBL phenotype. Diagn Microbiol Infect Dis 2007;57:341-4.
23. Karishma R Pardesi, Supriya P Yavankar, Balu A Chopade. Plasmid distribution and antimicrobial susceptibility patterns of Acinetobacter genospecies from healthy skin of a tribal population in western India. Indian J Med Res 2007;125:79-88.
24. Dheepa M, Vinitha L Rashme, Appalaraju B. Comparision of biofilm production and multiple drug resistance in clinical isolates of Acinetobacter baumanii from a tertiary care hospital in South India. Int J Pharm Biomed Sci 2011;2:103-7.
25. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999; 284:1318-22.