https://imcjms.com Ibrahim Medical College Journal of Medical Science https://imcjms.com/registration/journal_full_text/270 Tue, 31 Oct 2017 12:54:33 +0000 Abstract Background and objectives: Emergence of extended spectrum beta-lactamase (ESBL) producing bacteria is a major public health concern. Detection of multi drug resistant (MDR) ESBL producing organisms is necessary to prevent its spread and effective treatment. The purpose of the present study was to determine the magnitude of ESBL producing organism in hospital setting and to compare the suitability of double disc synergy test (DDST) and cefepime-clavulanate E-test method for the detection of ESBL producing organisms in routine microbiology laboratory. Materials and methods: The study was carried out in the Department of Microbiology, Sir Salimullah Medical College, Dhaka from January 2011 to December 2011. Clinical samples included urine and pus from patients with suspected urinary tract and wound infections respectively. Standard microbiological methods were employed for isolation and identification of the organisms. DDST and E-test were used to detect ESBL producing Gram negative organisms. Results: A total of 186 Gram-negative organisms were isolated from various samples. Among the 186 Gram negative bacteria, 120 (64.5%) were Esch. coli while 33 (17.7%), 20 (10.8%) and 11 (5.9%) were Pseudomonas sp, Klebsiella sp and Proteus sp respectively. Out of total 186 isolates, 77 (41.4%) and 73 (39.2%) isolates were found ESBL producers by DDST and E-test method (p=0.674) respectively. Compared to Escherichia coli, Pseudomonas and Proteus, significantly high (p<0.01) proportion of Klebsiella were ESBL positive by both DDST and E-test methods. The detection rate of ESBL producing organisms was not significantly different by DDST and E-test (41.4% vs 39.2%). Non-determinable result was obtained for 4 (2.2%) isolates by E-test method. Conclusion: In our present study, a substantially large number of clinical isolates were found ESBL producers. Compared to E-test, DDST was found as a reliable, convenient and inexpensive method for detection of ESBL producing organism in routine microbiology laboratory practice. IMC J Med Sci 2018; 12(1): 32-36. DOI: https://doi.org/10.3329/imcjms.v12i1.35176 Address for Correspondence: Dr. Mili Rani Saha, Assistant Professor, Department of Microbiology, BIRDEM General Hospital, 122, Kazi Nazrul Islam Avenue, Dhaka 1000, Bangladesh, Email: milisaha77@yahoo.com   Introduction Extended spectrum beta-lactamases are enzymes that confer resistance to the penicillin, cephalosporins and aztreonam by hydrolysis of the antibiotics. ESBL enzymes are inactivated by beta-lactamase inhibitors such as clavulanic acid [1]. Treatment of ESBL producing organisms is now a therapeutic challenge in hospitalized patients worldwide. Indiscriminate administration of extended spectrum cephalosporins, prolonged hospital stay, mechanical ventilation and catheterization are the major risk factors for colonization of ESBL producing bacteria [2]. Detection of ESBL producing organisms is necessary to prevent its spread. Several ESBL detection tests have been proposed by NCCLS [3]. The degree of resistance against extended spectrum cephalosporins can also be highly variable for the different ESBL enzymes. Thus, ESBL producing bacteria need reliable detection method [4]. Therefore, the present study was undertaken to determine the magnitude of ESBL producing organisms in tertiary hospitals and to compare the DDST with that of commercially available E-test to detect ESBL producing Gram negative organisms isolated from clinical samples. Materials and Methods Isolation and identification of organisms: Samples were inoculated onto blood agar and MacConkey’s agar media for isolation and identification of the organisms. All plates were incubated at 370C aerobically for 24-48 hrs. Suspected organisms were identified by standard biochemical tests [10]. Tests for the detection of ESBL producing organisms: All isolated organisms were tested for ESBL production by DDS test and E-test methods. a.  Double Disc Synergy Test: All isolated Gram-negative bacteria were tested for ESBL production by DDST using aztreonam (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg) and 20μg amoxicillin +10μg clavulanic acid discs. The four antibiotic discs were placed 20 mm apart from each other with amoxicillin /clavulanic acid disc at the center as shown in Fig-1a. ESBL production was considered positive when the zone of inhibition around any antibiotic disc was enhanced towards the amoxicillin/clavulanic acid disc [11].     Fig.1: Photograph showing DDST and E-test. 1a: DDST showing enhancement of zone of inhibition towards the aztreonam (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg) and cefotaxime (30 µg) discs. Amoxicillin/clavulanic acid disc is at the center. 1b: E-test strip showing formation ellipse at the cefepime/clavulanic end compared to on cefepime end; 1c: E-test with non-determinable result.   Results A total of 354 samples were included in the study. Total 186 Gram-negative organisms were isolated from various samples. Among the 186 Gram negative bacteria, 120 (64.5%) were Escherichia coli while 33 (17.7%), 20 (10.8%) and 11 (5.9%) were Pseudomonas sp, Klebsiella sp and Proteus sp respectively. All the isolates were tested for production of ESBL by DDST and E-test methods. Out of total 186 isolates, 77 (41.4%) and 73 (39.2%) isolates were found ESBL producers by DDST and E-test method (p=0.674) respectively (Table-I). Compared to Escherichia coli, Pseudomonas and Proteus species, significantly high (p<0.01) proportion of Klebsiella sp (65-75%) were ESBL positive by both DDST and E-test methods (Table-1). The detection rate of ESBL was not significantly different by DDST and E-test for each type of organism.  Non-determinable result was obtained in 4 isolates by E-test method. These four isolates did not show any zone of inhibition either at cefepime (PM) or at cefepime-clavulanate (PML) end of the test strip (Fig 1c).   Table-1: Comparative detection rate of ESBL producing organisms by DDST and E-test   Discussion      The study has demonstrated that a good proportion of Gram-negative organisms isolated from clinical samples were ESBL producers. Compared to expensive E-test, DDST is a reliable, convenient, relatively inexpensive and easy to perform method for detection of ESBL producing organisms in routine clinical laboratories.   Author’s contributions MRS performed the experiments analyzed the results and wrote the manuscript.  STJ conceived, designed and supervised the study.                            Competing interest Authors declare no conflict of interest.   Funding None   References 2.   Chaudhary U, Aggarwal R. Extended spectrum β-lactamases (ESBL) – An emerging threat to clinical therapeutics. Indian J Med Microbiol.  2004; 22(2): 75-80. 3.   Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard. Clinical and Laboratory Standards Institute Document M100-S9. PA, USA, 1999. 4.   Sturenburg E, Mack D. Extended spectrum β-lactamases: implications for the clinical microbiology laboratory, therapy and infection control. 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Rev Infect Dis. 1988; 10: 867-878. 13.  Livermore DM. β-lactamases-mediated resistance and opportunities for its control.  J Antimicrob Chemother. 1998; 41 suppl D: 25-41. 14.  Vercauteren E, Descheemaeker P, Ieven M, Sanders CC, Goossens H. Comparison of screening methods for detection of extended-spectrum β-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spp. in a Belgian teaching hospital. J Clin Microbiol. 1997; 35: 2191-2197. 15.  Wiegand I, Geiss HK, Mack D, Sturenburg E, Seifert H. Detection of extended-spectrum β-lactamases among Enterobacteriaceaeby use of semiautomated microbiology systems and manual detection procedures. J Clin Microbiol.2007;45: 1167-1174. 2026 Ibrahim Medical College. All rights reserved.