https://imcjms.com Ibrahim Medical College Journal of Medical Science https://imcjms.com/registration/journal_full_text/77 Tue, 02 Aug 2016 12:20:57 +0000   Carbapenem resistant Enterobacteriaceae (CRE) is becoming a major public health concern globally. Detection of carbapenem hydrolyzing enzyme carbapenemase in Enterobacteriaceae is important to institute appropriate therapy and to initiate preventive measures. This study was designed to determine the presence of carbapenemase producers among the CRE isolated from patients at Dhaka Medical College Hospital, Bangladesh. Twenty-nine CRE strains detected by disk diffusion technique were included in the study. Minimum inhibitory concentration of imipenem and tigecycline was determined by agar dilution method. Carbapenemase production was phenotypically detected by Modified Hodge test while MBL producers were detected by combined disk and double disk synergy tests. Genes encoding blaNDM-1, blaOXA-181, blaOXA-48, blaKPC, blaCTX-M-15, blaOXA-1-group were identified by polymerase chain reaction (PCR). The result of this study showed the presence of blaOXA-181/ blaOXA-48, blaNDM-1 positive strains in Bangladesh and colistin and tigecycline were the most effective drugs against carbapenemase producing Enterobacteriaceae (CPE). Epidemiological monitoring of carbapenemase producing organisms in Bangladesh is important to prevent their dissemination. Ibrahim Med. Coll. J. 2015; 9(2): 48-54     The emergence of carbapenem resistant Enterobacteriaceae (CRE) is a major concern worldwide. This is because of their importance as human pathogens especially within the hospital settings and its high transmissible nature and tendency for rapid spread.1 This resistance is mediated by the production of carbapenemases or hyper production of Amp C beta lactamase and up regulation of efflux pumps or by their combined mechanisms.2 Carbapenem hydrolyzing beta lactamases which belong to Ambler classes A, B and D have been reported worldwide in Enterobacteriaceae. The most clinically significant ones are class A enzymes such as KPC-types, class B enzymes such as IMP, VIM and NDM-1 types and class D enzymes such as OXA-48 and OXA-181 types. The genes encoding them are located on mobile genetic elements, which allow them to spread.2-4   The present cross-sectional study was conducted in the Department of Microbiology of Dhaka Medical College, Dhaka, Bangladesh, during July 2010 to June 2011. The research protocol was approved by the Research Review Committee (RRC) and Ethical Review Committee (ERC) of Dhaka Medical College. Written consent was obtained from each patient or their legal guardian before collection of samples. Bacterial isolates and identification: Twenty-nine Enterobacteriaceae isolates resistant to carbapenem by disk diffusion technique were included in the study. The organisms were isolated from various clinical specimens. The specimens included wound swab, endotracheal aspirate, blood and urine. All the organisms were identified by Gram stain, colony morphology, hemolytic criteria, pigment production and standard biochemical tests.9         Double-disk synergy test (DDST):Imipenem disc (10 µg) and a disk containing 20 µl of Tris-EDTA (1.0 M Tris-HCL, 0.1 M EDTA, pHapproximately 8.0) and 20 µl of 1:320 diluted 2-mercaptopropionic acid (MPA) were placed 10 mm apart in an inoculated Mueller-Hinton agar plate and incubated at 370 C for 24 hours. A clear extension of the edge of the inhibition zone of imipenem disk toward the Tris-EDTA-MPA disk was interpreted as MBLs production.15 Molecular characterization of carbapenemase producers:The presence of blaNDM-1, blaKPC, blaOXA-181, and blaOXA-48 among CRE were detected by polymerase chain reaction (PCR). In addition, ESBL encoding gene blaCTX-M-15 and blaOXA-1group were also identified among the CRE by PCR. The primers used are shown in Table 1.2,16-18 To prepare bacterial pellets, a loop full of bacterial colonies was inoculated into a Falcon tube containing trypticase soy broth. After incubation overnight at 370 C, the Falcon tubes were centrifuged at 4,000 g for 10 minutes, after which the supernatant was discarded. A small amount of sterile trypticase soy broth was added into the Falcon tubes with pellets and mixed evenly. Then an equal amount of bacterial suspension was placed into three 1.5 ml microcentrifuge tubes. The microcentrifuge tubes were then centrifuged at 4,000 g for 10 minutes and the supernatant was discarded. The microcentrifuge tubes containing bacterial pellets were kept at -200 C until DNA extraction. Bacterial DNA was extracted by boiling method.17Multiplex PCR was done for identification of blaNDM-1, blaOXA-48 and blaKPC. Multiplex PCR reaction cycle consisted of preheat at 940C for 10 minutes followed by denaturation at 940C for 30 seconds, annealing at 520C for 40 seconds, extension at 720C for 50 seconds with a final extension at 720C for 5 minutes. In case of OXA-181, CTX-M-15, OXA-1-group, PCR reaction consisted of initial denaturation at 950C for 10 minutes, then 35 cycles of denaturation at 950C for one minute, annealing at 550C for 45 seconds, extension at 720C for one minute and final extension at 720C for 10 minutes. The amplified DNA were loaded into a 1.5% agarose gel, electrophoresed at 100 volts for 35 minutes, stained with 1% ethidium bromide and visualized under UV light. Table-1: Primers used in this study.2,16-18   Result           The rate of resistance to different classes of antibiotics ranged from 63.2% to 100% except colistin and tigecycline (Table 4). All 19 isolates were sensitive to colistin. The rate of resistance to tigecycline and tetracycline was 5.3% and 63.2% respectively. Organism positive for OXA-181/OXA-48 had a low level of resistance to imipenem (MIC 1 - 4 μg/ml) while NDM-1 positive organisms had high level resistance to imipenem (MICs 16 - ³ 32 μg/ml (Table 2). MIC of tigecycline ranged from 2-0.5 μg/ml except one had MIC 8 μg/ml (Table 2). Out of 19 carbapenemase positive isolates, 12 (63.16%) were extensively drug-resistant (XDR) and were only sensitive to tigecycline and colistin. Discussion OXA-181 is a close relative of OXA-48 from which it differs by 4 amino acids.24 blaOXA-181 positive K. pneumonia infections were first described in India but imported cases have since been described in Oman, Netherlands and New Zealand.6,7,20 There are no reports of blaOXA-181 positive isolates in Bangladesh. However, this country borders India, which is a source of blaOXA-181 positive Enterobacteriaceae. These cases highlight potential problems that may arise from the rapidly increasing practice of traveling across international borders to obtain health care. The present study is the first report of the presence of blaOXA-181/blaOXA-48 genes in Enterobacteriaceae in Bangladesh. In this study, eleven OXA-181/OXA-48 producing organisms were isolated. Out of 11 OXA-181/OXA-48 producing organisms 6 were co-harbored with NDM-1 producing gene and showed high level of resistance to imipenem, 5 isolates which harbored only OXA-181/OXA-48 producing genes showed low level of resistance which correlates with one study where it was shown that co-producing NDM-1 and OXA-181 was fully resistant to carbapenem whereas all OXA-181 producing isolates showed an apparent susceptibility to carbapenem.5 This study evaluated the presence of ESBL encoding genes in CPE. All the CPE harbored ESBL producing gene blaCTX-M-15 except one C. freundii and nine isolates had blaOXA-1-group gene. In the present study, CDT was found to be the most sensitive test in detecting carbapenemase producing organisms compared to DDST and MHT. It was reported that, sensitivity of MHT was low for NDM-1 producers (50%) but was increased to 85.7% by adding ZnSO4 (100 µg/ml) in the culture medium.26 The effect of Zinc might be multiple such as, it acts by increasing stability of the enzyme or/and by modifying porin expression.27 But in the present study, MHT detected 81.82% OXA-181/OXA-48 producers. It appears that MHT is suitable for OXA-181/OXA-48 producers but not for NDM-1. No phenotypic method is adequate to detect carbapenemase producers. It may be concluded that multiple phenotypic methods and PCR could be the most reliable and acceptable approach for early and accurate identification of carbapenemase producers.   1.    Balm ND, Ngan G, Jureen R, Lin TP, Teo WP. OXA-181-producing Klebsiella pneumoniae establishing in Singapore. BMC Infect Dis 2013; 13: 58. 3.    Carrer A, Poirel L, Erakey H, Cagatay AA, Badur S, Nordmann P. Spread of OXA-48 positive carbapenem resistant Klebsiella pneumonia isolates in Istanbul, Turkey. Antimicrob Agents Chemother 2008; 52(8): 2950-54. 5.    Dortet L, Poirel L, Al Yaqoubi F, Nordmann P. NDM-1, OXA-48 and OXA-181 carbapenemase-producing Enterobacteriaceae in Sultanate of Oman. Clin Microbiol Infect 2012; 18: E144–E148. 7.    Koh TH, Cao DYH, Chan KS, Wijaya L, Low SBG, Lam MS, Ooi EE, Hsu LY. blaOXA-181-positive Klebsiella pneumoniae, Singapore. Emerg Infect Dis 2012; 18(9): 1524–1525. 9.    Cheesbrough M. Microscopical techniques used in Microbiology, culturing bacterial pathogens, biochemical tests to identify bacteria. District Laboratory Practice in Topical Countries, Part 2: Cambridge University Press: 35-70. 11.  Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012; 18: 268-281. 13.  Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 19th Informational Supplement. CLSI document. Wayne, PA: Clinical and Laboratory Standards Institute 2009; M100-S19. 15.  Kim SY, Hong SG, Moland ES, Thomson KS. Convenient test using a combination of chelating agents for detection of metallo-beta-lactamases in the clinical laboratory. J Clin Microbiol 2007; 45: 2798-2801. 17.  Guerra B, Soto SM, Arguelles JM, Mendoza MC. Multidrug resistance is medicated by large plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype. Antimicrob Agents Chemother 2001; 45: 1305-1308. http://dx.doi.org/10.1128/AAC.45.4. 1305-1308.2001 19.  Nordmann P, Naas T, Poirel L. Global spread of Carbapenemase producing Enterobacteriaceae. Emerg Infect Dis 2011; 17: 1791-1798. 21.  Kalpoe JS, Naiemi N, Poirel L, Nordmann P. Detection of an Ambler class D OXA-48-type b-lactamase in a Klebsiella pneumonia strain in the Netherlands. J Med Microbiol 2011; 60: 677–678. 23.  Paño-Pardo JR, Ruiz-Carrascoso G, Navarro-San Francisco C, Gómez-Gil R, Mora-Rillo M, Romero-Gómez MP. Infections caused by OXA-48 producing Klebsiella pneumoniae in a tertiary hospital in Spain in the setting of a prolonged hospital wide outbreak. J Antimicrob Chemother 2013; 68(1):     89-96. 25.  Kumar S, Bandyopadhyay M, Mondol S, Pal N, Ghosh T and Banerjee P. Tigecycline activity against metallo-β-lactamase-producing bacteria. Avicenna J Med 2013; 3(4): 92-96. 2026 Ibrahim Medical College. All rights reserved.