https://imcjms.com Ibrahim Medical College Journal of Medical Science https://imcjms.com/registration/journal_full_text/185 Tue, 11 Apr 2017 16:40:40 +0000 There are no standard methods for the detection of metallo-b-lactamase (MBL) production in gram negative organism in routine microbiology practice. The present study was undertaken to evaluate the screening tests like double disk synergy test (DDST) and disk potentiation test (DPT) using ceftazidime (CAZ) and imipenem (IPM) disks with chelating agents like EDTA, 2-mercaptopropionic acid (2-MPA). A total of 132 Pseudomonas and 76 Acinetobacter isolates were obtained from Bangabandhu Sheikh Mujib Medical University (BSMMU) and Bangladesh Institute of Research and Rehabilitation for Diabetes, Endocrine and Metabolic Disorders (BIRDEM) hospitals of Dhaka city. A total of 53 and 29 IPM resistant Pseudomonas and Acinetobacter isolates were selected. EDTA-IPM microdilution minimum inhibitory concentration (EDTA-IPM MIC) method detected MBL in 44 (83%) IPM resistant Pseudomonas and 19 (65.5%) Acinetobacter isolates. DDST with CAZ-0.1M EDTA and CAZ-2-MPA detected MBL in 73.6% and 67.9% of IPM resistant Pseudomonas and 55.2% and 48.3% of Acinetobacter isolates respectively. The detection rate was 67.9% and 66.1% in Pseudomonas and 51.7% and 44.8% in Acinetobacter isolates by EDTA-IPM and IPM-2-MPA methods respectively. In comparison to DDST, DPT with CAZ-0.1M EDTA showed higher sensitivity (89.7% ) and specificity (100%) for detection of MBL in Pseudomonas and Acinetobacter. The results showed that simple screening tests like DPT with 0.1M EDTA was able to detect MBL producing Pseudomonas and Acinetobacter from clinical samples with high sensitivity and specificity. Ibrahim Med. Coll. J. 2010; 4(1): 26-30 Address for Correspondence: Dr. Shaheda Anwar, Department of Microbiology, Ibrahim Medical College, 122 Kazi Nazrul Islam Avenue, Shahbag, Dhaka   Carbepenem, namely imipenem is the drug of choice for the treatment of infections caused by multidrug resistant gram-negative bacilli specially Pseudomonas and Acinetobacter. Recently, metallo-b-lactamase (MBL), a carbepenemase, has been reported to be involved in mediating resistance against imipenem.1 It is a class B beta-lactamase enzyme capable of hydrolyzing all b-lactams except monobactam and their catalytic activities are generally not inhibited by inhibitors like clavulanic acid, salbactam and tazobactam. MBLs are sensitive to metal chelators like EDTA and thiol based compounds and these inhibitors are exploited to detect MBL activities of the organisms.2 Currently, there is no recommended method for the detection of MBL in routine laboratory practice. Therefore, the aim of this study is to evaluate screening tests like DDST and DPT for the detection of MBL producing Pseudomonas and Acinetobacter isolated from clinical samples. Materials and Methods All the 208 isolates (132 Pseudomonas and 76 Acinetobacter) from sputum, urine, tracheal aspirate, blood, wound swab were obtained from the patient admitted in ICU, ward and outpatient department of BSMMU and BIRDEM hospitals. Samples were collected from January 2009 to December 2009. Identification and antimicrobial susceptibility testing All the isolates were tested for imipenem susceptibility by disk diffusion method using the Kirby-Bauer technique8 and as per the recommendations of the NCCLS.9 Imipenem (10µg) and ceftazidime (30µg) disks were obtained from Oxoid Ltd (Basingstoke, Hampshire, UK). Antibiotic potency of the disks were standardized against the reference Pseudomonas ATCC 25853 strain. Detection of MBL-production The EDTA-IPM microdilution MIC test was a modification of EPI microdilution MIC test as described by Migliavacca et al.10 MIC of IPM were determined with a standard microdilution assay in 96 well microtiter plates using Mueller Hinton broth (MHB) and a bacterial inoculum of 5x 104 CFU per well, in a final volume of 100µl. IPM concentrations in the range of 512 to 0.5 µg/ml were tested in the study. The MICs of IPM were determined with IPM alone and IPM plus 0.4mM EDTA. The best results of MIC of IPM were observed with a concentration of EDTA of 0.4mM. One well containing the bacterial suspension alone and another well containing 0.4mM EDTA alone were used as control. Results were recorded by visual inspection of microtiter plates after 18 hour of incubation at 37°C. A minimum fourfold reduction in the MIC of IPM in presence of EDTA in comparison to IPM alone was designated as the cutoff value for detection of MBL producers.10 The test has been used as gold standard for detection of MBLs production in this study. 2. Double disk synergy test (DDST)   DPT was performed according to the method described by Galani et al.12 Three CAZ (30µg) disks and three 10µg IPM disks were placed on the plates. The distance between every CAZ/IPM disk was about 3-4cm from center to center. 10µl of 0.1M EDTA was added to one CAZ/IPM disk and 10µl of 1:12 2-MPA was added to another CAZ/IPM disk. The plate was incubated at 37°C overnight. Enlargement of the diameter of growth inhibitory zone around CAZ/IPM+EDTA/2-MPA disk by ³7mm compared to CAZ/IPM alone was considered as positive for MBL. Result The details of the results of DDST and DPT are given in Table 1 and 2. In DDST, higher doubtful results were obtained by EDTA-IPM (5.6%) or IPM-2-MPA (7.5%) compared to CAZ plus EDTA/2MPA disks. Similarly, higher doubtful results were also obtained with EDTA-IPM (10.3%) or IPM-2-MPA (13.8%) for Acinetobacter compared to CAZ-EDTA and CAZ-2-MPA by DDST. But no doubtful results have been observed for either Pseudomonas or Acinetobacter in detecting MBL by DPT. Table-1: Comparison of detection of MBL positive Pseudomonas with DDST and DPT using CAZ/IPM with EDTA and 2-MPA     Discussion In this study, initially different concentrations of EDTA (0.1M and 0.5M) and 2-MPA (1:8,1:12) were used in DDST and DPT. The purpose of using different concentrations was to identify the most optimum concentration of these agents to detect MBL producing organism. For detection of MBL, 25 Pseudomonas and 19 Acinetobacter were tested with 0.1M and 0.5M EDTA by DDST. All gave positive results with 0.1M EDTA without any equivocal results but with 0.5M EDTA only 76% Pseudomonas and 73.7% Acinetobacter isolates were positive for MBL while 24% and 26.3% showed equivocal results respectively. Distances of 1, 1.5, 2, 2.5,3,4 cm (from center to center) between EDTA/2-MPA and CAZ/IPM were tested for DDST for detection of MBL production. It was found that 1-1.5 cm distance between EDTA/2-MPA and CAZ/IPM disks showed a clear and distinct synergistic zone towards EDTA/2-MPA in DDST. By comparing the sensitivity and specificity of DDST and DPT to detect MBL producing Pseudomonas and Acinetobacter isolates, DPT with 0.1M EDTA provided higher sensitivity and specificity results. DDST with 2-MPA showed the lowest specificity for detection of MBL. Franklin et al showed that DPT had 100% sensitivity and 98% specificity whereas DDST had a sensitivity of 79% and specificity of 98%.13 DPT is preferred because of its objective interpretation compared to DDST. Interpretation of DDST depends on the technician’s expertise in discriminating true synergism from intersection of the inhibitory zones.14 Also, it may be noted that the synergistic zone of inhibition sometimes may be masked if the resistance to CAZ is conferred by AmpC b-lactamase or ESBL.   1.   Nordmann P, and Poirel L. Emerging carbapenemases in Gram negative aerobs. Clin Microbiol Infect 2002; 8: 321-331. 3.   Walsh TR, Bolmstorm A, Gales A. Evaluation of a new E-test for detecting Metallo-b-lactamases in routine clinical testing. J Clin Microbiol 2002; 40: 2755-9. 5.   Walsh, T.R., Neville WA, Haran MH, Tolson D, Payne J, & Bateson JH. Nucleotide and amino acid sequences of the metallo-b-lactamases, ImiS, from Aeromonas veronii bv sorbia. Antimicrob Agents Chemother 1998; 42: 436-439 . 7.   Forbes BA, Sham DF, Weissfeld AS. Bailey and Scott’s diagnostic Microbiology, 10th Edition. Mosby, New York 1998; 167-87. 9.   National Committee for Clinical Laboratory Standards. Performance standards for Antimicrobial Susceptibility Testing: Eleventh informational supplement. NCCLS document M100-S11 NCCLS. Wayne, Pennsylvania 2001; USA. 11.Kim SY, Hong SG, Moland ES, & Thomson SK. Convenient Test Using a Combination of Chelating Agents for Detection of Metallo-b-lactamases in Clinical Laboratory. Journal of Clinical Microbiology 2007; 45(9): 2798-2801. 13.Franklin C, Liolios L, & Peleg A Phenotypic Detection of Carbapenem- Susceptible Metallo-b-lactamase Producing Gram-Negative Bacilli in the Clinical Laboratory. Journal of Clinical Microbiology 2006; 44(9): 3139-3144. 2026 Ibrahim Medical College. All rights reserved.