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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusions
 ~ Acknowledgment
 ~  References
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 30  |  Issue : 2  |  Page : 170-174
 

Comparison of disc and MIC reduction methods with polymerase chain reaction for the detection of metallo-β-lactamase in Pseudomonas aeruginosa


Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra, India

Date of Submission07-Nov-2011
Date of Acceptance10-Mar-2012
Date of Web Publication28-May-2012

Correspondence Address:
D K Mendiratta
Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.96683

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 ~ Abstract 

Purpose: The present study was undertaken to evaluate the screening antibiotic, confirmatory phenotypic test and agent against PCR as gold standard and to detect the prevalent MBL gene. Materials and Methods: Three hundred and twenty-six Pseudomonas aeruginosa isolates were screened for resistance to Imipenem (IPM), Meropemem (MEM) and Ceftazidime (CAZ) by disc diffusion. Isolates resistant to any of these were considered screen test-positive for MBL and were subjected to Double disc synergy test (DDST) and Disc potentiation test (DPT: Using IPM, MEM and CAZ alone and with EDTA), Minimum inhibitory concentration (MIC) reduction [four-fold or more reduction in MIC of IPM and MEM in presence of chelators: EDTA and 1,10-phenanthroline (EPI/EPM: EDTA-phenanthroline- Imipenem/Meropenem Broth Microdilution method)] and polymerase chain reaction (PCR) for blaIMP and blaVIM . Results: Screen test-positives by MEM and CAZ were 19.3% as against 17.8% by IPM. MEMDDST, DPT and EPM confirmed 100% screen-test positives as against 93.7% by CAZ DDST and DPT-2, 76.2% by CAZ DPT-1, 88.9% by IPM DDST, 85.7% by IPM DPT-1 and 92.1% by EPI. IPMand CAZ DDST together confirmed 100% while IPM and CAZ DPT-2 confirmed 96.8%. All 63 screen-test positives showed the presence of blaVIM . Conclusions: MEM was found to be the best screening and confirmatory agent for MBL detection and blaVIM was found to be the prevalent MBL gene in this part of the country.


Keywords: Double-disc synergy test, disc potentiation test, EDTA-phenanthroline- Imipenem/Meropenem Broth Microdilution, Metallo-β-lactamase, P. aeruginosa, PCR


How to cite this article:
Buchunde S, Mendiratta D K, Deotale V, Narang P. Comparison of disc and MIC reduction methods with polymerase chain reaction for the detection of metallo-β-lactamase in Pseudomonas aeruginosa. Indian J Med Microbiol 2012;30:170-4

How to cite this URL:
Buchunde S, Mendiratta D K, Deotale V, Narang P. Comparison of disc and MIC reduction methods with polymerase chain reaction for the detection of metallo-β-lactamase in Pseudomonas aeruginosa. Indian J Med Microbiol [serial online] 2012 [cited 2019 Jun 19];30:170-4. Available from: http://www.ijmm.org/text.asp?2012/30/2/170/96683



 ~ Introduction Top


Pseudomonas aeruginosa is a leading cause of nosocomial infections [1] and in the light of its numerous intrinsic and acquired mechanisms of drug resistance it is a cause of concern for treating physicians. Although the antibiotic resistance in P. aeruginosa is caused by multiple mechanisms, one growing factor leading to resistance is the production of carbapenemases.

Metallo-β-lactamases (MBL's) are carbapenemases which require zinc at the active site and are predominantly produced by P. aeruginosa. [2] They belong to Ambler's Class B and Bush-Jacoby Mederios Group 3 and hydrolyze virtually all β-lactam agents, including the carbapenems. Further, as the genes coding them are carried on highly mobile elements, their spread in recent years from P. aeruginosa to Enterobacteriaceae, has lead to a situation where a clinical scenario that could simulate the global spread of extended-spectrum β-lactamases appears to be developing. Till now seven main types of MBL have been described throughout the world - IMP, VIM, SPM, GIM, SIM, AIM-1 [2] and NDM-1. [3] Among them, bla IMP and bla VIM are the most common types of MBLs with worldwide distribution. [4] From India only bla VIM [5],[6] and NDM-1 [7] have been reported in P. aeruginosa in the past. VIM (Veronese Imipenemase) enzymes have been grouped into three main clusters designated VIM-1, VIM-2, and VIM-7. To date, VIM-2 is more widely spread among P. aeruginosa isolates, whereas VIM-1 is normally confined to Enterobacteriaceae isolates [8] The IMP sublineage has at least 27 unique variants (http://www.lahey.org/studies) differing by up to 22% amino acid sequence divergence (between IMP-9 and IMP-19) that exhibit important structural and functional differences from each other or from enzymes of other sublineages. IMP-type MBL determinants are mostly found in P. aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae isolates, although they have also been sporadically identified in other organisms (e.g., Pseudomonas putida) Clinical and Laboratory Standards Institute (CLSI) has not laid down any specific guidelines though there are several screening methods recommended for detection and confirmation of MBL production in P. aeruginosa. The selection criteria for confirmation of MBL producers is reduced susceptibility or resistance to carbapenems and/or ceftazidime. Currently, the most widely accepted standardized MBL confirmation method is the MBL Etest (AB BioDisk, Solna, Sweden). However, due to the high cost many clinical microbiology laboratories use alternative methods such as the double-disc synergy test (DDST) and the combined disk (CD)/disc potentiation test (DPT). Although the DDST and the DPT assay are simple to perform and cheaper than the Etest they have shown discordant results, depending on the employed methodology, β-lactam substrates, MBL inhibitors (IMBL), and bacterial genus tested.[9] As zinc ion is essential for the action of MBLs, chelating agent like Ethylene diamine tetra-acetic acid (EDTA) has been used commonly as in vitro inhibitor of MBLs. The genotypic methods viz. Polymerase chain reaction (PCR), random amplified polymorphic DNA, restriction fragment length polymorphism, though highly accurate and reliable, their accessibility is often limited to reference laboratories.

The present study was undertaken with the objectives of i) determining which of the antibiotic viz., meropenem, imipenem and ceftazidime was a good screening as also a confirmatory agent ii) evaluating DDST, DPT and MIC reduction assay using above antibiotics as confirmatory tests and iii) to detect the prevalent MBL gene in this part of the country.


 ~ Materials and Methods Top


Bacterial strains

A total of 326 P. aeruginosa strains, isolated from 50775 clinical specimens (Aspirate, Ascitic fluid, Blood, Biopsy, Cervical swab, Corneal scrapping, Conjunctival swab, CSF, Pus, Pleural fluid, Sputum, Semen, Throat swab, Urine, Vaginal swab, Wound swab) between July 2008 to August 2010 and identified as per the conventional standard methods were studied for MBL production.

Screening for MBLs

An isolate of P. aeruginosa was considered screen-test positive for MBL when it was resistant to Imipenem:10 μg (IPM) and/or Meropemem:10 μg (MEM) and/or Ceftazidime: 30 μg (CAZ) (Himedia). Antibiotic sensitivity was performed by the Kirby-Bauer disc diffusion method as per CLSI guidelines. [10]

Controls

P. aeruginosa (bla VIM and bla IMP positive: CMC, Vellore) and ATCC 27853 were used as positive and negative controls in all tests performed. All experiments were performed in duplicate.

Confirmation of MBL production: All screen-test positive isolates were subjected to:

Double disc synergy test

This test was performed as described by Lee et al. [11] with some modifications. IPM (10 mg), MEM (10 μg), CAZ (30 μg) discs were placed on the same Mueller Hinton agar (MHA) plate seeded with 0.5 McFarland (10 8 CFU/ml) of test organism with centre to centre distance of 20 mm from a disc containing 5 μl 0.5 M EDTA (930 μg). Plate was incubated at 37΀C for 18-20 hours. Enhancement of zone of inhibition around IPM and/or MEM and/or CAZ toward the EDTA disc in comparison with the zone of inhibition on the far side of corresponding antibiotic disc was interpreted as positive for MBL production.

Combined disc test/Disc Potentiation test

This test was performed as described by Yong D et al. [12] Two discs each of 10 μg IPM,10 μg MEM and 30 μg CAZ were placed on lawn culture of 0.5 McFarland test organism seeded on MHA. To one disk of IPM, MEM and CAZ 5 μl of 0.5 M EDTA (930 mg) was added. Plates were incubated at 37΀C for 18-20 hours. After incubation, zone of inhibition was measured using Hi-media scale. Organisms which showed increased zone of inhibition by 7 mm or more (DPT-1: Yong D et al.) [12] around any or all of the three disc with EDTA or showed increase in 5-28 mm inhibition around only CAZ-EDTA disc (DPT-2: Hemlatha et al.) [13] as compared to IPM, MEM and CAZ discs alone, respectively, were considered to be MBL producers. A blank disc of EDTA was tested as control.

EPM/EPI Microdilution test: EDTA phenanthroline+MEM/IPM Microdilution test [14]

MICs were determined in 96 well microtitre plates using 50 μl of Muller Hinton broth, 5 μl of bacterial inoculum (5 Χ 10 4 CFU: Soon after preparation) and 50 μl of corresponding concentration of the antibiotic per well as per CLSI guidelines. [10] IPM/MEM (Source-Piramal) concentrations in the range of 0.25 - 512 μg/ml were tested. Reduction in MIC of IPM/MEM were determined by adding 5-μl mixture of chelators EDTA 0.4 mM and 1,10-phenanthroline 0.04 mM to the second row, just before inoculation of wells with broth culture. Growth controls without IPM/MEM but with and without chelator mixtures were included. Results were recorded by visual inspection of microtitre plates after 18 hours of incubation at 37΀C as per CLSI guidelines. [10] The test was considered valid when acceptable growth (more or equal to 2-mm button or definite turbidity) occurred in the positive control well. Absence of turbidity or a button of less than 2 mm diameter in the test well was thus taken as the MIC of the organism under test. A ≥ four-fold IPM and/or MEM MIC reduction in presence of chelators as compared to MIC without them was taken as cutoff value for MBL production.

PCR for detection of MBL producing genes: blaIMP and bla VIM: [15]

Extraction

Fresh culture of the test organism and the control strains was suspended in 500 μl of saline and vortexed to get a uniform suspension. The cells were lysed by heating at 100΀C for 10 mins, and cellular debris was removed by centrifugation at 8000 rpm for 5 mins. The supernatant was used as a source of template.

PCR Master Mix (Total volume: 25 μl)

This included 2.5 μl of PCR buffer, 2.5 μl of MgCl 2 , 2.5 μl of DNTPs (Fermentas,Genetix Biotech Asia Pvt Ltd, N Delhi)), 8.2 μl of MiliQ water (Fermentas), 1 μl of each of the forward and reverse primers, 0.3 μl of Taq Polymerase (Fermentas) and 5 μl of the extracted DNA. Primers: (Sigma Aldrich, Statesman House, Barakhamba Road, N Delhi): VIM- Forward (5Ͳ-GTT TGG TCG CAT ATC GCA AC-3Ͳ) and VIM- Reverse (5Ͳ-AAT GCG CAG CAC CAG GAT AG-3Ͳ), which amplified a 382-bp amplicon. IMP- Forward (5Ͳ-GAA GGY GTT TAT GTT CAT AC-3Ͳ) and IMP- Reverse (5Ͳ-GTA MGT TTC AAG AGT GAT GC-3Ͳ), which amplified a 587 bp amplicon. Amplification: Initial denaturation step was at 94΀C for 2 mins followed by 30 cycles of DNA denaturation at 94΀C for 1 min, primer annealing at 54΀C for 1 min and primer extension at 72΀C for 15 mins with a holding temperature of 72΀C for 5 mins. After the last cycle, PCR products were stored at 4΀C. Electrophoresis: PCR products were analysed by electrophoresis with 1.5% agarose gels in TBE buffer. Staining: The gels were stained with ethidium bromide (75 μl in 500 μl of distilled water: Bangalore Genei). PCR products were visualized with UV light.

Statistical methods

Data was analysed using Epi info 6


 ~ Results Top


Out of 326 P. aeruginosa strains studied, 63 (19.3%) were resistant to MEM, 63 (19.3%) to CAZ and 58 (17.8%) to IPM (screen-test positive). IPM failed to pick up five isolates that were found resistant to MEM and CAZ [Table 1].
Table 1: Results of various tests

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MEM DDST and CAZ DDST confirmed all the 63 screen-test positives, while IPM DDST 88.9% of the screen-test positives. [Table 1].

Using criteria by Yong D et al. [12] for DPT (DPT-1), MEM confirmed 100% screen-test positive isolates, IPM 85.7% and CAZ 76.2% (P>0.05) [Table 1]. However, when criteria given by Hemlatha et al. [13] for CAZ DPT was used (DPT-2), CAZ was able to confirm additional 11 (17.4%) isolates, thereby raising the positivity of CAZ DPT to 59 (93.7%) [Table 1].

[Table 1] further shows that both IPM DDST and CAZ DDST, as also IPM DPT and CAZ DPT missed confirmation of some isolates found positive by the other antibiotic using the same technique. However, combination of IPM DDST with CAZ DDST confirmed all 63 screen-test positives, while IPM DPT-1 and CAZ DPT-2 together confirmed 96.8% of the screen-test positives.

All the 58 IPM and 63 MEM resistant screen-test positive isolates showed four-fold or more fall in MICs of IPM and MEM, respectively, in the presence of mixture of EDTA and 1,10-phenanthroline as compared to IPM and MEM alone. IPM MIC reduction ranged between 8 and 32-fold and MEM MIC 8 and 64-fold. With both carbapenems most isolates showed 8-fold reduction (IPM: 56.9% and MEM: 71.4%). The IPM MIC of four of the five screen test IPM-sensitive MEM-resistant isolates was in the sensitive range and that of the fifth in the intermediate range, but this isolate showed only one-fold reduction in IPM MIC in presence of chelators.

All the 63 screen-test positive isolates, including those not picked by IPM, were found to be positive for the presence of bla VIM using PCR.

Keeping PCR or EPM microdilution broth as gold standard, the sensitivity of CAZ DDST (93.7%) was higher than IPM DDST (88.9%) (P>0.05). The sensitivity of CAZ DPT-2 (93.7%) was higher than IPM DPT-1 (85.7%). However, the sensitivity status was reversed on using criteria given by Yong D et al. [12] (P>0.05). The sensitivity of CAZ DDST was higher (93.7%) than CAZ DPT (76.2%) using criteria by Yong D et al. [12] (P>0.05).


 ~ Discussion Top


In our study MEM and CAZ were each able to pick up 19.3% (63/326) of isolates for MBL confirmation as compared to IPM which picked up 17.8% (58/326) [Table 1]. CAZ has been recommended over IPM by many authors for screening of MBL producers. [13],[16],[17] This has been specially true for isolates showing a high MIC for CAZ and also for the fact that IPM is less stable than CAZ. [16] The CLSI 2009 [10] also considers IPM to be a poor screening agent for carbapenemases, but for members of Enterobacteriaceae using disc diffusion.

Keeping MEM DDST/DPT/MIC reduction and/or PCR as gold standard IPM DDST was found to be better (sensitivity 88.9%) than IPM DPT (sensitivity 85.7%) as also observed by Renu G et al., [18] because even a slight increase in the synergistic zone is taken as positive in DDST in contrast to zone difference of ≥7 mm taken as positive by DPT. However, S Sopasri et al. [19] and Behera et al. [1] from India and Qu TT et al. [9] from China found IPM DPT to be better than IPM DDST. Sakshi P Singh et al. [20] and Picao et al. [21] found both, IPM DPT and IPM DDST to be equally efficient.

CAZ DDST was better (sensitivity 93.7%) than CAZ DPT-1 (sensitivity 76.2%) in confirming isolates for MBL production though these differences were not statistically significant (P>0.05) [Table 1]. Nirav et al. [22] have recently reported CAZ DPT (85.2%) to be better than CAZ DDST (44.4%). Even in our study the sensitivity of CAZ DPT-2 [13] was as good as CAZ DDST (93.7%) [Table 1]. Franklin et al. [17] go a step further and suggest the use of zone enhancement in the combined disc test by 4 mm or more, as indicator of MBL production even in carbapenem susceptible MBL-producing organisms, for enhancing the utility and sensitivity of this test for MBL detection. They have emphasized that such carbapenem susceptible MBL-producing isolates carry hidden MBL genes and are a threat to infection control practices. Treatment of such cases results in poor clinical outcome when carbapenems are administered. However, they have further emphasized that such a hypotheses needs further evaluation.

In our study, evaluation of simple phenotypic tests viz., DDST vs DPT and antibiotics viz MEM vs IPM vs CAZ in confirming MBL production showed that MEM DDST or MEM DPT turned out to be the best followed by CAZ DDST/CAZ DPT-2 and IPM DDST/IPM DPT-1. Mendiratta et al. [23] have also reported CAZ DDST to be better than IPM DDST, though the number of isolates studied were few. Manoharan et al. [6] have reported higher sensitivity (87.8%) with IPM EDTA DPT as compared to CAZ and MEM EDTA DPT in a study on 176 P. aeruginosa isolates during a multicentric study between 2005 and 2007. S Sopasri et al. [19] have observed higher sensitivity (100%) using IPM EDTA DPT over MEM EDTA DPT (92.9%). However, Pitout et al. [15] have reported higher MEM EDTA DPT sensitivity over IPM EDTA DPT as also observed in our study. In absence of MEM, we observed that IPM and CAZ DDST (100% sensitivity) or IPM DPT-1 and CAZ DPT-2 (96.8% sensitivity) need to be performed together to obtain best results [Table 1]. Behera et al. [1] also advocate use of both CAZ and IPM, as according to them some strains may be missed when either is used alone, as also observed in our study [Table 1]. Picao et al. [21] insist that the use of two phenotypic methods, DDST and DPT should be based on genera to be tested as well as local prevalence of MBL producers.

Using the technique described by Migliavacca et al., [14] we successfully demonstrated the reduction in MIC of not only IPM but also MEM by four-fold or more titre for all isolates screen-test positive by IPM and MEM, respectively. The magnitude of IPM MIC reduction in our study ranged between 8 and 32-fold and most of the isolates (56.9%) showed 16-fold reduction. The magnitude of IPM MIC reduction has been reported to range between 4 and 512-fold. [13],[14] We could not find studies utilizing MEM MIC reduction for detection of MBL producers. The magnitude of MEM MIC reduction in our study ranged between 8 and 64-folds and most of the isolates (71.4%) showed 16-fold MIC reduction.

In the present study, all the 63 screen-test positive isolates were found to contain bla VIM by the use of broad primers directed to detect bla IMP and bla VIM . Using the same primers and the technique, Manoharan et al. [6] also reported only bla VIM type of MBL from 17 of 20 E test confirmed MBL-positive P. aeruginosa isolates collected from seven different centres of India. Similarly in a study carried out by M. Castanheira et al. [5] on 301 Pseudomonas isolates collected from 10 Indian hospitals in 2008 and showing elevated MICs (Broth Microdilution) for IPM or MEM (MIC≥8 μg/ml), only VIM-2 genes were demonstrated in 57 (53.2%) using multiplex PCR. Pitoutet al. [15] reported blaVIM in 43% and bla IMP in 2% of P. aeruginosa isolates collected between 2002-2004 from Canada using the primers that were used in our study. Recently NDM-1 has been reported in P. aeruginosa isolates from Mumbai. [7] Our study further strengthens the findings that bla VIM still continues to be the prevalent MBL enzyme in P. aeruginosa isolates in India.


 ~ Conclusions Top


MEM and CAZ were found to be good screening agents, MEM DDST, DPT and EPM best confirmatory tests and bla VIM the prevalent MBL gene in this part of the country.


 ~ Acknowledgment Top


The authors are grateful to the Kasturba Health Society, Sevagram for funding the study and BMPLIII, IDTRC, CMC, Vellore for technical support.

 
 ~ References Top

1.Behera B, Mathur P, Das A, Kapil A Sharma V. An eavluation of four different phenotypic techniques for detection of metallo-beta-lactamase producing Pseudomonas aeruginosa. Indian J Med Microbiol 2008;26:233-7.   Back to cited text no. 1
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2. Gupta V. Metallo beta lactamases in Pseudomonas aeruginosa and Acinetobacter species. Expert Opin Investig Drugs 2008;17:131-43.  Back to cited text no. 2
    
3.Struelens MJ, Monnet DL, Magiorakos AP, Santos O'Connor F, Giesecke J, the European NDM-1 Survey Participants. New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae: Emergence and response in Europe. Euro Surveill 2010;15.pii:19716.  Back to cited text no. 3
    
4.Kouda S, Ohara M, Onodera M, Fujiue Y, Sasaki M, Kohara T, et al. Increased prevalence and clonal dissemination of multidrug-resistant Pseudomonas aeruginosa with the blaIMP-1 gene cassette in Hiroshima. J Antimicrob Chemother 2009;64:46-51.  Back to cited text no. 4
    
5.Mariana Castanheira JM. Carbapenem Resistance among Pseudomonas aeruginosa Strains from India: Evidence for Nationwide Endemicity of Multiple Metallo-beta-lactamase clones (VIM-2,-5,-6 and -11 and the newly characterized VIM-18). Antimicrob Agents Chemother 2009;531225-7.  Back to cited text no. 5
    
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22.Pandya NP, Prajapati SB, Mehta SJ, Kikani KM, Joshi PJ. Evaluation of various methods for detection of metallo â lactamase (MBL) production in Gram negative bacilli. Int J Biol Med Res 2011;2:775-7.  Back to cited text no. 22
    
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2004 - Indian Journal of Medical Microbiology
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