Comparative evaluation of phenotypic tests for identification of metallo [beta]-lactamases producing clinical isolates of Pseudomonas aeruginosa.
Singh, Sakshi P.
|Publication:||Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2009 Indian Council of Medical Research ISSN: 0971-5916|
|Issue:||Date: June, 2009 Source Volume: 129 Source Issue: 6|
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Carbapenems have been the most successful [beta]-lactam antibiotics used in the treatment of infections caused by [beta]-lactam resistant Gram-negative bacteria. However, the clinical utility of these antimicrobials is under threat with the emergence of carbapenemases, particularly the Ambler class B metallo [beta]-lactamases (MBLs). MBLs can hydrolyze most [beta]-lactams except for monobactams and confer a broad-spectrum [beta]-lactam resistance phenotype to the bacterial host, which is not reversible by conventional therapeutic [beta]-lactamase inhibitors. The prevalence of MBLs has been increasing worldwide, notably among Pseudomonas aeruginosa and lately, amongst other Gram-negative bacteria as well (1).
All the methods for detection of MBL producing bacterial isolates depend on the principle, that MBLs are affected by the removal of zinc from their active site. Still, no single screening method has been found to be perfect. Currently, there is no Clinical Laboratory Standards Institute (CLSI) recommended method available. Also, no standard method is recommended by any other international committee for the detection of MBL producers.
Most of the studies from different parts of the world compared some of the available tests, but no study has ever been undertaken which has compared all the available tests. Such studies are lacking in India also. The present study thus, envisaged comparative evaluation of the various available phenotypic methods for detection of MBLs in P. aeruginosa in order to identify the most sensitive method for Indian clinical isolates.
One hundred non repetitive clinical isolates of P. aeruginosa randomly collected between March 2002 and December 2005 were screened for susceptibility to imipenem (IPM) using the CLSI disc diffusion method (2). Of these 100 isolates, 41 had been collected from Safdarjung Hospital, New Delhi, and 59 were from Vallabhbhai Patel Chest Institute, Delhi. All imipenem non susceptible isolates were tested for MBL production using a battery of phenotypic tests viz., modified Hodge test on Mueller-Hinton agar (MHT-MHA) (3), modified Hodge test on MacConkey agar (MHT-MCA) (4), imipenem-ethylenediaminetetraacetic acid + sodium mercapto acetic acid double disc synergy test (IPM-EDTA+SMA DDST) (3), combined disc test (CDT) (5), extended EDTA disc synergy test (eEDST) (6) and EDTA-IPM microbiological assay (EIM) (6).
Of the 100 clinical isolates of P. aeruginosa screened, 21 were non susceptible to IPM. All these 21 isolates were subjected to the phenotypic tests listed above. MHT-MHA identified 15 (71.4%) isolates, whereas MItT-MCA identified 19 (90.5%) isolates as MBL producers. Each of the three techniques viz., DDST, EIM assay, and eEDST was able to identify 20 (95.2%) isolates as MBL producers. Similarly, CDT could detect 20 (95.2%) isolates as positive. In eEDST, IPM disc synergy could identify 20 isolates; meropenem (MEM) disc synergy identified 18 while ceftazidime (CAZ) disc synergy could detect only 12 isolates as MBL producers.
Of the 21 IPM non susceptible isolates as many as, 20 could be identified as MBL positive by four techniques viz., EIM assay, DDST, CDT and eEDST-IPM. However, only 15 of 21 isolates were positive by all the tests employed in the study (Table). One isolate was negative by all the tests.
Varying prevalence rates of MBLs producing P. aeruginosa have been reported worldwide. As many as, 43.9 per cent of Brazilian and 39.1 per cent of Italian imipenem resistant isolates of P. aeruginosa were reported to be MBL producers (7). MBL positive P. aeruginosa constituted nearly 20 per cent of all nosocomial isolates in Korea (8). In India, a few studies have been carried out in different parts of the country in the recent past. The prevalence rates reported by these workers have ranged between 4.5 to 14 per cent (9-11). In our study, however, 20 per cent of the clinical isolates of P. aeruginosa were confirmed as MBL producers. The higher incidence recorded in our study could possibly be ascribed to use of multiple and sensitive techniques employed. It also indicated the alarming rise in incidence of MBL positive P. aeruginosa.
Conventionally, MHT is performed using MHA; however, Lee et al (4) reported better results using MCA instead of MHA. Our study corroborated the results reported by Lee et al (4) as the MHT-MCA detected MBLs in 19 (90.5%) isolates; while the conventional MHT-MHA detected only 15 (71.4%) isolates to be MBL positive.
A study incorporating the comparative evaluation of variations of CDT viz., CAZ + EDTA and IPM + EDTA showed the former to be more sensitive in detecting MBLs in P. aeruginosa (12). A study from India, reported detecting MBLs in 6 of 8 isolates of P. aeruginosa using CAZ + EDTA and in 5 of 8 using IPM +EDTA (10). However, we could detect MBLs in 38.1 per cent of the isolates using CAZ + EDTA, while, the IPM + EDTA combination detected MBLs in as many as 95.2 per cent of the isolates.
Authors thank Dr Rajni Gaind, Department of Microbiology, Safdarjung Hospital and Associated Vardhman Mahavir Medical College, New Delhi, for providing isolates of P. aeruginosa.
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Sakshi P. Singh, Malini Shariff
Tanushree Barua & S.S. Thukral *
Department of Microbiology, Vallabhbhai Patel Chest
Institute, University of Delhi, Delhi 110 007, India
* Present address:
Department of Microbiology, College of Medicine Sultan Qaboos University, Muscat, Oman
* For correspondence:
Table. Comparison of different phenotypic tests for detection of MBL producing isolates Phenotypic test Number of MBL positive isolates (n=21) Modified Hodge test on Mueller Hinton Agar 15 Modified Hodge test on MacConkey Agar 19 Combined Ceftazidime + EDTA 8 disc test Imipenem + EDTA 20 Double disc synergy test 20 EDTA-Imipenem microbiological assay 20 Extended EDTA Ceftazidime 12 disc synergy Imipenem 20 test Meropenem 18 n, number of screen positive isolates tested
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