Use of two double disk synergy tests to detect metallo beta lactamases in nonfermenters.
|Article Type:||Letter to the editor|
|Publication:||Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2008 Indian Council of Medical Research ISSN: 0971-5916|
|Issue:||Date: Nov, 2008 Source Volume: 128 Source Issue: 5|
|Product:||Product Code: 2834880 Bacteriostats NAICS Code: 325412 Pharmaceutical Preparation Manufacturing SIC Code: 2834 Pharmaceutical preparations|
A number of Gram negative bacilli especially belonging to family Enterobacteriaceae are rapidly acquiring resistance to the routinely used antimicrobial agents due to beta-lactamases production. This has led to an increase in the usage of newer drugs including carbapenems. Further, a number of bacteria belonging to the nonfermenter group--Pseudomonas aeruginosa and Acinetobacter species are being reported to be even resistant to this group of carbapenems due to metallo beta lactamases (MBL) production (1,2). Nonfermenter group of organisms is a group of aerobic, non-sporeforming Gram-negative bacilli that either do not utilize carbohydrates as a source of energy or degrade them through metabolic pathways other than fermentation (3). There are various reports on increasing prevalence of MBL worldwide including India (4,5).
Clinical and Laboratory Standards Institute (CLSI) has not laid down any specific guidelines for detection of MBL production in various organisms (6). Though there are several screening methods recommended for detection of MBL production, no single test, when used alone is specific for these enzymes. Polymerase chain reaction (PCR) is the most reliable diagnostic test but main difficulty is the prevalence of increasing types of MBLs requiring multiple primers. We thus evaluated two types of double disc diffusion tests (DDSTs): imipenem- ethylene diamine tetra acetic acid (IPMEDTA) method and ceftazidime- mercapto propionic acid (CAZ-MPA) method to detect MBL production in P. aeruginosa and Acinetobacter species.
The present study was undertaken on the 100 isolates of P. aeruginosa and Acinetobacter species in the Department of Microbiology, Govemment Medical College Hospital, Chandigarh, North India during the time period of July 2005 to December 2006. The selection criteria for MBL detection was reduced susceptibility to ceflazidime (inhibition zone diameter < 18 mm) and/or to imipenem (inhibition zone diameter <16 mm). MBL detection was done by Lee et al (1) modification of imipenem- EDTA (IPM-EDTA) disc method of Yong et al (1) by adding zinc sulphate to the medium to increase the sensitivity of the test, and ceftazidime- MPA (CAZ-MPA) disc method of Arakawa et al (8). All the MBL positive isolates were repeatedly checked for reproducibility and for susceptibility with aztreonam.
Of the total 100 isolates, 33 were P aeruginosa and 67 belonged to Acinetobacter species. Ali these isolates were ceftazidime resistant and when tested by CAZ-MPA method, MBL production was seen in 97 (97%) of them. Imipenem resistance was seen in only 72 isolates and by IPM-EDTA method, 62 (86.11%) of these were positive for MBL production. Ali were sensitive to aztreonam.
Of the 33 P aeruginosa isolates tested, 32 (96.97%) were found to be positive for MBL production by CAZ-MPA method. Of these, 25 isolates were also imipenem resistant and IPM-EDTA method was found to be positive for 21 (84%) of them.
Of the 67 isolates of Acinetobacter species tested by CAZ-MPA method 65 (97.01%) were positive for MBL production. Only 47 isolates were imipenem resistant and when tested by IPM-EDTA method, 41 were positive (87.23%). Of the 100 isolates tested, 97 per cent isolates showed MBL production considering either method. Of the 72 imipenem resistant isolates, 62 were positive by IPM-EDTA disc method, remaining 10 which did not show any increase in growth inhibition zone after addition of EDTA, may be belonging to IMP-1 category, as these were ceftazidime resistant also and positive by CAZ-MPA method. The reason for detection by CAZ-MPA method than IPM-EDTA method could be that IMP-1 isolates usually show high level resistance to CAZ. Furthermore, 2-MPA has also been found to block IMP-1 activity strongly even at low concentrations giving clear-cut growth inhibition zones (9).
Use of imipenem resistant strains alone can reduce the screening work greatly and imipenem with EDTA is found to give larger inhibition zones especially for VIM-2 isolates (7). Moreover, some of the resistant strains may also have another ceftazidime resistance mechanism than MBL production. In such cases, DDST with imipenem can show better results than ceftazidime disc. However, there are chances that some strains may be missed out by using imipenem alone. The previous studies show that for some of the nonfermenter organisms, minimum inhibitory concentration (MIC) to imipenem may not be very high (MIC <4 [micro]g/ml) and such organisms may appear susceptible to carbapenems by using current CLSI methods (2). The rate of resistance of Acinetobacter species to ceftazidime is also found to be more than that to imipenem at some places (7). Therefore, use of both CAZ-MPA method and IPM-EDTA method on ceftazidime resistant strains and imipenem resistant strains is being recommended.
Our study emphasizes the use of two different substrates with different inhibitors as each method individually has its own merits and demerits. This methodology can be adapted for MBL screening in routine diagnostic microbiology laboratories.
Varsha Gupta *, Nidhi Singla
& Jagdish Chander
Department of Microbiology
Govemment Medical College Hospital
Chandigarh 160 031, India
* For correspondence:
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(2.) Franklin C, Liolios L, Peleg AY. Phenotypic detection of carbapenem susceptible metallo B-lactamase producing Gram negative bacilli in the clinical laboratory. J Clin Microbiol 2006; 44: 3139-44.
(3.) Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC Jr. The non fermentative Gram negative bacilli. In: Color atlas and textbook of diagnostic microbiology, 5th ed. Philadelphia: Lippincott; 1997. p. 254.
(4.) Varaiya A, Kulkarni N, Kulkarni M, Bhalekar P, Dogra J. Incidence of metallo beta lactamases producing Pseudomonas aeruginosa in ICU patients. Indian d Med Res 2008; 127 : 398-402.
(5.) Gupta V, Datta P, Chander J. Prevalence of metallo-[beeta]-lactamase (MBL) producing Pseudomonas spp. andAcinetobacter spp. in a tertiary care hospital in India. d Infect 2006; 52 : 3 l 1-4.
(6.) Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 15th Informational Supplement. CLSI/NCCLS M 100- S 15. Wayne (PA); 2005.
(7.) Lee K, Lim YS, Yong D, Yum JH, Chong Y. Evaluation of the Hodge test and the Imipenem -EDTA double disk synergy test for differentiating metallo-B-lactamase- producing isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2003; 41 : 4623-9.
(8.) Arakawa Y, Shibata N, Shibayama K, Kurokawa H, Yagi T, Fujiwara H, et al. Convenient test for screening metallo[beta]-lactamase -producing Gram -negative bacteria using thiol compounds, d Clin Microbiol 2000; 35 : 40-3.
(9.) Goto M, Takahashi T, Yamashita F, Koreeda A, Mori H, Ohta M, et al. Inhibition of the metallo 13-1actamase produced from Serratia marcescens by thiol compounds. Biol Pharm Bull 1997; 20 : 1136-40.
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