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Year : 2015  |  Volume : 8  |  Issue : 3  |  Page : 334-336  

Screening for metallo-β-lactamase producing Pseudomonas aeruginosa in clinical isolates in a tertiary care hospital in North India

Department of Microbiology, Indira Gandhi Medical College, Shimla, Himachal Pradesh, India

Date of Web Publication15-May-2015

Correspondence Address:
Sunite A Ganju
House No 214/B, Sector 3, New Shimla, Shimla -171 009, Himachal Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-2870.150506

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Introduction: Pseudomonas aeruginosa has acquired a new metallo-β-lactamase (MBL) resistance gene responsible for increased resistance to fluoroquinolones, cephalosporins and carbapenems. Thus, it is essential to know the antibiotic sensitivity pattern and follow the antibiotic policy. Objectives: The objective of this study is to detect MBL production in clinical isolates by combined imipenem-ethylenediamine tetra acetic acid (IMP-EDTA) disc test. Materials and Methods: This study was conducted for a period of nine months from April 2011 to December 2011. A total of 66 consecutive isolates of P. aeruginosa were subjected to susceptibility testing by disc diffusion assay. IMP drug resistant strains were screened for MBL production by combined IMP-EDTA disc test. Results: Ciprofloxacin resistance was seen in 66.6% isolates followed by piperacillin in 51.5%. Resistance toward amikacin, ceftazidime, and cefoperazone were noted in 43.9%, 40.9%, and 37.8% isolates, respectively. In 37.8%, IMP resistance was observed. All IMP resistant strains (n = 25) were screened for MBL production. All the 25 isolates (100%) were MBL producers, exhibiting more than 7 mm zone size enhancement in IMP-EDTA combined disc test. Conclusion: Emergence of P. aeruginosa as MBL producer is becoming a therapeutic challenge. There is a need to implement routine antibiotic surveillance and judicious use of antibiotics.

Keywords: Imipenem, metallo-β-lactamase, Pseudomonas aeruginosa

How to cite this article:
Ganju SA, Guleria RC, Bhagra S, Kanga AK. Screening for metallo-β-lactamase producing Pseudomonas aeruginosa in clinical isolates in a tertiary care hospital in North India. Med J DY Patil Univ 2015;8:334-6

How to cite this URL:
Ganju SA, Guleria RC, Bhagra S, Kanga AK. Screening for metallo-β-lactamase producing Pseudomonas aeruginosa in clinical isolates in a tertiary care hospital in North India. Med J DY Patil Univ [serial online] 2015 [cited 2023 Sep 30];8:334-6. Available from:

  Introduction Top

Pseudomonas aeruginosa is a frequent cause of respiratory, surgical site and urinary tract infection, especially in patients admitted to intensive care units (ICU). [1] It has been shown to acquire a new metallo-β-lactamase (MBL) encoding gene responsible for the increase in resistance to fluoroquinolones, cephalosporins, and carbapenems. [2] The emergence of resistance is worrisome as it has led to increased mortality particularly among ICU patients besides increased hospital expenditure. [3] In this study, we document the MBL producing P. aeruginosa in clinical isolates from patients admitted to our tertiary care hospital.


To detect MBL producing P. aeruginosa in clinical isolates from hospitalized patients.

  Materials and Methods Top

This study was conducted in the Department of Microbiology, Indira Gandhi Medical College, Shimla, from April 2011 to December 2011. This a tertiary care teaching hospital where patients are referred from most parts of state of Himachal Pradesh. A total of 66 consecutive isolates of P. aeruginosa were isolated during the period of 9 months from various clinical samples. These include 35 isolates from pus, 11 isolates from urine, 7 isolates from ICU, and 8 isolates from burn patients. From sputum and blood samples, two isolates each were isolated, and one from stool sample was detected. All isolates were nonduplicate. These isolates were identified by conventional methods. [4] The routine antibiotic sensitivity was done by Kirby Bauer Method, according to Clinical Laboratory Standards Institute (CLSI) guidelines. [5]

All the strains showing resistance to anti-pseudomonal drugs were noted. Imipenem (IMP) resistant strains were further screened for MBL production by IMP-ethylenediamine tetra acetic acid (EDTA) combined disc test.

Imipenem-ethylenediamine tetra acetic acid combined disc test

The IMP-EDTA combined disc test was performed as described by Yong et al. [6] Test organisms were inoculated on to plates with Mueller Hinton media as recommended by CLSI. [5] Two 10 μg IMP discs (Becton Dickinson) were placed on the plate and appropriate amounts of 10 μl of EDTA (Hi-Media Pvt. Ltd.,) solution was added to one of them to obtain the desired concentration of 750 μg. The inhibition zones of the IMP and IMP-EDTA discs were compared after 16-18 h of incubation at 35°C. In the combined disc test, IMP-EDTA disc, increase in inhibition zone by >7 mm than the IMP disc alone was considered as MBL positive. [6] ATCC 27853 P. aeruginosa was used as a negative control. [4],[5],[6]

  Results Top

A total of 66 isolates of P. aeruginosa were included in this study. All the stains exhibited multiple resistances to most of the drugs. Of the 66 isolates, 66.6% (n = 44) where resistant to ciprofloxacin and 37.8% (n = 25) were resistant to cefoperazone. The pattern of resistance is shown in [Table 1]. Of these, 37.8% (n = 25) isolates were resistant to IMP.

All the 25 IMP resistant isolates were screened for MBL production. The isolates exhibited >7 mm zone size enhancement in IMP-EDTA combined disc test in all 100% (n = 25) isolates thereby proving MBL production as shown in [Figure 1].
Figure 1: Imipenem-ethylenediamine tetra acetic acid combined disc test: Metallo-â-lactamase positive Pseudomonas aeruginosa showing >7 mm zone size enhancement in the combined disc test

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  Discussion Top

Pseudomonas aeruginosa is known to cause severe nosocomial infections, like wound infection in burn patients, sepsis especially in patients from ICU and urinary tract infection. Carbapenems, the β-lactam antibiotics, once considered as lifesaving drugs in Pseudomonas species infection have now been shown to be ineffective due to the development of acquired multidrug resistance. The resistance toward carbapenems shown by P. aeruginosa is mainly due to the development of (i) impermeability due to loss of oprD porin, (ii) upregulation of an active efflux system, and (iii) production of MBLs. [7] Several countries have reported emergence of carbapenem hydrolyzing MBLs as the major mechanisms of carbapenem resistance. [7],[8] Reports from several authors both in India and other countries show MBL producing P. aeruginosa. [7],[9],[10] In our study, IMP resistance in P. aeruginosa was found to be in 37.8% almost similar to that reported by Gupta et al. being 30%. [9] Varaiya et al. reported that IMP resistance in 26%, [10] while higher IMP resistance was reported by other Indian studies being 59.52% and 67%. [7],[11] Emergence of drug resistant P. aeruginosa is a matter of great concern as the viable treatment option has narrowed down to potentially toxic polymixin-B and colistin. [7]

Different methods have been used to detect MBL production. These are MBL-E test, IMP-EDTA double disc synergy test and EDTA disc potentiation using ceftadzidime, ceftizoxime, cefepime and cefotaxime. These screening methods elucidate the mechanisms of antibiotic resistance. In our study, we used IMP-EDTA combined disc method. Though detection by polymerase chain reaction is highly accurate and a reliable method, but the availability in resource limited settings is not possible. IMP-EDTA is considered to be a relatively superior and a cost effective method for detection of MBL. [7] Our results, both negative and positive were clearly demarcated. Besides, the test is a simple screening method that can be easily incorporated into routine testing. [12]

Production of MBL by Pseudomonas species has dangerous therapeutic consequences. Injudicious or widespread use of broad spectrum antibiotics has increased incidence of MBL production in the community. Thus, adherence to antibiotic policy with effective infection control measures is the mainstay to restrict the evolving drug resistance.

  Conclusion Top

The increase in the rates of antibiotic resistance is becoming a global concern. It is a looming threat within the community and hospital. Detection by simple screening methods is essential to find out the local prevalence of MBL producers, so as to limit intra hospital dissemination of such strains and to devise an appropriate hospital antibiotic policy.

  References Top

Streit JM, Jones RN, Sader HS, Fritsche TR. Assessment of pathogen occurrences and resistance profiles among infected patients in the intensive care unit: Report from the SENTRY Antimicrobial Surveillance Program (North America, 2001). Int J Antimicrob Agents 2004;24:111-8.  Back to cited text no. 1
National Nosocomial Infections Surveillance System. National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued October 2004. Am J Infect Control 2004;32:470-85.  Back to cited text no. 2
Zavascki AP, Barth AL, Gonçalves AL, Moro AL, Fernandes JF, Martins AF, et al. The influence of metallo-beta-lactamase production on mortality in nosocomial Pseudomonas aeruginosa infections. J Antimicrob Chemother 2006;58:387-92.  Back to cited text no. 3
Franklin C, Liolios L, Peleg AY. Phenotypic detection of carbapenem-susceptible metallo-beta-lactamase-producing gram-negative bacilli in the clinical laboratory. J Clin Microbiol 2006;44:3139-44.  Back to cited text no. 4
Clinical and Laboratory Standards Institute (CLSI). Performance Standard for Antimicrobial Susceptibility Testing. 16 th Informational Supplement. CLSI Document M2-A9. Wayne, PA: CLSI; 2006.  Back to cited text no. 5
Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002;40:3798-801.  Back to cited text no. 6
Behra B, Mathur P, Das A. Kapil A, Sharma V. An evaluation of 4 different phenotypic techniques for detection of metallo-β-lactamase producing Pseudomonas aeruginosa. Indian J Med Microbiol 2008;26:233-7.  Back to cited text no. 7
Hancock RE. Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria. Clin Infect Dis 1998;27 Suppl 1:S93-9.  Back to cited text no. 8
Gupta E, Mohanty S, Sood S, Dhawan B, Das BK, Kapil A. Emerging resistance to carbapenems in a tertiary care hospital in north India. Indian J Med Res 2006;124:95-8.  Back to cited text no. 9
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Varaiya A, Kulkarni M, Bhalekar P, Dogra J. Incidence of carbapenem-resistant Pseudomonas aeruginosa in diabetes and cancer patients. Indian J Med Microbiol 2008;26:238-40.  Back to cited text no. 10
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Bhalerao DS, Roushani S, Kinikar AG, Akther I. Study of metallo-beta-lactamase producing Pseudomonas aeruginosa in Pravara Rural Hospital. Pravara Med Rev 2010;2:16-9.  Back to cited text no. 11
Yan JJ, Wu JJ, Tsai SH, Chuang CL. Comparison of the double-disk, combined disk, and Etest methods for detecting metallo-beta-lactamases in gram-negative bacilli. Diagn Microbiol Infect Dis 2004;49:5-11.  Back to cited text no. 12


  [Figure 1]

  [Table 1]


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