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 Table of Contents  
Year : 2016  |  Volume : 3  |  Issue : 4  |  Page : 263-267

Prevalence of carbapenemase-producing organisms in a tertiary care hospital in Ludhiana

1 Department of Microbiology, Christian Medical College and Hospital, Ludhiana, Punjab, India
2 Department of Medicine, Christian Medical College and Hospital, Ludhiana, Punjab, India

Date of Web Publication14-Sep-2016

Correspondence Address:
Dr. Aroma Oberoi
Department of Microbiology, Christian Medical College and Hospital, Ludhiana - 141 008, Punjab
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2348-3334.190574

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Aim: This study was done to determine the minimum inhibitory concentration (MIC) of imipenem for multidrug-resistant (MDR) clinical isolates and identify carbapenemase-producing organisms among these MDR isolates. Materials and Methods: The antibiotic susceptibility of clinical isolates was determined by Kirby–Bauer disc diffusion method. MDR isolates showing resistance or reduced susceptibility to carbapenems were further tested for MIC with imipenem and carbapenemase production by Modified Hodge test (MHT). Results: A total of 65 MDR isolates were tested, of which 46 (70.77%), 15 (23%), and 4 (6.15%) had MIC in resistant, sensitive, and intermediate range, respectively. MHT was positive for 37 (57%) isolates. The most common carbapenemase producers in order of frequency were Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter aerogenes, Escherichia coli, Pseudomonas spp. Conclusion: Phenotypic tests such as MHT are simple, cost-effective, and easy to perform and hence can be used in any microbiology laboratory to detect carbapenemase production and applied clinically to guide the antimicrobial therapy, especially in severe and life-threatening infections.

Keywords: Carbapenemase production, carbapenem minimum inhibitory concentration, Modified Hodge test, multidrug resistant

How to cite this article:
Mathias A, Oberoi A, John M, Alexander VS. Prevalence of carbapenemase-producing organisms in a tertiary care hospital in Ludhiana. CHRISMED J Health Res 2016;3:263-7

How to cite this URL:
Mathias A, Oberoi A, John M, Alexander VS. Prevalence of carbapenemase-producing organisms in a tertiary care hospital in Ludhiana. CHRISMED J Health Res [serial online] 2016 [cited 2023 Apr 1];3:263-7. Available from: https://www.cjhr.org/text.asp?2016/3/4/263/190574

  Introduction Top

The term “carbapenem” is defined as the 4:5 fused ring lactam of penicillins with a double-bond between C-2 and C-3 but with the substitution of carbon for sulfur at C-1. Thienamycin was the first carbapenem discovered and demonstrated potent broad-spectrum antibacterial and β-lactamase inhibitory activity but was chemically unstable. Imipenem, an N-formimidoyl derivative of thienamycin, was the first carbapenem that became available for the treatment of complex microbial infections. It demonstrated high affinity for penicillin-binding proteins (PBPs) and stability against β-lactamases.[1]

Carbapenems are broad-spectrum antibiotics with activity against both Gram-positive organisms such as Staphylococcus spp., Streptococcus spp., Enterococcus spp., and Nocardia spp. and Gram-negative organisms such as Pseudomonas spp., Acinetobacter spp., and Enterobacteriaceae such as Klebsiella spp.,  Escherichia More Details coli, and Enterobacter spp.

Carbapenem resistance in Gram-negative bacteria can be the result of production of β-lactamase enzyme known as carbapenemase, expression of efflux pumps, porin loss, and alterations in PBPs.[2] Recently, organisms belonging to the family Enterobacteriaceae such as E. coli, Klebsiella sp., and Enterobacter sp. and the nonfermenter group mainly Pseudomonas aeruginosa and Acinetobacter species have increased their potential to become extensively drug resistant by acquiring resistance to carbapenems, due mainly to carbapenemases and metallo beta lactamase production,[3],[4] respectively.

Clinically important carbapenemases include Class A – most common type isolated clinically is Klebsiella pneumoniae carbapenemase (KPC), Class B – metallo-β-lactamases having a Zn 2+ ion at its core, e.g., New Delhi metallo-beta-lactamase - 1 (NDM-1), Verona integron-encoded metallo-β-lactamase (VIM) type, and Imipenemase (IMP) type, Class D – oxacillin-hydrolyzing β-lactamases or oxacillinases which have a serine molecule at its core, e.g. OXA-48, OXA-181.[5] Discovered in 2008 in Sweden from an Indian patient hospitalized previously in New Delhi, NDM-1-positive Enterobacteriaceae has become the focus of worldwide attention.

This study was done to determine the minimum inhibitory concentration (MIC) of imipenem for multidrug-resistant (MDR) clinical isolates and identify carbapenemase-producing organisms among these MDR isolates.

  Materials and Methods Top

Inclusion criteria and selection of samples

This descriptive study was conducted in the Department of Microbiology at a tertiary care hospital in North India over a period of 2 months. Nonrepeat samples from admitted patients only were included in the study. Various samples included were blood, urine, sputum, endotracheal tube (ET) tip, bronchoalveolar lavage (BAL) and Mini-BAL, pus, and body fluids such as ascitic and pleural fluids. Samples were processed using the standard microbiological protocol, and various biochemical tests were put up for identification of the organisms up to the species level.

Antibiotic susceptibility testing and minimum inhibitory concentration determination

The antimicrobial susceptibility was performed using Kirby–Bauer disc diffusion method, and the MIC of imipenem for the isolates was determined by broth dilution method for concentrations 16, 8, 4, 2, 1, and 0.5 µg/ml with P. aeruginosa ATCC 27853 strain as the control organism. The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The sensitive, intermediate, and resistant MIC concentrations for Enterobacteriaceae were taken as ≤1 µg/ml, 2 µg/ml, and ≥4 µg/ml and for P. aeruginosa and Acinetobacter species as 2 µg/ml, 4 µg/ml, and ≥8 µg/ml, respectively.[6] MDR isolates [7] showing resistance or reduced susceptibility to carbapenems by disc diffusion and MIC were further tested for carbapenemase production by Modified Hodge test (MHT).

Modified Hodge test

MHT was performed according to the CLSI guidelines for phenotypic detection of carbapenemase production by the isolate [Figure 1]. An inoculum of E. coli ATCC 25922 standardized to 0.5 McFarland was uniformly swabbed onto Mueller-Hinton agar, and 10 µg imipenem disc was placed at the center of the plate. The test isolate was streaked as a straight line of at least 20–25 mm length from the edge of the disk to the edge of the plate, and the plate was incubated at 37°C in ambient air for 16–20 h and thereafter examined for enhanced growth around the test organism streak, at the intersection of the streak and the zone of inhibition. Enhancement of growth was considered as positive result and no enhancement of growth was considered as negative result for carbapenemase production.[6],[8]K. pneumoniae BAA ATCC 1705 and K. pneumoniae BAA ATCC 1706 strains were used as positive and negative controls for MHT as recommended by the CLSI.
Figure 1: Clover leaf-shaped enhancement of growth of Escherichia coli ATCC 25922 at the intersection of zone of inhibition due to imipenem and streak of test organism

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  Results Top

A total of 65 MDR clinical isolates that were resistant either to imipenem or meropenem or both were studied. These included 23 E. coli (urine - 18, blood - 2, pus - 1, ET tip - 1, wound swab - 1), 12 K. pneumoniae (urine - 6, sputum - 1, ET tip - 1, Mini-BAL - 1, ascitic fluid - 1, blood - 2), 8 Pseudomonas spp. (urine - 4, pus - 1, ET tip - 1, sputum - 1, BAL - 1), 15 Acinetobacter baumannii (blood -1, Mini-BAL - 1, sputum - 3, ET tip - 8, pleural fluid - 1, ear discharge - 1), and 7 Enterobacter aerogenes (urine - 2, sputum - 1, ET tip - 2, ulcer tissue - 1, blood - 1) isolates [Table 1].
Table 1: Summary of multidrug-resistant Gram-negative Bacilli clinical isolates

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The susceptibility pattern of the isolates for carbapenems by Kirby–Bauer disc diffusion method and MIC of imipenem is shown in [Table 2]. Among these isolates, the MIC was in the resistant range for 46 (70.77%), sensitive for 15 (23%), and intermediate for 4 (6.15%) organisms [Figure 2]. The imipenem MIC was >16 µg/ml for 20 isolates, 16 µg/ml for 13 isolates, 8 µg/ml for 13 isolates, 8 µg/ml for 4 isolates, 2 µg/ml for 6 isolates, 1 µg/ml for 1 isolate, 0.5 µg/ml for 4 isolates, and <0.5 µg/ml for 2 isolates [Table 3]. The MHT for carbapenemase production was positive for 37/65 (57%) isolates [Figure 3] which included 9 (39%) E. coli, 9 (75%) K. pneumoniae, 3 (37.5%) Pseudomonas spp., 13 (86.7%) A. baumannii, and 3 (42.8%) E. aerogenes [Table 2].
Table 2: Susceptibility to carbapenems by disc diffusion method and imipenem minimum inhibitory concentration and results of modified Hodge test

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Figure 2: Minimum inhibitory concentration of imipenem in multidrug-resistant Gram-negative Bacilli

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Table 3: Minimum inhibitory concentration of imipenem in multidrug-resistant Gram-negative Bacilli

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Figure 3: Modified Hodge test result

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  Discussion Top

Carbapenems such as imipenem, meropenem, and ertapenem are used as reserve drugs in the treatment of life-threatening infections in either severely ill or immunocompromised patients or those hospitalized for prolonged duration. These patients are more prone to acquire MDR infections. Carbapenems are needed for treating extended spectrum β-lactamase-producing Enterobacteriaceae which along with Pseudomonas species and Acinetobacter species are predominantly isolated pathogens from clinical samples. Production of carbapenemases by Enterobacteriaceae has led to emergence of carbapenem-resistant Enterobacteriaceae. Carbapenemase production in these organisms has reduced the clinical utility of carbapenem group of drugs posing a major challenge in the treatment of severe infections.[9] Therefore, a rapid, simple, and reliable method for detection of carbapenemase production is needed.

Khajuria et al., in a study in carbapenem-resistant isolates of A. baumannii, have reported MIC values for imipenem and meropenem ranging from 16 to 64 mg/L in 83.87% isolates and 60% isolates positive for carbapenemase production by MHT.[10] High MIC values of imipenem (MIC50 = 64 µg/ml and MIC90 = 256 µg/ml) and meropenem (MIC50 = 32 µg/ml and MIC90 = 256 µg/ml) were also reported in a study involving carbapenem-resistant isolates of P. aeruginosa.[11]

Datta et al., in a study in North India, have reported phenotypic carbapenemase production in 51% K. pneumoniae and 15% E. coli bloodstream infection isolates.[8] Another study in South India has reported carbapenemase production as 14.3% in A. baumannii and 28.1% in P. aeruginosa by MHT.[12] A study done in a tertiary care center in Italy has reported that MHT correctly identified 95/101 carbapenemase producers with sensitivity and specificity of 94% and 100%, respectively.[13] Arnold et al. also have reported MHT to be 100% sensitive for detection of carbapenemases though not very specific for KPC production.[14]

The limitations of this study are the relatively small sample size, short duration of the study, and its restriction to phenotypic detection of carbapenemase production. Genotypic characterization which confirms the presence of carbapenemase-producing genes in the organism was not done.

  Conclusion Top

Carbapenemase-producing pathogens cause infections that are difficult to treat and have high mortality rates, due to their appearance in MDR pathogens such as K. pneumoniae, P. aeruginosa, and Acinetobacter spp. Their occurrence in outbreak settings is being reported increasingly. The recommended effective therapeutic options for such MDR infections are polymyxins, tigecycline, and aminoglycosides. Evaluating effective antibiotic options, combination antibiotic therapy, and rigorous infection control measures will help in controlling spread of carbapenemase-producing MDR organisms in hospitals.

Phenotypic tests for carbapenemase detection such as MHT are simple, cost-effective, and easy to perform and hence can be used in any microbiology laboratory to detect carbapenemase production and applied clinically to guide the antimicrobial therapy, especially in severe and life-threatening infections.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Papp-Wallace KM, Endimiani A, Taracila MA, Bonomo RA. Carbapenems: Past, present, and future. Antimicrob Agents Chemother 2011;55:4943-60.  Back to cited text no. 1
Bedenic B, Plecko V, Sardelic S, Uzunovic S, Godic Torkar K. Carbapenemases in gram-negative bacteria: Laboratory detection and clinical significance. Biomed Res Int 2014;2014:841951.  Back to cited text no. 2
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. 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
Queenan AM, Bush K. Carbapenemases: The versatile beta-lactamases. Clin Microbiol Rev 2007;20:440-58.  Back to cited text no. 5
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. M100–S24. Wayne(PA): Clinical and Laboratory Standards Institute; 2014.  Back to cited text no. 6
Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268-81.  Back to cited text no. 7
Datta S, Wattal C, Goel N, Oberoi JK, Raveendran R, Prasad KJ. A ten year analysis of multi-drug resistant blood stream infections caused by Escherichia coli & Klebsiella pneumoniae in a tertiary care hospital. Indian J Med Res 2012;135:907-12.  Back to cited text no. 8
[PUBMED]  Medknow Journal  
Sood S. Identification and differentiation of carbapenemases in Klebsiella pneumoniae : A phenotypic test evaluation study from Jaipur, India. J Clin Diagn Res 2014;8:DC01-3.  Back to cited text no. 9
Khajuria A, Praharaj AK, Kumar M, Grover N. Molecular characterization of carbapenem resistant isolates of Acinetobacter baumannii in an intensive care unit of a tertiary care centre at central India. J Clin Diagn Res 2014;8:DC38-40.  Back to cited text no. 10
Rizek C, Fu L, Dos Santos LC, Leite G, Ramos J, Rossi F, et al. Characterization of carbapenem-resistant Pseudomonas aeruginosa clinical isolates, carrying multiple genes coding for this antibiotic resistance. Ann Clin Microbiol Antimicrob 2014;13:43.  Back to cited text no. 11
Noyal MJ, Menezes GA, Harish BN, Sujatha S, Parija SC. Simple screening tests for detection of carbapenemases in clinical isolates of nonfermentative gram-negative bacteria. Indian J Med Res 2009;129:707-12.  Back to cited text no. 12
[PUBMED]  Medknow Journal  
Bartolini A, Frasson I, Cavallaro A, Richter SN, Palù G. Comparison of phenotypic methods for the detection of carbapenem non-susceptible Enterobacteriaceae. Gut Pathog 2014;6:13.  Back to cited text no. 13
Arnold RS, Thom KA, Sharma S, Phillips M, Kristie Johnson J, Morgan DJ. Emergence of Klebsiella pneumoniae carbapenemase-producing bacteria. South Med J 2011;104:40-5.  Back to cited text no. 14


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]

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