Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 22  |  Issue : 1  |  Page : 31-34

Rapid detection of carbapenemase production in Enterobacteriaceae by different phenotypic methods


Department of Microbiology, R.G. Kar Medical College and Hospital, Kolkata, West Bengal, India

Date of Submission11-Jul-2019
Date of Decision07-Nov-2019
Date of Acceptance19-Nov-2019
Date of Web Publication20-May-2020

Correspondence Address:
Post Graduate Trainee Tulika Majumder
Department of Microbiology, R.G. Kar Medical College and Hospital, Kolkata, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomt.jomt_24_19

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  Abstract 


Introduction: Carbapenemase producing enterobacteriaceae (CPE) isolates have now emerged worldwide. Resistance to carbapenems is mainly due to production of beta-lactamases inactivating carbapenems (carbapenemases) included in classes A, B, C, or D of the Ambler classification. In Enterobacteriaceae carbapenemase encoding genes are often located on plasmids that contribute to a rapid spread among clinically relevant gram negative bacteria. Rapid and reliable detection of carbapenemase production is needed for therapeutic and control reasons. Aims: To compare the different phenotypic methods of carbapenemase detection namely, the modified paper strip carba NP method, the CLSI Carba NP method, the carbapenem inactivation method, and for rapid detection of CPE. Materials and methods: A total of 200 CPE from urine, pus, and blood cultures sent to the Microbiology Laboratory, R.G. Kar Medical College and Hospital (RGKMCH), were tested for the modified paper strip carba NP method, the CLSI Carba NP method, and the carbapenem inactivation method. Results and analysis: Out of 200 isolates, 108 isolates of Klebsiella spp and 92 isolates of Escherichia coli were compared for carbapenemase production by various phenotypic methods. In total, 100(93%) isolates of Klebsiella spp and 88 (95%) isolates of Escherichia coli showed positive results by paper strip and carba NP methods. A total of 96 (89%) isolates of Klebsiella spp and 84 (91%) isolates of Escherichia coli showed positive results by CLSI carba NP method. A total of 104 (96%) isolates of Klebsiella spp and 92 (100%) isolates of Escherichia coli showed positive results by CIM method. Conclusion: Rapid and accurate detection of carbapenemase producers are important for preventing their spread in health care settings. Although genotypic tests remain the gold standard but cannot practically be conducted because they are highly expensive and results are limited by the targets. The different phenotypic methods used in this study were inexpensive, rapid, highly sensitive, and specific. The modified paper strip Carba NP method in this study is a simple and rapid method compared to those performed by the CLSI method.

Keywords: Carba NP, carbapenem inactivation method, enterobacteriaceae


How to cite this article:
Kumar S, Bandyopadhyay M, Majumder T, Gupta SD. Rapid detection of carbapenemase production in Enterobacteriaceae by different phenotypic methods. J Med Trop 2020;22:31-4

How to cite this URL:
Kumar S, Bandyopadhyay M, Majumder T, Gupta SD. Rapid detection of carbapenemase production in Enterobacteriaceae by different phenotypic methods. J Med Trop [serial online] 2020 [cited 2020 May 31];22:31-4. Available from: http://www.jmedtropics.org/text.asp?2020/22/1/31/284636




  Introduction Top


Since carbapenems have a broad antibacterial spectrum, these antibiotics are the last resort for controlling infections caused by gram negative bacteria. However, recently, carbapenemase producing Enterobacteriaceae (CPE) that inactivates these antibiotics have emerged worldwide.[1] The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) is an issue of great clinical and public health concern.[2]

The mechanism underlying carbapenem resistance in Enterobacteriaceae is complex and includes both the production of carbapenem-hydrolyzing beta-lactamases (carbapenemase) as well as resistance due to the presence of combination of other factors such as hyperproduction of AmpC beta-lactamases or extended-spectrum beta-lactamases (ESBLs) combined with altered membrane permeability.[2] Carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) are associated with significant morbidity and mortality. A variety of carbapenemase genes have been described that are either plasmid or chromosomally encoded including blaKPC, blaSME, blaNDM, blaIMI, blaVIM, blaIMP, blaOXA-48.[3]

Early and accurate identification of CP-CRE is essential to prevent their dissemination within healthcare settings. Molecular methods such as PCRs for the detection of carbapenemase genes are costly, may require significant expertise, and are limited by the targets included in the assay. Over the last decade a number of phenotypic-based assays have been available. The Carba NP assay is a rapid phenotypic method that allows the detection of carbapenemase within 2 h, but many modifications of the test procedure have been described. The CLSI has come up with a standardized Carba NP procedure (CLSI CNP test) using microcentrifuge tubes and recommended the color change to be interpreted as positive (yellow, dark yellow, or light orange), negative (red or red-orange), or invalid (orange) using a color figure. Many studies have demonstrated that the cost is relatively high and only a few false positive test results have been reported. Some groups have reported difficulties with the Carba NP test, especially with the mucoid isolates or isolates with weak carbapenemases such as OXA-48. In these cases, only a subtle color change is observed that can easily be missed. The carbapenem inactivation method (CIM) is another cost-effective and highly sensitive method to detect carbapenemase activity in Enterobacteriaceae. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemase along with OXA-23.[4] The sensitivity of the CIM for the detection of NDM carbapenemase and OXA-48 type carbapenemase was found to be higher than the Carba NP test. But a long turnaround time (18 to 24 h) is a major limitation. Recently, a modification of the Carba NP test by use of filter paper strip (strip CNP test) indicates that it may be possible to shorten the assay time from 2 h to 5 min. [4],[5]


  Aims Top


This study was done to detect the CP-CRE by rapid method in comparison to other phenotypic methods.


  Materials and methods Top


Bacterial isolates: A total of 200 isolates of Enterobacteriaceae isolated from urine, pus, and blood culture specimens sent to the Microbiology Laboratory, R.G. Kar Medical College and Hospital (RGKMCH). Isolates were selected using the UCLA CRE definition, that is, not susceptible (intermediate or resistant, MICs 2-4 μg/ml) to meropenem or imipenem or ertapenem (MIC 2 μg/ml) as defined by the current CLSI M100, 29th ed. Standard.[6] Disk diffusion method was used to determine the susceptibility of isolates to ertapenem, imipenem, and meropenem. Etest was used to determine the imipenem MIC for the subset of CPE isolates.[5].

Carba NP test by CLSI: All tested isolates were subcultured twice using blood agar plates before testing by the test. Carba NP test was performed according to the CLSI procedure.[7] Solution A (phenol red solution of ph 7.8) and B (solution A +6 mg/ml imipenem) were freshly prepared on each day of testing. 100 μl of solution A and solution B was dispensed on tube A and tube B, respectively. A 1 μl loopful of bacteria from an overnight blood agar plate used as the inoculums and B-PER-2 (Thermo scientific, Rockford, IL, USA) was used for protein extraction and reagent mixtures were vortexed for 5 s. The tubes were incubated at 37°C and results were examined after 2 h. Color change in tube B was interpreted as positive (yellow, dark yellow, light orange), negative (red or red-orange), or invalid (orange) using the color figure from CLSI [Figure 1].[5],[7]
Figure 1: CLSI Carba NP method showing positive results in 1 and 4 no isolates

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CIM: The CIM method was performed according to the methods described by van der Zwaluw and colleagues with some modification. Briefly 400 μl of sterile double-distilled water or trypticase soy broth (TSB, BD, Sparks MD) was added to a 1.5 ml of sterile microfuge tube. For each CRE isolate, a heaping 10 μl loopful of organism from an 18–24 h culture on a blood agar plate was added to the tube and vortexed. To this, a 10 μg meropenem disk was added, ensuring the disk was immersed in the suspension. The suspension was then incubated at 37°C for 2 h. After incubation the disk was removed from the suspension. It was then applied to a lawn culture of Escherichia coli ATCC 25922 on a MHA plate that had been inoculated as required for routine disk diffusion test using a suspension equivalent to a 0.5 McFarland. Plates were incubated at 35°C for 18–20 h, after which time the zone of growth inhibition surrounding the meropenem disk was measured using reflected light. A zone size of ≤15 mm was considered as positive for carbapenemase production and a clear zone of ≥19 mm was considered negative [Figure 2].[4]
Figure 2: CIM method showing carbapenemase production in no-1 and no-4isolate

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The paper strip CNP test: The strip CNP was performed using the same preparations of solution A and B for the CLSI CNP test. Modifications to the test procedures described by Srisrattakaran et al. were made.[9] Filter papers (Whatman no 2., Maidstone, England) cut to size of 15 × 15 mm were taped onto petri dish. Each strip was moistened with 50 μl of either solution A (control strip) or solution B (test strip). Afterwards, 1 μl loopful of each test organism from an overnight blood agar plate was applied on each of the control and test strips by rubbing in a circular fashion of 5–7 mm in diameter. The plates were covered with lids to prevent rapid strip drying and incubated at 37°C. Results were read at 1 min and 5 min. Isolates giving any color change (yellow or light yellow) on test strip are interpreted as positive. If the test strips remain red after 5 min then the test was considered negative. If the control strips turn orange then test was considered invalid [Figure 3]. [5]
Figure 3: Paper strip Carba NP method showing carbapenemase production in no-1 and no-4 isolate

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  Results and analysis Top


Out of 200 isolates in this study, 108 isolates of Klebsiella spp and 92 isolates of Escherichia coli were compared for carbapenemase production by various phenotypic methods. In total, 100 (93%) isolates of Klebsiella spp and 88 (95%) isolates of Escherichia coli showed positive results by paper strip and CNP method. A total of 96 (89%) isolates of Klebsiella spp and 84 (91%) isolates of Escherichia coli showed positive results by CLSI CNP method. A total of 104 (96%) isolates of Klebsiella spp and 92 (100%) isolates of Escherichia coli showed positive results by CIM method.

The sensitivity of the assays for the detection of CPE ranged from 85% for the CLSI CNP method to 96% for the CIM method. The sensitivity of paper strip CNP method was 93% and this test can detect CPE within 5 min instead [Table 1].
Table 1: Showing number of isolates of Enterobacteriaceae detected by rapid method and other phenotypic methods

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  Discussion and conclusion Top


A selection of the isolates subjected to CIM has also been tested with CLSI CNP and paper strip CNP. The application of all the three methods in this study shows that it is capable of detecting carbapenemase production in Enterobacteriaceae. There was a high concordance between PCRs that detect carbapenem-encoding genes and carbapenemase activity detected by CIM. By the CLSI CNP test, 70% CPE isolates were positive at end of 2 h, and an additional 19% were positive at 4 h. By the paper strip CNP test only 15% CPE isolates were positive at 1 min, and rest of 80% were positive at 5 min.[4],[5] Recently the CIM was described as a highly sensitive and specific method for the detection of CP-CRE. In contrast to these preliminary reports, we identified limited sensitivity of the CIM for detecting both NDM and OXA-48 type producer. The mCIM was developed further with some modification (2 ml of TSB in contrast to 400 μl water) of CIM. The modification made the CIM more sensitive and detect both NDM and OXA-48 producer. The costs were also low, perhaps the biggest drawback is the time requirement. It’s taking approximately 18–24 h in contrast to the CLSI CNP test taking 2 h only. But many false negative and invalid results were obtained by CLSI CNP test. [3] The paper strip CNP test was also a sensitive method for detecting metallo-β-lactamase and possible KPC production in CP-CRE within 5 min instead. The strip test is convenient and easy to perform. The reagent requirement per test is minimal, and bacterial lysis using protein extraction is not required.[8]Rapid and accurate detection of carbapenemase producers is important for preventing their spread in health care settings. Although genotypic tests remain the gold standard but cannot practically be conducted because highly expensive and results are limited by the targets. The different phenotypic methods used in this study were inexpensive, rapid, highly sensitive, and specific. The paper strip CNP method in this study is a simple and inexpensive method and assay time superior to those performed by CLSI procedures. It holds promise for routine implementation in low-resource health care settings. Overall, the ability to detect CPE varied across phenotypic tests. The local epidemiology of CP genotypes, accuracy of the assay, turnaround time, and ease of incorporation into the laboratory workflow should all be considered when selecting a phenotypic test for clinical use.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Yamada K, Kashiwa M, Arai K, Nagano N, Saito R. Comparison of the Modified-Hodge test, Carba NP test, and carbapenem inactivation method as a screening methods for carbapenemase-producing Enterobacteriaceae. Journal of Microbiological Methods 2016;128:48-51  Back to cited text no. 1
    
2.
Pierce VM, Simner PJ, Lonsway DR et al. The modified carbapenem inactivation method (mCIM) for phenotypic detection of carbapenemase production among Enterobacteriaceae. J Clin Microbiol. Doi:10.1128/JCM. 00193-17  Back to cited text no. 2
    
3.
Tamma PD, Opene BNA et al. A comparison of eleven phenotypic assays for the accurate detection of carbapenemase-producing Enterobacteriaceae. J Clin Microbiol doi:10.1128/JCM.02338-16.  Back to cited text no. 3
    
4.
Van der Zwaluw K, de Haan A, Pluister GN, Bootsma HJ, de Neeling AJ, Schouls LM. The carbapenem inactivation method (CIM), a simple and low-cost alternative for the carba NP test to assess phenotypic carbapenemase activity in Gram Negative rods. PLoS ONE 201;10:e0123690. doi:10.1371/journal.pone.0123690  Back to cited text no. 4
    
5.
Ho P-L, Wang Y, Wing-Sza Tse C, Fung KS-C, Cheng VC-C, Lee R, To W-K, Lal RW-M, Luk W-K, Que T-L, Tsang DN-C. Rapid detection of carbapenemase production in Enterobacteriaceae by use of a modified paper strip Carba NP method. J Clin Microbiol 2018;56:e01110-17  Back to cited text no. 5
    
6.
Miller SA, Hindler JA, Chengcuenca A, Humphires RM. Use of ancillary carbapenemase tests to improve specificity of phenotypic definitions for carbapenemase producing Enterobacteriaceae. J Clin Microbial doi: 10.1128/JCM. 00157-17  Back to cited text no. 6
    
7.
Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing, 28th ed. CLSI M100. Clinical and Laboratory Standards Institute, Wayne, PA; 2018.  Back to cited text no. 7
    
8.
Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol Med 2012;18:263-72.  Back to cited text no. 8
    
9.
Srisrattakarn A, Lulitanond A, Wilailuckana C, Charoensri N, Wonglakorn L, Piyapatthanakul S et al. Modification and evaluation of the Carba NP test by use of paper strip for simple and rapid detection of carbapenemase-producing Enterobacteriaceae. World J Microbiol Biotechnol 2016;32:117.  Back to cited text no. 9
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]



 

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