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 Table of Contents  
ORIGINAL ARTICLES
Year : 2022  |  Volume : 9  |  Issue : 2  |  Page : 149-153

Phenotypic detection of metallo-beta-lactamase production in clinical isolates of Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital


Department of Microbiology, MGM Medical College and Hospital, Aurangabad, Maharashtra, India

Date of Submission07-Feb-2022
Date of Acceptance05-Apr-2022
Date of Web Publication17-May-2022

Correspondence Address:
Dr. Smita S Kulkarni
Department of Microbiology, MGM Medical College and Hospital, Aurangabad 431003, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mgmj.mgmj_12_22

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  Abstract 

Introduction: Carbapenem resistance due to metallo-beta-lactamase (MBL)-producing bacteria is an emerging threat worldwide. This study aimed to detect the MBL production in clinical isolates of E. coli and Klebsiella pneumoniae species in our hospital setting and to evaluate the efficiency of two phenotypic methods for the detection of MBL production. Materials and Methods: The present study was carried out in the Department of Microbiology, MGM Medical College and Hospital, Aurangabad, Maharashtra, for a period of 2 years from April 2018 to March 2020. From a total of 12,324 various clinical specimens, 393 isolates of E. coli and Klebsiella pneumoniae species were tested for MBL production. MBL was detected by two different phenotypic methods, i.e., combined disc test and E-test. Results: Out of 393 isolates, 130 (33.07%) isolates were resistant to imipenem on screening of which 71 (18.06%) were Klebsiella pneumoniae and 59 (15.01%) were E. coli. About 43.66% Klebsiella pneumoniae isolates and 40.67% E. coli isolates were MBL-positive by the combined disc test. Using the E-test, MBL production was found to be 46.47% and 45.76% in Klebsiella pneumoniae and E. coli, respectively. Conclusion: Routine screening of MBL-producing organisms should be performed in diagnostic laboratories to control the spread of resistance and for the proper management of antibiotic therapy. E-test is better than the combined disc test for the detection of MBL-producing gram-negative bacilli.

Keywords: Carbapenem, combined disc test, E-test, imipenem, metallo-beta-lactamase


How to cite this article:
Kulkarni SS, Mulay MV. Phenotypic detection of metallo-beta-lactamase production in clinical isolates of Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital. MGM J Med Sci 2022;9:149-53

How to cite this URL:
Kulkarni SS, Mulay MV. Phenotypic detection of metallo-beta-lactamase production in clinical isolates of Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital. MGM J Med Sci [serial online] 2022 [cited 2022 Dec 7];9:149-53. Available from: http://www.mgmjms.com/text.asp?2022/9/2/149/347683




  Introduction Top


Carbapenems are often used as the last resort antibiotics for treating serious infections caused by multidrug-resistant bacteria.[1] These agents have the broadest antibacterial spectrum compared with other beta-lactam classes. Although antibiotic resistance is caused by multiple mechanisms, the resistance to carbapenem antibiotics due to carbapenem hydrolyzing enzymes is now a worldwide issue.[2] Carbapenem hydrolyzing enzymes belong to classes A, B, and D according to the molecular classification and are called carbapenemases. However, the carbapenemases in class B require one or two zinc ions for their full catalytic activity, and these enzymes are called metallo-beta-lactamase (MBL).[3] MBL-producing gram-negative bacilli produce severe infections leading to high morbidity and mortality. The emergence of MBLs in gram-negative bacilli is becoming a therapeutic challenge as these enzymes render all penicillins, cephalosporins, and carbapenems ineffective.[4] MBL was reported for the first time in 1991 in Japan, and since then, nosocomial outbreaks due to MBL-producing gram-negative bacilli are being increasingly reported from different parts of the world.[5]

Various phenotypic methods are used for MBL detection such as the combined disc method, double-disc synergy method, and Epsilometer test (E-test). MBL E-test is considered the phenotypic standard method for MBL detection, but the test is expensive.[6] Double-disc synergy and combined disc tests (CDTs) are economical and simple to perform.[6] It is essential to detect MBL-producing organisms to reduce the further spread and to initiate appropriate therapy. Hence, we conducted this study to detect MBL production in clinical isolates of E. coli and Klebsiella pneumoniae species in our hospital setting and to evaluate the efficiency of two phenotypic methods for the detection of MBL production.


  Materials and methods Top


This prospective observational study was conducted in the Department of Microbiology, MGM Medical College and Hospital, Aurangabad, Maharashtra, India, for a period of 2 years from April 2018 to March 2020. Routine clinical specimens such as blood, urine, pus, sputum, endotracheal (ET) tube aspirates, and wound swabs received in the Microbiology department during the study period were included in the study. All the samples were processed by standard microbiological procedures. Organisms were identified by using an automated system (Vitek 2 Compact System; Biomerieux). From a total of 12,324 various specimens,393 nonrepeat isolates of E. coli and Klebsiella pneumoniae were subjected to antibiotic susceptibility testing as per the Clinical and Laboratory Standards Institute guidelines.[7]

Screening of imipenem resistance

The screening of clinical isolates was performed with a disc of imipenem (10 µg). The isolates showing reduced susceptibility to imipenem (zone diameter ≤ 21 mm) were imipenem screen positive. Imipenem screen positive isolates were subjected to the confirmation of MBL production by two different phenotypic methods such as CDT and E-test.

CDT

A lawn culture of the 0.5 McFarland turbidity matched test organism was inoculated onto Muller Hinton agar (MHA). Two 10 µg imipenem discs were placed 30 mm apart from center to center on the surface of the agar plate, and 10 µL of 0.5 M ethylenediamine tetraacetic acid (EDTA) solution was added to one imipenem disc. The inhibition zones of the imipenem and imipenem-EDTA discs were compared after 16–18 hours of incubation at 37°C. An increase in the zone size of ≥7 mm around the imipenem-EDTA disc as compared to the imipenem disc alone was considered as MBL positive[8],[9] [Figure 1].
Figure 1: Combined disc test for the detection of MBL producer. The test showing enhanced inhibition zone of >7 mm around IPM + ETDA disc indicating MBL production

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MBL Epsilometer test

MBL E-test strips (Himedia Laboratories) containing a double-sided seven-dilution range of imipenem (4–256 µg/mL) and imipenem (1–64 µg/mL) in combination with a fixed concentration of EDTA were used for the confirmation of MBL production. A lawn culture of the 0.5 McFarland turbidity matched test organism was inoculated onto an MHA plate. Once dried, an E-test MBL strip was applied onto the plate, which was then incubated at 37°C for 16–18 hours. The minimum inhibitory concentration (MIC) endpoints were read where the inhibition ellipse intersected the strip. The MIC ratio of Imipenem (IPM)/IPM + EDTA of ≥8 or the reduction of imipenem MIC by ≥3nlog dilutions was interpreted as being suggestive of MBL production[10],[11] [Figure 2].
Figure 2: MBL imipenem/imipenem with inhibitor E-test demonstrating a reduction in MIC in the presence of EDTA of >8 fold indicating MBL activity

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  Results Top


In the present study, the maximum isolation of E. coli and Klebsiella pneumoniae was obtained from pus specimens (34.86%), followed by urine (29.51%) and wound swabs (8.3%) [Table 1].
Table 1: Number of organisms isolated from various clinical samples

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About 33.07% (130/393) isolates were resistant to imipenem, which were considered MBL screening positive. Out of which, 54.61% (71/130) were Klebsiella pneumoniae and 45.38% (59/130) were E. coli [Table 2].
Table 2: Distribution of imipenem-resistant and imipenem-sensitive isolates of E. coli and Klebsiella pneumoniae (n = 393)

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All 130 imipenem-resistant isolates were further processed for the confirmation of MBL production by two different phenotypic methods, E-test and CDT.

Evaluation of phenotypic tests

E-test detected MBL production in 46.47% of Klebsiella pneumoniae and 45.76% of E. coli. By using CDT, 43.66% of Klebsiella pneumoniae and 40.67% of E. coli isolates found MBL producers [Table 3].
Table 3: Results of CDT and E-test for the detection of MBL producers among imipenem-resistant isolates

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Considering both the tests, out of 393 isolates, 60 (15.26%) isolates were positive for MBL production. The rate of MBL production in Klebsiella pneumoniae was 8.39% (33/393), and in E. coli, it was 6.87% (27/393).


  Discussion Top


Bacterial resistance due to MBL production is increasing day by day.[12] MBL-producing gram-negative bacteria can cause several severe infections. MBLs spread easily via plasmids, so they rapidly disseminate and lead to poor outcomes when infection occurs.[13],[14] The detection of MBL-producing gram-negative bacteria is very important to limit the spread of the MBL gene among bacteria.[15]

The samples used for the test were pus, urine, wound swabs, sputum, blood, body fluids, and ET secretions. The maximum number of Klebsiella pneumoniae and E. coli isolates was obtained from pus specimens (34.86%) followed by urine specimens (29.51%).

In the present study, 33.07% of clinical isolates of Klebsiella pneumonia and E. coli were found to be imipenem screen positive. Kumarasamy et al. (24%)[16] and Panchal et al. (28%)[17] also reported a higher carbapenem resistance rate. Saranraj et al.[18] and Datta et al.[19] have reported much lower rates of resistance to carbapenems.

The resistance to imipenem was found to be more in Klebsiella pneumoniae (18.06%) than in E. Coli (15.01%). Similarly, a high prevalence of resistance to carbapenems in Klebsiella spp (18.55%) than in E. coli (12.9%) has been reported by Agarwal et al.[20] Chauhan et al.[21] also found a slightly lower rate of resistance among E. coli and a slightly higher rate among Klebsiella isolates.

Polymerase chain reaction (PCR) analysis is the gold standard method for the detection of MBL producers, but it is not suitable for daily testing in diagnostic microbiology laboratories because of the higher cost and inconvenience.[22]

The sensitivity of the MBL E-test is considered to be 100%, and the same findings were observed in the studies conducted by Walsh et al.[23] and Khosravi et al.[24] Hence, in this study, MBL E-test was taken as a gold standard test for MBL detection in clinical isolates of E. coli and Klebsiella pneumoniae.

In our study, the E-test detected MBL production in more isolates of Klebsiella pneumoniae (46.47%) and E. coli (45.76%) as compared to CDT. By using CDT, we found MBL production in 43.66% of Klebsiella pneumoniae and 40.67% of E. coli.

Pandya et al.[9] and Adhikary et al.[25] have reported that CDT (IPM-EDTA) is the most sensitive test for the detection of MBL production in gram-negative bacilli.

Overall, using both the tests, we could confirm MBL production in 15.26% of clinical isolates of Klebsiella pneumoniae and E. coli, which is to the study done by Panchal et al. (19.62%)[17] and Wadekar et al. (18.00%).[26] In contrast to our study, Fazlul et al. (52.6%)[12] and Marie et al. (53%)[27] have found a high prevalence of MBL in their studies.

In the present study, MBL production was found to be more in Klebsiella pneumoniae than in E. coli. Similar findings have been reported by many authors.[19],[25],[26],[28]


  Conclusion Top


Thus to conclude, MBL-producing organisms are emerging rapidly in our region. It is important to perform routine screening of MBL-producing organisms in diagnostic laboratories to reduce the dissemination of such strains in hospital patients and the management of antibiotic therapy. Though the imipenem-EDTA CDT is a sensitive and simple method for the detection of MBL producers, positive isolates may further be confirmed by the MBL E-test or PCR method.

Ethical consideration

Approval from the Institutional Ethics Committee of MGM Medical College and Hospital, Aurangabad, Maharashtra, India has been obtained to undertake the study on “Phenotypic detection of metallo-beta-lactamase production in clinical isolates of  Escherichia More Details coli and Klebsiella pneumoniae in a tertiary care hospital” vide their letter no. MGM-ECRHS/201 dated April 7, 2017.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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