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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 24  |  Issue : 3  |  Page : 40-46

Phenotypic detection and molecular characterization of carbapenem-resistant Enterobacteriaceae at a tertiary care center


1 Manipal Centre for Virus Research, MAHE, Manipal, Karnataka, India
2 Department of Lab Medicine, Command Hospital (CC), Lucknow, Uttar Pradesh, India
3 Department of Microbiology, Dr. DY Patil Medical College, Pune, Maharashtra, India
4 Department of Lab Sciences, Army Hospital (R and R), New Delhi, India
5 Department of Pathology, 92 Base Hospital, Srinagar, Jammu and Kashmir, India
6 Department of Lab Medicine, Military Hospital, Ahmednagar, Maharashtra, India

Date of Submission04-Nov-2020
Date of Decision30-Apr-2021
Date of Acceptance18-May-2021
Date of Web Publication01-Apr-2022

Correspondence Address:
Lt Col (Dr.) Kundan Tandel
Command Hospital, Central Command, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jmms.jmms_163_20

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  Abstract 


Background: Carbapenem-resistant Enterobacteriaceae (CRE) is of utmost clinical importance because they challenge the armamentarium of the treating clinicians, hampering current treatment strategies. Aim: The aim of the study is to detect carbapenemase-producing Enterobacteriaceae by phenotypic and molecular methods. Materials and Methods: Nonrepetitive isolates of Enterobacteriaceae were identified by conventional phenotypic methods from various clinical samples. All these isolates were screened for carbapenem resistance by meropenem (10μg) or ertapenem (10μg) disc. All the isolates which were found to be carbapenem-resistant by screening test were subjected to phenotypic confirmatory test in the form of modified Hodge test (MHT). These isolates were then subjected to polymerase chain reaction for the detection of various carbapenemase-producing genes, namely New Delhi metallo-β-lactamase 1 (NDM), verona integron-encoded metallo-β-lactamase (VIM), imipenem-resistant Pseudomonas (IMP), KPC, and OXA-48. Results: Out of 1254 isolates of Enterobacteriaceae, 230 isolates (18.3%) were found to be positive for carbapenemase production by screening test. A total of 150 out of 230 isolates (65.2%) tested positive for carbapenemase production by MHT. Out of these 150 phenotypically confirmed carbapenemase-producing isolates, blaNDM gene was found in 85, blaVIM in 32, and blaIMP in 22 isolates. blaOXA-48 and blaKPC genes were not found in any isolate. Moreover, there were 19 isolates, in which no gene was detected. Conclusion: The prevalence of phenotypically confirmed carbapenemase resistance among Enterobacteriaceae is 11.96% (150/1254). Genes responsible for carbapenemase production are widely prevalent in Enterobacteriaceae Routine detection will help in the management of these broadly-resistant pathogens and implementation of appropriate infection control measures, thus limiting their spread.

Keywords: Carbapenem-resistant Enterobacteriaceae, modified Hodge test, polymerase chain reaction


How to cite this article:
Bhatt P, Tandel K, Das NK, Grover N, Ranjan P, Rathi K R. Phenotypic detection and molecular characterization of carbapenem-resistant Enterobacteriaceae at a tertiary care center. J Mar Med Soc 2022;24, Suppl S1:40-6

How to cite this URL:
Bhatt P, Tandel K, Das NK, Grover N, Ranjan P, Rathi K R. Phenotypic detection and molecular characterization of carbapenem-resistant Enterobacteriaceae at a tertiary care center. J Mar Med Soc [serial online] 2022 [cited 2022 Aug 9];24, Suppl S1:40-6. Available from: https://www.marinemedicalsociety.in/text.asp?2022/24/3/40/342378




  Introduction Top


Resistance to majority of available antimicrobials has become a major public health problem because the morbidity and mortality associated with these drug-resistant bacterial infections are on the rise all over the world.[1] The highest risk to public health is possessed by the bacteria belonging to the family Enterobacteriaceae, because of their multidrug resistance and rapid dissemination of resistance to other bacterial strains and species.[1],[2]

β-lactams are diverse group of antibiotics that are classified into different classes according to their chemical structure. Both Gram-positive and Gram-negative bacteria are killed by Carbapenems as they possess exceptionally wide spectrum of action. Due to emergence and rapid dissemination of extended-spectrum β-lactamase (ESBL) producers, which inactivates all antimicrobials covered under the β-lactams group except the carbapenems, these agents are being increasingly used in hospitals as an empirical therapy for the treatment of life-threatening infections.[1],[3],[4] However, in the recent past, due to indiscriminate use of carbapenems, reports of increasing carbapenem resistance have been accumulating, which are quite alarming.[5]

Carbapenem-resistant Enterobacteriaceae (CRE) was first described in 1993 when carbapenemase-producing Enterobacter cloacae were isolated. Initially, the carbapenem resistance was more often observed in nonfermenters, and isolation of CRE occurred sporadically throughout 1990s. However, in the recent past, the isolation rate of CRE has been increasing at an alarming rate.[1],[2],[6]

The two mechanisms responsible for carbapenem resistance in Enterobacteriaceae are: (1) production of carbapenemases and (2) reduced expression of porins along with the overproduction of β-lactamases possessing weak carbapenemase activity.[3] Because the resistance is not transferable, carbapenem resistance by reduced membrane permeability is considered less important.[7] Different types of carbapenemases have been identified among Enterobacteriaceae. They are divided into 3 classes of β-lactamases which are Ambler classes A, B and D β-lactamases.

A number of Class A carbapenemases has been identified; some are chromosomally encoded like not metalloenzyme carbapenemase A, Serratia marcescens enzyme, IMI-1 (imipenem-hydrolyzing β-lactamase), Serratia fonticola carbapenemase-1, with the others being plasmid encoded—KPC (KPC-2 to KPC-13), IMI (IMI-1 to IMI-3), derivatives (Guiana extended spectrum [GES-1] to GES-20) of GES. These enzymes actively hydrolyze carbapenems and are partially inhibited by clavulanic acid.

The most common Class B metallo-β-lactamase families include the New Delhi metallo-β-lactamase 1 (NDM-1), imipenem-resistant Pseudomonas (IMP)-type carbapenemases, verona integron-encoded metallo-β-lactamase (VIM), German imipenemase, and Seoul imipenemase. They hydrolyze carbapenems but are susceptible to inhibition by ethylenediaminetetraacetic acid (EDTA), a chelator of Zn2+ and other divalent cations.

Class D carbapenemases are of the OXA enzyme type and have a weak activity against carbapenems. These enzymes are serine-β-lactamases poorly inhibited by EDTA or clavulanic acid. These enzymes are found primarily in nonfermenter organisms such as Acinetobacter baumannii, Pseudomonas aeruginosa, and rarely in isolates of Enterobacteriaceae family.[1],[3]

Among the class A carbapenemases, KPCs have spread worldwide and caused outbreaks in many countries. KPC carbapenemases are very potent and hydrolyze β-lactams of all classes, with the most efficient hydrolysis observed for nitrocefin, cephalothin, cephaloridine, benzylpenicillin, ampicillin, and piperacillin. Imipenem and meropenem, as well as cefotaxime and aztreonam, were hydrolyzed 10-fold-less efficiently than the penicillins and early cephalosporins. Weak but measurable hydrolysis was observed for cefoxitin and ceftazidime, giving the KPC family a broad hydrolysis spectrum that includes most-lactam antibiotics.[1],[2]

In view of the rapid increase in resistance to carbapenems, which are often considered the last resort drug in the treatment of life-threatening infections caused by Enterobacteriaceae, this study was carried out in a tertiary care hospital in India with an aim to detect carbapenemase-producing Enterobacteriaceae isolates by phenotypic methods and perform their molecular characterization.


  Materials and Methods Top


The present cross-sectional study was carried out from June 2014 to March 2015 in Microbiology laboratory of an urban tertiary care hospital.

Isolation and identification

In this study, a total of 1254 nonrepeat isolates of Enterobacteriaceae were first identified by conventional phenotypic methods from various clinical samples and then further characterized.

Screening for carbapenem resistance (carbapenemase production)

All the isolates of Enterobacteriaceae were screened for carbapenem resistance by using meropenem (10 μg) or ertapenem (10 μg) disc. CLSI guidelines were followed for interpreting the results.[8] A zone size of <22 mm for ertapenem or <23 mm for meropenem was considered to be carbapenem resistant. Escherichia coli ATCC 25922 was used as the control strain for quality control.

Confirmatory test for carbapenemase production

All the isolates which were found to be carbapenem resistant by screening test were subjected to phenotypic confirmatory test in the form of modified Hodge test (MHT). A 1:10 diluted 0.5 McFarland standard suspension of E. coli ATCC 25922 (indicator organism) was made and lawn culture done on Mueller-Hinton agar (MHA). The plates were allowed to dry for 3–10 min. An ertapenem (10 μg) or meropenem (10 μg) disc was applied in the center of the plate. Using a 10 μl loop, 3–5 colonies of the test or quality control organisms (MHT positive–Klebsiella pneumoniae ATCC BAA-1705, MHT negative–K. pneumoniae ATCC BAA-1706) were picked with a straight wire and inoculated in a straight line out from edge of the disc. The streak should be at least 20–25 mm in length. The plates were incubated at 37°C for 16–20 h. Following incubation, the MHA plates were observed for enhanced growth around the test or QC organism streak at the intersection of the streak and the zone of inhibition. The organism was considered carbapenemase producer, if there was enhanced growth.[8]

Polymerase chain reaction for detection of carbapenemase-producing genes

The genes analyzed in this study for carbapenemase production were KPC, NDM, VIM, IMP, and OXA-48.

DNA extraction

DNA extraction was performed from all the carbapenemase-producing isolates confirmed by MHT using the QIAamp DNA mini kits (QIAGEN, Germany). DNA extraction was performed according to manufacturer's instructions.

Primers

Previously published primers were used as described by van der Zee, et al. and are shown in [Table 1].[9]
Table 1: Primers used for polymerase chain reaction to detect genes responsible for Carbapenemase production

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Polymerase chain reaction mixtures

Two polymerase chain reactions (PCRs) (each 50 μl reaction) were carried out which included one multiplex PCR for detecting NDM, VIM, IMP, KPC, and OXA-48 and the other two uniplex PCRs for detecting NDM and VIM genes separately. The amplicon size for NDM and VIM genes were 83 bp and 94 bp, respectively. Due to very similar amplicon size, it was difficult to differentiate between these two genes on multiplex PCR. Hence, whenever any sample gave amplicon size of around 90 bp, it can have either NDM or VIM gene. The accurate identification among these two was carried out by uniplex PCR to differentiate between NMD and VIM gene. The reagents and their concentration used in the multiplex PCR mix has been shown in [Table 2]. PCR mixtures were prepared under laminar flow under strict precautions to prevent cross contamination.
Table 2: Various reagents used in multiplex polymerase chain reaction mixture (50 μl)

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Amplification in both PCRs was carried out with the following thermal cycling profile: initial denaturation for 5 min at 95°C, 35 cycles of amplification consisting of 30 s at 95°C, 30 s at 45°C, and 30 s at 72°C, with 5 min at 72°C for the final extension.

Gel electrophoresis

Gel electrophoresis was carried out in 0.5X Tris-borate-EDTA using 1.5% agarose gel.

Results and interpretation

The gel was viewed under ultraviolet transilluminator and was documented with the help of digital camera attached to the computer. Specific bands at particular gene amplicon size were considered as positive PCR reaction, with no band in negative control.


  Results Top


In the present study, a total of 1254 isolates of Enterobacteriaceae were characterized. The maximum number of isolates were E. coli (724) followed by K. pneumoniae (406) and Proteus spp. (50), Enterobacter aerogenes (40), S. marcescens (18), Citrobacter freundii (14), and Raoultella ornithinolytica (02).

All these isolates were screened for carbapenemase production by Kirby-Bauer disc diffusion method using either meropenem or ertapenem disc, depending upon the availability of the disc [Figure 1]. Out of these 1254 isolates, 230 isolates (18.3%) were found to be positive for carbapenemase production by screening test. A total of 52 out of 724 E. coli (7.2%), 138 out of 406 K. pneumoniae (34%), 6 out of 50 Proteus spp. (3%), 20 out of 40 E. aerogenes (50%), 8 out of 18 S. marcescens (44.4%), 5 out of 14 C. freundii (35.7%), and 1 out of 02 R. ornithinolytica (50%) were screening test positive.
Figure 1: Screening test for carbaenem resistance by using ertapenem (10 μg) or imipenem (10 μg) disc

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All the 230 isolates which tested positive by screening test were subjected to carbapenemase production by phenotypic confirmatory testing method MHT as shown in [Figure 2]. A total of 150 out of 230 isolates (65.2%) tested positive for carbapenemase production by MHT, out of which, 35 were E. coli, 90 were K. pneumoniae, 3 were Proteus spp., 13 were E. aerogenes, 6 were S. marcescens, 2 were C. freundii, and 1 was R. ornithinolytica. The species wise distribution of screening test positive and MHT positive isolates is shown in [Table 3].
Table 3: Species distribution of carbapenemase production screening test and modified Hodge test positive isolates

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Figure 2: Modified Hodge test for phenotypic confirmation of carbapenemase production

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All the 150 phenotypically confirmed carbapenemase-producing isolates were subjected to two PCRs – one multiplex PCR for detecting NDM, VIM, IMP, KPC, and OXA-48 and the other two uniplex PCR for detecting NDM and VIM genes separately. [Figure 3] displays the gel electrophoresis analysis of amplified product of multiplex PCR, which shows bands at particular base pair denoting various genes. The most common gene found in these isolates was NDM, which was present in 85 isolates. The next most common gene was VIM, present in 32 isolates followed by IMP, which was present in 22 isolates. These also included a few isolates with multiple genes, the most common combination being NDM + IMP in 05 isolates, followed by NDM + VIM, which were present in 3 isolates. OXA-48 and KPC genes were not found in any isolate. Moreover, there were 19 phenotypically confirmed carbapenemase-producing isolates, in which no gene was detected. [Table 4] shows the distribution of these genes responsible for carbapenemase production in 150 isolates.
Figure 3: Analysis of polymerase chain reaction product by gel electrophoresis showing bands at various bp size denoting New Delhi metallo-β-lactamase 1 (83 bp), verona integron-encoded metallo-β-lactamase (94 bp), and imipenem-resistant pseudomonas (101 bp) genes A–100 bp molecular marker B, K–Band at 83 bp denoting New Delhi metallo-β-lactamase 1 gene G, J–No band denoting no gene C-H– Band at 94 bp denoting verona integron-encoded metallo-β-lactamase gene L– Band at 101 bp denoting imipenem-resistant pseudomonas gene

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Table 4: Distribution of various carbapenemase-producing genes among phenotypically confirmed carbapenemase-producing isolates of Enterobacteriaceae

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  Discussion Top


The emergence of multidrug-resistant organisms at an alarming rate is a cause for concern, as they are responsible for both health care-associated and community acquired infections. The members of the family Enterobacteriaceae are the most commonly implicated organisms of this multidrug resistance. Carbapenems are the drug of choice and in fact the last option left to deal with these multidrug-resistant organisms. However, the ever-increasing resistance to these highly potent agents among Enterobacteriaceae has now spread extensively, mainly as a result of acquisition of carbapenemase-producing genes. Given the frequency with which Enterobacteriaceae cause infections, the high morbidity and mortality associated with these infections and the potential for widespread dissemination of carbapenem resistance through mobile genetic elements, the management of infections caused by CRE is particularly challenging.[10],[11]

Detection of CRE is of utmost importance, as carbapenemases are commonly accompanied by various other resistance determinants also, which leads to multidrug resistance and even pan-drug resistance. Tigecycline and colistin were the only two microbials showing sensitivity to the majority of isolates that were MHT positive. Many other studies have also reported blaNDM-1 producing Enterobacteriaceae exhibiting resistance to other group of antimicrobials including fluoroquinolones and aminoglycosides, except to tigecycline and nitrofurantoin.[12],[13],[14] In our study, we found similar findings wherein most NDM producers exhibited sensitivity to tigecycline and nitrofurantoin, and a few isolates were sensitive to cotrimoxazole and chloramphenicol. A recent study reported that only tigecycline and colistin were susceptible to among NDM harboring K. pneumoniae and E. coli and released global alert on CRE.[2] One study from Pune has also shown emergence pan drug-resistant organisms in health-care settings.[15]

A plethora of phenotypic tests is available for the detection of carbapenemase-producing organisms with varying sensitivity and specificity. Most commonly used and recommended phenotypic test is MHT.[16],[17],[18] It has good sensitivity for the detection of carbapenemase production among organisms harboring KPC and OXA-48 genes. However, it requires at least 24–48 h for interpretation of results and it is labor intensive. The other disadvantage of MHT is that false-positive results have been observed among isolates harboring for high-level AmpC or CTX-M gene. Moreover, the results of NDM detection are also not very sensitive. However, the sensitivity for detecting NDM producing organisms can be considerably enhanced on addition of zinc into the culture medium.[19] MHT is recommended as the first-line test for detecting organisms producing carbapenemases. However, MHT cannot discriminate among different types of carbapenemases.[19]

The results of MHT in the present study are in line with those published earlier. Miriagou V et al. have reported very high sensitivity for MHT with recommendation to be used as a confirmatory test.[20] However, false-positive result can be seen, especially among ESBLs or isolates having AmpC with decreased porins as discussed earlier.[18] In addition, false-negative results can also be seen among organisms having MBLs like NDM.[17],[21]

The prevalence of carbapenem resistance among Enterobacteriaceae, based on MHT, in our institute is 11.96% (150/1254). The resistance to carbapenems observed in this study is pretty low in comparison to other studies from India. In the study done by Wattal et al., at a tertiary care hospital in Delhi, higher prevalence of carbapenem resistance ranging from 13 to 51% in E. coli and Klebsiella spp. was reported.[22] The other study by Gupta et al. also reported a higher prevalence ranging from 17% to 22% among Enterobacteriaceae isolates.[23]

Accurate and timely detection of CRE is crucial not only for suitable and adequate antibiotic therapy but also for strict implementation of sufficient infection control measures. The phenotypic tests are time taking and may give false-positive as well as false-negative results. Moreover, they do not differentiate between different mechanisms of carbapenem resistance.[19],[24] In addition, it may fail to detect OXA-48 harboring organisms as they show lower lever of carbapenem resistance and they can also miss ESBL producing organisms with reduced permeability. Moreover, phenotypic tests though specific do not differentiate between chromosomal and plasmid-encoded genes. Thereby, genotypic methods such as PCR is taken as gold standard for their detection. In this study, a multiplex PCR was carried out for the detection of commonly reported genes, namely NDM, KPC, VIM, IMP, and OXA-48. The main goal of this study was to find the prevalence of various carbapenemase-producing genes at our center.

All the 150 phenotypically confirmed carbapenemase-producing isolates were subjected to multiplex PCR for the detection of NDM, VIM, IMP, KPC, and OXA-48 genes. NDM was found to be the most common gene and present in 85/150 isolates. K. pneumoniae was having the highest percentage of NDM positivity of 60% (54/90). After NDM, the second most common gene found was VIM (29/150). Eight (08) isolates possessed combination of these genes. The most common combination was NDM + IMP which was found in 5 isolates. KPC and OXA-48 were not found in any of the isolates in this period under study. Class D carbapenemase, OXA-48, is commonly present in Acinetobacter spp. and only occasionally in Enterobacteriaceae. Nineteen isolates under this study did not show the presence of any of the genes, although they were positive on screening as well as on MHT. The presence of one or combination of many other carbapenem resistance genes is a possibility in these 19 isolates.

In our study, the prevalence of NDM gene harbored by Enterobacteriaceae is 6.8% (85/1254). Species-wise, the prevalence of NDM gene in E. coli, K. pneumoniae, E. aerogenes, S. marcescens, Proteus spp., C. freundii, and Raoultela ornithinolytica is 2.2% (16/724), 14% (57/406), 17.5% (7/40), 11.1% (2/18), 2% (1/50), 7% (1/14), and 50% (1/2), respectively. In a study conducted in India, the percentage of Enterobacteriaceae harboring the NDM gene was 30% in Chennai and 13% in Haryana with E. coli and K. pneumoniae being in majority.[2] In another study from India, 14.7% of E. coli isolates were NDM-1 positive.[25]

It is clear from our study that the percentage of NDM genes detected was significantly higher among K. pneumoniae as compared to E. coli. Similarly, the presence of VIM and IMP genes was also found to be significantly higher among K. pneumoniae as compared to E. coli.

In our study, the fact that NDM gene was detected in at least seven species, namely E. coli, K. pneumoniae, E. aerogenes, Serratia marcescenes, Proteus spp, C. freundii, and R. ornithinolytica suggests that the real problem is endemic spread within the species by transferable mobile genes carried on plasmids and high usage of ineffective antimicrobials in turn, selecting out both ESBL and MBL producers and helping the spread.

The study highlights the importance of detection of CRE and its implications in clinical practice. The advantage of this study is that it discusses in detail the steps and techniques used in the laboratory tests for screening and confirmation of CRE. However, the limitation of the study is that neither the clinical profile of the patients has been discussed nor the patients were followed up after detection of carbapenem resistance.


  Conclusion Top


To conclude, the prevalence of phenotypically confirmed carbapenemase resistance among Enterobacteriaceae is 11.96%, which is quite high. MHT is a useful confirmatory test for the detection of carbapenem resistance. However, molecular detection is the gold standard for the identification of carbapenemase-producing isolates and various genes responsible for it.

Acknowledgments

  • Laboratory Technician Kishore Mohanta
  • Laboratory Assistant Sabyasachi Mondal.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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[PUBMED]  [Full text]  


    Figures

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