| Abstract|| |
Context: Carbapenemase production is an important mechanism responsible for carbapenem resistance. Aims: Phenotypic detection and differentiation of types of carbapenemase in carbapenem resistant Enterobacteriaceae is important for proper infection control and appropriate patient management. Settings and Design: We planned a study to determine the occurrence of Class A Klebsiella pneumoniae carbapenemase (KPC type) and Class B Metallo-β-lactamase (MBL type) carbapenemase in hospital and community. Materials and Methods: Clinical isolates of Escherichia coli and Klebsiella species and simultaneously evaluate different phenotypic methods for detection of carbapenemases. Results: It was observed that 20.72% clinical isolates of E. coli and Klebsiella spp. were resistant to carbapenem on screening of which, 14.64% were E. coli and 29.69% were Klebsiella spp. Using phenotypic confirmatory tests the occurrence of carbapenemase production was found to be 87.01% in E. coli and 91.51% in Klebsiella spp. using both modified Hodge test (MHT) and combined disk test (CDT) using imipenem-ethylenediaminetetraacetic acid. Conclusions: Both MBL and KPC type carbapenemases were seen among clinical isolates of E. coli and Klebsiella spp. CDT is simple, rapid and technically less demanding procedure, which can be used in all clinical laboratories. Supplementing MHT with CDT is reliable phenotypic tests to identify the class A and class B carbapenemase producers.
Keywords: Escherichia coli, Klebsiella pneumoniae carbapenemase, metallo-β-lactamases, phenotypic detection
|How to cite this article:|
Chauhan K, Pandey A, Asthana AK, Madan M. Evaluation of phenotypic tests for detection of Klebsiella pneumoniae carbapenemase and metallo-beta-lactamase in clinical isolates of Escherichia coli and Klebsiella species. Indian J Pathol Microbiol 2015;58:31-5
|How to cite this URL:|
Chauhan K, Pandey A, Asthana AK, Madan M. Evaluation of phenotypic tests for detection of Klebsiella pneumoniae carbapenemase and metallo-beta-lactamase in clinical isolates of Escherichia coli and Klebsiella species. Indian J Pathol Microbiol [serial online] 2015 [cited 2019 Jun 25];58:31-5. Available from: http://www.ijpmonline.org/text.asp?2015/58/1/31/151168
| Introduction|| |
Gram-negative bacteria pose a therapeutic problem not only in the hospital settings, but also in the community as they have acquired resistance to multiple antibiotics. Emergence of carbapenemases in Enterobacteriaceae and nonfermentative bacteria poses a serious therapeutic problem in hospitals because carbapenems are often antibiotics of last resort for the treatment of serious infections caused by multidrug-resistant Gram-negative bacteria. These bacteria have the potential to spread rapidly within the hospital environment and also across the continents.  Resistance to carbapenem is mostly due to production of enzymes-carbapenemases that hydrolyze carbapenems and other β-lactams. Acquired carbapenemases belong to group A (IMI, NMC, SME GES, and Klebsiella pneumoniae carbapenemase [KPC]), group B (Metallo-β-lactamase [MBLs] of VIM, IMP, GIM, NDM, SIM, and DIM series), and group D (carbapenem hydrolyzing oxacillinases).  The various mechanisms of drug resistance in Gram-negative bacteria include extended spectrum beta-lactamases (ESBL) production, AmpC beta-lactamase production, efflux mechanism and porin loss.  These enzymes are usually present in integrons on plasmids and pose a serious threat of massive dissemination among the Gram-negative fraternity.  Although reported with increasing frequency, the true rate of occurrence of carbapenemase production in different members of Enterobacteriaceae, remains unknown. The isolates showing a reduced susceptibility to meropenem (MRP) (zone diameter <21 mm) was used to screen isolates and used modified Hodge test (MHT) as a confirmatory test for isolates that harbor KPC (class A) carbapenemase. The current clinical and laboratory standard institute (CLSI) guidelines  do not describe any method for detection of isolates producing MBLs (class B) enzymes in members of Enterobacteriaceae. The aim of the present study was to detect the occurrence of class A KPC β-lactamase and class B MBLs in clinical isolates of Escherichia More Details coli and Klebsiella spp. both from inpatient units and outdoor samples in our tertiary care hospital and simultaneously evaluate two different phenotypic methods that is the MHT  and combined disk test (CDT) using imipenem (IPM)-ethylenediaminetetraacetic acid (EDTA)  for its detection.
| Materials and Methods|| |
A total of 883 non-repeat clinical isolates of E. coli and Klebsiella spp. obtained from different clinical samples (urine, endotracheal aspirate, pus, sputum etc.) received in Clinical Microbiology Laboratory over a period of 1-year from various inpatient units and outpatient departments was processed. The isolates were identified as per standard bacteriological technique.  Antimicrobial susceptibility testing was carried out as per CLSI recommendations. E. coli ATCC 25922, K. pneumoniae ATCC BAA-1705 (MHT positive) and K. pneumoniae ATCC BAA-1706 (MHT negative) were used for quality control.
Screening of carbapenem resistance
Screening of clinical isolates of E. coli and Klebsiella spp. was performed with MRP disk (10 ug) (Hi-media, Mumbai, India).  The isolates showing a reduced susceptibility to MRP (zone diameter <21 mm) was MRP screen positive. Minimum inhibitory concentration (MIC) of MRP was also determined by VITEK 2C (bioMerieux, France) for screen positive isolates (all screen positive isolates had MIC >64 μg/ml). The MRP screen positive isolates were subjected for confirmation of carbapenemases by two different phenotypic tests MHT and CDT.
Detection of carbapenemases
Modified hodge test
Carbapenemase production was detected by MHT as per the recommendation of CLSI.  The isolate showing a clover leaf-like indentation of E. coli 25922 growing along the test organism growth streak within the disk diffusion zone was considered as MHT positive [Figure 1] and the isolate showing no growth of E. coli 25922 along the test organism growth streak within the disk diffusion zone was taken as MHT negative [Figure 2].
|Figure 1: Modified Hodge test positive: Isolate showing a clover leaf-like indentation (arrow) of the Escherichia coli 25922 growing along the test organism growth streak within the disk diffusion zone, indicating serine carbapenemase producer (Klebsiella pneumoniae carbapenemase type)|
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|Figure 2: Modified Hodge test negative: Isolate showing no indentation of the Escherichia coli 25922 along the test organism growth streak within the disk diffusion zone, indicating serine carbapenemase non producer (Klebsiella pneumoniae carbapenemase type)|
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Combined disk test
Metallo-β-lactamase production was detected by CDT method on disk containing EDTA as per the method used by Yong et al.  Briefly, a 0.5 M EDTA (Hi-Media) solution was prepared by dissolving 18.61 g of EDTA in 100 ml of distilled water and adjusted it to pH 8.0 by using NaOH and sterilized by autoclaving. Turbidity of 0.5 McFarland standard was made from overnight subculture of test organism and spread as lawn culture on the surface of a Muller Hinton agar plate. Two IPM disks (10 μg) (Hi-media) were placed on the agar, and 10 μl 0.5M EDTA solution was added to one of the disks. Inoculated plates were incubated overnight at 35°C. An organism demonstrating a zone diameter around the disk containing IPM-EDTA ≥ 7 mm than the zone diameter around the IPM disk alone was considered as an MBL producer [Figure 3].
|Figure 3: Combined disk test positive: Isolate showing an increase in zone diameter of ≥7 mm around the around the imipenem-ethylenediaminetetraacetic acid disk as compared to that of the imipenem disk alone, indicating Metallo-β-lactamase producer|
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| Results|| |
Out of the total 883 clinical isolates of E. coli and Klebsiella spp. screened for carbapenem resistance, 20.72% (183/883) isolates were MRP screen positive. Out of which, 77/183 (14.64%) were E. coli and 106/183 (29.69%) were Klebsiella spp. (P < 0.05) [Table 1].
|Table 1: Distribution of carbapenem resistant and carbapenem sensitive isolates of E. coli and Klebsiella species (n = 883)|
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Phenotypic detection of carbapenemases
Carbapenemase production was detected phenotypically in 28.57% and 32.07% of clinical isolates of E. coli and Klebsiella spp. using MHT and in 45.45% of E. coli and 38.67% of Klebsiella spp. using CDT [Table 2]. Moreover, in 12.99% of E. coli and 20.75% of Klebsiella spp. carbapenemase production could be detected both by MHT and CDT. However, carbapenemase production could not be detected using both MHT and CDT in 12.99% and 8.49% of E. coli and Klebsiella spp., which were MRP screen positive.
|Table 2: Phenotypic differentiation of clinical isolates of E. coli and Klebsiella spp. resistant to carbapenem (n = 183)|
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Distribution of carbapenemase producers and nonproducers
Out of the total 20.72% (MRP screen positive) clinical isolates of E. coli and Klebsiella spp. carbapenemase production could be detected in 87.01% of E. coli and 91.51% of Klebsiella spp. isolated from our hospital using both the tests [Table 3]. The difference between carbapenemase producers and nonproducers was found to be statistically significant using Z-test (Z cal > 2.58) (P < 0.05).
|Table 3: Distribution of carbapenemase producers and nonproducers in carbapenem resistant isolates of E. coli and Klebsiella species (n = 183)|
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Evaluation of phenotypic tests
Combined disk test could definitely detect enzymes in more number of isolates of E. coli (58.44%) and Klebsiella spp. (52.94%) as compared to MHT 41.56% in E. coli and 47.06% in Klebsiella spp. [Table 4]. The difference was, however, found to be statistically not significant at 5% level of significance (χ2 = 0.5722, P = 0.4501).
|Table 4: Comparison of phenotypic tests in carbapenemase producing isolates of E. coli and Klebsiella spp.|
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Antibiotic susceptibility pattern and site of localization
The carbapenem resistant clinical isolates of E. coli (64/77) and Klebsiella spp. (100/106) both were isolated more frequently from samples received from inpatient units than from samples received from outdoor patients. E. coli was isolated predominantly from the urine sample followed by pus, endotracheal aspirate and sputum. However, Klebsiella spp. was isolated predominantly from endotracheal aspirate followed by urine and pus. The clinical isolates of E. coli and Klebsiella spp. showed resistance to multiple antimicrobial agents. The hospital isolates of E. coli showed maximum resistance to ciprofloxacin (73.13%) followed by gentamicin (58.20%) and amikacin (8.95%). Least resistance was observed with tigecycline (2.98%). However, all the E. coli isolated from samples received from outdoor patients was sensitive to tigecycline. On the contrary, 9.27% and 1.03% of Klebsiella spp. isolated from samples received from inpatient units and outdoor samples respectively, showed resistance to tigecycline. High level of resistance to ciprofloxacin (84.53%), cotrimoxazole (81.44%), gentamicin (46.39%) and amikacin (17.52%) was seen in hospital isolates of Klebsiella spp. However, all the clinical isolates of E. coli and Klebsiella spp. isolated both from samples received inpatient units, and outdoor samples were 100% sensitive to colistin.
| Discussion|| |
Carbapenems belong to the β-lactam group of antibacterial agents. The emergence and proliferation of bacteria resistant to this important group of drug are jeopardizing the use of carbapenems.  The prevalence of carbapenemase production among Gram-negative bacilli varies greatly from country to country and among different institutions within the country. Therapeautic options for infections caused by Gram-negative bacteria expressing carbapenemases are limited. This emphasizes the need for detecting carbapenemases harboring isolates so as to avoid therapeautic failure and nosocomial outbreaks.
In the present study, 20.72% of clinical isolates of E. coli and Klebsiella spp. were found to be MRP screen positive. Resistance to carbapenem was found to be more in Klebsiella spp. (29.69%) than in E. coli (14.64%) [Table 1]. Similarly, a high prevalence of resistance to carbapenems 2-13% in E. coli and 31-51% in Klebsiella spp. has been reported from Delhi.  Another study has reported carbapenem resistance (17-22%) in different strains of Enterobacteriaceae from North India.  Overall, using both the tests we could confirm carbapenemase production in of 87.01% of clinical isolates of E. coli and 91.51% of clinical isolates of Klebsiella spp. (P < 0.05) [Table 3]. A comparatively higher occurrence of carbapenemase producers in the present study may be partly attributed to the fact that our hospital being a tertiary care center. Carbapenemase production was more in Klebsiella spp. compared to E. coli in the present study. Similar findings have been reported. ,, However, 12.99% of E. coli and 8.49% of Klebsiella spp. which were MRP screen positive were both KPC and MBL production test negative [Table 2]. This negative result could be due to other important causes of carbapenem resistance among Enterobacteriaceae such as overproduction of ESBL or AmpC enzyme with porin loss and group D (carbapenem hydrolyzing oxacillinases). , Various studies done during the period of 2004-2012 have also reported similar findings. ,,
Combined disk test could definitely detect more number of cases both in E. coli (58.44%) and Klebsiella spp. (52.94%) compared to MHT (41.56% in E. coli and 47.06% in Klebsiella spp.) [Table 4]. Currently, CLSI documents do not indicate the screening and confirmatory tests that are optimal for detection of these beta-lactamases. In 2009, CLSI approved MHT for the detection of carbapenemases, especially KPC's from members of Enterobacteriaceae but not in nonfermenters. However, the current guidelines do not describe any method for detection of MBL enzymes in members of Enterobacteriaceae. In spite of many phenotypic tests, molecular methods like polymerase chain reaction, DNA hybridization and sequencing are the gold standard for detection of carbapenemase production, which will help us to know the actual prevalence of these enzymes and characterize them for epidemiological purpose. But these methods are of limited practical use for daily application in clinical laboratories because of the cost and are used in research settings.  The most common mechanism for carbapenem resistance in Klebsiella spp. is the production of carbapenemases belonging to Ambler class A, especially KPC, or Ambler class B, MBLs such as IMP and VIM types. Unfortunately, in the present study, we were unable to perform the genotypic tests due to limited resources. However, the finding of the present study shows the presence of carbapenemases in this area, future study may be carried out in more number of samples employing genotypic methods to know the actual prevalence of carbapenemase enzyme.
Our study, also demonstrates the wide-spread presence of MBLs in members of Enterobacteriaceae (58.44% E. coli and 52.94% Klebsiella spp.) isolated from our tertiary care center. Similar findings have been reported from hospitals in east Delhi.  Carbapenem resistance among clinical isolates of Enterobacteriaceae, especially E. coli and K. pneumoniae, is largely conferred by MBL.  The early detection of MBL-producing isolates would be important for the reduction of mortality rates and also to avoid the intra-hospital dissemination of such strains. Pandya et al.  have reported that CDT (IPM-EDTA) is the most sensitive test that can be used as a convenient screening method for detection of MBL production in Gram-negative bacilli in routine microbiological practice. CDT had an advantage over MHT in being less time consuming, technically less demanding, therefore, less cumbersome to perform in routine clinical laboratory where workload is more. Similar observations about CDT have also been reported by other workers.  In a nutshell, we can say that the need of the hour is simple, rapid and cost effective tests, which will be able to identify and distinguish resistant pathogens for better patient outcome, facilitating efficient infection control and reducing the escalation of resistance.
It has been reported that supplementing MHT with at least one of the screening methods increases the likelihood of picking up such isolates that may be missed by the MHT.  Therefore, in the present study, we used both the phenotypic methods and observed that CDT could definitely detect more number of cases. Similar observation was made by Jesudason et al.  who concluded that EDTA disk synergy test detected more strains not detected by MHT. MHT was found to be useful for the phenotypic detection of KPC enzymes in hospitals where these beta lactamases are endemic, the test cannot discriminate between KPCs and other carbapenemases and its positive predictive value for KPC detection is low in regions where other carbapenem-hydrolyzing enzymes, like MBLs, are also prevailing.  Thus, MHT may not be a useful screening test for carbapenemases as many MBL producing isolates were not detected by this test. 
In our study, the carbapenem resistant strains were isolated more frequently from sample received from inpatient units than from outdoor clinics. Detection of carbapenem resistance in an organism isolated from samples received from outdoor patients is a matter of concern as such strains may spread into the community rapidly and may cause therapeautic problem. To the contrary, few studies have shown that carbapenemase producers are restricted to hospitalized patients only, with nosocomial spread as their main mode of dissemination. , The clinical isolates of E. coli and Klebsiella spp. showed varied resistance to other antimicrobial agents. The carbapenem resistant isolates of E. coli and Klebsiella spp. were 100% susceptible to colistin. However, low level of resistance was observed with tigecycline (9.27% in Klebsiella spp. and 2.98% in E. coli). Resistance to tigecycline in Klebsiella spp. has also been reported. , Both the drugs, that is colistin and tigecycline are used as treatment options in multidrug-resistant carbapenemase producing organisms.  Thus, appropriate detection of carbapenem resistant Enterobacteriaceae (CRE) is vital for patient care in order to institute correct therapeautic options, that is, colistin and polymixin B. Urban et al.  observed that carbapenemase producing E. coli and K. pneumoniae were uniformly susceptible to polymyxin B and tigycycline. Similarly, various workers have reported polymyxin B as the most sensitive drug for MBL producers.  In a recent study in 2012 Charan et al.  reported that polymyxins (colistin) and glycylcyclines (tigecycline) are effective against NDM 1 harboring Enterobacteriaceae.
Enterobacteriaceae contribute to a major part of the gut flora. Just like other bacteria, blaNDM producing Enterobacteriaceae are capable of colonizing the gut of patients. They in turn serve as reservoirs for spreading infection or contaminating the environment and fomites, especially in healthcare settings. In order to control the spread, disinfection measures need to be followed as contact isolation of these infected/colonized patients is not routine and may not be feasible in all healthcare institutions, especially in the developing countries. Microbiological surveillance of the rectal flora at the time of admission (especially in patients who have already been exposed to antibiotics and healthcare interventions) and contact isolation of potentially colonized/infected patients will go a long way in preventing contamination of the environment and spread to other patients. In addition, appropriate use of carbapenems will also prevent selecting resistant bacteria in a given geographical area. Carbapenem is always used as a reserve drug in multidrug-resistant Gram-negative pathogens in Intensive Care Units and inwards. Siegel et al.  showed antimicrobial stewardship to be an important part of efforts to control multidrug resistant organisms.
To conclude, the occurrence of carbapenemase producers in clinical isolates of E. coli and Klebsiella spp. was relatively high in our setting. Thus, there is a need for the correct and reliable phenotypic test to identify beta-lactamases and to discriminate between KPC type and MBL type enzymes. Detection of carbapenemase has important implications for infection control and for the epidemiological purpose. CDT had an advantage over MHT in being less time consuming, technically less demanding, therefore, less cumbersome to perform in routine clinical laboratory where workload is more. However, supplementing MHT with CDT are rapid and reliable phenotypic tests to identify the class A and class B carbapenemase producers. Detection tests are still evolving hindered by the hetrogenicity of both enzymes and hosts which confer different levels of carbapenem susceptibility. The focus is on a microbiology laboratory that will detect CRE accurately, timely and cost effectively for better patient outcome, facilitating efficient infection control and reducing the escalation of resistance.
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Dr. Anita Pandey
Department of Microbiology, Subharti Medical College, Swami Vivekanand Subharti University, Meerut - 250 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]