|Year : 2012 | Volume
| Issue : 3 | Page : 357-360
|Phenotypic method for differentiation of carbapenemases in Enterobacteriaceae: Study from north India
Priya Datta, Varsha Gupta, Shivani Garg, Jagdish Chander
Department of Microbiology, Government Medical College Hospital, Chandigarh, India
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|Date of Web Publication||29-Sep-2012|
| Abstract|| |
Aims: Carbapenems are usually the choice of antimicrobials in infections caused by Enterobacteriaceae bacteria-producing ESBL (extended spectrum β-lactamases) and Amp C. Resistance to carbapenems is mostly due to production of enzymes - Carbapenemases, which are divided into Ambler Classes A, B and D. Phenotypic detection and differentiation of types of Carbapenemases in carbapenem-resistant Enterobacteriaceae (CRE) is important for proper infection control and appropriate patient management. Materials and Methods: The present study done in a tertiary care hospital from North India differentiates Class A (KPC type) and B (MBL type) carbapenemases among Enterobacteriaceae isolates by simple phenotypic method that uses both the inhibitors EDTA and phenylboronic acid. Results: Total of 330 strains of Enterobacteriaceae were included in the study. Out of these 330 strains, 26 strains were resistant to carbapenems. The prevalence of CRE in our Institute is 7.87% (26/330). Conclusions: The prevalence of Enterobacteriaceae strains producing MBL type carbapenemase in our health care setup is 5.75% (19/330). None of the strains among the carbapenem-resistant bacterial isolates showed production of KPC enzyme. The need of the hour is simple, rapid and cost effective tests which will be able to identify and distinguish resistant pathogens for improved patient outcome, facilitating efficient infection control and reducing the escalation of resistance.
Keywords: Carbapenemase, enterobacteriaceae, phenotypic detection
|How to cite this article:|
Datta P, Gupta V, Garg S, Chander J. Phenotypic method for differentiation of carbapenemases in Enterobacteriaceae: Study from north India. Indian J Pathol Microbiol 2012;55:357-60
|How to cite this URL:|
Datta P, Gupta V, Garg S, Chander J. Phenotypic method for differentiation of carbapenemases in Enterobacteriaceae: Study from north India. Indian J Pathol Microbiol [serial online] 2012 [cited 2019 Jun 17];55:357-60. Available from: http://www.ijpmonline.org/text.asp?2012/55/3/357/101744
| Introduction|| |
In the treatment of infections due to extended spectrum β-lactamases (ESBL) and Amp C enzymes producing strains of Enterobacteriaceae the antimicrobial of choice is carbapenem. However, the emergence and proliferation of bacteria resistant to this important group of drug is jeopardizing the use of carbapenems.  Resistance to carbapenem mostly is due to production of enzymes-Carbapenemases that hydrolyse carbapenems and other β-lactams. Another important cause among carbapenem-resistant Enterobacteriaceae (CRE) is over production of ESBL or Amp C enzyme in organism with porin loss. 
Carbapenemase enzymes fall into Ambler classification - A, B and D.  Class A (serine carbapenemase) enzymes include enzymes such as KPC, IMI, SME, etc and are commonly present in members of Enterobacteriaceae and rarely in Pseudomonas species and Acinetobacter baumanii. , These classes of enzyme usually hydrolyse penicillin and cephalosporins more efficiently than carbapenems and are inhibited by boronic acid and less by clavulanate. KPCs are usually found in Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia More Details coli, Citrobacter species and Enterobacter species. 
Class B enzymes are metallo-Beta lactamases (MBL) including VIM, IMP and SPM. These occur in various genera of Enterobacteriaceae and non-fermenters. These enzymes have a wide spectrum of activity against carbapenem, penicillin and extended spectrum cephalosporins and not aztreonam. In addition they resist hydrolyses by clavulanate and are inhibited by chelating agents such as EDTA. 
Class D carbapenemase belong to OXA family and are commonly present in Acinetobacter spp. and occasionally in Enterobacteriaceae and Pseudomonas spp. These enzymes hydrolyze carbapenems weakly and are poorly inhibited by clavulanate. 
Appropriate detection of CRE is vital for patient care in order to institute correct therapeutic options i.e. colistin and polymyxin B. Molecular techniques can easily differentiate between these classes of carbapenemases but need of the hour is a rapid, practical, phenotypic method which can differentiate KPCs and MBLs in Enterobacteriaceae. Another important epidemiological implication is that both MBLs and KPCs are spread by transposon and/or integron-encoded determinants, that can also carry non-β-lactam resistance determinants, which can disseminate to other enterobacterial strains. 
In our Institute members of CREs are being increasingly isolated. The present study detects and differentiates Class A and B carbapenemases among Enterobacteriaceae isolates by simple phenotypic method that uses both the inhibitors EDTA and phenylboronic acid.
| Material and Methods|| |
The study was a prospective study carried out in the Department of Microbiology, at our tertiary care hospital, North India, from January 2011 to July 2011. The clinical samples included in the study were pus, wound swabs, body fluids, sputum, throat swab and endotracheal secretions collected from various patients admitted in wards and ICUs. The bacterial isolates were identified according to standard microbiological procedure.  All isolates belonging to Enterobacteriaceae family like Escherichia coli, Klebsiella species and Citrobacter species were included in the study.
All these isolates were tested for susceptibility to ertapenem (10 μg)/ meropenem (10 μg) (BD, Diagnostics) by disc diffusion method according to CLSI (Clinical Laboratory Standard Institute) criteria. Those strains which showed reduced susceptibility to ertapenem/meropenem were confirmed to be carbapenem resistant by E-test (bioMérieux). Resistance of Enterobacteriaceae strains to carbapenem was reported if MIC to ertapenem was ≥1 μg/ml and/or MIC to meropenem was ≥4 μg/ml. 
Use of Inhibitor for Differentiation of KPC and MBL
Use of inhibitor phenylboronic acid (PBA), EDTA or both along with meropenem disc was used for detection of KPC and MBL, respectively. 
The stock solution of PBA in the concentration of 20 mg/ml was prepared by dissolving PBA (Sigma-Aldrich, Germany) in DMSO. Twenty microliters (400 μg of PBA) from this solution was dispensed onto meropenem discs. The stock solution of EDTA was prepared by dissolving anhydrous EDTA (Sigma-Aldrich) in distilled water at a concentration of 0.1 M. Ten microliters (292 μg of EDTA) from this solution was dispensed onto meropenem discs. The meropenem discs with inhibitor added was dried and used within 60 min. 
On Mueller Hinton agar plate inoculated with test strain, four discs of meropenem were used. One disc of meropenem was without any inhibitor, one disc had PBA (400 μg) only, one disc had EDTA (292 μg) only and fourth disc of meropenem had both PBA plus EDTA. The agar plates were incubated at 37° C overnight and the diameter of the growth inhibitory zone around these meropenem discs with inhibitor added was compared with that around the plain meropenem disc. 
The isolate was considered KPC producing when the growth-inhibitory zone diameter around the meropenem disc with PBA and the meropenem disc with both PBA and EDTA was increased ≥5 mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone.
The isolate was considered MBL producing when the growth-inhibitory zone diameter around the meropenem disc with EDTA and the meropenem disc with both PBA and EDTA was increased ≥5 mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone.
The isolate was considered producing both KPC and MBL enzymes when the growth-inhibitory zone diameter around the meropenem disc with both PBA and EDTA was increased ≥5 mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone while the growth-inhibitory zone diameters around the meropenem disc with PBA and the meropenem disc with EDTA were increased <5 mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone.
The isolate was considered negative for MBL and KPC production, when none of the three combined-disc tests was positive. 
- The concentration of PBA and EDTA employed in the present study did not show any detectable effect on bacterial growth.
- Positive control for KPC - Klebsiella pneumoniae ATCC BAA-1705
Modified Hodge Test
All CRE isolates detected by disc diffusion and MIC were also tested by Modified Hodge test. Modified hodge test (MHT) is recommended by CLSI as isolates of Enterobacteriaceae producing KPC-type carbapenemase have a high level of sensitivity (>90%) and specificity. 
A 0.5 McFarland standard suspension of E. coli ATCC 25922 was prepared in broth. A Mueller Hinton agar plate was inoculated as for the routine disk-diffusion procedure. The plate was allowed to dry for 10 minutes. Meropenem disk was placed in the center of the plate. Using a 10 μL loop, three to five colonies of test organism grown overnight on a blood agar plate were picked and inoculated in a straight line out from the edge of the disk. The streak was at least 20-25 mm in length. Following incubation, Mueller Hinton agar was examined for enhanced growth around the test streak at the intersection of the streak and the zone of inhibition. 
Result: Enhanced growth = positive for carbapenemase production.
No enhanced growth = negative for carbapenemase production.
K. pneumoniae ATCC BAA-1705-MHT positive
K. pneumoniae ATCC BAA-1706-MHT negative
| Results|| |
Total of 330 strains of Enterobacteriaceae were included in the study. These included 200 strains of E. coli, 100 Klebsiella spp. and 30 Citrobacter spp. Twenty-six strains were resistant to carbapenem by both disc-diffusion and E test. There was no discordance between these two phenotypic methods. These included seven resistant strains of E. coil, 12 strains of Klebsiella spp. and 7 strains of Citrobacter spp. The test using inhibitor for differentiation of KPC and MBL showed;
None of the isolates were KPC producers i.e., none of these 26 strains showed the growth-inhibitory zone diameter around the meropenem disc with PBA and the meropenem disc with both PBA and EDTA to be more than ≥5 mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone [Table 1].
|Table 1: Phenotypic differentiation of Enterobacteriaceae strains resistant to carbapenem|
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19 isolates were MBL producers i.e., 19 strains showed the growth-inhibitory zone diameter around the meropenem disc with EDTA and the meropenem disc with both PBA and EDTA to be more than ≥5 mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone [Table 1].
None of the isolates were considered producing both KPC and MBL i.e ., no isolate showed the growth-inhibitory zone diameter around the meropenem disc with both PBA and EDTA to be more than ≥5 mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone while the growth-inhibitory zone diameters around the meropenem disc with PBA and the meropenem disc with EDTA were increased <5 mm compared with the growth-inhibitory zone diameter around the disc containing meropenem alone.
Seven isolates were considered negative for MBL and KPC production, as none of the three combined-disc tests was positive [Table 1].
Modified Hodge Test
All the 26 isolates which were resistant to carbapenem were also tested by MHT. None of these isolates showed positive result in MHT.
| Discussion|| |
The prevalence of CRE in our Institute is 7.87% (26/330). This resistance to carbapenem is low compared to studies by various authors from India. Wattal C et al. reported high prevalence of resistance to carbapenems ranging from 13 to 51% in E. coli and Klebsiella spp. from ICUs and wards from a tertiary care hospital in Delhi. Gupta E et al. also reported high prevalence of resistance varying from 17 to 22% to various carbapenems among Enterobacteriaceae strains.
In our hospital Antibiotics Stewardship Programme has been in place for last 4 years, which may be a contributing factor for low prevalence of CRE. Carbapenem is always used as a reserve drug in multidrug-resistant Gram-negative pathogens in ICUs and in wards. Siegel et al. showed antimicrobial stewardship to be an important part of efforts to control multidrug resistant organisms (MDO).
Additionally vancomycin and broad spectrum antimicrobial agents like ciprofloxacin are used only when required as its indiscriminate use allows growth of MDO. Hussein et al. and Wiener WY et al. also showed use of fluoroquinolones and vancomycin to be an important risk factor in patients with infections or carriage of CRE.
The prevalence of Enterobacteriaceae strains producing MBL-type carbapenemase in our health care setup is 5.75% (19/330). No strains among the carbapenem-resistant bacteria isolates showed production of KPC enzyme both by phenotypic inhibition and by MHT. There was 100% concordance between inhibitor based phenotypic method for detection of KPCs and MHT. Tsakris A et al. in his study showed that use of PBA and EDTA for phenotypic detection of carbapenemase to be 100% sensitive, using PCR as gold standard. One drawback of our study is the inability to compare the phenotypic methods with PCR for detection for CRE. Molecular methods like PCR, DNA hybridization and sequencing are the gold standard for detection of carbapenemase production. But these are used in research settings and not in routine diagnostic laboratories. The need of the hour is simple, rapid and cost effective tests which will be able to identify and distinguish resistant pathogens for improved patient outcome, facilitating efficient infection control and reducing the escalation of resistance. 
Seven strains (7/330) were resistant to carbapenem but both MBL and KPC production test were negative in these isolates. Since another important cause of carbapenem resistance among Enterobacteriaceae could be overproduction of ESBL or Amp C enzyme with porin loss, this could be the reason in these seven bacterial strains.
Detection of carbapenemase has important implications for infection control and for epidemiological purpose.  There is coexistence of non-β-lactam resistance determinants along with carbapenemase enzyme. Also, since this enzyme can easily be transmitted via transposon and or integron, there is possibility of widespread dissemination among susceptible gram negative bacterial isolates in the hospital. Additionally detection and surveillance of CRE has become a matter of major importance for the selection of appropriate therapeutic schemes and implementation of infection control measures. , The evolution of carbapenemases in terms of host range is rapid and there is potential for emergence of new variants because the genes are associated with mobile genetic elements. Detection tests are still evolving, hindered by the heterogeneity of both enzymes and hosts, which confer different levels of carbapenem susceptibility. The need of the hour is microbiological laboratory that detects CRE accurately and timely for better patient outcomes.
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Department of Microbiology, Government Medical College Hospital, Sector 32, Chandigarh-160 030
Source of Support: None, Conflict of Interest: None
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