Original Research Article http://doi.org/10.18231/j.ijmr.2019.027 Indian Journal of Microbiology Research, April-June, 2019;6(2):126-130 126 Detection of carbapenem resistant enterobacteriaecae and the comparison between phenotypic methods and multiplex PCR Rubhana Qadri 1* , Suhail Ahmed 2 , Dalip K Kakru 3 , Gulnaz Bashir 4 , Bashir Fomda 5 1 Senior Resident, 2 Lecturer, 3 Professor and Head, 4,5 Associate Professor, Dept. of Microbiology, 1,3-5 SKIMS Medical College, Bemina, Srinagar, Jammu and Kashmir, GB Panth Hospital GMC Srinagar, Jammu and Kashmir, India *Corresponding Author: Rubhana Qadri Email: rubhana.qadri@gmail.com Abstract Introduction: Carbapenem resistant Enterobacteriacea (CRE) has created a remarkable health distress. Definitive detection of Carbapenemase producing CRE is the first step in combating this problem. Objective: To detect and compare CP- CRE both by phenotypic and molecular methods. Materials and Methods: A total of 52 carbapenem resistant clinical isolates were screened for the presence of carbapenemase genes by routine phenotypic methods like modified hodge test and combined disc test as well by multiplex PCR. Results: Out of the total 52 meropenem resistant isolates, 35 were modified hodge test positive and 33 were combined disc test positive. 42 isolates were found harbouring one or more than one gene. blaKPC alone was present in 38 isolates, while as blaKPC with blaNDM were present in 1 isolate and blaKPC with blaIMP was seen in 1 isolate. blaNDM alone in 2 isoates, blaIMP and blaVIM alone in none of the isolates. Conclusion: Accurate detection of carbapenemase producing genes by molecular methods overcomes the problem related to CRE. Though there is no signal method that is ideal for all situations. Keywords: Modified hodge test, Combined disc test, Multiplex PCR, Carbapenem resistant Enterobacteriaecae. Introduction Enterobacteriaceae, are the most common causes of community-acquired and as well as nosocomial infections. These bacteria acquire resistance which in turn complicates the treatment. They can acquire genes that encodes for multiple antibiotic resistance mechanisms, including ESBLs, Amp Cs, and carbapenemases. Carbapenemases are clinically important because they destroy carbapenems as they are the antibiotic of last resort. 1 CRE are often resistant to β lactam drugs and can carry the resistance to other antimicrobial classes, thus limiting treatment options. Inappropriate treatment of severe infections caused by CP- CRE and lag in the detection is associated with increased mortality. So, they should be screened routinely for susceptibility to atleast one carbapenem. Several phenotypic methods are available for detection of carbapenemases like Modified Hodge test, combined disc test and Inhibitor based E test. The Modified Hodge test which is recommended by CLSI, is cheap and, simple to perform. However, its subjective, and it cannot distinguish among the different carbapenemase classes. Combined disc s tests have been extensively used because of low cost. Recently, various molecular methods like multiplex PCR have been shown to be sensitive and scrupulous method for identification of genes. Both sensitivity and specificity of multiplex PCR assay is 100% and can determine class of β-lactamase present. 2 Materials and Methods Study Design Prospective study was done for a period of one and a half year in the department of microbiology SKIMS institute soura. Aims and Objectives Comparing phenotypic methods like Modified Hodge test and Combined Disc test with multiplex PCR (VIM, IMP, KPC and NDM 1) for the detection of carbapenemases in CRE. Isolates of Enterobacteriaceae obtained from blood of patients of all age groups admitted at SKIMS or attending the OPD were included in this study. Isolates other than Enterobacteriaecae were not included. Blood was processed for the recovery of bacterial pathogens as per standard microbiological techniques. 3 Further identification and susceptibility testing were done on the vitek 2 compact. So vitek 2 was the screening test. All clinically significant CRE isolates were included in this study. Confirmation of MBL Production All screen test positive isolates were subjected to combined disc test (CDT) using imipenem, meropenem and ceftazidime disc along with EDTA. The CDT was performed as described by Yong D et al. 4 Confirmation of KPC Carbapenemase (KPC) production was confirmed by Modified hodge test. The test isolate producing the carbapenemase enzyme will allow the growth of a carbapenem susceptible strain (E. coli ATCC 25922) towards a carbapenem disk. Combined Disc Test (CDT) For the test, imipenem (IMP) 10 μg, meropenem (MRP) 10μg and ceftazidime (CAZ) 30 μg discs was used. In addition to this 0.5mM EDTA solution was used. The EDTA impregnated discs were stored at -20°C in airtight vials after being dried in an incubator, till further use. 2 Increase in the zone size of 5-10 mm between inhibition