COMPARISON OF PHENOTYPIC AND GENOTYPIC METHODS FOR DETECTION OF CARBAPENEM RESISTANCE IN AN INDIAN TERTIARY CARE HOSPITAL.

Polymerase chain reaction:-DNA Extraction: Molecular identification of KPC-producing Klebsiella pneumoniae was performed by bla KPC PCR using bacterial lysates from overnight broths prepared by removal of 200 μl of broth culture, centrifugation (12,000 × g ; 2 min), resuspension in 200 μl of molecular-grade water, boiling at 95°C for 10 min, and discarding the cellular debris by centrifugation (12,000 × g ; 2 min at 4°C).PCR analysis for bla KPC was performed with 1 μl of cell lysates, using the following primers designed to identify all bla KPC genes ( bla KPC-1 through bla KPC-7 ): KPC forward (ATGTCACTGTATCGCCGTCT). KPC reverse (TTTTCAGAGCCTTACTGCCC). The Reaction was set up in a PCR vial, after adding the master mix, the forward and reverse primers and the extracted DNA. 25μl of Master Mix contained 10X Taq buffer, 2mM Mgcl2, 0.4mM dNTPs mix, and 2U Proofreading Taq DNA polymerase. (Thermo SCIENTIFIC ,USA) Lysates derived from Escherichia coli ATCC 25922 and bla KPC carrying K. pneumoniae strain 1705 were used as negative and positive controls,respectively, in each PCR. The PCR conditions were as follows: 15 min at 95°C and 38 cycles of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C, followed by an extension step of 10 min at 72°C.The PCR products were subjected to electrophoresis on 2% agarose gel stained with ehidium bromide and visualized with UV light . The blaKPC gene gave band at 893bp


ISSN: 2320-5407
Int. J. Adv. Res. 6(5), 1452-1457 1453 the carbapenem resistance criteria to ensure that KPC-producing organisms were not misclassified. This change eliminated the need for secondary testing by MHT. So,a lower MIC for antibiotic resistance was established for CREs and these criteria were again revised in 2012. [12] Polymerase chain reaction confirms the detection of a particular gene.So the present study was conducted to compare MIC with blakpc gene detection in meropenem resistant Klebsiella pneumoniae isolates.

Material and methods:-
Study design:-This study was conducted in the Department of Microbiology, Sher-i-Kashmir Institute of Medical Sciences, Srinagar; over a period of one year and three months.

Methodology:-
The study included all isolates of Klebsiella pneumoniae recovered from blood culture of the patients. The identification and antimicrobial susceptibility of the isolates was done on Vitek 2. Isolates that are resistant to Meropenem were included for phenotypic(MIC) and genotypic(PCR) testing of KPC. MIC was determined by broth microdilution for meropenem. [13] These isolates were then tested for blaKPC gene by conventional polymerase chain reaction. [14] MIC was done by Broth Microdilution method as under:-Preparation of antibiotic stock solution for Meropenem:-Stock solution was prepared using the formula:-1000/P x V x C = W Where, P= potency given by manufacturer (µg/mg),V = volume required (ml),C = final concentration of solution (mg/ L) and W = weight of antibiotic (mg) to be dissolved in volume V (ml).
The stock solution was prepared in such a way that its concentration was 1mg/ml or greater .Meropenem stock solution was prepared by dissolving 55.43 mg of the antibiotic powder in 1ml of distilled water. Microbroth dilution method:-Using a micropipette 50 μl of Muller Hinton broth was dispensed into all wells of a microtitre plate leaving the first column unfilled.After this 100μl of working antibiotic solution (concentration 256μg/ml) was added to the wells of the first column.From the first well 50μl of the working antibiotic solution was pipetted out and added to the second well,already containing 50 μl of MH broth .From the second well 50 μl of solution was added into the next well and so on and so forth till the well well number 10 was reached from which 50 μl of solution was discarded .The final concentration in the wells ranged from 256-0.5μg/ml.The last two columns served as growth control and sterility control respectively.
The turbidity of the bacterial inoculum was adjusted to 0.5 McFarland standards and 50 μl of it was dispensed into all the wells of microtitre plate.Finally the plates were incubated at 37ºC overnight and read the other day.
Results were recorded by visual inspection of the microtitre plates after overnight incubation at 37ºC as per CLSI guidelines. The test was considered valid when acceptable growth (more or equal to 2mm button or definite turbidity ) was seen in the positive control well.Absence of turbidity or a button of less than 2mm diameter in the test well was thus taken as the MIC of the organism under test .
Polymerase chain reaction:-DNA Extraction: Molecular identification of KPC-producing Klebsiella pneumoniae was performed by blaKPC PCR using bacterial lysates from overnight broths prepared by removal of 200 μl of broth culture, centrifugation (12,000 × g; 2 min), resuspension in 200 μl of molecular-grade water, boiling at 95°C for 10 min, and discarding the cellular debris by centrifugation (12,000 × g; 2 min at 4°C).PCR analysis for blaKPC was performed with 1 μl of cell lysates, using the following primers designed to identify all blaKPC genes (bla KPC

Discussion:-
Out of the total isolates 55(78.5%) were meropenem resistant and 15(21.5%) were meropenem sensitive .our study results are similar with study conducted by Marquez P et al according to which 83% isolates were carbapenem resistant Klebsiella pneumonia. [15] According to a study conducted by Shanmugam P et al.(2013), Forty three (93.4%) out of the 46 isolates were resistant to Meropenem. [16] Seibert et al in their study in Brazil found K. pneumoniae was the microorganism that presented with the greatest resistance to carbapenems ( 62.0%). [17] Praveen et al., 2010 studied K. pneumoniae, from 134 clinical isolates about 43.6% percent isolates were resistant to Meropesnem. (Praveen et al., 2010). [18] On comparison of MIC with PCR for blaKPC gene findings it was seen that out of 20 isolates with MIC ≥256 , 18(90%) were positive for blaKPC gene , among isolates with MIC 128 µg/ml, 61% were positive for blaKPC gene . whereas isolates with MIC 64µg/ml, 72.7% were positive for this gene and isolates with MIC 32 µg/ml, 66.7% were positive for the blaKPC gene . According to a study conducted by Giani et al ,Carbapenem MICs of the 234 carbapenem-nonsusceptible K. pneumoniae, MIC of >32 μg/ml was found in 94% isolates with ( KPC) carbapenemase, whereas isolates with (VIM ) carbapenemase ,MIC range was 2 to >32 , and those with (OXA) type carbapenemase MIC was 1 to 8 μg/ml. [19] Also according to Parveen et al. 43.6% were resistant to meropenem. The MIC of meropenem showed a varied range. Eight isolates showed a maximum MICs of>128μg/mL, nine were MIC at 128μg/mL. Similarly 6 isolates showed MIC of 16μg/mL and five had MIC of 32μg/mL. None of the K. pneumoniae was found to produce MBL by EDTA-meropenem disk approximation test. [18] As per a study conducted by Castenheira [21] Also Spyros Pournaras et al investigated Meropenem heteroresistance in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemasenegative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 μg/ml. Heteroresistant colonies had significantly elevated expression of the bla KPC gene compared with the native populations. [22] Conclusion:-All the Klebsiella pneumoniae resistant isolates by vitek 2 were also having MIC in resistant range by BMD method and isolates positive for blaKPC gene are having higher MIC ranges, as all resistant isolates were having MIC ≥32μg/ml.