Detection of Klebsiella pneumoniae Carbapenemase (KPC) Producing Enterobacteriaceae Isolates from Various Clinical Samples in a Rural Health Setup

Carbapenem resistance is increasing and emerging as a public health threat. The Indian subcontinent serves as a reservoir for all 3 types of carbapenemases: KPC, OXA-181 and NDM. The present study was done to determine the antibiotic resistance pattern of Gram negative bacilli (GNB) belonging to family Enterobacteriaceae and molecular detection of blaKPC gene among the Carbapenem resistant Enterobacteriaceae (CRE). Antibiotic sensitivity pattern of 301 gram negative isolates, members of Enterobacteriaceae (E. coli, K. pneumoniae, K. oxytoca, Proteus species, Citrobacter species, Enterobacter species and Serratia species) was determined and those resistant to carbapenem (ertapenem) were further processed in the study. Modified Hodge Test (MHT) was performed to phenotypically confirm the presence of carbapenemases (blaKPC) and Polymerase Chain Reaction (PCR) was done to identify blaKPC gene. Of the total 301 isolates, 45 were resistant to ertapenem. Out of these 45 isolates 32 were MHT positive and three of these 32 MHT positive strains harboured blaKPC gene.


INTRODUCTION
The members of family Enterobacteriaceae are Gram negative bacilli (GNB) which are a part of human normal flora. They are also a source of community and hospital acquired infections 1 . Carbapenems belong to a group of beta lactam antibiotics which have a broad spectrum activity. They are being used for treatment of severe life threatening infections caused by Multidrug resistant (MDR) GNB. These agents have become ineffective due to misuse and development of resistance against them 2 .
Resistance has been developing against the antibiotics soon after they have been put to use. Alexander Fleming the discoverer of penicillin, while accepting his Noble Prize in 1945 warned about the possibility of drug resistance. Resistance to carbapenems was first demonstrated in the North Carolinan strain of Klebsiella pneumoniae that produced an enzyme Klebsiella pneumoniae carbapenemase (KPC). The gene coding for resistance was found on plasmid. It was then when carbapenem resistance arrived 3 .
KPCs are beta lactam enzymes produced by GNB (K. pneumoniae, E. coli, Enterobacter species) belonging to Enterobacteriaceae family harboring bla KPC gene. These enzymes hydrolyze and inactivate a wide range of beta lactam antibiotics such as penicilllins, cephalosporins and carbapenems. There are different variants of bla KPC gene (KPC-2 to KPC-15) which are seen in non fermenting bacteria also 4 .
In order to contain the spread of bacteria harboring bla KPC gene effective control measures and judicious antibiotic usage in hospitalized patients must be implemented.
The study focused on KPC producers because they are an important resistance mechanism for wide range of GNB which is not only limited to K. pneumoniae. KPC producing isolates can be misidentified by routine tests and should be suspected by ertapenem resistance. The study was done to identify bla KPC and not other carbapenemases as no other study has been done previously to detect bla KPC gene in a hospital situated in budhera village in Haryana.

MATERIALS AND METHODS
The study was conducted in the microbiology laboratory of SGT Medical College and Hospital for a period of 8 months (March-October 2018) on the patient samples received from various clinical departments.
The institutional ethical committee approval was taken prior to commencement of this study.

Phenotypic Test for detection of Carbapenemases Modified Hodge Test (MHT)
Mueller hinton agar (MHA) plate was inoculated as a lawn culture with 0.5 Mc Farland E. coli ATCC 25922. The plate was dried for 3 to 10 minutes and ertapenem disc was applied on the centre of the plate. Using a 10-μL loop, 3 to 5 colonies of test and Quality Control organism which were grown overnight on a blood agar plate were inoculated in straight lines perpendicular to each other. After overnight incubation, the plate was looked for the presence of a "clover-leaf'' indentation in the zone of inhibition. The isolates showing clover-leaf like indentation around the zone of inhibition of E. coli ATCC 25922 were considered as positive and absence of indentation was considered as negative. Quality control strains used were: 1. K. pneumoniae ATCC BAA-1705 (MHT positive) 2. K. pneumoniae ATCC BAA-1706 (MHT negative).

bla KPC gene detection
DNA Extraction, PCR and Electrophoresis were performed according to Shanmugam et al 6 with minor modifications. The following primers were used to amplify the bla KPC gene: Forward Primer: 5'-GCT CAG GCG CAA CTG TAA G-3' and Reverse Primer: 5'-AGC ACA GCG GCA GCA AGA AAG-3'.

PCR Reaction
To make 25µl reaction mix. 12.5µl of HiChrom PCR Master Mix was added. To it 0.75µl of Forward Primer, 0.75µl of Reverse Primer, 2.5µl of Template DNA and 8.5µl of Molecular Biology Grade water was added. The PCR cycles followed were: 1.
Initial denaturation was done at 94 o C for 3 minutes.

2.
It was followed by 30 cycles of denaturation at 94 o C for 1 minute.

3.
Annealing was done at 60 o C for 1 minute.

4.
Extension at 72 o C for 1 minute which was followed by final extension for 5 minutes at 72 o C. Gel electrophoresis was performed on 2% agarose gel at 100volts for 15 minutes. The gel was observed under UV Transilluminator.
Of the 45 CRE, 32 (71.1%) were resistant to meropenem, 36 (80%) were resistant to imipenem. The resistance pattern of these isolates is given in table 1. All carbapenem (ertapenem) resistant isolates were subjected to Modified Hodge Test. MHT was positive for 32(71.1%) of 45 ertapenem resistant isolates confirming the presence of significant carbapenems hydrolyzing activity (Fig. 1). PCR for detection of bla KPC gene was carried out for MHT positive isolates. Three (9.3%) of the 32 isolates were found to harbor bla KPC gene. Band formation was seen at 90 basepair as shown in Fig. 2. All the KPC positive isolates were K. pneumoniae and they showed resistance to both meropenem and imipenem. Two of the bla KPC isolates were isolated from blood and one from pus sample from patients admitted in Surgery and ICU ward. Twenty nine (90.6%) of 32 MHT positive isolates were bla KPC negative.  However, various studies conducted in India before 2006 failed to show any evidence of resistance for E. coli and K. pneumoniae to carbapenems 10 , Indian subcontinent serves as a reservoir for all 3 types of carbapenemases: KPC, OXA-181 and NDM 8 .
Two different studies from Western Rajasthan reported 31.7% and 37% CRE respectively 11,12 . Whereas, the present study showed 14.9% CRE, this is lesser than the other two studies. However, it is in accordance to a study conducted in a Tertiary Care Hospital, North India 15 which showed 14.7% CRE.
In the present study, CRE isolates exhibited increased prevalence of multidrug resistance to various antibiotics ranging from 66.6% to 100% which is comparable to a study from Northeast India with resistance rate ranging from 85.7% to 100% 14 .
MHT was positive in 32 (71.1%) isolates, whereas, only 3(9.3%) showed the presence of bla KPC gene by conventional PCR. However, this non specificity of MHT was discussed by Tsakris et al 15 .Their report states that the false positive results of MHT could be due to presence of CTX-M ESBL positive or AmpC-hyperproducing Enterobacteriaceae. This also suggests the involvement of other resistance mechanisms, such as production of carbapenemases (OXA, NDM, MBL) alone or in combination with porins loss, ESBL (TEM, SHV, CTX-M) 7 .
KPC producing strains in the current study were 100% resistant to ertapenem, imipenem and meropenem. The first report of KPC producer was from South India (Pondicherry) in 2010 16 . It reported six (5.8%) out of 103 isolates as bla KPC positive, that were 100% resistant to carbapenem (imipenem, ertapenem and meropenem). However, this is the first study conducted in SGT medical college and hospital to detect the prevalence of bla KPC gene.
The present study reported 9.3% KPC isolates which is more than the findings of a study conducted at Safdarjung Hospital, New Delhi 17 that reported 2.1% KPC producers. However, absence of bla KPC was reported in a study conducted in North India by Mohan et al 18 that is contrary to the present study. Although, there are a few studies from India that shows the prevalence of CRE in clinically relevant isolates but not many laboratories follow the standard protocol for CRE identification.

CONCLUSION
The emergence of carbapenem resistance globally and in India is a cause of concern because of the limited treatment options for carbapenem resistant organisms. More studies should be undertaken in different regions of the country to avail the information on prevalence of KPC. Early diagnosis of KPC can improve the patient outcome. Whereas, the diagnostic technique to be introduced in the laboratory workflow is a choice which should be done carefully, as per the resources available and personnel in each hospital.