Rising threat of OXA-48 and other Carbapenemase encoding Genes among Carbapenem Resistant Enterobacteriaceae in india

Members of Enterobacteriaceae family are responsible for both community and hospital acquired infections. Because of development of antimicrobial resistance carbapenem has remained as last resort of drug for treatment of infections caused by these bacteria.Mechanism for development of this resistance in carbapenem resistant Enterobacteriaceae (CRE) may due to production of carbapenemases, efflux mechanism or loss of outer membrane porins.The most common carbapenemase enzymes are Class A – KPC, Class B – NDM, VIM and IMP and Class D oxacillinase(OXA-48 like enzymes).In India, most prevalent carbapenemase encoding gene is NDM-1but there is rising threat of OXA-48 prevalence. Unlike the phenotypic methods, the genotypic methods are useful to discriminate the type of carbapenemase enzyme, specifically for OXA-48 like enzymes. Total 170 CRE isolates were subjected for multiplex PCR study for their molecular characterization. Of the 170 CRE isolates,68.2 % (n=116) were positive for NDM-1 gene while 44.1 % (n= 75) of the isolates showed presence of OXA-48 gene. VIM (2.3%), KPC (1.7 %) were responsible for carbapenemase production while none of the isolates showed presence of IMP gene. NDM-1 and OXA-48 coexisted in 21.2 % (n=36) of the total isolates. OXA-48 causes weak hydrolysis of carbapenem because of which it is under reported with routine diagnostic methods. Early detection of OXA-48 and other carbapenemase encoding genes, helps for contact precautions and effective therapy which prevents further escalation and horizontal spread of CRE.

Initially PCR was carried out to standardize the protocol for individual genes. For this PCR mixture used was as deionized distilled water -15µl ,buffer-2 µl ,dNTP-0.5 µl, forward primer -0.5 µl, reverse primer 0.5 µl, Taq DNA polymerase -0.5 µl, Template DNA -1 making total volume 20 µl. The PCR programs used for amplification were as shown in Table 2.
Klebsiella pneumoniae ATCC BAA-2146 was used as control for NDM -1 gene and Klebsiella pneumoniae ATCC BAA-1705 was used for KPC 21 . For VIM and OXA-48, in house control strains confirmed by gene sequencing were used as control during PCR study. For detection of bla IMP conventional PCR method was used. After standardization of PCR, all DNA plasmids were subjected for multiplex PCR study 22 . (Qiagen Multiplex PCR Kit). This was used for detection of bla NDM-1, bla KPC , bla  , and bla VIM . For this reaction mixture was prepared as mastermix (Qiagen) -10 µl, primers NDM-1 , KPC ,OXA-48, VIM -forward and reverse-0.5 µl each, RNAse free water-3 µl, Q buffer -2µl , template-1 µl making 20 µl total reaction volume. PCR program used to carry multiplex PCR reaction is as shown in Table 3.
The amplification product of multiplex PCR were analyzed by electrophoresis in 2.0% agarose gel at 100 V for 1 h in 1X Tris acetate EDTA (TAE) buffer stained with 0.01 mg/ml ethidium bromide. For molecular weight marker 1 Kb DNA ladder was loaded in a gel along with the samples to confirm specific size of the corresponding gene. For staining of the gel ethidium bromide (10mg/ ml) was used and UV transilluminator was used for visualization and results were photographed in gel documentation system (Bio-Rad Laboratories).

DNA sequencing
DNA sequencing was done for OXA-48 and VIM gene as no control strains were available for the same. Two clinical isolates from the study encoded with likely OXA-48 and VIM gene with PCR amplification of 307 bp fragments and 523 bp fragments respectively were used for sequencing purpose. PCR products were first purified and were sent for DNA sequencing by direct cycle sequencing 23 . DNA sequencing was carried out using ABI PRISM 310 Analyzer from commercial source by dideoxy nucleotide chain termination method. DNA sequencing of the core region was  The analysis of derived nucleotide sequences for identification of sequence similarity was performed using the NCBI/BLAST network service of National Centre for Biotechnology Information System (NCBI) website (www.ncbi. nlm.nih.gov/BLAST). The obtained DNA sequences from isolates were aligned with representative of the sequences for identification of sequence similarity from gene bank database (NCBI blast search) with the help of nucleotide alignment program. The DNA sequence of both OXA-48 and VIM genes were sent for publication in National Centre for Biotechnology Information (NCBI) database and were assigned with GenBank accession number MK183750.2 and MK183751.2 respectively 24,25 .

Statistical Analysis
Data were filled in the MS Excel Software. Analyzed results were expressed as percentage and p values by Chi square test using GraphPad Instat Software. If the probability is less than 0.05, the association or difference is said to be significant.

OBSERVATION AND RESULTS
Of the total 170 CRE isolates,158 (93 %) of Total 170

D i s C U s s i O N
Members of Enterobacteriaceae got main focus of attention especially around year 2000 because of sudden rise of Extended Spectrum β-Lactamase (ESBL) producing isolates. This resistance scenario got worsened with rapid spread of CTX-M enzyme producing Enterobacteriaceae isolates. But in last decade or so the focus has shifted to the rise of carbapenem resistance caused by carbapenemase producing Enterobacteriaceae isolates which inactivates almost all types of β-lactam antimicrobials 26 .
Molecular methods are gold standard method for detection of carbapenemase production 3 . Also it discriminates different types of carbapenemase production unlike phenotypic methods which fails to discriminate the types of carbapenemase 3,27 . Of the 170 CRE isolates 158 isolates were encoded by at least one carbapenemase producing gene. According to Centre for Disease Control (CDC) definition,remaining 12 isolates were CRE 14 but negative for bla NDM-1 , bla OXA -48 , bla KPC , bla VIM , bla IMP

14
. These 12 isolates might be having other mechanism for carbapenem resistance like target site mutation, OMP alteration and efflux pumps 28 . Chromosomal mediated genes like bla IMI , bla SME , bla NMC are also having carbapenem resistant activity 29 . In the present project, plasmid mediated genes were focused and because of low prevalence of other mechanisms, they were not studied. Multiplex PCR study is one of the reference method for simultaneous detection of genes encoded for carbapenemases of different classes 21 . NDM-1 has been predominant carbapenemase in Indian CRE isolates 8  In life threatening or invasive infections carbapenems are most used antimicrobials because of their bactericidal effect which is concentration independent 36,37 . Also because of their broad spectrum activity which includes action against Gram-positive, Gram-negative bacteria including anaerobes, their use is widespread 28 . The widespread use, selection pressure of antimicrobial surviving resistant strains, horizontal plasmid spread among Enterobacteriaceae isolates might be contributing factors for raised prevalence of NDM-1 and OXA-48 in the present study. Also being tertiary care centre many patients referred have already received antibiotics, β -lactams being preferred one.
There respectively. Higher percentage of multiple gene co-occurrence in NDM-1 strains specifically in combination with OXA-48 in the present study is a cause of concern.

CONClUsiON
Though NDM-1 is leading carbapenemase encoded gene in carbapenem resistant Enterobacteriaceae isolates, the spread of OXA-48 is also quite noticeable. As there is no well defined phenotypic method available for detection of OXA-48-like producer emphasis should be on its molecular detection. This will help in contact precautions and to prevent further escalation of carbepenem resistance. Further considering a raised prevalence of coexistence of OXA-48 and NDM-1 gene,a study on role of NDM-1in transfer of plasmid mediated transfer of OXA-48 is warranted.