The NDM-1 producing E. coli isolates (n = 17) were collected from different clinical specimens (stool, n = 3; surgical wound, n = 1; urine, n = 3; pus, n = 5; throat swab, n = 1; ear swab, n = 1; endotracheal aspirates, n = 1; cerebrospinal fluid, n = 1; blood, n = 1) of seventeen patients who were admitted in different wards or attended to outpatient departments (OPDs) of Silchar Medical College and Hospital (Silchar, India) from March till September 2013. The isolates were identified by standard biochemical characterization and 16 s rDNA sequencing . Presence of bla
NDM was determined by PCR assay using primers (NDM-F 5/-GGGCAGTCGCTTCCAACGGT-3/and NDM-R 5/-GTAGTGCTCAGTGTCGGCAT-3/ . The amplified products were purified using MinElute PCR Purification Kit (Qiagen, Germany) and were sequenced.
Transcriptional expression analysis of bla
Transcriptional expression of bla
NDM-1 in response to imipenem, meropenem and ertapenem stress was determined by inoculating the organisms harboring bla
NDM-1 in Luria Bertani broth (Hi-media, Mumbai, India) with and without antibiotics. Antibiotic concentration used was 1 μg/ml. For a period of 16 h, total RNA was isolated at the interval of 1 h using Qiagen RNease Mini Kit (Qiagen, Germany), immediately reverse transcribed into cDNA by using QuantiTect® reverse transcription kit (Qiagen, Germany). The cDNA was quantified by Picodrop (Pico 200, Cambridge, UK) and real time PCR was performed using Power Sybr Green Master Mix (Applied Biosystem, Warrington, UK) in Step One Plus real time detection system (Applied Biosystem, USA) using a set of primer (NDM-F 5/-GGGCAGTCGCTTCCAACGGT-3/and NDM-RT-R 5/-CGACCGGCAGGTTGATCTCC-3/). The relative expression of bla
NDM-1 in each interval with and without carbapenem pressure was determined by ΔΔCt method . Relative quantification was done using a transformant (E. coli DH5α harboring bla
NDM; PEC-611) grown for 4 h without any antibiotic pressure.
Transformation and Conjugation assay
Transformation was performed by heat shock method  using E. coli DH5α as a recipient and the transformants were selected on Luria Bertani agar (Hi-Media, Mumbai, India) containing 0.25 μg/ml of imipenem. Conjugation experiment was carried out using bla
NDM-1 harboring clinical strains as donors and a streptomycin resistant E. coli recipient strain B (Genei, Bangalore, India). The MIC of clinical isolates against streptomycin was pre-determined to optimize the agar for selection of transconjugants. Both the donor and recipient cells were cultured in Luria Bertani Broth (Hi-Media, Mumbai, India) till it reach an O.D. of 0.8–0.9 at A600. Cells were mixed at 1:5 donor-to-recipient ratios and transconjugants were selected on agar plates containing imipenem (0.25 μg/ml) and streptomycin (1000 μg/ml). The E. coli strain B is chromosomally resistant to streptomycin which can grow on media containing streptomycin at a concentration of 1000 μg/ml. However, the donors although resistant to aminoglycoside had the minimum inhibitory concentration ranging from 100-200 μg/ml. Therefore, selection of transformants in 1000 μg/ml rules out false selection of donor strains. The accuracy of conjugation was further cross checked by typing all the transconjugants by enterobacterial repetitive intergenic consensus PCR  and pulsed field gel electrophoresis using Xba1 restriction enzyme.
Replicon typing and plasmid stability analyses
Incompatibility type of the plasmid encoding bla
NDM-1 was determined by PCR based replicon typing targeting 18 different replicons viz. FIA, FIB, FIC, HI1, HI2, I1/Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F and FIIA as described previously . Also IncX types i.e. IncX1, IncX2, IncX3 and IncX4 were also targeted . Purified plasmid DNA was used as template for the reaction.
Plasmid stability analysis of parent strains and transformants was done by serial passage method for consecutive 100 days at 1:1000 dilutions without any antibiotic pressure . After each passage, 1 ml of the culture was diluted in normal saline (1:1000) and 40 μl of the diluted sample was spread on to the LB agar plate. After overnight incubation, 50 colonies from the agar plates were randomly picked and subjected to phenotypic detection of MBL and further confirmed by PCR assay for the presence of bla
NDM-1 using primers (NDM-F 5/-GGGCAGTCGCTTCCAACGGT-3/and NDM-R 5/-GTAGTGCTCAGTGTCGGCAT-3/.
Copy number determination of plasmid encoding bla
Clinical isolates of Escherichia coli harboring bla
NDM-1 carried by plasmids of incompatibility groups IncFIC, IncA/C or IncK were selected for determining the copy number under exposure of different concentrations of carbapenem antibiotics. Single colony of each incompatibility type was inoculated into LB broth containing 0.5 μg/ml, 1 μg/ml, 2 μg/ml and 4 μg/ml of each imipenem, meropenem and ertapenem and also without any antibiotic (considered as a reference), was incubated at 37 °C for 5–6 h until the OD reached 0.9 at A600. Transformants with different bla
NDM-1 carrying plasmid types (IncFIC, A/C & K) were used as control (without any antibiotic pressure). Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen, Germany). Quantitative Real Time PCR was performed using Step One Plus real time detection system (Applied Biosystem, USA) to estimate the relative copy number of bla
NDM-1 for different concentrations of each antibiotic for three different incompatibility types. The copy number of bla
NDM-1 within the wild type plasmid of different incompatibility types were also determined to know the type of Inc group where copy number of bla
NDM-1 gene was maintained in high number. Quantitative real time PCR reaction was carried out using 10 μl of SYBR® Green PCR Master Mix (Applied Biosystem, Warrington, UK), 4 ng plasmid DNA as template and 3 μl of each primer (10 Picomol) in a 20 μl reaction under a reaction condition of initial denaturation at 94 °C for 5 min, 40 cycles of denaturation 94 °C for 20 s, annealing 52 °C for 40 s and extension at 72 °C for 30 s. The relative fold change was measured by ΔΔCT method and Ct value of each sample was normalized against a housekeeping gene rpsel of E. coli .
Antimicrobial susceptibility testing and MIC determination
Antibiotic susceptibility of bla
NDM-1 harboring parent strains, transformants and transconjugants were determined by Kirby Bauer disc-diffusion method including piperacillin-tazobactam (100/10 μg), co-trimoxazole (25 μg), amikacin (30 μg), gentamicin (10 μg), ciprofloxacin (5 μg), polymyxin B (300units), netilmicin (30 μg), carbenicillin (100 μg), tigecycline(30 μg) and faropenem (5 μg) (Hi-Media, Mumbai, India). MICs of imipenem, meropenem, ertapenem, cefepime, aztreonam, gentamicin, amikacin, ciprofloxacin, piperacillin-tazobactam & polymixin-B were determined for parent strains harboring bla
NDM-1, as well as transformants and transconjugants by agar dilution method. Each stock solution for the corresponding antibiotic was made at 1 mg/ml concentration in nuclease free water and was stored at −80 °C. The quality control for stock solution was checked each time against E. coli ATCC 25922. The result of the susceptibility testing was interpreted as per CLSI guidelines . However, for polymyxin B, faropenem and carbenicillin, the organisms were considered as non susceptible if the MIC value was higher and diameter of the zone of inhibition was lower than the values given in CLSI guidelines for respective antibiotics against E. coli ATCC 25922.
Typing of bla
NDM-1 harboring isolates
NDM-1 harboring E. coli isolates were typed by pulsed field gel electrophoresis (PFGE), genomic DNA was prepared in agarose blocks and digested with the restriction enzyme Xba1 (Promega, Madison, USA) and the DNA fragments were separated with a CHEF-DR III (Bio-Rad, USA) for 24 h at 6 V/cm with a pulses at 1200 angle in a 10–40 s pulse time .