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Transcriptional analysis of bla NDM-1 and copy number alteration under carbapenem stress
Antimicrobial Resistance & Infection Control volume 6, Article number: 26 (2017)
New Delhi metallo beta-lactamase is known to compromise carbapenem therapy and leading to treatment failure. However, their response to carbapenem stress is not clearly known. Here, we have investigated the transcriptional response of bla NDM-1 and plasmid copy number alteration under carbapenem exposure.
Three bla NDM-1 harboring plasmids representing three incompatibility types (IncFIC, IncA/C and IncK) were inoculated in LB broth with and without imipenem, meropenem and ertapenem. After each 1 h total RNA was isolated, immediately reverse transcribed into cDNA and quantitative real time PCR was used for transcriptional expression of bla NDM-1. Horizontal transferability and stability of the plasmids encoding bla NDM-1 were also determined. Changes in copy number of bla NDM-1 harboring plasmids under the exposure of different carbapenems were determined by real time PCR. Clonal relatedness among the isolates was determined by pulsed field gel electrophoresis.
Under carbapenem stress over an interval of time there was a sharp variation in the transcriptional expression of bla NDM-1 although it did not follow a specific pattern. All bla NDM-1 carrying plasmids were transferable by conjugation. These plasmids were highly stable and complete loss was observed between 92nd to 96th serial passages when antibiotic pressure was withdrawn. High copy number of bla NDM-1 was found for IncF type plasmids compared to the other replicon types.
This study suggests that the single dose of carbapenem pressure does not significantly influence the expression of bla NDM-1 and also focus on the stability of this gene as well as the change in copy number with respect to the incompatible type of plasmid harboring resistance determinant.
Since the discovery of New Delhi metallo-β-lactamase (bla NDM) in 2008 from a Swedish patient of Indian origin in New-Delhi, India , this enzyme is known for several reasons including treatment failure, emergence of new variants and lateral transfer of the gene coding this enzyme within diverse host range of Gram negative bacilli [2, 3]. The bla NDM is known for its ignominious nature being linked with other resistance determinants along with various mobile elements like plasmid, insertion sequences & transposons which facilitates its horizontal dissemination [2, 4]. In many studies bla NDM-1 was found to be associated with ISAba125 [2, 5]. However, there were also reports of other insertion elements like ISCR1, ISCR16, IS26, IS1, ISEc33 and IS903 associated with this gene . Additionally, the transposons Tn3 and Tn125 were reported to be linked with this resistance determinant and horizontal transfer of bla NDM-1 is often facilitated by plasmids of IncF, IncA/C, IncL/M, IncH, IncN and more recently by IncX type . Among Enterobacteriaceae, bla NDM was detected in Escherichia coli in many countries worldwide (Australia, France, Germany, Japan, UK and the USA) . E. coli is the most common pathogen associated with nosocomial and community acquired infections and also been considered as a potent host for this resistance determinant . Dissemination of bla NDM-1 through E. coli has become a global concern  and also in India there were several reports of NDM-producing E. coli in all parts of the country [8–14]. Treatment of infections with NDM-producers is restricted due to their multidrug resistance phenotype . Several studies have highlighted the hydrolytic activity of NDM-1 to carbapenems [2, 16]. However, it is not known how bacteria harboring this resistance gene will respond when carbapenem therapy is initiated to a patient. The present study was designed to investigate the transcriptional response of bla NDM-1 in vitro under single dose carbapenem exposure, and also to investigate the transmission dynamics within clinical isolates of Escherichia coli in a single center study from India.
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 NDM-1
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 NDM-1
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
All bla 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 .
During the study period (March-September), 17 isolates were obtained carrying bla NDM-1, collected from different clinical samples mostly associated with surgical wound infection from surgery ward of the hospital (Table 1). Transcriptional expression of bla NDM-1 with or without carbapenem stress is shown in Fig. 1. It was observed that at the initial stage, under meropenem pressure the transcriptional level of bla NDM-1 was low. However, there was a sharp increase from 12th hour of incubation for meropenem and ertapenem (approximately 2 fold and 4 fold respectively), whereas imipenem did not cause any alteration in transcriptional response. Overall the transcriptional expression did not show any specific pattern of response.
Plasmids carrying bla NDM-1 were selected in the medium containing imipenem and could be conjugatively transferred from all 17 clinical E. coli isolates into recipient E. coli strain B. The transformation experiment revealed that the size of the transferable plasmids was approximately of 50-60 kb. Replicon typing showed that FIC was the predominant replicon type (n = 7) followed by A/C (n = 4) and K (n = 3) whereas 3 isolates were untypeable (Table 1). The copy number of bla NDM-1 was found to be variable. The copy number of bla NDM-1 gene within IncFIC and IncA/C type of plasmids showed an increasing trend when increasing concentrations of imipenem and meropenem were added whereas for ertapenem, the case was reverse (Figs. 2 & 3). For IncK type plasmids, the copy number of bla NDM-1 consistently raised when meropenem concentration was increased whereas with the increasing concentration of imipenem and ertapenem, the copy number of bla NDM-1 reduced (Fig. 4). The overall copy number of F-Inc type was six fold higher compared to IncA/C and K type (Fig. 5). Complete loss of plasmids for all the isolates containing bla NDM-1 was observed between 92nd to 96th serial passages when antibiotic pressure was withdrawn.
Antimicrobial susceptibility result showed that the 17 bla NDM-1 harboring isolates were resistant to co-trimoxazole, ciprofloxacin, carbenicillin and faropenem whereas very few isolates were found to be susceptible to polymyxin B (n = 4) and tigecycline (n = 3) (Additional file 1: Table S1). MIC results revealed that the parent strains carrying bla NDM-1 showed MIC range above the breakpoint for all three carbapenems (≥64 μg/ml), third generation cephalosporin (≥256 μg/ml), piperacillin/tazobactam (≥32 μg/ml), polymyxin-B (≥1 μg/ml) aminoglycosides, quinolone and monobactam (≥64 μg/ml) (Additional file 1: Table S1). Transformants and transconjugants carrying bla NDM-1 were also resistant to cephalosporin, piperacillin/tazobactam, aminoglycosides, quinolone and all carbapenems (Additional file 1: Table S2). PFGE analysis revealed the presence of six different E. coli clones with clone 2 (pulsotype 2) as the most frequent one (n = 6) (Additional file 2: Figure S1). However, the replicon types of the bla NDM-1 carrying plasmids were different in this clone (IncFIC, n = 3; IncA/C, n = 1; IncK, n = 1; untypeable, n = 1).
Resistance to carbapenems due to the production of New Delhi metallo-β-lactamase among enterobacterial isolates has become a very common phenomenon and the expansion of bla NDM-1 among the members of Enterobacteriaceae is increasing and in consequence this resistance determinant has been reported across the globe . Earlier studies demonstrated that the sub-inhibitory concentrations of antibiotics interfere the expression of the genes, colonization and motility of the cell . Therefore, we have investigated the transcriptional response of NDM-1 against carbapenem antibiotics below the inhibitory concentration level. Under the pressure of imipenem, no significant change was observed in the pattern of transcriptional level for 16 h duration, which is in contrast to the previous report of Liu et al. 2012 , as they reported that under the pressure of imipenem bla NDM-1 gene was expressed (0.83 times higher) than that of the control. In this study, a possible down regulated expression of bla NDM-1 took place under the exposure of meropenem, however to support our data no existing literature is available till date. This study has pointed that no specific or defined transcriptional response is initiated for bla NDM-1 when carbapenem stress is created and the overall response is partially chaotic. Thus, there could be other inducing factors which trigger its response in order to synthesis this carbapenemase. The study isolates showed resistance to almost all the antibiotics especially high rate of polymyxin resistance was also observed. The emergence of different E. coli clones with pulsotype 2 as the most common, indicates a possible clonal spread but different replicon types within this clone are uncommon and require further detailed analyses in future studies.
Plasmids encoding bla NDM-1 gene were successfully transferred to the recipient E. coli strain B by conjugation indicating potential horizontal transmission through diverse incompatible plasmid types such as IncFIC, IncA/C and IncK in this hospital setting. Association of bla NDM-1 with IncK type of plasmid in the present study is not commonly reported as coexisting data recorded recent spread of bla NDM-1 in India has been associated with IncA/C type, IncF1/FII-type, or unknown types of plasmids . An earlier study  suggested that copy number of bla NDM-1 is affected by the concentration of imipenem. In contrast we observed that plasmid copy number is not only related with high concentration of imipenem but also depends on the replicon type of the bla NDM-1 carrying plasmids. This could be supported by the high copy number of bla NDM-1 within IncF type plasmids compared to the other replicon types (e.g. IncA/C or Inc K).
The expression of bla NDM-1 could predict the bacterial response in different time interval when a single carbapenem exposure is applied. Additionally, this study could underscore that irrespective of plasmid types, bla NDM-1 is highly stable within a host of clinical origin. However, it was also evident from this study that different Inc types of plasmids have a specific pattern in copy number alteration under concentration gradient carbapenem stress. Thus, the study came up with epidemiological knowledge of a stable bla NDM-1 mediated carbapenem resistance in E. coli and further investigation is required to evaluate the risk for their dissemination in health care systems in this geographical part of the world.
- bla NDM-1 :
New-Delhi metallo β-lactamase
Complementary deoxyribonucleic acid
Clinical laboratory standard institute.
- Ct :
- Inc type:
Minimum inhibitory concentration
Polymerase chain reaction
Pulsed field gel electrophoresis
Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K. Characterization of a New Metallo-β-Lactamase Gene, bla NDM-1, and a Novel Erythromycin Esterase Gene Carried on a Unique Genetic Structure in Klebsiella pneumoniae Sequence Type 14 from India. Antimicrob Agents Chemother. 2009;53:5046–54.
Mishra S, Sen MR, Upadhyay S, Bhattacharjee A. Genetic linkage of bla NDM among nosocomial isolates of Acinetobacter baumanii from a tertiary referral hospital in northern India. Int J Antimicrob Agents. 2013;41:452–6.
Pagano M, Poirel L, Martins AF, Rozales FP, Zavascki AP, Barth AL, et al. Emergence of NDM-1-producing Acinetobacter pittii in Brazil. doi:10.1016/j.ijantimicag.2014.12.011.
Bennett PM. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol. 2008;153:S347–57.
Toleman MA, Spencer J, Jones L, Walsh TR. bla NDM-1 is a chimera likely constructed in Acinetobacter baumannii. Antimicrob Agents Chemother. 2012;56:2773–6.
Wailan AM, Paterson DL, Kennedy K, Ingram PR, Bursle E, Sidjabat HE. Genomic characteristics of NDM-producing Enterobacteriaceae isolates in Australia and their bla NDM genetic contexts. Antimicrob Agents Chemother. 2016;60:136–41.
Johnson AP, Woodford N. Global spread of antibiotic resistance: the example of New Delhi metallo-β-lactamase (NDM) mediated carbapenem resistance. J Med Microbiol. 2013;62:499–13.
Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis. 2010. doi:10.1016/S1473-3099(10)70143-2.
Kumari S, Sen MR, Upadhyay S, Bhattacharjee A. Dissemination of the New Delhi metallo-β-lactamase-1 (NDM-1) among enterobacteriaceae in a tertiary referral hospital in north India. J Antimicrob Chemother. 2011. doi:10.1093/jac/dkr180.
Kumar M, Dutta R, Saxena S, Singhal S. Risk factor analysis in clinical isolates of ESBL and MBL (including NDM-1) producing Escherichia coli and Klebsiella species in a tertiary care hospital. J Clin Diagn Res. 2015;9:8–13.
Paul D, Bhattacharjee A, Ingti B, Choudhury NA, Maurya AP, Dhar D, et al. Occurrence of bla NDM-7 within IncX3-type plasmid of Escherichia coli from India. J Infect Chemother. 2016. http://0-dx.doi.org.brum.beds.ac.uk/10.1016/j.jiac.2016.12.009.
Ranjan A, Shaik S, Mondal A, Nandanwar N, Hussain A, Semmler T, et al. Molecular epidemiology and genome dynamics of New Delhi metallo-β-lactamase producing extraintestinal pathogenic Escherichia coli strains from India. Antimicrob Agents Chemother. 2016;11:6795–805.
Datta S, Roy S, Chatterjee S, Saha A, Sen B, Pal T et al. A five-year experience of carbapenem resistance in enterobacteriaceae causing neonatal septicaemia: predominance of NDM-1. PLoS ONE 10(9):e0134079.
Hussein A, Ranjan A, Nandanwar N, Babbar A, Jadhav S, Ahmed N. Genotypic and phenotypic profiles of Escherichia coli isolates belonging to clinical sequence type 131 (ST131), clinical non-ST131, and fecal non-ST131 lineages from India. Antimicrob Agents Chemother. 2014;58:7240–9.
Paul D, Maurya AP, Chanda DD, Sharma GD, Chakravarty A, Bhattacharjee A. Carriage of bla NDM-1 in Pseudomonas aeruginosa through multiple Inc type plasmids in a tertiary referral hospital of northeast India. Indian J Med Res. 2016;143:826–9.
Li T, Wang Q, Chen F, Li X, Luo S, Fang H, et al. Biochemical characteristics of New Delhi Metallo-β-Lactamase-1 show unexpected difference to other MBLs. PLoS ONE. 2013;8(4):e61914. doi:10.1371/journal.pone.0061914.
Woo PCY, Leung PKL, Leung KW, Yuen KY. Identification by 16 s ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient. J Clin Pathol-Mol Pathol. 2000;53:211–5.
Paul D, Dhar Chanda D, Maurya AP, Mishra S, Chakravarty A, Sharma GD, Bhattacharjee A. Co-Carriage of bla KPC-2 and bla NDM-1 in Clinical Isolates of Pseudomonas aeruginosa Associated with Hospital Infections from India. PLoS ONE. 2015;10(12):e0145823. doi:10.1371/journal.pone.0145823.
Swick MC, Morgan-Linnell SK, Carlson KM, Zechiedrich L. Expression of multidrug efflux pump genes acrAB-tolC, mdfA and norE in Escherichia coli clinical isolates as a function of fluroquinolone and multidrug resistance. Antimicrob Agents Chemother. 2011;55:921–4.
Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1991;19:6823–31.
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219–28.
Johnson TJ, Bielak EM, Fortini D, Hansen LH, Hasman H, Debroy C, Nolan LK, Carattoli A. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug resistant enterobacteriaceae. Plasmid. 2012;68:43–50.
Locke JB, Rahawi S, LaMarre J, Mankin LS, Shawa KJ. Genetic Environment and Stability of cfr in Methicillin-resistant Staphylococcus aureus CM05. Antimicrob Agents Chemother. 2012;56:332–40.
Institute CLS. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement; M100-S21.CLSI. CLSI: Wayne, USA; 2011.
Tato M, Coque TM, Ruiz-Garbajosa P, Pintado V, Cobo J, Sader HS, et al. Complex clonal and plasmid epidemiology in the first outbreak of enterobacteriaceae infection involving VIM-1 metallo-β-lactamase in Spain: toward endemicity? Clin Infect Dis. 2007;45:1171–8.
Cornaglia G, Giamarellou H, Rossolini GM. Metallo-β-lactamases: a last frontier for β-lactams. Lancet Infect Dis. 2011;11:381–93.
Romero D, Traxler MF, Lopez D, Kolter R. Antibiotics as signal molecules. Chem Rev. 2011;111:5492–505.
Liu W, Zou D, Wang X, Li XL, Zhu L, Yin Z, et al. Proteomic analysis of clinical isolate of Stenotrophomonas maltophilia with bla NDM-1, bla L1 and bla L2 β-lactamase genes under imipenem treatment. J Proteome Res. 2012;11:4024–33.
Huang TW, Chen TL, Chen YT, Lauderdale TL, Liao TL, Lee YT, et al. Copy number change of the NDM-1 sequence in a multidrug resistant Klebsiella pneumoniae clinical isolate. Plos One. 2013;8:1–12.
The authors sincerely acknowledge the financial support provided by Council of Scientific and Industrial Research (CSIR) to carry out the work.
Council of Scientific and Industrial Research (CSIR Grant number 37(1632)/14/EMR-II) and Deepjyoti Paul is a Senior Research Fellow in the Department of Microbiology, Assam University, Silchar and receives CSIR Senior Research Fellowship under the grant number 37(1632)/14/EMR-II.
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All the relevant data and information are presented in the manuscript.
DP Performed the experimental work, data collection & analysis and prepared the manuscript. AB Supervised the research work and participated in designing the study and drafting the manuscript. DB Analysis of the data. APM Participated in sample collection and part of experiments. DD & AC Participated in experiment designing and manuscript correction. All authors read and approved the final manuscript.
The authors have declared that no competing interests exist.
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All the authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
The work was approved by Institutional Ethical committee of Assam University, Silchar vide Reference Number: IEC/AUS/C/2014-001. The authors confirm that participants provided their written informed consent to participate in this study.
Table S1. Antimicrobial profile of bla NDM-1 harboring Escherichia coli isolates SXT: Trimethoprim/sulfamethoxazole, TGC: Tigecycline, FAR: Faropenem, CIP: Ciprofloxacin, CAR: Carbenicillin, PMB: Polymixin B, AMK: Amikacin, GEN: Gentamicin, NET: Netilmicin, TZP: Piperacillin/tazobactum, IPM: Imipenem, ETP: Ertapenem, MEM: Meropenem, FEP: Cefepime, ATM: Aztreonam. Table S2. Susceptibility pattern of transformants and transconjugants carrying bla NDM-1 IPM: Imipenem, ETP: Ertapenem, MEM: Meropenem, FEP: Cefepime, ATM: Aztreonam, GEN: Gentamicin, AMK: Amikacin, CIP: Ciprofloxacin, TZP: Piperacillin/tazobactum, PMB: Polymixin B T.F (transformants) = recipient E. coli DH5α carrying plasmid encoding bla NDM-1 T.C (transconjugants) = recipient E. coli strain B strain carrying plasmid encoding bla NDM-1. (DOC 139 kb)
PFGE analysis showed six pulsotypes of Escherichia coli harboring bla NDM-1 Lane 1: High range ladder; Lane 2: EC51; Lane 3: EC54; Lane 4: EC61; Lane 5: EC75; Lane 6: EC177; Lane 7: EC178; Lane 8: EC255; Lane 9: EC355; Lane 10: EC456; Lane 11: EC472; Lane 12: EC477; Lane 13: EC489; Lane 14: EC492; Lane 15: EC571; Lane 16: EC611; Lane 17: EC639; Lane 18: EC678. (TIF 4160 kb)
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Paul, D., Bhattacharjee, A., Bhattacharjee, D. et al. Transcriptional analysis of bla NDM-1 and copy number alteration under carbapenem stress. Antimicrob Resist Infect Control 6, 26 (2017). https://0-doi-org.brum.beds.ac.uk/10.1186/s13756-017-0183-2
- Escherichia coli
- Plasmid stability
- Transcriptional expression