Transcriptional analysis of bla NDM-1 and copy number alteration under carbapenem stress
Paul et al. Antimicrobial Resistance and Infection Control
Transcriptional analysis of bla and NDM-1 copy number alteration under carbapenem stress
Deepjyoti Paul 0
Amitabha Bhattacharjee 0
Dibyojyoti Bhattacharjee 2
Debadatta Dhar 1
Anand Prakash Maurya 0
Atanu Chakravarty 1
0 Department of Microbiology, Assam University , Silchar , India
1 Department of Microbiology, Silchar Medical College and Hospital , Silchar , India
2 Department of Statistics, Assam University , Silchar , India
Background: 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 blaNDM-1 and plasmid copy number alteration under carbapenem exposure. Methods: Three blaNDM-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 blaNDM-1. Horizontal transferability and stability of the plasmids encoding blaNDM-1 were also determined. Changes in copy number of blaNDM-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. Results: Under carbapenem stress over an interval of time there was a sharp variation in the transcriptional expression of blaNDM-1 although it did not follow a specific pattern. All blaNDM-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were highly stable and complete loss was observed between 92nd to 96th serial passages when antibiotic pressure was withdrawn.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 blaNDM-1 was found for IncF type plasmids compared to the other replicon types. Conclusion: This study suggests that the single dose of carbapenem pressure does not significantly influence the expression of blaNDM-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.
Escherichia coli; Plasmid stability; Transcriptional expression; Transferability
Since the discovery of New Delhi metallo-β-lactamase
(blaNDM) 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 blaNDM 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 blaNDM-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 blaNDM-1 is often
facilitated by plasmids of IncF, IncA/C, IncL/M, IncH, IncN
and more recently by IncX type . Among
Enterobacteriaceae, blaNDM 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
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resistance determinant . Dissemination of blaNDM-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
blaNDM-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 blaNDM was determined by PCR assay
using primers (NDM-F 5/-GGGCAGTCGCTTCCAAC
GGT-3/and NDM-R 5/-GTAGTGCTCAGTGTCGGC
AT-3/ . The amplified products were purified using
MinElute PCR Purification Kit (Qiagen, Germany) and
Transcriptional expression analysis of blaNDM-1
Transcriptional expression of blaNDM-1 in response to
imipenem, meropenem and ertapenem stress was
determined by inoculating the organisms harboring blaNDM-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/-GGGCAGTCGCTTC
CAACGGT-3/and NDM-RT-R 5/-CGACCGGCAG
GTTGATCTCC-3/). The relative expression of blaNDM-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 blaNDM; 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 blaNDM-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 blaNDM-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 blaNDM-1 using
primers (NDM-F 5/-GGGCAGTCGCTTCCAACGGT-3/
and NDM-R 5/-GTAGTGCTCAGTGTCGGCAT-3/.
Copy number determination of plasmid encoding blaNDM-1
Clinical isolates of Escherichia coli harboring blaNDM-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 blaNDM-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 blaNDM-1
for different concentrations of each antibiotic for three
different incompatibility types. The copy number of
blaNDM-1 within the wild type plasmid of different
incompatibility types were also determined to know the
type of Inc group where copy number of blaNDM-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
Antibiotic susceptibility of blaNDM-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,
piperacillintazobactam & polymixin-B were determined for parent
strains harboring blaNDM-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 blaNDM-1 harboring isolates
All blaNDM-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 blaNDM-1, collected from
different clinical samples mostly associated with surgical
wound infection from surgery ward of the hospital
(Table 1). Transcriptional expression of blaNDM-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 blaNDM-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 blaNDM-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 blaNDM-1 was
found to be variable. The copy number of blaNDM-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 blaNDM-1 consistently
raised when meropenem concentration was increased
whereas with the increasing concentration of imipenem
and ertapenem, the copy number of blaNDM-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
blaNDM-1 was observed between 92nd to 96th serial passages
when antibiotic pressure was withdrawn.
Antimicrobial susceptibility result showed that the
17 blaNDM-1 harboring isolates were resistant to
cotrimoxazole, 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 blaNDM-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
blaNDM-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 blaNDM-1
carrying plasmids were different in this clone (IncFIC, n = 3;
IncA/C, n = 1; IncK, n = 1; untypeable, n = 1).
Fig. 1 Transcriptional response of blaNDM-1 against carbapenem exposure at different time interval
Fig. 2 Copy number of blaNDM-1 within IncFIC plasmid. 0 μg/ml (control) = copy number of blaNDM-1 without any antibiotic pressure. 0.5, 1, 2 and
4 μg/ml = change in copy number of blaNDM-1 under 0.5, 1, 2 and 4 μg/ml exposure of imipenem (blue bar), meropenem (red bar) and ertapenem
(green bar) pressure. The error bars represent the standard deviation of the three replicates of one sample
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 blaNDM-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
subinhibitory 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 blaNDM-1 gene was expressed
(0.83 times higher) than that of the control. In this
study, a possible down regulated expression of blaNDM-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 blaNDM-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
Fig. 3 Copy Number of blaNDM-1 within IncA/C plasmid. 0 μg/ml (Control) = Copy number of blaNDM-1 without any antibiotic pressure. 0.5, 1, 2
and 4 μg/ml = Change in copy number of blaNDM-1 under 0.5, 1, 2 and 4 μg/ml exposure of imipenem (blue bar), meropenem (red bar) and
ertapenem (green bar) pressure. The error bars represent the standard deviation of the three replicates of one sample
Fig. 4 Copy Number of blaNDM-1 within IncK plasmid. 0 μg/ml (Control) = Copy number of blaNDM-1 without any antibiotic pressure. 0.5, 1,
2 and 4 μg/ml = Change in copy number of blaNDM-1 under 0.5, 1, 2 and 4 μg/ml exposure of imipenem (blue bar), meropenem (red bar)
and ertapenem (green bar) pressure. The error bars represent the standard deviation of the three replicates of one sample
replicon types within this clone are uncommon and
require further detailed analyses in future studies.
Plasmids encoding blaNDM-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 blaNDM-1
with IncK type of plasmid in the present study is not
commonly reported as coexisting data recorded recent spread
of blaNDM-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
blaNDM-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
Fig. 5 Relative copy number of IncF, A/C and K plasmid. The error
bars represent the standard deviation of the three replicates of
depends on the replicon type of the blaNDM-1 carrying
plasmids. This could be supported by the high copy
number of blaNDM-1 within IncF type plasmids compared
to the other replicon types (e.g. IncA/C or Inc K).
The expression of blaNDM-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, blaNDM-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 blaNDM-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.
Additional file 1: Table S1. Antimicrobial profile of blaNDM-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 blaNDM-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 blaNDM-1 T.C (transconjugants) =
recipient E. coli strain B strain carrying plasmid encoding blaNDM-1. (DOC 139 kb)
Additional file 2: Figure S1. PFGE analysis showed six pulsotypes of
Escherichia coli harboring blaNDM-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)
blaNDM-1: New-Delhi metallo β-lactamase; cDNA: Complementary
deoxyribonucleic acid; CLSI: Clinical laboratory standard institute.;
Ct: Threshold cycle; DNA: Deoxyribonucleic acid; Inc type: Incompatibility
type; LB: Luria bertani; MIC: Minimum inhibitory concentration; O.D: Optical
density; OPD: Outpatient department; PCR: Polymerase chain reaction;
PFGE: Pulsed field gel electrophoresis; RNA: Ribonucleic acid
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.
Availability of data and materials
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.
Consent for publication
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.
1. Yong D , Toleman MA , Giske CG , Cho HS , Sundman K , Lee K. Characterization of a New Metallo-β-Lactamase Gene, blaNDM-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 .
2. Mishra S , Sen MR , Upadhyay S , Bhattacharjee A. Genetic linkage of blaNDM among nosocomial isolates of Acinetobacter baumanii from a tertiary referral hospital in northern India . Int J Antimicrob Agents . 2013 ; 41 : 452 - 6 .
3. 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.
4. Bennett PM . Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria . Br J Pharmacol . 2008 ; 153 : S347 - 57 .
5. Toleman MA , Spencer J , Jones L , Walsh TR . blaNDM-1 is a chimera likely constructed in Acinetobacter baumannii . Antimicrob Agents Chemother . 2012 ; 56 : 2773 - 6 .
6. Wailan AM , Paterson DL , Kennedy K , Ingram PR , Bursle E , Sidjabat HE. Genomic characteristics of NDM-producing Enterobacteriaceae isolates in Australia and their blaNDM genetic contexts . Antimicrob Agents Chemother . 2016 ; 60 : 136 - 41 .
7. 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 .
8. 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 .
9. 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.
10. 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 .
11. Paul D , Bhattacharjee A , Ingti B , Choudhury NA , Maurya AP , Dhar D , et al. Occurrence of blaNDM-7 within IncX3-type plasmid of Escherichia coli from India . J Infect Chemother . 2016 . http://dx.doi.org/10.1016/j.jiac. 2016 .12.009.
12. 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 .
13. 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 .
14. 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 .
15. Paul D , Maurya AP , Chanda DD , Sharma GD , Chakravarty A , Bhattacharjee A. Carriage of blaNDM-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 .
16. 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.
17. 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 .
18. Paul D , Dhar Chanda D , Maurya AP , Mishra S , Chakravarty A , Sharma GD , Bhattacharjee A. Co-Carriage of blaKPC-2 and blaNDM-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.
19. 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 .
20. 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 .
21. 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 .
22. 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 .
23. 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 .
24. Institute CLS . Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement; M100-S21 .CLSI. CLSI: Wayne , USA ; 2011 .
25. 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 .
26. Cornaglia G , Giamarellou H , Rossolini GM. Metallo-β-lactamases: a last frontier for β-lactams . Lancet Infect Dis . 2011 ; 11 : 381 - 93 .
27. Romero D , Traxler MF , Lopez D , Kolter R. Antibiotics as signal molecules . Chem Rev . 2011 ; 111 : 5492 - 505 .
28. Liu W , Zou D , Wang X , Li XL , Zhu L , Yin Z , et al. Proteomic analysis of clinical isolate of Stenotrophomonas maltophilia with blaNDM-1, blaL1 and blaL2 β-lactamase genes under imipenem treatment . J Proteome Res . 2012 ; 11 : 4024 - 33 .
29. 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 .