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 ~  Abstract
 ~ Introduction
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 ~ Results
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  Table of Contents  
Year : 2019  |  Volume : 37  |  Issue : 4  |  Page : 527-530

Adaptation of blaNDMthrough IncP plasmid within broad host range

1 Department of Microbiology, Assam University, Silchar, Assam, India
2 Department of Microbiology, Silchar Medical College and Hospital, Silchar, Assam, India

Date of Submission04-Feb-2020
Date of Acceptance09-Apr-2020
Date of Web Publication18-May-2020

Correspondence Address:
Dr. Amitabha Bhattacharjee
Assam University, Silchar, Assam
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_20_48

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 ~ Abstract 

Introduction: It was also known that the IncP-1 plasmids are ubiquitous in environmental bacteria and those reside in soil, sewage, marine sediments and in manure. The blaNDMis associated with resistance determinants along with various mobile elements such as plasmid, insertion sequences and transposons, which facilitates its horizontal dissemination. These plasmids, if tracked, can be a starting point for the control of infection due to multidrug-resistant pathogens. The aim of the study was to investigate that IncP-type plasmids carrying blaNDMis adapted in different hosts. Materials and Methods: Thirteen of the isolates were harbouring IncP-type plasmid and they all were Escherichia coli isolated from hospitalised patients of Silchar Medical College and Hospital, India. The isolates were checked for susceptibility test, and the stability was assessed by a serial passage. These isolates were further subjected to transcriptional analysis of NDM gene as well as plasmid copy number alteration. Results: The study isolates were highly stable, and the resistance gene (blaNDM) was retained within isolates till 55th subsequent serial passages. Plasmid copy number alteration was random in isolates when exposed to carbapenem antibiotics, whereas increasing trend in transcriptional expression was observed with the increase in imipenem concentration. Conclusion: This study was able to underscore the presence of IncP plasmid that was harbouring blaNDMand was maintained within diverse host. The finding also highlights the adaptation of the broad-host-range plasmid that responds in terms of transcriptional expression under antibiotic exposure.

Keywords: Broad host range, Escherichia coli, IncP, plasmid

How to cite this article:
Choudhury NA, Paul D, Das BJ, Dhar(Chanda) D, Bhattacharjee A. Adaptation of blaNDMthrough IncP plasmid within broad host range. Indian J Med Microbiol 2019;37:527-30

How to cite this URL:
Choudhury NA, Paul D, Das BJ, Dhar(Chanda) D, Bhattacharjee A. Adaptation of blaNDMthrough IncP plasmid within broad host range. Indian J Med Microbiol [serial online] 2019 [cited 2020 Sep 30];37:527-30. Available from:

 ~ Introduction Top

From the very discovery of New Delhi metallo-β-lactamase (blaNDM) in 2008 from a Swedish patient of Indian origin in New Delhi, India,[1] 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]blaNDM is associated with resistance determinants along with various mobile elements such as plasmid, insertion sequences and transposons, which facilitates its horizontal dissemination.[2],[4] Plasmids thus facilitate the rapid adaptation of bacteria to changing environments by mediating horizontal gene transfer, as seen in research articles in an alarming rate of multidrug-resistant pathogens.[5] A property which is universally inherited by plasmids and which is more suitable for classification is incompatibility. This is the inability of two plasmids to be propagated stably in the same cell line. Incompatibility is a manifestation of relatedness: the sharing of common elements involved in plasmid replication control.[6],[7] It was also recognised that the IncP-1 plasmids are ubiquitous in environmental bacteria residing in soil, sewage, marine sediments and manure.[8]

These plasmids are either broad host range, that is, they are propagated within the broad host range or narrow host range which is not known to be transferred among a wide range of bacteria. In Indian perspective, Inc FrepB and IncK were reported, carrying blaOXA-23 within Escherichia coli.[9] Furthermore, it has been already reported that IncX3 plasmid type was responsible for disseminating blaNDM-7 and blaNDM-4 in the same health setting.[10],[11] However, no information is available on the IncP plasmid and association with NDM in this country. Therefore, the present study was aimed to investigate the IncP plasmid carrying blaNDM within diverse host range and their transcriptional response and copy number alteration under carbapenem exposure.

 ~ Materials and Methods Top

Bacterial sample

E. coli-harbouring NDM gene was taken for the study which was isolated from the patient attended in Silchar Medical College and Hospital, India. Plasmid incompatibility was determined by polymerase chain reaction (PCR)-based replicon typing assay, targeting 18 different replicons, namely FIA, FIB, FIC, HI1, HI2, I1/Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F and FIIA.[2] The presence of blaNDM gene was further determined by PCR assay using NDM-F 5-GGGCAGTCGCTTCCAACGGT-3 and NDM-R 5-GTAGTGCTCAGTGTCGGCAT-3 as primer for the study.[6] The isolates were verified by different tests, namely, Gram staining method, standard biochemical characterization tests including IMViC test, urease test, triple sugar iron test, sugar fermentation test and nitrate reduction test and followed by 16s rDNA sequencing of isolate.

Plasmid preparation and transmission assay

Plasmids encoding blaNDM were extracted by the QIAprep Spin Miniprep Kit (Qiagen, Germany) as per manufacturer's instruction. Isolated plasmids were subjected to transformation assay. Transformation was carried out by heat shock method.[7] The recipient strains used were E. coli JM107, E. coli DH5α, Klebsiella pneumonia and Pseudomonas aeruginosa of clinical origin. Transformants were selected on Luria Bertani Agar (Hi-Media, Mumbai, India) plates containing gentamicin (100 μg/ml) and ampicillin (100 μg/ml).

Conjugation experiment was carried out using blaNDM harbouring clinical strains as donors and a streptomycin-resistant E. coli recipient strain B (Genei, Bangalore, India). The minimal inhibitory concentration 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 reaches an OD 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 ampicillin (100 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. 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.

Plasmid stability within different hosts

Plasmid stability analysis of blaNDM producers in parent strain (E. coli) and transformants in different hosts, that is, K. pneumonia, P. aeruginosa, E. coli DH5α and E. coli JM107 was performed by the serial passage method for consecutive 55 days at 1:1000 dilutions without any antibiotic pressure.[8] After each passage, 1 ml of the culture was diluted into 103 dilution with normal saline, and 40 μl of the diluted sample was spread onto the LB agar plate. After overnight incubation, 50 colonies from plates were randomly picked and subjected to the phenotypic detection of MBL and further confirmed genotypically by PCR assay for the presence of NDM which was harbouring IncP type, after the confirmation by plasmid-based incompatibility test.

Plasmid copy number alteration and transcriptional expression of NDM harbouring IncP within broad host range against concentration gradient imipenem stress: clinical isolates of E. coli and other transformants of different hosts, i.e., K. pneumonia, P. aeruginosa, E. coli DH5α, E. coli JM107 co-harbouringblaNDM and plasmids of incompatibility group IncP were selected for determining the copy number under the exposure of different concentrations of carbapenem antibiotics. Single colony of each incompatibility type was inoculated into LB broth containing 1 μg/ml, 2 μg/ml and 4 μg/ml and 8 μg/ml of imipenem, 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 blaNDM carrying plasmid type (IncP) 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 StepOnePlus Real-Time Detection System (Applied Biosystem, USA) to estimate the relative copy number of blaNDM for different concentrations of antibiotic for each host types. The copy number of blaNDM within the wild-type plasmid of IncP incompatibility type 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 normalised against a housekeeping gene rpsel of E. coli for E. coli and Klebsiella and rpsL P for Pseudomonas aeruginosa.[12]

Susceptibility testing

The antibiotic susceptibility of IncP-harbouring parent strains as well as transformants was done by Kirby–Bauer disc diffusion method against antibiotics as piperacillin-tazobactam (100/10 μg), amikacin (30 μg), gentamicin (10 μg), ciprofloxacin (5 μg), ampicillin (30 μg), co-trimoxazole (10 μg) and carbenicillin (100 μg) (Hi-Media, Mumbai, India). The minimum inhibitory concentration was performed using agar dilution method against imipenem, meropenem, cefepime and aztreonam, co-trimoxazole, ampicillin and ciprofloxacin, and the results were compared with standard Clinical and Laboratory Standards Institute guidelines.[13]

 ~ Results Top

During the study, 13 isolates were obtained, harbouring IncP-type plasmid which was carrying blaNDM. Transformation assay was done with the isolates containing IncP type group which was found to be transferable when selected in gentamicin and ampicillin screen agar. IncP plasmid was stable within all four recipients' hosts, that is, K. pneumonia, P. aeruginosa, E. coli DH5α and E. coli JM107 till 55th serial passages. Plasmids carrying blaNDM were selected in the medium-containing imipenem and could be horizontally transferred from all 13 clinical E. coli isolates into recipient hosts.

On analyzing the copy number of IncP-type plasmids, it was observed that plasmid copy number increases with the increase of imipenem concentration in E. coli mostly, but in all other hosts, including the wild-type expression level decreased with the increase in gradient concentration of imipenem [Figure 1]. The transcriptional expression with IncP marker was random but was stable in all hosts and maintained throughout. There was a consistent level of transcriptional response for wild type with and without concentration gradient of imipenem stress [Figure 2].
Figure 1:Copy number alteration of different host with concentration gradient of imipenem

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Figure 2:Transcriptional expression of bla NDM under concentration gradient carbapenem (imipenem) exposure

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The copy number of blaNDM-1 was found to be variable. The copy number of blaNDM gene within IncP type of plasmids showed an increasing trend when increasing concentrations of imipenem in all the hosts transferred.

Antimicrobial susceptibility result showed that the 13 blaNDM-harbouring isolates were resistant to co-trimoxazole, ciprofloxacin and carbenicillin.

 ~ Discussion Top

Incompatible plasmids were found to carry various antibiotic resistance genes, for example PMQR genes, ESBL genes (blaTEM-52, blaCTX-M and blaSHV-12), carbapenemase genes (blaNDM) and others.[14],[15] The findings as indicate that this plasmid type can carry a diverse range of resistance genes in enterobacteriaceae.

This study also reports the presence of blaNDM within IncP-type plasmid. Thus, analysis of this plasmid by determining the copy number alteration is of utmost importance. The study could highlight that plasmid copy number of IncP type is maintained under carbapenem stress in diverse host range. The finding is quite unique to the earlier studies where IncP is regarded as having broad host range.[16] Plasmid copy number is dependent on the type of organism which acts as host of that plasmid and the origin of replication. It is also reported that mutation can bring high copy number.[16] The current study showed antibiotic pressure helps in maintenance and adaptation of IncP-type plasmid within diverse host range although there was no significant alteration of plasmid copy number. The study establishes a linkage among selection pressure, stability and copy number of plasmids encoding resistance genes. The study also investigates the analysis of transcriptional expression of blaNDM encoded within IncP type within different hosts, and it was observed that the gene was transcriptionally expressed in all the host ranges. This could be due to the adaptation of this plasmid in an unknown host machinery, so its importance with respect to future infectious diseases risk assessment, evaluating and minimizing the selective pressure in clinical settings, thereby slowing down the horizontal transmission of multidrug resistance. The expression of blaNDM could predict the bacterial response in different time interval when a single carbapenem exposure is applied. In addition, this study could underscore that irrespective of plasmid types, blaNDM 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.

 ~ Conclusion Top

This study underscores the presence of IncP plasmid-harbouring blaNDM which was maintained within diverse host range. The finding also highlights the adaptation of the broad-host-range plasmid that responds in terms of transcriptional expression under antibiotic exposure. Thus, the study came up with epidemiological knowledge of a stable blaNDM-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.

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Conflicts of interest

There are no conflicts of interest.

 ~ References Top

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 Klebsiellapneumoniae Sequence Type 14 from India. Antimicrob Agents Chemother 2009;53:5046-54.  Back to cited text no. 1
Mishra S, Sen MR, Upadhyay S, Bhattacharjee A. Genetic linkage of blaNDM among nosocomial isolates of Acinetobacter baumannii from a tertiary referral hospital in northern India. Int J Antimicrob Agents 2013;41:452-6.  Back to cited text no. 2
Huttner A, Von Dach E, Renzoni A, Huttner BD, Affaticati M, Pagani L, et al. Augmented renal clearance, low β-lactam concentrations and clinical outcomes in the critically ill: an observational prospective cohort study. International journal of antimicrobial agents 2015;45:385-92.  Back to cited text no. 3
nnett PM. Plasmid encoded antibiotic resistance: Acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol 2008;153 Suppl 1:S347-57.  Back to cited text no. 4
Wellington EM, Boxall AB, Cross P, Feil EJ, Gaze WH, Hawkey PM, et al. The role of the natural environment in the emergence of antibiotic resistance in gram-negative bacteria. Lancet Infect Dis 2013;13, 155-65.  Back to cited text no. 5
Nordstrom K, Molin S, Light J. Control of replication of bacterial plasmids: Genetics, molecular biology and physiology of the plasmid Rl system. Plasmid 1984;12:71-90.  Back to cited text no. 6
Novick RP. Plasmid incompatibility. Microbiol Rev 1987;51:381-95.  Back to cited text no. 7
Heuer H, Smalla K. Plasmids foster diversification and adaptation of bacterial populations in soil. FEMS Microbiol Rev 2012;36:1083-104.  Back to cited text no. 8
Paul D, Ingti B, Bhattacharjee D, Maurya AP, Dhar D, Chakravarty A, et al. An unusual occurrence of plasmid-mediated blaOXA-23 carbapenemase in clinical isolates of Escherichia coli from India. Int J Antimicrob Agents 2017;49:642-5.  Back to cited text no. 9
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 2017;23:206-10.  Back to cited text no. 10
Choudhury NA, Paul D, Chakravarty A, Bhattacharjee A, Dhar Chanda D. IncX3 plasmid mediated occurrence of blaNDM-4 within Escherichia coli ST448 from India. J Infect Public Health 2018;11:111-4.  Back to cited text no. 11
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 fluoroquinolone and multidrug resistance. Antimicrob Agents Chemother 2011;55:921-4.  Back to cited text no. 12
Clinical and Laboratory Standard Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. Wayne, USA: Clinical and Laboratory Standard Institute; 2011. p. M100-S21.  Back to cited text no. 13
Johnson TJ, Bielak EM, Fortini D, Hansen LH, Hasman H, Debroy C, et al. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae. Plasmid 2012;68:43-50.  Back to cited text no. 14
Paul D, Dhar Chanda D, Maurya AP, Mishra S, Chakravarty A, Sharma GD, et al. Co-carriage of blaKPC-2 and blaNDM-1in clinical isolates of Pseudomonas aeruginosa associated with hospital infections from India. PLOS One 2015;10:e0145823.  Back to cited text no. 15
Das N, Valjavec-Gratian M, Basuray AN, Fekete RA, Papp PP, Paulsson J, et al. Multiple homeostatic mechanisms in the control of P1 plasmid replication. Proc Natl Acad Sci U S A 2005;102:2856-61.  Back to cited text no. 16


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