Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-1, NDM-5, NDM-6 and...

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ARTICLE IN PRESSG ModelNTAGE-4299; No. of Pages 8

International Journal of Antimicrobial Agents xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents

j o ur nal ho me pag e: ht tp : / /www.e lsev ier .com/ locate / i jant imicag

revalence and molecular characterisation of New Delhietallo-�-lactamases NDM-1, NDM-5, NDM-6 and NDM-7 inultidrug-resistant Enterobacteriaceae from India

ohibur Rahmana,b, Sanket Kumar Shuklaa, Kashi Nath Prasada,∗, Cristina M. Ovejeroc,inod Kumar Pati a, Aparna Tripathia, Avinash Singha, Ashwini K. Srivastavab,runo Gonzalez-Zornc

Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow 226014, IndiaDepartment of Biosciences, Integral University, Lucknow, IndiaDepartment of Animal Health and VISAVET, Universidad Complutense de Madrid, Madrid, Spain

r t i c l e i n f o

rticle history:eceived 21 September 2013ccepted 7 March 2014

eywords:nterobacteriaceaeSBLetallo-�-lactamaseethylaseDM-1DM variants

a b s t r a c t

The growing prevalence of carbapenem resistance in Enterobacteriaceae worldwide is a major con-cern. New Delhi metallo-�-lactamase (NDM)-mediated carbapenem resistance has been identified inEnterobacteriaceae from numerous countries including those of the Indian subcontinent. Currently,seven NDM �-lactamase variants (NDM-1 to -7) have been identified. This study evaluated the detec-tion and molecular characterisation of NDM variants in Enterobacteriaceae at a tertiary care hospital inIndia. A total of 464 isolates were tested; 57 (12.3%) were resistant or showed reduced susceptibilityto imipenem and meropenem. All carbapenem-resistant isolates were blaNDM-positive by PCR, but 13isolates bore variants that differed in sequence from blaNDM-1. NDM-5, NDM-6 and NDM-7 were identi-fied in two, eight and three isolates, respectively. blaNDM variants were located on plasmids of >100 kbwith IncF, IncA/C and untypeable replicon types. Genes encoding the 16S rRNA methyltransferases RmtB,RmtC and ArmA as well as those for AmpC �-lactamases were also located on the same plasmids asblaNDM in different combinations. The prevalence of NDM-5 to -7 variants was significantly higher inEscherichia coli (P = 0.015) and they were more frequently isolated from the urology ward (P = 0.037)
than NDM-1. The mortality rate was comparable between patients infected with isolates positive forblaNDM-1 and blaNDM variants [25% (11/44) vs. 23% (3/13)]. Expression of blaNDM variants in E. coli usingthe same promoter showed that NDM-7 conferred higher resistance to imipenem. The diverse geno-typic features of blaNDM indicate rapid evolution of NDM resulting from their wide spread in the Indiansubcontinent.
© 2014 Published by Elsevier B.V.

. Introduction

Carbapenems, most notably imipenem and meropenem, haveeen recognised as the most potent �-lactams against multidrug-esistant Gram-negative bacteria because of their high affinityor penicillin-binding proteins, their stability against extended-pectrum �-lactamases (ESBLs) and their permeability of the

Please cite this article in press as: Rahman M, et al. Prevalenlactamases NDM-1, NDM-5, NDM-6 and NDM-7 in multidrug-resistanhttp://dx.doi.org/10.1016/j.ijantimicag.2014.03.003

acterial outer membranes [1]. However, as carbapenemasesegan to emerge worldwide, the effectiveness of this ‘last-line’ntibiotic class was challenged. New Delhi metallo-�-lactamase-1

∗ Corresponding author. Tel.: +91 5222494265; fax: +91 5222668017.E-mail addresses: [email protected], [email protected] (K.N. Prasad).

ttp://dx.doi.org/10.1016/j.ijantimicag.2014.03.003924-8579/© 2014 Published by Elsevier B.V.

(NDM-1), a new type of metallo-�-lactamase, was first reportedin Klebsiella pneumoniae and Escherichia coli recovered from aSwedish patient who was previously admitted to a hospital in NewDelhi (India) [2]. Since 2009, the emergence and dissemination ofNDM-positive isolates has been reported in almost all continentsexcept Antarctica [3]. The NDM gene (blaNDM) has been identi-fied in various Enterobacteriaceae and also in Acinetobacter andPseudomonas spp. [4,5]. An environmental study using direct PCRshowed the presence of blaNDM-1 in seepage and drinking watersamples. Two positive drinking water samples and 12 positive

ce and molecular characterisation of New Delhi metallo-�-t Enterobacteriaceae from India. Int J Antimicrob Agents (2014),

seepage samples yielded growth of blaNDM-1-positive E. coli, K.pneumoniae, Citrobacter freundii, Shigella boydii, Vibrio cholerae andAeromonas caviae [6]. In 2011, Kaase et al. reported for the firsttime a novel variant of blaNDM that had a C → G substitution at

dx.doi.org/10.1016/j.ijantimicag.2014.03.003


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2 M. Rahman et al. / International Journal of Antimicrobial Agents xxx (2014) xxx–xxx

Table 1Amino acid substitutions at various positions among NDM variants.

NDM type Proline 28 Valine 88 Aspartate 95 Aspartate 130 Methionine 154 Alanine 233 Source organism(s) GenBankaccession no.

References

NDM-1 – – – – – – Klebsiella pneumoniae,Escherichia coli

FN396876 [2]

NDM-2 Alanine – – – – – Acinetobacterbaumannii

JF703135 [7]

NDM-3 – – Asparagine – – – E. coli JQ734687 [8]NDM-4 – – – – Leucine – E. coli JQ348841 [9]

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NDM-5 – Leucine – – LeucinNDM-6 – – – – –

NDM-7 – – – Asparagine Leucin

osition 82 and this variant was designated blaNDM-2 [7]. Likewise,ther variants such as blaNDM-3, blaNDM-4, blaNDM-5, blaNDM-6 andlaNDM-7 have also been reported (Table 1).

Data on the prevalence of blaNDM variants in Enterobacteriaceaerom Indian hospitals are lacking. The present study was thereforeesigned to evaluate the identity and molecular characterisationf Enterobacteriaceae isolates positive for blaNDM-1 and its vari-nts recovered at a tertiary care referral hospital in India. The studylso analysed genotypic characterisation, the susceptibility of thesolates to different antibiotics, and patient information.

. Materials and methods

.1. Bacterial isolates

This study was conducted at Sanjay Gandhi Post Graduate Insti-ute of Medical Sciences (Lucknow, India), an 800-bed tertiary careeferral centre located in northern India. A total of 464 non-repeatnterobacteriaceae isolates from various clinical specimens such asrine, sputum, blood, pus, cerebrospinal fluid and tracheal aspirateere included in the study. Specimens were obtained from 412ospitalised and 52 non-hospitalised patients between February012 and July 2012. Standard microbiological techniques weresed for isolation and identification of the isolates [11]. Further

dentification of the isolates was confirmed by Phoenix automateddentification and sensitivity systems (BD Biosciences, San Jose,A). Prior to testing, all of the isolates were stored in Luria–BertaniLB) broth (Difco, BD Biosciences) supplemented with 15% glycerolSigma–Aldrich, St. Louis, MO) at −80 ◦C.

.2. Antimicrobial susceptibility testing

Initial susceptibility testing of the Enterobacteriaceae iso-ates to different �-lactam and non-�-lactam antibiotics waserformed by the Kirby–Bauer disk diffusion method. The min-

mum inhibitory concentration (MIC) was determined by EtestbioMérieux, Marcy-l’Étoile, France) and was further confirmedy the broth microdilution method following Clinical and Labo-atory Standards Institute (CLSI) guidelines [12]. Antibiotic disksnd media were procured from Difco (BD Biosciences) and purentibiotic powders were from Sigma–Aldrich. E. coli ATCC 25922as used as a quality control strain.

.3. DNA amplification and analysis

Total DNA was extracted by boiling. Briefly, a fresh bacterialolony was suspended in 150 �L of sterile distilled water andas boiled at 100 ◦C for 10 min. After centrifugation at 15 000 × g


or 15 min at 4 ◦C, the supernatant was removed and was storedt −20 ◦C until further analysis. Multiplex and simplex PCR waserformed for the detection of genes encoding acquired carbapen-mases (NDM, IMP, VIM, SIM, GIM, SPM, OXA-48/-181, BIC, DIM and

– E. coli JN104597 [19]Valine E. coli JN967644 [20]– E. coli JX262694 [10]

KPC) following previously described methods [13,14]. Isolates werealso examined for the presence of additional plasmid-encodedgenes for �-lactamases such as TEM, SHV, CTX-M and AmpC aswell as genes encoding for aminoglycoside resistance 16S rRNAmethylases ArmA and RmtA–RmtF by PCR as described elsewhere[15–18].

2.4. Sequencing

For the detection of blaNDM-1 and its variants, DNA frag-ments amplified for the blaNDM region were purified usinga QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany)and were sequenced using blaNDM-specific primers. Sequenc-ing was performed by Amnion Biosciences Pvt. Ltd. (Bengaluru,India) and sequences were analysed using the BLAST pro-gramme at the National Centre for Biotechnology Information(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Amino acid sequences ofNDM �-lactamases were predicted by the standard code andwere aligned using the ExPASy online tool for translation(http://www.expasy.org).

2.5. Comparative analysis of blaNDM variants

To evaluate and compare the susceptibility of NDM-1 and otherNDM �-lactamase variants to different antibiotics, cloning of theblaNDM-1 and blaNDM variants was performed using a Zero Blunt®

TOPO® PCR Cloning Kit (Invitrogen, Carlsbad, CA) followed byexpression in E. coli TOPO10 (Invitrogen). PCR amplicons of 984 bpencompassing the entire blaNDM gene with the same promoter wereamplified using blaNDM full-length specific primers as describedpreviously [7,19].

2.6. Plasmid manipulation and analysis

Plasmid DNA of the donors, transconjugants and transformantswas extracted using a QIAGEN Plasmid Midi Kit according tothe manufacturer’s instructions and was separated by agarosegel electrophoresis. The localisation of blaNDM was predicted byPCR using gel-purified plasmid DNA as template and transformingthe same into E. coli TOPO10. NDM �-lactamase transformantswere selected on LB agar (Difco, BD Biosciences) supplementedwith meropenem (0.5 mg/L) (Sigma–Aldrich) or ceftazidime (1, 2and 4 mg/L) (Sigma–Aldrich). Conjugation experiments were car-ried out in LB broth with sodium-azide-resistant E. coli J53AzRas the recipient. Cultures of donor and recipient cells in log-arithmic phase (0.5 mL each) were added to 4 mL of fresh LBbroth and were incubated overnight without shaking. Transcon-

ce and molecular characterisation of New Delhi metallo-ˇ-t Enterobacteriaceae from India. Int J Antimicrob Agents (2014),

jugants were selected on MacConkey agar (Difco, BD Biosciences)supplemented with meropenem (0.5 mg/L) or ceftazidime (1,2 and 4 mg/L) with sodium azide (100 mg/L) (Sigma–Aldrich)[20].


http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://www.expasy.org/

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Table 2Demographic and clinical data of patients infected with isolates positive for blaNDM-1

and its variants.a

Factor NDM-1 (n = 44) NDM variants(n = 13)

Age (years) (mean ± S.D.) 48.12 ± 12.23 45.83 ± 18.83Sex

Male 29 (66) 11 (85)Female 15 (34) 2 (15)

SpecimensBlood 3 (7) 0Urine* 3 (7) 4 (31)Pusb 17 (39) 6 (46)Tracheal aspirate 19 (43) 3 (23)Cerebrospinal fluid 2 (5) 0

OrganismsKlebsiella pneumoniae 20 (45) 2 (15)Escherichia coli** 14 (32) 9 (69)Citrobacter spp. 4 (9) 0Providencia spp. 4 (9) 2 (15)Enterobacter spp. 2 (5) 0

Hospital wardICU 4 (9) 2 (15)Nephrology 2 (5) 0Urology† 1 (2) 3 (23)Critical care medicine 3 (7) 1 (8)Transplant unit 9 (20) 0Neurosurgery 4 (9) 0Endocrine surgery 1 (2) 1 (8)Pulmonary medicine 1 (2) 0Surgical gastroenterology 13 (30) 5 (38)Gastroenterology 0 1 (8)Radiotherapy 1 (2) 0Endocrinology 3 (7) 0Neurology 1 (2) 0Paediatric superspecialty 1 (2) 0

PatientsHospitalised 41 (93) 13 (100)Non-hospitalised 3 (7) 0

OutcomeRecovered 29 (66) 9 (69)Improved 5 (11) 1 (8)Death 11 (25) 3 (23)

Predisposing factorMechanical ventilation 16 (36) 2 (15)Urinary catheter 1 (2) 1 (8)

Co-morbiditiesDiabetes mellitus 7 (16) 3 (23)Hypertension 2 (5) 3 (23)Addictionc 7 (16) 0Lung disease 6 (14) 3 (23)Haemodialysis 4 (9) 0 (0)

S.D., standard deviation; ICU, intensive care unit.a Data are n (%) unless otherwise stated.b Includes pus, debrided tissue, aspirated bile and surgical wound swab.c To tobacco chewing, smoking and alcohol.

ARTICLENTAGE-4299; No. of Pages 8

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.7. PCR-based replicon typing

All of the transconjugants and transformants were subjected toyping by a previously described PCR method based on replicons ofhe major plasmid incompatibility groups among Enterobacteria-eae [21].

.8. Nucleotide sequence accession numbers

The nucleotide sequence data reported in the present studyave been assigned EMBL/GenBank nucleotide accession nos.F284076–KF284132.

.9. Pulsed-field gel electrophoresis (PFGE)

Genomic DNA of the isolates positive for blaNDM variantsrom Enterobacteriaceae was prepared in agarose blocks and wasigested with restriction enzyme XbaI for K. pneumoniae and E. colind with NotI for Providencia spp. DNA fragments were separatedsing a CHEF II D-Mapper XA PFGE system (Bio-Rad, Hercules, CA)ith running conditions as described previously [16,22].

.10. Statistical analysis

Statistical analysis was performed using SPSS Statistics for Win-ows v.17.0 (SPSS Inc., Chicago, IL). Categorical variables expresseds numbers and percentages were compared by �2 test or Fisher’sxact test as appropriate. A P-value of <0.05 was considered statis-ically significant, and all the tests were two-tailed.

. Results

.1. Demographic and clinical data of patients infected withsolates positive for blaNDM-1 and its variants

Clinical and demographic data were compared between patientsnfected with isolates positive for blaNDM-1 (n = 44) and blaNDM vari-nts (n = 13) (Table 2). A higher number of isolates positive forlaNDM variants compared with blaNDM-1 was recovered from urine31% vs. 7%; P = 0.04). The distribution of the 44 blaNDM-1-positivesolates was as follows: K. pneumoniae, 20; E. coli, 14; Citrobac-er spp., 4; Providencia spp., 4; and Enterobacter spp., 2. However,laNDM variants were only detected in K. pneumoniae (n = 2), E. colin = 9) and Providencia spp. (n = 2). blaNDM variants were more fre-uently detected in E. coli than blaNDM-1 (69% vs. 32%; P = 0.015). Theighest number of blaNDM-positive isolates was recovered from theurgical gastroenterology ward (18/57; 32%). The frequency of iso-ates positive for blaNDM variants was significantly higher in therology ward compared with blaNDM-1 (23% vs. 2%; P = 0.03). All7 patients infected with blaNDM-positive isolates received broad-pectrum antibiotics before and after hospital admission.

.2. Presence of additional antibiotic resistance genes in isolatesositive for blaNDM-1 and its variants

All blaNDM-1-positive isolates were resistant to aminoglycosidesMIC > 256 mg/L) and different �-lactams, �-lactam/�-lactamasenhibitor combinations and carbapenems (MIC range, 2 mg/L to512 mg/L) but were susceptible to colistin and tigecycline, exceptor one isolate (KNKp6a) that had a MIC of 1.5 mg/L to tigecycline.he MIC range of all 13 isolates positive for blaNDM variants areiven in Table 3.


The frequency of the presence of additional bla genes (overallr in combination) in blaNDM-positive isolates is shown in Table 4.he overall frequency of blaKPC was significantly higher in isolatesositive for blaNDM-1 compared with blaNDM variants (45% vs. 15%;

* P = 0.04 (NDM variants vs. NDM-1).** P = 0.015 (NDM variants vs. NDM-1).† P = 0.03 (NDM variants vs. NDM-1).

P = 0.04). The presence of genes encoding AmpC, carbapenemase,ESBL and 16S rRNA methylase family were also analysed betweenisolates positive for blaNDM-1 and its variants; these gene combi-nations were significantly higher in isolates positive for blaNDMvariants (P = 0.003) (Table 4). The different ESBL genes detected inisolates positive for blaNDM-1 and its variants are shown in Table 4.Besides blaNDM, PCR detected blaIMP and blaVIM genes; however, itfailed to detect other metallo-�-lactamase genes such as blaSPM-1,blaGIM-1, blaSIM-1, blaAIM-1, blaBIC, blaDIM and 16S rRNA methyltrans-ferase family genes such as rmtD, rmtE and rmtA.


3.3. Prevalence of blaNDM variants

A total of 57 isolates (12.3%) were resistant to carbapenemsbased on their reduced susceptibility to meropenem and imipenem



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Table 4Presence of additional antibiotic resistance genes in clinical isolates positive forblaNDM-1 and its variants.

Overall gene frequency NDM-1 (n = 44) NDM variants(n = 13)

armA 22 (50) 6 (46)rmtB 12 (27) 4 (31)rmtC 20 (45) 6 (46)rmtF 8 (18) 3 (23)blaCTX-M 23 (52) 9 (69)blaTEM 42 (95) 11 (85)blaSHV 38 (86) 10 (77)blaAmpC 7 (16) 5 (38)blaIMP 7 (16) 5 (38)blaVIM 2 (5) 0blaOXA-48/-181 11 (25) 2 (15)blaKPC

* 20 (45) 2 (15)Gene combinations

CarbapenemasesOnly blaIMP 2 (5) 4 (31)Only blaKPC 11 (25) 2 (15)Only blaOXA-48/-181 3 (7) 1 (8)blaVIM + blaKPC 2 (5) 0blaIMP + blaKPC 1 (2) 0blaIMP + blaOXA-48/-181 2 (5) 1 (8)blaKPC + blaOXA-48/-181 4 (9) 0

blaIMP + blaKPC + blaOXA-48/-181

2 (5) 0

ESBLsOnly blaCTX-M 1 (2) 2 (15)blaSHV + blaTEM 20 (45) 4 (31)blaCTX-M + blaTEM 4 (9) 1 (8)

blaCTX-M + blaTEM + blaSHV

18 (41) 6 (46)

MethylasesOnly armA 12 (27) 3 (23)Only rmtB 8 (18) 2 (15)Only rmtC 9 (20) 2 (15)Only rmtF 3 (7) 0armA + rmtB 3 (7) 1 (8)armA + rmtC 6 (14) 2 (15)rmtB + rmtC 1 (2) 0rmtC + rmtF 4 (9) 2 (15)rmtF + rmtB 0 1 (8)armA + rmtF 1 (2) 0

Only AmpC 4 (9) 0

AmpC + carbapenemase + ESBL + 16SrRNA methylase**

3 (7) 5 (38)

ESBL, extended-spectrum �-lactamase.
* P = 0.04 (NDM-1 vs. NDM variants).
** P = 0.003 (NDM variants vs. NDM-1).

by Etest and broth microdilution. All 57 isolates harboured blaNDM;other carbapenemase genes such as blaVIM, blaIMP, blaKPC andblaOXA-48/-181 were also detected (Table 4). After sequence analy-sis, 13 of 57 isolates differed from blaNDM-1 by point mutations.Among the blaNDM variants, two isolates with point mutation at 88(Val → Leu) and 154 (Met → Leu) sites were inferred to be blaNDM-5.Likewise, eight isolates with point mutation at 233 (Ala → Val) andthree isolates with point mutations at 130 (Asp → Asn) and 154(Met → Leu) were inferred to be blaNDM-6 and blaNDM-7, respec-tively.

3.4. Case summary of patients infected with isolates positive forblaNDM variants

Two blaNDM-5-positive isolates (KNEc5a and KNEc5b) wererecovered from deep-seated surgical wound infections of patients


admitted to the surgical gastroenterology ward. The patientinfected with KNEc5a had choledocholithiasis and underwentcholecystectomy with exploration of the common bile ductand stone removal. The other patient, infected with KNEc5b,


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ad gangrenous jejunal perforation peritonitis following proxi-al jejunal resection with end jejunostomy. Both patients had

ospital-acquired infection with hypertension as a co-morbidity.Three blaNDM-7-positive isolates (KNEc7a, KNEc7b and KNEc7c)

ere recovered from the gastroenterology ward (KNEc7a), surgi-al gastroenterology ward (KNEc7c) and intensive care unit (ICU)KNEc7b). The patient admitted to the gastroenterology ward had

diagnosis of idiopathic chronic pancreatitis with biliary stric-ure. This patient developed urinary tract infection 5 days afterdmission, and E. coli (KNEc7a) was isolated from urine. The secondatient, admitted to the ICU, had acute pancreatitis with sepsis andulti-organ dysfunction. He was on ventilator support, and endo-

racheal aspirate showed heavy growth of E. coli (KNEc7b). Thisatient had diabetes mellitus as a co-morbidity and died due toulti-organ failure. The third patient, infected with KNEc7c, had

holedocholithiasis. He developed surgical site infection followingholecystectomy and stone removal from the common bile duct.ll patients infected with blaNDM-7-positive isolates had hospital-cquired infection.

Of the eight blaNDM-6-positive isolates, three (KNPd6b, KNKp6bnd KNEc6c) were from urine of post-operative patients admit-ed to the urology ward with right percutaneous nephrolithotomy,pen cystolithotomy and penopubic fistula, respectively. Two iso-ates (KNEc6b and KNKp6a) from the surgical gastroenterology

ard were from pus samples; one patient had appendicular perfo-ation peritonitis and the other was on T-tube drainage followingpen subtotal cholecystectomy. The isolate from the endocrineurgery ward (KNEc6a) was from debrided tissue of a patient withn infected diabetic foot ulcer. Isolates from the ICU (KNPd6a) andritical care medicine (KNEc6d) were from endotracheal aspiratesf patients with severe sepsis and multi-organ dysfunction syn-rome. Demographic and clinical details of patients infected with

solates positive for blaNDM variants are given in Table 5.

.5. Pulsed-field gel electrophoresis and plasmid analysis ofsolates positive for blaNDM variants

PFGE was performed on 13 isolates positive for blaNDM variantsnd all of the strains had a different PFGE band pattern. A conju-ation experiment and plasmid analysis were performed for E. colilaNDM-1 (KNEc1d as control) and all 13 isolates positive for blaNDMariants. Transconjugants containing blaNDM plasmids were suc-essfully obtained from the representative blaNDM-1 E. coli donorKNEc1d). The conjugation experiment was successful only in threelaNDM-6-positive bacterial isolates (E. coli KNEc6a and KNEc6b and. pneumoniae KNKp6b) with frequencies between 10−4 and 10−6.he conjugation experiments failed in the rest of the isolates withDM variants. However, electrotransformation experiments were

uccessful in all 13 isolates positive for blaNDM variants. All recipient. coli TOPO10 transformants contained a single plasmid >100 kb inize following electrotransformation. These plasmids belonged toncompatibility groups IncF (n = 8), IncA/C (n = 2) and untypeablen = 3) (Table 5). All transconjugants and transformants wereighly resistant to amikacin and gentamicin (MICs ≥ 256 �g/mL).6S rRNA methyltransferase genes rmtB, rmtC and armA and the-lactamase gene blaAmpC were also found located on the samelaNDM plasmid in different combinations (Table 5).

.6. Comparative analysis of the susceptibility of isolates positiveor blaNDM-1 and other blaNDM variants to different antibiotics

E. coli clinical isolates KNEc1d, KNEc5a, KNEc6b and KNEc7a


ositive for different blaNDM variants (NDM-1, NDM-5, NDM-6 andDM-7, respectively) were selected for full-length gene cloning in. coli TOPO10 that gave rise to E. coli TOPO10 carrying recombi-ant plasmid pblaNDM. The MICs for carbapenems (imipenem and

PRESStimicrobial Agents xxx (2014) xxx–xxx 5

meropenem) and different �-lactams and �-lactam/�-lactamaseinhibitor combinations were compared (Table 6). E. coli TOPO10carrying pblaNDM-7 conferred higher resistance to imipenem, fol-lowed by E. coli TOPO10 with pblaNDM-5; however, there was nodifference in MIC range between E. coli TOPO10 carrying pblaNDM-6and pblaNDM-1 for imipenem, indicating that blaNDM-6 and blaNDM-1conferred similar resistance to imipenem.

4. Discussion

NDM-1 �-lactamase-producing Enterobacteriaceae are increas-ingly reported from various parts of the world. Although NDM-1appears to be endemic in the Indian subcontinent, other reportssuggest that the Balkan countries may act as another reservoirof NDM-1-producers [23,24]. Currently, seven variants of NDM(NDM-1 to -7) have been identified (Table 1). All of these isolateswere recovered from patients who had a history of prior admis-sion to healthcare facilities in the Indian subcontinent. It is likelythat identification of NDM variants may signal an ongoing andrapid evolution of the gene encoding NDM resulting from theirextensive spread. However, on literature search there was no large-scale clinical validation for the presence of NDM variants in Indianhealthcare centres. To the best of our knowledge, we report herefor the first time NDM-1 along with NDM variants (NDM-5, -6 and-7) in Enterobacteriaceae isolated from various clinical samples.In the urology ward, isolation of blaNDM variants was significantlyhigher than blaNDM-1-positive isolates (23% vs. 2%; P = 0.03). In anearlier study, the urology unit was identified as a vulnerable spe-cialty for NDM-1 �-lactamase-producers because of the nature ofthe procedures involved such as endoscopy of the urinary tract,which has the potential for direct exposure and transmission of theorganisms that commonly cause urinary tract infection [25]. Over-all, isolation of blaNDM-positive isolates was higher from surgicalgastroenterology (32%), which is in concordance with an earlierIndian study [26]. Some of the variants conferred higher resistanceto carbapenems as evidenced from the MIC study; possibly thesevariants might have evolved from NDM-1 owing to the use of higherdoses of carbapenems in these patients.

Of the 13 blaNDM variants, 9 were found in E. coli. The frequencyof blaNDM variants NDM-5, -6 and -7 in E. coli was higher thanblaNDM-1 (P = 0.015). Several other studies have also reported differ-ent blaNDM variants in E. coli (Table 1). Two isolates of Providenciaspp. were found to harbour blaNDM-6. To our knowledge, this is thefirst report of blaNDM variants detected in Providencia spp. All ofthe patients infected with blaNDM-positive isolates had receivedbroad-spectrum antibiotics prior to hospital admission as well asduring their hospital stay. It is likely that the use of broad-spectrumantibiotics might exert a selective pressure for the developmentof resistance and evolution of blaNDM and its variants. Moreover,all of the isolates were found to harbour more than one antibi-otic resistance gene other than blaNDM. Among carbapenemases,KPC was significantly associated (P = 0.04) with blaNDM-1-positiveisolates. Other studies from India have also reported the coexis-tence of KPC and NDM-1 carbapenemases [27,28]. Interestingly, thecombined presence of genes encoding all four groups of antibioticresistance enzymes (AmpC, carbapenemase, ESBL and 16S rRNAmethylase) was significantly associated with isolates positive forblaNDM variants (P = 0.003). This is the first report showing a signif-icant association of four different groups of resistance genes withblaNDM variants. Some of these genes [blaAmpC and 16S rRNA methy-lases (armA, rmtB and rmtC)] were located on the same plasmidsalong with the blaNDM variant.


To the best of our knowledge, this is the first comparative studyon the ability to confer resistance to carbapenems and severalbulky cephalosporins by NDM-1 and its several variants (NDM-5, -6 and -7) expressed in E. coli TOPO10. The ability to confer


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Table 5Demographic and clinical features of patients infected with isolates positive for blaNDM variants (n = 13) and their molecular characterisation.

KNEc5a KNEc5b KNEc6a KNEc6b KNPd6a KNKp6a KNPd6b KNEc6c KNKp6b KNEc6d KNEc7a KNEc7b KNEc7c

NDM type NDM-5 NDM-5 NDM-6 NDM-6 NDM-6 NDM-6 NDM-6 NDM-6 NDM-6 NDM-6 NDM-7 NDM-7 NDM-7Age (years) 28 52 49 40 60 65 37 03 55 55 37 68 56Sex Female Male Male Female Male Male Male Male Male Male Male Male MaleDepartment Surgical

gastroenterologySurgical gas-troenterology

Endocrinesurgery

Surgical gas-troenterology

ICU Surgical gas-troenterology

Urology Urology Urology Critical caremedicine

Gastroenterology ICU Surgicalgastroenterology

Specimen Intra-operative pus Intra-operativepus

Debridedtissue

Aspirated bile Endotrachealaspirate

Peritoneal pus Right PCNurine

Urine Urine Endotrachealaspirate

Urine Endotrachealaspirate

Surgical woundswab

Diagnosis Choledocholithiasis Gangrenousjejunalperforationperitonitis

Infected leftdiabetic footulcer

Cholelithiasis Severe sepsiswithmulti-organdysfunction

Perforationperitonitis

Nephrolithiasis Post-operativepenopubicfistula

Urolithiasis Severe acutepancreatitis,severe sepsis,multi-organdysfunction

Idiopathic chronicpancreatitis

Acutepancreatitis

Choledocholithiasis

Hospitalisationperiod

19 June–5 July 2012 21 Feb–2 May2012

29 May–12June 2012

19 June–9 July2012

4 May–4 June2012

12 March–8April 2012

21–29 April2012

23–26 April2012

11–15 July2012

8 April–14May 2012

15 March–15 April2012

10–28 June2012

19 April–2 May2012

Procedure (if any) Cholecystectomy withexploration of CBD

Proximaljejunalresection withendjejunostomy

Wounddebridement

Open subtotalcholecystec-tomy andT-tubedrainage ofCBD

Mechanicalventilation

Laparotomylavage andappendicec-tomy

Right PCN Dilatation andcatheterisationthrough fistula

Open cys-tolithotomy

Laparotomywithnecrosectomy

– Mechanicalventilation

Cholecystectomy

Species Escherichia coli E. coli E. coli E. coli Providencia sp. Klebsiellapneumoniae

Providencia sp. E. coli K. pneumoniae E. coli E. coli E. coli E. coli

Infection HA HA CA HA HA HA HA HA HA HA HA HA HACo-morbidity Hypertension Hypertension Diabetes

mellitus– Diabetes

mellitusStructural lungdiseases

Structural lungdiseases

Structural lungdiseases

Hypertension – – Diabetesmellitus

–

Outcome Recovered Recovered Recovered Recovered Death Improved Recovered Recovered Recovered Death Recovered Death RecoveredCarbapenemase

genesNDM-5, IMP,OXA-48/-181

NDM-5,OXA-48/-181

NDM-6 NDM-6, IMP NDM-6 NDM-6, KPC NDM-6 NDM-6, IMP NDM-6, KPC NDM-6 NDM-7 NDM-7, IMP NDM-7, IMP

ESBL genes CTX-M, TEM TEM, SHV CTX-M, TEM,SHV

CTX-M, TEM,SHV

CTX-M, TEM,SHV

CTX-M, TEM,SHV

CTX-M,TEM,SHV

TEM, SHV TEM, SHV TEM, SHV CTX-M, TEM, SHV CTX-M CTX-M

Methylase genes armA, rmtC rmtB, rmtF rmtC, rmtF rmtC, rmtF armA rmtC armA rmtC armA armA, rmtC armA, rmtB rmtB rmtBAmpC + − − + − − − − + − − + +Replicon type F F F F Untypeable Untypeable Untypeable F A/C F A/C F FCo-resident genes

on NDM plasmidarmA, ampC rmtB rmtC rmtC, ampC armA – armA rmtC armA, ampC armA rmtB rmtB, ampC rmtB, ampC

Accession no. KF284078 KF284079 KF284087 KF284088 KF284082 KF284080 KF284083 KF284089 KF284081 KF284090 KF284084 KF284085 KF284086

ICU, intensive care unit; PCN, percutaneous nephrolithotomy; CBD, common bile duct; HA, hospital acquired; CA, community acquired; ESBL, extended-spectrum �-lactamase.



ARTICLE ING ModelANTAGE-4299; No. of Pages 8

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resistance to carbapenems in this study was in the followingorder: NDM-7 > NDM-5 > NDM-6 = NDM-1. Whether the amino acidsubstitutions unique to these NDM variants could enhance thehydrolytic activity in the same order of MICs as found here needs tobe assessed in kinetic studies using the purified enzyme. Recently,the role of Leu154 in NDM-7 in elevating carbapenem MICs hasbeen reported [10].

5. Conclusion

The molecular epidemiology of blaNDM-1-positive Enterobacte-riaceae is rapidly evolving. In the present study, besides blaNDM-1,other variants such as blaNDM-5, blaNDM-6 and blaNDM-7 were iden-tified in different species of Enterobacteriaceae. Isolates positivefor blaNDM variants also harbour genes encoding four other differ-ent groups of resistance enzymes (AmpC, other carbapenemases,ESBLs and 16S rRNA methyltransferases). The coexistence of NDMwith other antibiotic resistance genes is a major clinical problembecause of limited treatment options. Hence, further epidemiologi-cal studies are required to develop strategies in order to contain theemergence of such multidrug-resistant isolates and to limit theirtransmission.

Acknowledgment

The authors are grateful to the Department of Animal Health,Universidad Complutense de Madrid (Madrid, Spain) for providingpositive controls, including sodium-azide-resistant E. coli J53AzR.

Funding: This study was supported by an Indo-Spanish researchgrant from the Department of Science and Technology (New Delhi,India) [DST/INT/SPAIN/P-28/2011-C] and by the Spanish Ministryof Science and Innovation [PRI-PIBIN-2011-0915].

Competing interests: None declared.Ethical approval: This study was approved by the Institutional

Ethics Committee of Sanjay Gandhi Post Graduate Institute of Med-ical Sciences (Lucknow, India) [code 2012-69-EMP-61].

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Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-1, NDM-5, NDM-6 and...

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