Occurrence of bla NDM-1 and bla NDM-5 in a Tertiary Referral Hospital of North India

Abstract

Antimicrobial resistance poses a great challenge to clinicians leaving very limited treatment options available. A panel of carbapenem-resistant bacterial isolates was selected based on Rapidec Carba NP test from a total of 900 samples, which were collected from different specialities hospitals of Kanpur, India. Carba NP-positive isolates were screened for carbapenemases, extended-spectrum beta-lactamases (ESBLs), quinolone resistance, and 16s methyltransferase genes. Presence of diverse bla_NDM (bla_NDM-1 and bla_NDM-5) were detected and horizontal transferability was determined by transformation and conjugation assay. Elimination of bla_NDM-1 and bla_NDM-5 harboring plasmid was done by treating the isolates with sodium dodecyl sulfate. The transcriptional response of bla_NDM-1 and bla_NDM-5 under the exposure of imipenem, meropenem, and ertapenem stress was determined by quantitative real-time polymerase chain reaction. bla_NDM harboring isolates were found to be horizontally transferable through IncF_repB and K type plasmid and could be successfully eliminated after the single treatment with sodium dodecyl sulfate. A distinct pattern of transcriptional response was observed for bla_NDM-1 and bla_NDM-5 under the pressure of carbapenem antibiotics where an upregulated expression of both bla_NDM-1 and bla_NDM-5 was observed. Minimum inhibitory concentration (MIC) results revealed that bla_NDM harboring strains showed a high MIC range against imipenem, meropenem, ertapenem, cefepime, and aztreonam. Thus, prudent action should be taken to control the spread of these multidrug-resistant determinants or at least slowing down their lateral transfer in the hospital settings.

Introduction

Production of New Delhi metallo-β-lactamase-1 enzyme is one of the most common antibiotic resistance mechanisms found in Gram-negative bacteria against carbapenem group of antibiotics and this resistant determinant has been reported among members of enterobacteriaceae family, Acinetobacter and Pseudomonas from various countries like India, Australia, the United Kingdom, the United States, and New Zealand.¹ The presence of bla_NDM-1 within plasmid and other mobile genetic elements facilitate the transfer and spread of this resistance determinant within broad host range. As a result, the variants of bla_NDM-1 are also emerging within different hosts causing increased treatment failure and potential global health threat.²

Currently, at least 16 NDM variants has been reported that differ by very few amino acid substitution from one another (www.lahey.org/studies/other.asp#table 1). Furthermore, most of the NDM producing isolates coexisted with other β-lactam and non β-lactam resistance determinants that confer resistance to different classes of antibiotics.³ It was reported that bla_NDM-4 and bla_NDM-5 confer higher beta-lactam resistance to the harboring organisms than bla_NDM-1. So, to better understand the impact of bla_NDM-1 and its variant in carbapenem resistance, this study was designed to characterize bla_NDM-1 and bla_NDM-5 and their transcriptional response under the exposure of different carbapenem antibiotics.

Materials and Methods

Phenotypic and genotypic detection of carbapenemase

The clinical specimens for this study were collected from patients admitted at L.L.R & Associated Hospitals (10 hospitals of different specialities), G.S.V.M Medical College, Kanpur from January 2013 to December 2014. The clinical samples included urine, blood, cerebrospinal fluid, endotracheal aspirates, pus, stool, and other sterile body fluids. The samples were inoculated on sheep blood agar and MacConkey agar and incubated at 37°C overnight. The isolated bacteria were identified by standard biochemical characterization and 16s rDNA sequencing and antibiotic sensitivity pattern of the isolates was determined by Kirby Bauer disc diffusion testing.

Carbapenem-resistant bacterial isolates were selected and Rapidec Carba NP test (BioMérieux, La Balme-les-Grottes, France) was performed as per manufacturer's guidelines to detect carbapenemase production.⁴ Multiplex polymerase chain reaction (PCR) for genotypic detection of bla_NDM, bla_VIM, bla_IMP, bla_SIM, bla_SMB, bla_GIM, and bla_SPM genes was performed subsequently.^5–7

Sequencing of bla_NDM

For sequencing the bla_NDM a common primer was designed (Table 1) from the flanking region of bla_NDM to amplify the whole gene. The amplified product was purified using MinElute PCR Purification Kit (Qiagen, Germany) and was ligated into pGEM-T Vector (Promega, Madison) and was sequenced.

Table 1.

Oligonucleotides Used in This Study

Serial number	Primer pairs	Target	Sequence	Product size	Reference
1	NDM-F	NDM	5′-GGGCAGTCGCTTCCAACGGT-3′	476	⁵
	NDM-R		5′-GTAGTGCTCAGTGTCGGCAT-3′
2	NDM-FW	NDM whole gene	5′-TGCTACAGTGAACCAAATTAAG-3′	1,090	This study
	NDM-RW		5′-CATCGAAATCGCGATGGCAG-3′
3	VIM-F	VIM	5′-GATGGTGTTTGGTCGCATA-3′	390	⁵
	VIM-R		5′-CGAATGCGCAGCACCAG-3′
4	IMP-F	IMP	5′-TTGACACTCCATTTACDG-3′	139	⁵
	IMP-R		5′-GATYGAGAATTAAGCCACYCT-3′
5	SIM-F	SIM	5′-TACAAGGGATTCGGCATCG-3′	571	⁵
	SIM-R		5′-TAATGGCCTGTTCCCATGTG-3′
6	GIM-F	GIM	5′-GCTCAGGGTCATAAACCGCT-3′	614	⁵
	GIM-R		5′-CTTCCAACTTTGCCATGCCC-3′
7	SMB-F	SMB	5′-CAGCAGCCATTCACCATCTA-3′	492	⁶
	SMB-R		5′-GAAGACCACGTCCTTGCACT-3′
8	SPM-F	SPM	5′-CTGCTTGGATTCATGGGCGC-3′	784	⁷
	SPM-R		5′-CCTTTTCCGCGACCTTGATC-3′
9	KPC-F	KPC	5′-CATTCAAGGGCTTTCTTGCTGC-3′	538	⁸
	KPC-R		5′-ACGACGGCATAGTCATTTGC-3′
10	IMI/NMC-F	IMI/NMC	5′-CCATTCACCCATCACAAC-3′	440	⁸
	IMI/NMC-R		5′-CTACCGCATAATCATTTGC-3′
11	SME-F	SME	5′-AACGGCTTCATTTTTGTTTAG-3′	831	⁸
	SME-R		5′-GCTTCCGCAATAGTTTTATCA-3′
12	OXA-23-F	OXA-23	5′-GATCGGATTGGAGAACCAGA-3′	501	⁹
	OXA-23-R		5′-ATTTCTGACCGCATTTCCAT-3′
13	OXA-48-F	OXA-48	5′-GATTATCGGAATGCCTGCGG-3′	845	⁹
	OXA-48-R		5′-CTACAAGCGCATCGAGCATCA-3′
14	OXA-58-F	OXA-58	5′-CGATCAGAATGTTCAAGCGC-3′	529	⁹
	OXA-58-R		5′-ACGATTCTCCCCTCTGCGC-3′
15	TEM-F	TEM	5′-ATGAGTATTCAACATTTCCG-3′	867	⁵
	TEM-R		5′-CTGACAGTTACCAATGCTTA-3′
16	SHV-F	SHV	5′-AGGATTGACTGCCTTTTT-3′	392	⁵
	SHV-R		5′-ATTTGCTGATTTCGCTCG-3′
17	PER-F	PER	5′-AATTTGGGCTTAGGGCAGAA-3′	920	⁵
	PER-R		5′-ATGAATGTCATTATAAAAGC-3′
18	OXA-2-F	OXA	5′-AAGAAACGCTACTCGCCTGC-3′	478	⁵
	OXA-2-R		5′-CCACTCAACCCATCCTACCC-3′
19	CTX-M-F	CTX-M	5′-CGCTTTGCGATGTGCAG-3′	550	⁵
	CTX-M-R		5′-ACCGCGATATCGTTGGT-3′
20	armA-F	armA	5′-GGTGCGAAAACAGTCGTAGT-3′	1,153	¹⁰
	armA-R		5′-TCCTCAAAATATCCTCTATGT-3′
21	rmtB-F	rmtB	5′-GGAATTCCATATGAACATCAACGATGCCCT-3′	756	¹⁰
	rmtB-R		5′-CCGCTCGAGTCCATTCTTTTTTATCAAGTA-3′
22	npmA-F	npmA	5′-CGGGATCCAAGCACTTTCATACTGACG-3′	981	¹⁰
	npmA-R		5′-CGGAATTCCAATTTTGTTCTTATTAGC-3′
23	rmtA-F	rmtA	5′-CTAGCGTCCATCCTTTCCTC-3′	635	¹⁰
	rmtA-R		5′-TTTGCTTCCATGCCCTTGCC-3′
24	rmtC-F	rmtC	5′-CGAAGAAGTAACAGCCAAAG-3′	1,000	¹⁰
	rmtC-R		5′-GCTAGAGTCAAGCCAGAAAA-3′
25	rmtD-F	rmtD	5′-TCATTTTCGTTTCAGCAC-3′	744	¹⁰
	rmtD-R		5′-AAACATGAGCGAACTGAAGG-3′
26	qnrA-F	qnrA	5′-TTCAGCAAGAGGATTTCTCA-3′	628	¹¹
	qnrA-R		5′-GGCAGCACTATTACTCCCAA-3′
27	qnrB-F	qnrB	5′-ATGACGCCATTACTGTATAA-3′	546	¹¹
	qnrB-R		5′-GTTTGCTGCTCGCCAGTCGA-3′
28	qnrS-F	qnrS	5′CAATCATACATATCGGCACC-3′	675	¹¹
	qnrS-R		5′TCAGGATAAACAACAATACCC-3′
29	qepA-F	qepA	5′-GCAGGTCCAGCAGCGGGTAG-3′	260	¹¹
	qepA-R		5′-CACGATACTCGGGCAGGAAG-3′
30	qnrC-F	qnrC	5′-GGGTTGTACATTTATTGAATC-3′	447	¹¹
	qnrC-R		5′TCCACTTTACGAGGTTCT-3′
31	qnrD-F	qnrD	5′-CGAGATCAATTTACGGGGAATA-3′	582	¹¹
	qnrD-R		5′-AACAAGCTGAAGCGCCTG-3′
32	aac(6^′)Ib-cr-F	aac(6^′)Ib-cr	5′-ATGACTGAGCATGACCTTGC-3′	519	¹¹
	aac(6^′)Ib-cr-R		5′-TTAGGCATCACTGCGTGTTC-3′

Coexistence of other resistance determinants

Coexistence of class A and class D carbapenemase genes were determined by performing two sets of multiplex PCR targeting bla_KPC, bla_IMI/NMC, and bla_SME⁸ and bla_OXA-23, bla_OXA-48, and bla_OXA-58.⁹ The presence of extended-spectrum beta-lactamase (ESBL) genes were also detected by PCR assays targeting bla_PER, bla_TEM, bla_OXA-2, bla_SHV, and bla_CTX-M.⁵ The coexistence of 16s methyltransferase genes were determined by multiplex PCR targeting armA, rmtB, and npmA in first multiplex and rmtA, rmtC, and rmtD in second multiplex.¹⁰ The co-occurrence of quinolone resistance determinants were also investigated targeting qnrA, qnrB, qnrS, qepA, qnrC, qnrD, and aac(6^′)Ib-cr¹¹ and all the amplified products were sequenced. The oligonucleotides used in this study are mentioned in Table 1.

Determination of horizontal transferability of bla_NDM

Plasmids harboring bla_NDM were extracted by QIAprep Spin Miniprep Kit (Qiagen, Germany) and analyzed. Isolated plasmids were subjected to transformation by heat shock method using Escherichia coli JM107 as recipient. Conjugation assay was performed using parent strains and transformants (only for the non E. coli isolates harboring NDM) as donor and E. coli strain B (Genei, Bangalore) as a recipient strain.

Plasmid incompatibility typing

Plasmid harboring bla_NDM-1 and bla_NDM-5 was characterized by PCR-based replicon typing (PBRT) to identify the different incompatibility (Inc.) types. PBRT targets 18 different replicon types viz. FIA, FIB, FIC, HI1, HI2, I1-Iy, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA performing 5 multiplex PCR and 3 simplex PCR as described previously.⁶ Additionally, IncX-types (IncX1, IncX2, IncX3, and IncX4) were also targeted.¹²

Plasmid elimination assay

Plasmid curing experiment was performed with wild-type isolates harboring bla_NDM-1 and bla_NDM-5 using three different concentration of sodium dodecyl sulfate (SDS) treatment.¹³ For this, 100 μl of bacterial suspension was inoculated in 5 ml of fresh Luria Bertani broth and to which 50 μl of 8%, 10%, and 12% SDS was added separately and was incubated overnight. Meropenem resistance was used as the selectable marker for this plasmid curing assay. After treatment, 30 μl bacterial suspension was grown on Luria-Bertani (LB), agar with and without meropenem (2 mg/L) and was incubated overnight. Plasmid elimination was calculated comparing the number of cells grown on LB agar with and without meropenem. The absence of bla_NDM encoding plasmid in treated strains was determined by performing PCR assay by selecting 50 random colonies from the plate. The cured mutants and the wild strains (non-treated) were subjected to antibiotic susceptibility testing.

Transcriptional expression of bla_NDM-1 and bla_NDM-5

The transcriptional response of bla_NDM-1 and bla_NDM-5 under the exposure of imipenem, meropenem, and ertapenem was determined by quantitative real-time PCR. Organisms harboring bla_NDM-1 and bla_NDM-5 were inoculated in Luria Bertani broth (Hi-media, Mumbai, India) with and without antibiotic (1 mg/L). After 12 hr of incubation, total RNA was isolated using Qiagen RNease Mini Kit (Qiagen, Germany), immediately reverse transcribed into cDNA by using QuantiTect^® reverse transcription kit (Qiagen, Germany) and the cDNA was quantified by Picodrop (Pico 200, Cambridge, United Kingdom).

Quantitative real-time PCR was performed using Power Sybr Green Master Mix (Applied Biosystem, Warrington, United Kingdom) in Step One Plus real-time detection system (Applied Biosystem). The housekeeping gene rpsel of E. coli was used as an internal standard.¹⁴ The reaction condition followed was 95°C for 2 min, 40 cycles of 95°C for 20 sec, 52°C for 40 sec, and 72°C for 30 sec. The relative expression of bla_NDM-1 with and without carbapenem pressure was determined by ΔΔC_t method. Relative quantification was done using a transformant grown for 5 hr without any antibiotic pressure. The same experiment was performed in transconjugants and clones (bla_NDM-1 and bla_NDM-5 along with native promoters was cloned on P^GEM-T vector, Promega) encoding bla_NDM-1 and bla_NDM-5 respectively.

Minimum inhibitory concentration

Minimum inhibitory concentration for bla_NDM harboring strains was determined by agar dilution method against imipenem (Merck, France), meropenem (AstraZeneca, United Kingdom), ertapenem (MSD France), cefotaxime (Alkem, India), and aztreonam (Aristo, India) and the results were interpreted as per CLSI guideline.¹⁵

Molecular typing

All the bla_NDM producing E. coli and Proteus mirabilis isolates were typed by pulsed-field gel electrophoresis. Total genomic DNA was prepared in agarose blocks and digested with the restriction enzyme Xba1 (Promega, Madison) as suggested by the manufacturer. DNA fragments were separated in 1.5% molecular biology grade agarose (Hi-media, Mumbai, India) with 0.5 × TBE buffer in a pulsed field gel electrophoresis (PFGE) system CHEF-DR III (Bio-Rad) for 24 hr at 4 V/cm with a pulse at 120° angle in a 10–40 sec pulse time.⁵

Results

A total of 900 nonrepetitive samples were collected and Gram-negative bacteria grew in 320 (35%). Most common isolate was E. coli (40%) followed by Pseudomonas aeruginosa and Acinetobacter spp. Thirty-five carbapenem-resistant bacteria were isolated and Rapidec Carba NP test was performed of which 30 isolates were found to be positive for carbapenemase production.

Of the 18 clinical isolates harboring bla_NDM, fourteen were carrying bla_NDM-1 and the rest were found to harbor bla_NDM-5 (E. coli = 3, Stenotrophomonas maltophilia = 1). The other twelve Carba NP-positive isolates were positive for VIM-2 (n = 7), OXA-48 (n = 4), and SPM (n = 1) carbapenemases. The clinical details of these bla_NDM harboring organisms are illustrated in Table 2. Screening for ESBL genes revealed that bla_NDM carrying isolates were co-harboring bla_CTX-M-15, bla_TEM, bla_OXA-2, and bla_SHV. Presence of multiple quinolone resistance determinants such as qnrB2, aac(6^′)Ib-cr, and qnrD were found in five of them. The combination of these resistance determinants harboring within the isolates were explained in Table 2. Both bla_NDM-1 and bla_NDM-5 were conjugatively transferable to recipient E. coli strain B. Replicon typing results F_repB and K Inc., type of plasmids having an approximate size of 65 and 40 kb respectively were present within transconjugants whereas it was untypeable for three transformants (Table 2).

Table 2.

Clinical Details and Coexisting Resistance Genes in bla_NDM Harboring Strains

Strain ID	bla_NDM variant	Bacteria	Sample origin	Patient's sex/age (years)	Ward	Clinical infection/condition	Co-occurrence of ESBL genes	Quinolone resistance profile	Location	Inc., type
N-1	NDM-1	Escherichia coli	Urine	M/25	Gynecology	UTI	—	—	Plasmid	F_repB and K
N-2	NDM-5	E. coli	Urine	F/58	Medicine	Renal stones with UTI	CTX-M-15	qnrB2 + aac(6^′)-Ib-cr + qnrD	Plasmid	F_repB
N-3	NDM-1	E. coli	Pus	M/18	Surgery	Post trauma	—	—	Plasmid	F_repB
N-4	NDM-1	Proteus mirabilis	Pus	M/59	Orthopedics	Diabetic foot with gangrene	—	—	Plasmid	Untypeable
N-5	NDM-5	E. coli	Urine	F/21	Gynecology	Postoperative catheterized patient	—	—	Plasmid	F_repB and K
N-6	NDM-5	S. maltophila	Endotracheal aspirate	M/44	ICU	Post trauma on ventilator	—	—	Plasmid	K
N-7	NDM-1	P. mirabilis	Wound swab	F/35	ICU	Intra abdominal pus	—	—	Plasmid	K
N-8	NDM-1	Klebsiella pneumoniae	Pleural fluid	M/58	Chest	COPD	SHV	qnrB2 + aac(6′)-Ib-cr	Plasmid	K
N-9	NDM-1	E. coli	Endotracheal aspirate	F/19	ICU	Post trauma on ventilator	—	—	Plasmid	F_repB and K
N-10	NDM-5	E. coli	Blood culture	M/6	Pediatrics	Septicemia	OXA-2	aac(6′)-Ib-cr + qnrD	Plasmid	F_repB
N-11	NDM-1	P. mirabilis	Wound swab	F/49	Plastic surgery	Burn	TEM	—	Plasmid	F_repB
N-12	NDM-1	E. coli	Urine	M/53	Neurosurgery	Trauma	—	—	Plasmid	Untypeable
N-13	NDM-1	E. coli	Pus	M/2	Pediatric surgery	Postoperative for congenital malformation	—	—	Plasmid	F_repB and K
N-14	NDM-1	E. coli	Blood culture	M/22	ICU	Multi organ failure following poisoning	OXA-2	aac(6′)-Ib-cr + qnrD	Plasmid	K
N-15	NDM-1	P. mirabilis	Urine	F/34	Uro surgery	UTI with pelvic inflammatory disease	—	—	Plasmid	Untypeable
N-16	NDM-1	P. mirabilis	Endotracheal aspirate	F/72	ICU	Burn patient on ventilator	—	qnrB2 + aac(6′)-Ib-cr	Plasmid	K
N-17	NDM-1	E. coli	Pleural Fluid	F/27	Chest	Complicated case of MDR tuberculosis	CTX-M-15	—	Plasmid	F_repB
N-18	NDM-1	Pseudomonas aeruginosa	Endotracheal aspirate	M/33	ICU	Neuro trauma	—	—	Plasmid	F_repB

ESBL, extended-spectrum beta-lactamase.

Plasmids encoding bla_NDM-1 and bla_NDM-5 could be successfully eliminated after the single treatment with SDS with an elimination rate of 100%, which was confirmed by PCR assay. Cured mutants were susceptible to all the group of antibiotics. Distinct pattern of response was observed for bla_NDM-1 and bla_NDM-5 under the pressure of carbapenem antibiotics where an upregulated expression of both bla_NDM-1 and bla_NDM-5 was observed. The expression level of bla_NDM-1 was found to be maximal under the exposure of meropenem antibiotic, which was approximately fourfold high (relative quantification [RQ] = 4.249) from the control (RQ = 1). An approximate of twofold upregulated expression of bla_NDM-1 was also observed when this resistance determinant was exposed against imipenem (RQ = 1.924) and for ertapenem the fold change was 1.69 compared to the control (Fig. 1). In case of bla_NDM-5, a different pattern of response was observed where the expression level of bla_NDM-5 was found to be extremely high with an RQ value of 9.67 under the pressure of ertapenem antibiotic when compared to imipenem and meropenem where the fold change noticed was 1.056 and 4.859-fold high respectively (Fig. 1). Similar expressional trend was observed with the transconjugants and clones carrying bla_NDM-1 and bla_NDM-5 (Figs. 2 and 3) and the results were consistent as repeated thrice. Minimum inhibitory concentration (MIC) results revealed that these bla_NDM harboring strains showed a high MIC range against imipenem (32 to ≥256 mg/L), meropenem (64 to ≥256 mg/L), ertapenem (16 to ≥256 mg/L), and against cefepime (128 to ≥256 mg/L) and aztreonam (16 to ≥128 mg/L). While analyzing clonal relationship six pulsotypes of E. coli and four pulsotypes of P. mirabilis were observed as per PFGE analysis.

FIG. 1.

Transcriptional response of bla_NDM-1 and bla_NDM-5 in parental strain under exposure of different carbapenems with reference to control. The error bars represent the standard deviation of the three replicates of one sample.

FIG. 2.

Transcriptional response of transconjugants encoding bla_NDM-1 and bla_NDM-5 under exposure of different carbapenems with reference to control. The error bars represent the standard deviation of the three replicates of one sample.

FIG. 3.

Transcriptional response of clones carrying bla_NDM-1 and bla_NDM-5 under exposure of different carbapenems with reference to control. The error bars represent the standard deviation of the three replicates of one sample.

Discussion

Since their first report, the bla_NDM-1 is known to become pandemic and many reports have highlighted their global spread³. In India, this resistance determinant is expanded within environmental isolates and has been reported in sewage and tap water as well.¹⁶ This is quite alarming considering their persistence within nonpathogenic strain, which in turn act as reservoir of this gene. This particular factor probably has a major role in the evolution of different variants of New Delhi metallo beta-lactamases in this subcontinent. A previous study from this country has reported NDM-1, NDM-5, NDM-6, and NDM-7 from a single center,¹⁷ indicates their parallel propagation within hospital environment. In consistence with that study we have also observed occurrence of bla_NDM-5 in four study isolates. To our knowledge, this is the first study reporting the presence of bla_NDM-5 within Stenotrophomonas maltophilia. This further highlights how this variant is emerging in this part of the world and adapting new host system. However, in our study none of Acinetobacter spp. showed carriage of bla_NDM.

In this study, we tried to analyze the plasmid loss in the presence of SDS and elimination rate was better compared to other studies.¹⁸ As described previously,¹⁸ we also agree that plasmid elimination strategy can be an option for infection control and minimizing the spread of resistant plasmids. It was observed that two different Inc., type plasmids that is, K and FrepB were invariably encoding the two resistance determinants. The carriage of bla_NDM-1 has been reported on various plasmid types, namely IncA/C, IncF, IncL/M, IncH, IncN, and recently in IncX type.¹⁹ However, presence of two different Inc., types of plasmids encoding bla_NDM gene variants underscore multiple sources of origin and acquisition and also suggests their parallel propagation in the study hospital.

While analyzing the transcriptional response of the two NDM variants against carbapenem drugs, although there was ∼4.5-fold increase of expression in both the variants (parental strains) against meropenem, the fold increase in expression was more for NDM-5 against ertapenem. However, there is no study till date to support the data. But it leaves a speculation whether the new variants of NDM is attaining inducibility against carbapenem drugs. Seven NDM-producing isolates in the study were co-harboring other resistance determinants like ESBLs, qnr, and 16s methyltransferases and probably they were encoded within the same plasmid since the transformants selected on imipenem plates also showed PCR positive for other resistant genes.

Thus, prudent action should be taken to control the spread of these multidrug-resistant determinants or at least slowing down their lateral transfer in the hospital settings.

Footnotes

Acknowledgment

The authors sincerely acknowledge the financial support provided by Council of Scientific and Industrial Research (CSIR) and Department of Biotechnology, Government of India to carry out the work.

Financial Support

Council of Scientific and Industrial Research (CSIR Grant number 37(1632)/14/EMR-II) and Department of Biotechnology (DBT-NER Twinning Scheme number BT/215/NE/TBP/2011 dtd.15/11/2011) Government of India. Deepjyoti Paul is a Senior Research Fellow in the Department of Microbiology, Assam University and receives CSIR Senior Research Fellowship under the grant number 37(1632)/14/EMR-II.

Disclosure Statement

No competing financial interests exist.

References

Williamson

D.A.

, Sidjabat

H.E.

, Freeman

J.T.

, Roberts

S.A.

, Silvey

, Woodhouse

, Mowat

, Dyet

, Paterson

D.L.

, Blackmore

, Burns

, and Heffernan

. 2012. Identification and molecular characterization of New Delhi metallo-β-lactamase-1 (NDM-1) and NDM-6 producing enterobacteriaceae form New Zealand hospitals. Int. J. Antimicrob. Agents, 39:529–533.

Huang

Li.

, Hu

, Zhou

, Yang

, Qiao

, Wang

, Yu

, Cui

, Zhang

X.E.

, and Wei

. 2014. Rapid detection of New Delhi Metallo-β-lactamase gene and variants coding for carbapenemases with different activities by use of a PCR-based in vitro protein expression method. J. Clin. Microb., 52:1947–1953.

Karthikeyan

, Thirunarayan

M.A.

, and Krishnan

. 2010. Coexistence of bla_OXA-23 with bla_NDM-1 and armA in clinical isolates of Acinetobacter baumanii from India. J. Antimicrob. Chemother., 65:2253–2254.

Garg

, Garg

, Upadhyay

G.C.

, Agarwal

, and Bhattacharjee

. 2015. Evaluation of the RAPIDEC CARBA NP test kit for detection of carbapenemase producing gram negative bacteria. Antimicrob. Agents Chemother., 59:7870–7872.

Paul

, Dhar

, Maurya

A.P.

, Mishra

, Sharma

G.D.

, Chakravarty

, and Bhattacharjee

. 2016. Occurrence of co-existing bla_VIM-2 and bla_NDM-1 in clinical isolates of Pseudomonas aeruginosa from India. Ann. Clin. Microbiol. Antimicrob., 15:31.

Wachino

J.I.

, Yoshida

, Yamane

, Suzuki

, Matsui

, Yamagishi

, Tsutsui

, Konda

, Shibayama

, and Arakawa

. 2011. SMB-1, a novel subclass B3 metallo-β-lactamase, associated with ISCR1 and a class 1 integron, from a carbapenem-resistant Serratia marcescens clinical isolate. Antimicrob. Agents Chemother., 55:5143–5149.

Poirel

, Magalhaes

, Lopes

, and Nordmann

. 2004. Molecular analysis of metallo-β-lactamase gene bla_SPM-1 surrounding sequences from disseminated Pseudomonas aeruginosa isolates in Recife, Brazil. Antimicrob. Agents Chemother., 48:1406–1409.

Paul

, Dhar Chanda

, Maurya

A.P.

, Mishra

, Chakravarty

, Sharma

G.D.

, and Bhattacharjee

. 2015. Co-carriage of bla_KPC-2 and bla_NDM-1 in clinical isolates of Pseudomonas aeruginosa associated with hospital infections from India. PLoS One, 10:e0145823.

Evans

B.A.

, Amyes

S.G.B.

. 2014. OXA β-Lactamases. Clin. Microbiol. Rev., 27:241–263.

10.

, Zhang

, Han

, Sun

, and Ni

. 2009. Plasmid-mediated 16S rRNA methylases in aminoglycoside-resistant enterobacteriaceae isolates in Shanghai, China. Antimicrob. Agents Chemother., 53:271–272.

11.

Strahilevitz

, Jacoby

G.A.

, Hooper

D.C.

, and Robicsek

. 2009. Plasmid-mediated quinolone resistance: a multifaceted threat. Clin. Microbiol. Rev., 22:664.

12.

Johnson

T.J.

, Bielak

E.M.

, Fortini

, Hansen

L.H.

, Hasman

, Debroy

, Nolan

L.K.

, and Carattoli

. 2012. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug resistant enterobacteriaceae. Plasmid, 68:43–50.

13.

El-Mansi

, Anderson

K.J.

, Inche

C.A.

, Knowles

L.K.

, and Platt

D.J.

. 2000. Isolation and curing of the Klebsiella pneumoniae large indigenous plasmid using sodium dodecyl sulphate. Res. Microbiol., 151:201–208.

14.

Swick

M.C.

, Morgan-Linnell

S.K.

, Carlson

K.M.

, and Zechiedrich

. 2011. 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., 55:921–924.

15.

Clinical Laboratory Standard Institute. 2011. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. M100-S21.CLSI, Wayne.

16.

Zhang

, Qiu

, Wang

, Qi

, Hao

, Liu

, Shi

, Hu

, An

, Li

, Wang

, Cui

, Wang

, Huang

, Klena

J.D.

, and Song

. 2013. Higher isolation of NDM-1 producing Acinetobacter baumanii from the sewage of the hospitals in Beijing. PLoS One, 8:e64857.

17.

Rahman

, Shukla

S.K.

, Prasad

K.N.

, Ovejero

C.M.

, Pati

B.K.

, Tripathi

, Singh

, Srivastava

A.K.

, and Gonzalez-Zorn

. 2014. Prevalence and molecular characterization of New Delhi metallo-β-lactamase NDM-1, NDM-5, NDM-6 and NDM-7 in multidrug resistant enterobacteriaceae from India. Int. J. Antimicrob. Agents, 44:30–37.

18.

Sun

, Liu

, Chen

, Song

, Liu

, Guo

, Zhu

, Ji

, Xu

, Zhou

, Qian

, and Feng

. 2014. Characterization and plasmid elimination of NDM-1-producing Acinetobacter calcoaceticus from China. PLoS One, 9:e106555.

19.

Wailan

A.M.

, Paterson

D.L.

, Kennedy

, Ingram

P.R.

, Bursle

, and Sidjabat

H.E.

. 2016. Genomic characteristics of NDM-Producing Enterobacteriaceae isolates in Australia and their bla_NDM genetic contexts. Antimicrob. Agents Chemother., 60:136–141.

Occurrence of bla NDM-1 and bla NDM-5 in a Tertiary Referral Hospital of North India

Abstract

Introduction

Materials and Methods

Phenotypic and genotypic detection of carbapenemase

Sequencing of blaNDM

Coexistence of other resistance determinants

Determination of horizontal transferability of blaNDM

Plasmid incompatibility typing

Plasmid elimination assay

Transcriptional expression of blaNDM-1 and blaNDM-5

Minimum inhibitory concentration

Molecular typing

Results

Discussion

Footnotes

Acknowledgment

Financial Support

Disclosure Statement

References

Sequencing of bla_NDM

Determination of horizontal transferability of bla_NDM

Transcriptional expression of bla_NDM-1 and bla_NDM-5