Outbreak of Efficiently Transferred Carbapenem-Resistant bla NDM -Producing Gram-Negative Bacilli Isolated from Neonatal Intensive Care Unit of an Indian Hospital

Abstract

The emergence of bla_NDM particularly in Gram-negative bacteria is a burden on the health care system in developing countries. Hence, this study was initiated to screen New Delhi Metallo-β-lactamase (NDM)-producing Gram-negative bacterial strains from neonatal intensive care unit (NICU) of an Indian Hospital. A total of 18 bla_NDM-producing isolates were detected in the present study. Out of 18 bla_NDM variant isolates, 6 were Klebsiella pneumoniae, 4 Escherichia coli, 2 Enterobacter aerogenes, 1 Acinetobacter lwoffii, 1 Enterobacter cloacae, 3 Acinobacter baumannii, and 1 Cedecea davisae from NICU, showing resistance against all antibiotics, except colistin and polymixin. The transferability of resistance determinants was tested by conjugation. Transfer of bla_NDM-producing strains was successful in all 18 strains. In the case of transconjugants, the minimum inhibitory concentration values were found to decrease. The bla_NDM-producing isolates contained detectable plasmids of size 66, 38, and 6 kb. Plasmi/d-based replicon typing revealed the incompatibility types Inc (A/C, FIIA, FIC, K, F, W, FIA, P, X, FIB, B/O) in bla_NDM-carrying isolates. This study revealed the outbreak of multiple variants of bla_NDM (13 NDM-1, 4 NDM-5, and 1 NDM-7). Moreover, other resistance markers, viz. bla_OXA-1, bla_C_MY-1, bla_VIM-1, and bla_SHV-1 coassociated with bla_NDM were also found. In this study, we reported NDM–producing C. davisae as a first report to the best of our knowledge. This study is an attempt to reveal the dissemination of bla_NDM isolated from neonates in NICU and their efficient transferability among Gram-negative bacilli through horizontal gene transfer.

Introduction

Multidrug-resistant organisms are the main cause of early and late neonatal sepsis in India and are nearly certainly widespread in the community.¹ Overall, multidrug-resistant organisms have affected patients in hospital settings, where exposure to antibiotics, long-term hospitalization, and host factors provide risks for acquisition and use of indwelling devices. Over the past years, infections and colonization due to carbapenemases-producing Enterobacteriaceae (CPE) have increased nationally and internationally. Carbapenemase-producing Enterobacteriaceae caused infections that are extremely difficult to treat due to their marked resistance to a wide range of antibiotics.² Carbapenemase belongs to β-lactamases classes A, B, and D of Ambler classification system and are classified by their molecular structures; the Ambler class A (SHV, TEM type), class B Metallo-β-lactamases (VIM, IMP [imipenem], and NDM [New Delhi Metallo-β-lactamase] enzymes), and class D (OXA type). Moreover, class C β-lactamases, such as CMY type are weak carbapenemase, which may lead to carbapenem-resistance, especially in combination with other resistance mechanisms.³ The gene encoding NDM is often carried by plasmids and therefore easily transfers to other microorganisms through horizontal gene transfer, thereby the probability of emergence of drug-resistant strains of pathogenic microorganisms increase rapidly.⁴ NDM, one of the significant carbapenemases, and their rapid spread creates a major global public health problem.⁵ NDM-1 was first detected in Klebsiella pneumonia from a Swedish patient previously hospitalized in India in 2008.⁶ Although, NDM-1 has now been found worldwide, leading to the serious attention because of its severe resistance to almost all types of antibiotics and its repeated association with other resistant markers.⁷ The NDM-1 gene is located on self-transmissible plasmids that carry a significant number of other antibiotic resistance genes.⁸ NDM-producing Gram-negative bacteria are mostly carrying conjugative plasmids ranging from 4 to 154 kb in size belonging to several incompatibility types, such as IncFIA, IncFIC, IncK, IncB/O, IncFIB, IncHI1, IncF, IncP, IncY, IncFIIA, IncI, and IncN.⁹ In Enterobacteriaceae, NDM variants evolved by single and/or double amino acid residue substitutions at different positions.^10,11A total of 24 known variants of NDM have been identified so far.

In this study, we characterized NDM-producing Gram-negative bacilli isolated from neonatal intensive care unit (NICU) of a North Indian hospital for the period of (May 2017 to September 2017), which can efficiently transfer to multiple Gram-negative bacilli through horizontal gene transfer.

The aim of this study was to reveal the dissemination of bla_NDM isolated from neonates in NICU and their efficient transferability among Gram-negative bacilli through horizontal gene transfer.

Materials and Methods

Sample collection

From May 2017 to September 2017, the neonates hospitalized in the NICU were screened for NDM-producing Gram-negative strains. This study was carried out in the NICU of JN Medical College and Hospital, AMU (Aligarh, India). JNMCH is a tertiary care hospital located in Aligarh, India with a 1,300-bed capacity. A single rectal swab was collected from each patient with the help of Hi-Culture collection device. Neonates enrolled in the study were those who enrolled in the active surveillance system (NICU stay 48 hours and weekly surveillance swabs taken at least once). Neonates admitted before May 2017 and/or discharged after September 2017 were excluded.

The samples were spread over Luria Bertani agar (HiMedia Laboratories Pvt. Ltd.) plate and incubated at 37°C for 16–20 hours. The colonies were isolated by the streak plate method using a sterile loop. All the pure culture isolates were cryopreserved at −80°C in 70% glycerol.

Identification of isolates

Bacterial species were identified by using BD Phoenix TM⁻¹⁰⁰ Automated Microbiology System (Becton, Dickinson & Co., Franklin Lakes, NJ).

Antimicrobial susceptibility tests and minimum inhibitory concentration assay

Antimicrobial susceptibility test was determined by the standard disk diffusion method with the antibiotic discs placed on MH agar plates (HiMedia Laboratories Pvt. Ltd.). The antibiotic discs used were carbapenems (imipenem 10 μg, meropenem 10 μg), aminoglycosides (gentamicin 10 μg, amikacin 30 μg), monobactam (aztreonam 30 μg), cephalosporin (cefoxitin 30 μg, ceftazidime 30 μg), fluoroquinolones (ciprofloxacin 5 μg), colistin (10 μg), co-trimoxazole (25 μg), and ticarcillin/clavulanic acid (75/10 μg; Hi-Media, Pvt. Ltd., Mumbai, India). The results of this test were interpreted according to Clinical and Laboratory Standard Institute guidelines (Clinical and Laboratory Standards Institute 2015).¹² AST was also done by BD Phoenix TM⁻¹⁰⁰ Automated Microbiology System. Minimum Inhibitory Concentrations (MICs) for antimicrobial agents were determined by broth microdilution method, according to the guidelines of the CLSI.¹²

Molecular analysis

Whole-cell DNA from strains was prepared by taking pure colonies from a culture plate, mixing in 100 μL of nuclease-free water, and was incubated at 95°C for 15 minutes in a thermal block followed by centrifugation at 10,000 rpm at 4°C for 10 minutes. Genes were amplified by polymerase chain reaction (PCR) on a Veriti^™ 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA) using the gene-specific primers for the detection of bla_NDM, bla_VIM, bla_OXA, bla_SHV, and bla_CMY as described earlier.¹³ PCR products were mixed with 5 μL of gel loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol) run on 1% agarose gels (Sigma) and was prepared in 1 × TAE (40 mM Tris-acetate, 1 mM EDTA) buffer. Ethidium bromide (EtBr) was added to a final concentration of 0.5 μg/mL and the images were documented using a gel documentation system (Gel Doc). Amplicons of NDM were purified from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Inc., Waltham, MA) following the manufacturer's protocol. The purified DNA fragments were sequenced at SciGenom Labs Pvt. Ltd. (Cochin, India).

Plasmid analysis

Plasmid incompatibility group was determined by a PCR-based replicon typing method. Plasmid DNA was amplified by five multiplex and three simplex PCRs using 18 pairs of primers as reported previously¹⁴ that are recognized as incompatibility replicon types: FIA, FIB, FIC, HI1, HI2, I1-Ic, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. Plasmid DNA extraction and molecular size of multiple plasmids were recognized by Kieser method.¹⁵

Conjugation experiment

Conjugation assay was performed between clinical isolates as donors and azide-resistant Escherichia coli J53 strain as the recipient¹⁶ using a selection based on cefoxitin (25 mg/L; Sigma-Aldrich) and sodium azide (250 mg/L; Hi-Media Laboratories). The PCR amplification confirmed the transconjugants having resistant markers.

Results

Bacterial isolates

For this study, 65 samples were collected from neonates having bloodstream infection and low birth weight with an average age of 6 days admitted in the NICU, of JN Medical College and Hospital, Aligarh, India. Seventy-seven bacterial strains were isolated from these samples. Out of the 77 isolates, 31 isolates were carbapenem-resistant. Of 31 isolates, 18 were found to carry bla_NDM variants. Among 18 bla_NDM-positive isolates, 6 Klebsiella pneumoniae, 4 E. coli, 2 Enterobacter aerogenes, 1 Acinetobacter lwoffii, 1 Enterobacter cloacae, 3 Acinobacter baumannii, and 1 Cedecea davisae were identified.

Antimicrobial susceptibility tests and MIC testing

All the 18 NDM-producing isolates were highly resistant to frequently used antibiotics in clinical settings, including carbapenems (imipenem), aminoglycosides (gentamicin, amikacin, and streptomycin), monobactam (aztreonam), cephalosporin (cefoxitin, ceftazidime, and cefotaxime), tetracycline, fluoroquinolones (ciprofloxacin), and colistin. The MIC data revealed high values against all tested antibiotics, which were found in the range of 0.25 ≥ 4,096 μg/mL (Table 1). The MICs of tetracycline, fluoroquinolones (ciprofloxacin), and colistin were found to be less than other antibiotics as used in the study. The risk factors for NDM-producing strains include the carbapenem exposure and ampicillin usage. The average time period of admitted neonates was about 2 weeks and they were diagnosed with prematurity, congenital heart defects, chronic pulmonary diseases, and metabolic diseases.

Table 1.

Minimum Inhibitory Concentrations of NDM-Producing Gram-Negative Bacilli, Its Transconjugants, and Escherichia coli J53

Strain name	MIC (μg/mL)
Strain name	GEN	CTX	FOX	CAZ	STP	TE	CIP	IMP	AZT	AM	CL
AK-151	≥4,096	≥4,096	≥4,096	≥4,096	2,048	64	64	≥1,024	≥4,096	≥4,096	1,024
E. coli J53+AK-151	≥4,096	512	1,024	≥4,096	1,024	64	32	512	2,048	≥4,096	256
AK-152	≥4,096	≥4,096	2,048	≥4,096	2,048	512	≥4,096	≥1,024	≥4,096	≥4,096	16
E. coli J53+AK-152	2,048	1,024	512	≥4,096	1,024	64	≥4,096	256	≥4,096	≥4,096	4
AK-153	≥4,096	2,048	2,048	2,048	2,048	256	64	≥1,024	≥4,096	≥4,096	8
E. coli J53+AK-153	≥4,096	2,048	1,024	2,048	2,048	128	32	512	≥4,096	≥4,096	2
AK-154	≥4,096	2,048	2,048	≥4,096	2,048	256	64	≥1,024	≥4,096	≥4,096	0.5
E. coli J53+AK-154	≥4,096	1,024	2,048	≥4,096	2,048	256	64	≥1,024	≥4,096	≥4,096	0.25
AK-155	≥4,096	512	512	≥4,096	1,024	64	64	≥1,024	≥4,096	≥4,096	0.5
E. coli J53+AK-155	1,024	128	128	2,048	512	32	32	256	512	1,024	0.25
AK-165	≥4,096	2,048	≥4,096	≥4,096	≥4,096	128	2,048	≥1,024	≥4,096	≥4,096	4
E. coli J53+AK-165	2,048	1,024	1,024	2,048	1,024	32	512	256	1,024	1,024	2
AK-166	≥4,096	≥4,096	2,048	≥4,096	1,024	512	2,048	≥1,024	≥4,096	≥4,096	256
E. coli J53+AK-166	1,024	2,048	512	2,048	256	64	512	256	1,024	1,024	64
AK-167	≥4,096	2,048	1,024	≥4,096	≥4,096	128	64	≥1,024	≥4,096	≥4,096	64
E. coli J53+AK-167	≥4,096	2,048	1,024	2,048	2,048	64	32	≥1,024	≥4,096	≥4,096	16
AK-168	≥4,096	≥4,096	2,048	≥4,096	2,048	1,024	1,024	≥1,024	≥4,096	≥4,096	1
E. coli J53+AK-168	2,048	2,048	512	1,024	1,024	512	256	512	1,024	2,048	0.5
AK-169	≥4,096	≥4,096	2,048	≥4,096	256	512	≥4,096	≥1,024	≥4,096	≥4,096	2
E. coli J53+AK-169	2,048	1,024	256	1,024	64	128	2,048	256	1,024	2,048	0.5
AK-170	≥4,096	≥4,096	1,024	≥4,096	≥4,096	512	≥4,096	≥1,024	≥4,096	≥4,096	1
E. coli J53+AK-170	2,048	≥4,096	512	≥4,096	1,024	128	2,048	128	≥4,096	≥4,096	0.25
AK-171	≥4,096	≥4,096	≥4,096	≥4,096	512	128	1,024	≥1,024	≥4,096	≥4,096	1
E. coli J53+AK-171	≥4,096	≥4,096	≥4,096	2,048	128	64	256	128	≥4,096	≥4,096	0.5
AK-172	≥4,096	≥4,096	≥4,096	≥4,096	≥4,096	512	256	≥1,024	≥4,096	≥4,096	128
E. coli J53+AK-172	2,048	2,048	1,024	2,048	1,024	128	64	256	2,048	2,048	32
AK-173	≥4,096	≥4,096	1,024	≥4,096	512	128	≥4,096	≥1,024	≥4,096	≥4,096	2
E. coli J53+AK-173	≥4,096	1,024	512	2,048	128	64	512	256	≥4,096	≥4,096	0.5
AK-175	≥4,096	≥4,096	2,048	≥4,096	2,048	512	≥4,096	≥1,024	≥4,096	≥4,096	1
E. coli J53+AK-175	≥4,096	≥4,096	1,024	≥4,096	≥4,096	256	1,024	256	2,048	≥4,096	0.25
AK-176	≥4,096	≥4,096	2,048	≥4,096	2,048	2,048	≥4,096	≥1,024	≥4,096	≥4,096	1
E. coli J53+AK-176	2,048	1,024	512	1,024	2,048	512	1,024	256	≥4,096	≥4,096	0.5
AK-534	≥4,096	≥4,096	≥4,096	≥4,096	128	≥4,096	256	≥1,024	≥4,096	≥4,096	1
E. coli J53+AK-534	≥4,096	≥4,096	2,048	≥4,096	128	≥4,096	128	128	≥4,096	≥4,096	0.25
AK-536	≥4,096	≥4,096	2,048	≥4,096	≥4,096	≥4,096	≥4,096	≥1,024	≥4,096	≥4,096	1
E. coli J53+AK-536	1,024	2,048	512	1,024	2,048	1,024	2,048	256	1,024	512	0.5
E. coli J53	0.06	0.06	0.06	0.06	0.25	0.5	0.06	0.06	0.06	0.25	0.03

AM, amikacin; ATM, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; CL, colistin; CTX, cefotaxime; FOX, cefoxitin; GEN, gentamicin; IMP, imipenem; MIC, minimum inhibitory concentration; NDM, New Delhi Metallo-β-lactamase; STP, streptomycin; TE, tetracycline.

Detection of antibiotic resistance genes

PCR amplification and sequencing confirmed that all the 18 isolates harbored bla_NDM variants (thirteen NDM-1, four NDM-5, and one NDM-7) and the sequences were submitted to the NCBI database. Accession numbers of these sequences are shown in Table 2. Furthermore, bla_OXA-1 was detected in seven isolates (AK-151, AK-153, AK-165, AK-167, AK-171, AK-173, and AK-534), bla_CMY-1 in five isolates (AK-151, AK-153, AK-173, AK-176, and AK-534), bla_VIM-1 in two isolates (AK-154, AK-155), and bla_SHV-1 in one isolate (AK-173). The conjugation experiment confirmed that resistance determinants remained present on the plasmid in respective isolates.

Table 2.

Genetic Features Associated with bla_NDM Among Gram-Negative Bacilli with Respect to Replicon Types, Associated Resistance Determinants and Plasmid Size

Isolate name and transconjugate	NDM variants/accession No.	Organism name	Associated markers				Plasmid size (kb)	Replicon types (Inc)
Isolate name and transconjugate	NDM variants/accession No.	Organism name	OXA-1	CMY-1	VIM-1	SHV-1	Plasmid size (kb)	Replicon types (Inc)
AK-151	NDM-1/MH211124	Klebsiella pneumoniae	+	+	−	−	38, 6	A/C, FIIs
AK-151.T		Escherichia coli J53	+	+	−	−	38	A/C, FIIs
AK-152	NDM-1/MH064494	Cedecea davisae	−	+	−	−	38	FIC, F, K
AK-152.T		E. coli J53	−	+	−	−	38	FIC, F, K
AK-153	NDM-1/MH211125	Acinobacter baumannii	+	+	−	−	38, 6	A/C, W, K
AK-153.T		E. coli J53	−	+	−	−	38, 6	A/C, W, K
AK-154	NDM-5/MH211136	A. baumannii	−	−	+	−	66, 38	F, K
AK-154.T		E. coli J53	−	−	+	−	66, 38	F, K
AK-155	NDM-1/MH211126	A. baumannii	−	−	+	−	38	FIA, A/C, FIIs, F, W
AK-155.T		E. coli J53	−	−	+	−	38	FIA, W, A/C, F
AK-165	NDM-1/MH211127	K. pneumoniae	+	−	−	−	38	P, A/C, FIIs, F
AK-165.T		E. coli J53	+	−	−	−	38	P, A/C, FIIs, F
AK-166	NDM-1/MH211128	K. pneumoniae	−	−	−	−	38	X, FIA, FIB, A/C, FIIs, F
AK-166.T		E. coli J53	−	−	−	−	38	X, FIA, FIB, A/C, FIIs, F
AK-167	NDM-1/MH211129	K. pneumoniae	+	−	−	−	38	FIB, P, A/C, FIIs, F
AK-167.T		E. coli J53	+	−	−	−	38	FIB, P, A/C
AK-168	NDM-1/MH211130	K. pneumoniae	−	−	−	−	38	FIA, FIB, A/C, F
AK-168.T		E. coli J53	−	−	−	−	38	FIA, FIB, A/C, F
AK-169	NDM-1/MH211131	K. pneumoniae	−	−	−	−	38	FIA, FIB, A/C, FIIs, F
AK-169.T		E. coli J53	−	−	−	−	38	FIA, FIB, A/C, FIIs, F
AK-170	NDM-1/MH211132	Enterobacter aerogenes	−	−	−	−	38, 6	X, FIA, FIB, A/C, FIIs, F
AK-170.T		E. coli J53	−	−	−	−	38, 6	X, FIA, FIB, A/C, FIIs
AK-171	NDM-1/MH211133	Acinetobacter lwoffii	+	−	−	−	38	FIA, FIB, A/C, K
AK-171.T		E. coli J53	+	−	−	−	38	FIA, FIB, A/C, K
AK-172	NDM-5/MH211137	E. coli	−	−	−	−	38, 6	FIA, FIB, A/C, F
AK-172.T		E. coli J53	−	−	−	−	38, 6	FIA, FIB, A/C, F
AK-173	NDM-7/MH211140	E. coli	+	+	−	+	38	FIA, FIB, A/C, F, B/O
AK-173.T		E. coli J53	−	+	−	+	38	FIA, FIB, A/C, F, B/O
AK-175	NDM-1/MH211134	E. aerogenes	−	−	−	−	38	X, FIA, FIB, A/C, F, B/O
AK-175.T		E. coli J53	−	−	−	−	38	X, FIA, FIB, A/C, F, B/O
AK-176	NDM-1/MH211135	Enterobacter cloacae	−	+	−	−	38	FIA, FIB, A/C, FIIs, F, B/O
AK-176.T		E. coli J53	−	+	−	−	38	FIA, FIB, A/C, FIIs, F, B/O
AK-534	NDM-5/MH211138	E. coli	+	+	−	−	38, 6	X, FIC, K
AK-534.T		E. coli J53	+	+	−	−	38, 6	X, FIC, K
AK-536	NDM-5/MH211139	E. coli	−	−	−	−	38	FIA, FIB, K
AK-536.T		E. coli J53	−	−	−	−	38	FIA, FIB, K

Inc, incompatability; T, transconjugants.

Conjugation experiment

The plasmid location of resistant markers was determined by conjugation, using an azide-resistant E. coli J53 strain as the recipient and isolates as donors.¹⁶ Transconjugants were obtained at the frequencies of 10⁻³–10⁻⁵ cells, showing that plasmid from the donors (K. pneumoniae, E. coli, E. aerogenes, A. lwoffii, E. cloacae, A. baumannii, and C. davisae) were transferred and found stable in E. coli J53.

Plasmid analysis

In our study, NDM-producing isolates contained detectable plasmid size (6, 38 and 66 kb) as shown in Table 2. The number of plasmids found in the isolates were 66 kb (n = 1), 38 kb (n = 18), and 6 kb (n = 5). The isolates were found to carry plasmid incompatibility types IncA/C, IncFIIA, IncFIC, IncK, IncF, IncW, IncFIA, IncP, IncX, IncFIB, IncB/O. IncA/C (n = 14), IncFIIA (n = 8), IncFIC (n = 2), IncK (n = 6), IncF (n = 13), IncW (n = 2), IncFIA (n = 11), IncP (n = 2), IncX (n = 4), IncFIB (n = 11), and IncB/O (n = 3), as shown in Table 2. The incompatibility plasmid types were transferred successfully through horizontal gene transfer to the recipient strain (Table 2).

Discussion

Dissemination of NDM producing among Gram-negative strain has become a serious global concern. The bla_NDM not only confers resistance to most β-lactams but also accompanies with multiple resistance gene determinants of different groups of antimicrobials in the same strain, which enables pathogens to become multidrug resistant. The primary treatment of serious infections caused by ESBL-producing Gram-negative bacteria is carbapenem antibiotics. Therefore, the issue of carbapenem resistance in Gram-negative bacilli deserves special attention. In this study, we reported carbapenem-resistant NDM-producing Gram-negative bacterial strain isolated from NICU of one of the Indian hospitals. In this study, NDM-producing K. pneumoniae, E. coli, E. aerogenes, E. cloacae, A. lwoffi, and A. baumannii were mainly identified from neonatal wards. Recently, a study on bla_NDM variants among Enterobacteriaceae from a NICU of a Northern India hospital has recently been published by our group.⁹

This study revealed the outbreak of multiple variants of bla_NDM (13 NDM-1, 4 NDM-5, and 1 NDM-7). NDM variants in E. coli are bla_NDM-5 and bla_NDM-7 associated with bla_OXA-1, bla_CMY-1, and bla_SHV-1. It has also been shown in earlier studies in E. coli carrying bla_NDM-5 and bla_NDM-7.^17,18 Of 4 E. coli, bla_NDM-7 coassociated with bla_OXA-1, bla_CMY-1, and bla_SHV-1 in AK-173and bla_NDM-5 coassociated with bla_OXA-1 and bla_CMY-1 in AK-534. Furthermore, AK-172 and AK-536 carrying bla_NDM-5 were found not to carry any other associated markers. Replicon typing analysis revealed that E. coli was carrying different incompatibility plasmid types (FIA, FIB, FIC, A/C, F, B/O, X, and K). It has been reported in the earlier study that E. coli strains were found to harbor different plasmid types (IncK, IncF, and IncX3).^19–21

NDM in K. pneumoniae, is bla_NDM-1 coassociated with bla_OXA-1 and bla_CMY-1, carrying the incompatibility plasmid group (A/C, FII, F, FIA, P, X, FIB). It was also shown in a recently published study in K. pneumoniae carrying bla_NDM-1.²² NDM-1 among E. aerogenes, E. cloacae, A. lwoffi, and A. baumannii harboring different plasmid sizes (6, 66, 38) kb, carry different plasmid types A/C, FIIs, F, K, W, FIC, FIA, P, X, B/O. Previously published report showed bla_NDM-1-producing E. cloacae in China and Turkey.^23,24 Earlier research showed that bla_NDM-1 had been spread in the NICU through different Gram-negative bacteria (E. cloacae, A. baumannii, and K. pneumoniae).²⁵ Previously bla_NDM-1 harboring IncF, IncA/C and IncX were reported.^13,26

Moreover, for the first time, we have identified bla_NDM-1-producing C. davisae in AK-152 strain coassociated with bla_CMY-1 and carried plasmid types IncFIC, IncF, and IncK. It has been reported in earlier studies that carbapenem-resistant C. davisae produced a novel AmpC β-lactamase.²⁷ Furthermore, in a previous study, bla_NDM-1-producing Cedecea lapagei in the presence of bla_CMY has been reported from a neonate admitted to pediatric intensive care unit of an Indian Hospital.²⁸

In our study, bla_NDM variants were found to be stable for conjugal transfer when mated with sodium azide-resistant E. coli J53 and cotransfered bla_OXA-1, bla_SHV-1, bla_CMY-1, and bla_VIM-1 from donor, the transferable ability indicating the alarming potential of these resistance determinants to spread among Gram-negative bacteria. All replicon types, which were identified in parental isolates, were transferred successfully through horizontal gene transfer to a recipient strain, except IncFIIs in AK-155 and AK-167, whereas IncF in AK-170 as shown in Table 2. Previously, bla_NDM was found to carry two or more incompatibility plasmid types, which were transferred successfully to the recipient through conjugation.²⁹ The presence of resistance genes on plasmid size ranges 6–66 kb were identified in this study. The MICs for those transconjugants were lower than those obtained for clinical strains, showing that multiple carbapenem resistance mechanisms are possibly present in those clinical isolates.

Conclusion

This study indicated the emergence of NDM-producing Gram-negative bacteria in the NICU. Through molecular characterization, epidemiological studies and observation may permit for timely approaches in the prevention of this global public health threat. Hospital environment is the main site of Carbapenem resistant Enterobacteriaceae (CRE)-based infections. These organisms can be transferred to the neonates through medical equipment such as ventilators and personal contact between the caretakers and the infants hospitalized in the same ward. Therefore, the nosocomial surveillance system should play an important role in the infection control to limit the spread of NDM-producing pathogens.

Footnotes

Acknowledgment

The Internal funds of Biotechnology Unit are highly acknowledged for the support of this study.

Disclosure Statement

No competing financial interests exist.

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