Occurrence of blaNDM Variants Among Enterobacteriaceae From a Neonatal Intensive Care Unit in a Northern India Hospital

Carbapenem-resistance among enterobacteriaceae has become a global health concern. The objective of this study was to understand NDM producing enterobacteriaceae and their genetic basis of resistance, spreading in neonatal intensive care unit. Carbapenem resistant NDM producing enterobacteriaceae isolates were recovered from rectal swab and blood sample of infants admitted in NICU. These were determined by using Carba-NP test. All isolates were identified using BD PhoenixTM−100 and MICs were determined by broth microdilution method. The blaNDM and associated resistant markers were checked by PCR followed by sequencing. Moreover, ERIC-PCR and genetic environment of blaNDM gene were also performed for the analysis of clonal relationship and genetic surrounding of the strains. We characterized 44 isolates with blaNDM variants in Escherichia coli (45.5%), Klebsiella pneumoniae (40.9%), Citrobacter freundii (4.5%), Citrobacter braakii (2.3%), Klebsiella oxytoca (2.3%), Enterobacter cloacae (2.3%), Enterobacter aerogenes (2.2%) from NICU, showing resistance against all antibiotics except colistin and polymixin B. ISAba125 and bleomycin gene were found surrounding all blaNDM variants, besides class I integron on plasmid. (ERIC)-PCR data revealed non-clonal relatedness among most of the isolates. The transfer of resistant markers was confirmed by conjugation experiment. The PCR-based replicon typing was carried out using DNA of transconjugants. These isolates carried NDM-1 (20.45%), NDM-4 (36.36%), NDM-5 (38.64%), NDM-7 (4.55%), along with OXA, CMY, and SHV variants on conjugative plasmid of IncFIA, IncFIC, IncF, IncK, IncFIB, IncB/O, IncHI1, IncP, IncY, IncFIIA, IncI1, and IncN types. An increased number of carbapenem-resistant NDM producing enterobacteriaceae isolates recovered from NICU which is alarming signal for health workers and policy makers. Hence, it is utmost important to think about infection control measures.


INTRODUCTION
Emergence of New-Delhi Metallo-β-lactamase (NDM) producers is a matter of concern. The spread of MBL-producing enterobacteriaceae has increased from 2008 onward with the discovery of an ST14 Klebsiella pneumoniae with a new MBL gene, bla NDM−1 , from a 59-years old Swedish patient who received healthcare in New Delhi, India (Yong et al., 2009). Indian subcontinent are the most endemic region for the spread of NDM-type MBLs and prevalence rates of NDM-producing enterobacteriaceae were found in range of 5-18.5% in Indian and Pakistan hospitals (Perry et al., 2011;Bharadwaj et al., 2012). In other regions (except the Balkan and Middle East countries), NDM-type MBLs are described mostly as periodic occurrences (Dortet et al., 2014). Carbapenem-resistant microorganisms have become an alarming phenomenon in children (Logan, 2012). A recently published study in USA reported that the frequency of carbapenem resistance increased from 0% in 1999-2000 to 0.47% in 2010-2011 among Enterobacteriaceae isolates in children (Logan et al., 2015). To date, 19 variants of NDM-type carbapenemases (NDM-1 to  have been identified (http://www.lahey. org/Studies/other.asp#table1). These variants were identified in expanded species of Gram-negative bacteria and were found to have variation either by multiple residues at different positions or by replacing single amino acid. Recently, an NDM-4, NDM-5, and NDM-7 producing Enterobacter aerogenes from NICU of Indian hospital were reported by our group (Ahmad et al., 2017a). The most widespread variants were found in Indian subcontinent, are NDM-1, NDM-4, NDM-5, NDM-6, and NDM-7 (Khan et al., 2017). Whereas, several types of carbapenemases, such as KPC, IMP, OXA-48, VIM, and New Delhi metallo-βlactamase (NDM), have been identified globally (Pitout et al., 2015;Logan and Weinstein, 2017).
NDM producing bacteria are resistant to almost all antibiotics, except polymyxins (Kumarasamy et al., 2010). But, the hope of colistin and polymyxins as treatment option has become limited after the discovery of MCR-1 gene in human and animals (Liu et al., 2016). The indiscriminate nature of the gene encoding NDM-1 has made major problem in neonatal intensive care units (NICU). In NICU, high consumption of antimicrobial agents, numerous indwelling devices, and staff rotativity, may further complicate the problem (Zaidi et al., 2005).
In enterobacteriaceae, bla NDM−1 is generally located on conjugative plasmids, ranging from 50 to 200 kb in size and belongs to several incompatibility groups, such as IncL/M, IncHI1, IncFIIs, IncF, or untypable, enabling transfer, and rapid dissemination of multidrug resistance (Poirel et al., 2011).
Our study was designed to evaluate retrospectively the spread of NDM producing Enterobacteriaceae and their genetic basis in neonatal intensive care unit of one of the north Indian tertiary care hospital.

Collection of Bacterial Strains and Hospital Setting
A total of 750 Enterobacteriaceae clinical isolates were screened from blood and rectal swab of 1,140 neonates admitted in neonatal intensive care unit (NICU) of Jawaharlal Nehru Medical College and Hospital (JNMCH), Aligarh Muslim University, Aligarh, India, during the period, December 2015 to January 2017 in which 308 isolates were found to be carbapenem resistant. It is a tertiary care hospital of 1,300 bed capacity, in which 90 beds were allotted for pediatric patients and 35 beds for the NICU. Patients enrolled in the study were those who enrolled in the active surveillance system (NICU stay 48 h and weekly surveillance swabs taken at least once). Neonates admitted to the ward before December 2015 and/or discharged after January 2017, were excluded.

Ethical Approval
A formal consent from the institutional ethical committee was taken and clearance was obtained from the institute's ethics committee. Participants/guardians had provided written, informed consent to participate in the study. We have a specific format to get the consents of patients/ parents of minors. These formats were made according to the Institutional ethics committee's guidelines. These forms are confidential and cannot be disclosed as per the guide lines. Institutional ethical committee has already approved. The name of committee/board is "Institutional Ethical Committee of Interdisciplinary Biotechnology Unit [Biot/307/01.06.13], " Aligarh Muslim University, Aligarh, India.
Antimicrobial Susceptibility, Metallo-β-Lactamase (MBL), and MICs Testing Antimicrobial susceptibility was determined by the standard disc diffusion method using Mueller Hinton agar plate as per the Clinical and Laboratory Standards Institute guidelines (CLSI, 2016). More than 05 colonies were picked from MH agar plate for antimicrobial susceptibility testing and MBL detection. Detection of metallo-β-lactamase activity was performed, using two imepenem discs (10 µg), one containing 10 µl of 0.1M anhydrous Ethylene Diamine Tetra-Acetic Acid (EDTA). The discs were placed 25 mm distance (center to center) on Mueller-Hinton agar plates (Ahmad et al., 2017b). Minimum Inhibitory Concentrations (MICs) for antimicrobial agents were determined using broth micro dilution method, according to the guidelines of the CLSI.

Carba NP Test for Detection of Carbapenemase
Carba NP test is a biochemical method used for the detection of carbapenemase activity in enterobacteriaceae isolates, performed as described earlier ).

Isolate Identification
The species level identification of isolates were performed by using BD Phoenix TM−100 automated microbiology system using panel NMIC/ID-55 (Gram negative susceptibility card) and further validated by 16s rRNA sequencing using primer as described previously (Shemesh et al., 2012).

Polymerase Chain Reaction (PCR) Amplification and Sequence Analysis
PCR (Applied Biosystems model-9902 Verity thermo cycler) amplification was performed using primers as described previously (Poirel et al., 2011;Ali et al., 2014) for bla NDM and other resistant marker (bla VIM , bla OXA−1 , bla OXA−9 , bla CMY , bla TEM , bla SHV , and bla KPC ). Amplicons of NDM were purified from the gel using gel extraction kit (Thermo Fisher Scientific), following manufacturers' protocol and then sequenced for DNA sequencing at Sci Genom Labs Private Ltd, Cochin, India. The nucleotide and deduced protein sequences were analyzed with software available at the National Centre for Biotechnology Information Website (www.ncbi.nlm.nih.gov).

Molecular Characterization of Plasmid
Plasmid DNA extraction and molecular size of multiple plasmids were identified by Kieser method (Kieser, 1984). Plasmid incompatibility group was determined by a PCR-based replicon typing (PBRT) method. Plasmid DNA was amplified by five multiplex and three simplex PCRs using 18 pair of primers as reported previously (Carattoli et al., 2005)

Genetic Environment Analysis
It was performed to identify the genes present at upstream and downstream of bla NDM variants as described previously (Poirel et al., 2011).

Integron Analysis
The transconjugants of all the isolates, with bla NDM , were subjected to undergo integron analysis, using PCR amplification of 3 ′ /5 ′ conserved segment along with Int1 and Sul1 as reported earlier .

Molecular Genotyping of Isolates
The clonally relatedness between NDM producing isolates were investigated by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) using the primers ERIC-Forward (5 ′ ATGT AAGCTCCTGGGGATTAAC-3 ′ ) and ERIC-Reverse (5 ′ AAGTAAGGACTGGGGTGAGCG-3 ′ ), was performed as described earlier (Versalovic et al., 1991). Bio-Red Gel Doc system was used to scan gel image and analyzed the bands by PyElph version 1.4 Software to generate a dendrogram by the un weighted pair group method using arithmetic averages (UPGMA) clustering (PyElph) (Pavel and Vasile, 2012).

Carbapenemase Production
All 44 NDM-producing enterobacteriaceae isolates were found positive for Carba-NP test, indicating the production of a carbapenemase as shown in Table 1.

Conjugation
The plasmidic location of resistant markers was determined by conjugation, using an azide-resistant E. coli J53 strain as the recipient [12]. Transconjugants were obtained at the frequencies of 10 −3 to 10 −5 cells, showing that plasmid from the donors (E. coli, K. pneumoniae, C. freundii, C. braakii, K. oxytoca, E. cloacae, E. aerogenes), were found stable in E. coli J53.

Integron Analysis
The transconjugants of all isolates harbored plasmid carrying class 1 integron, except two isolates (AK-90 and AK-103) which were confirmed by PCR amplification of 5 ′ /3 ′ CS, IntI, and SulI genes. We further confirmed that no resistant marker was present in the integron cassette as shown by a PCR using amplicon of 5 ′ /3 ′ CS as template.
Complete ISAba125 sequence was observed at upstream of bla NDM in most of the isolates implies that this factor may play a main role in horizontal gene transfer of the bla NDM among enterobacteriaceae members (Poirel et al., 2011). In all bla NDM variants, ble MBL was found at it downstream. The occurrence of ble MBL, associated with bla NDM gene, suggests that they might have mobilized simultaneously from same progenitor and is thought to protect bla NDM  . These results suggest that the plasmids encoding for carbapenem resistant NDM variants can easily spread among the enterobacteriaceae isolates. These results are in conformity with previous reports that clarified the horizontal transfer of plasmids encoding for carbapenemases among enterobacteriaceae including K. pneumoniae (Dortet et al., 2014;Jin et al., 2015).

CONCLUSIONS
Carbapenem resistance among enterobacteriaceae has been considered as one of the most significant menaces to the global healthcare, and the prevalence of NDM variants in enterobacteriaceae has further increased the threat. Therefore, the early detection of the bla NDM possessing enterobacteriaceae isolates with any decreased sensitivity to the carbapenems is crucial for the choice of the most appropriate antibiotic therapy and the application of efficient infection control measures. The emergence of such resistance patterns may be reduced by the restricted implementation of antibiotics, especially for carbapenems and cephalosporins. Moreover, a strong infection control management in the hospital is necessary to check such infection.

AUTHOR CONTRIBUTIONS
NA: performed experiments, wrote draft manuscript; SK: performed experiments; SA: provided samples, and interpreted clinical data; AK: designed study and checked draft manuscript.