Molecular Detection of New Delhi Metallo Beta Lactamase 1 (NDM-1) Producing Bacterial Isolates in Kano- Northwestern Nigeria

New Delhi Metallo Beta Lactamase 1 (NDM-1) is an enzyme with zinc ions at its active site that cleaves the amide bond of β -lactam ring and provides resistance against major classes of β lactam antibiotics. The molecular detection of NDM-1 producing bacterial isolates from tertiary Hospitals in Kano was investigated. A total of 500 bacterial isolates of Enterobacteriaceae and Pseudomonas aeruginosa from samples of blood, urine, catheter tip were screened for NDM-1 over a period of 12 months. The isolates were screened preliminarily for carbapenemases using meropenem (10 µg) and imipenem (10 µg) by disc diffusion technique. Isolates of 23 mm and 21 mm for meropenem and imipenem respectively were confirmed by modified Hodge test then EDTA Disc Synergy Test using two meropenem discs, one with MEM (10 µg), and other containing 10 µl of 0.1 M anhydrous EDTA (292 µg) for Metallo Beta Lactamase (MBLs) and finally seventeen MBLs isolates were screened with NDM-1 specific primers by PCR then four PCR positive products were sequenced for bla NDM-1 gene. Of the 500 clinical bacterial isolates screened, 162(32.4%), 43(8.6%) and 4(0.8%) were found to produce carbapenemase, MBLs and NDM-1 respectively. The highest frequency of NDM-1 producers was found among Escherichia coli 3(1.6%) followed by Klebsiella pneumoniae 1(0.5%) . Based on clinical samples, blood (25.0%) was found to have highest prevalence of MBLs followed by catheter tips (21.0%), wound swabs (11.1%) and urine (6.3%). Conclusively, NDM-1 was first detected in Kano, Nigeria.


INTRODUCTION
New Delhi Metallo Beta Lactamase-1 (NDM-1) is a newly described Metallo Beta Lactamase (MBLs) that was first identified in 2009 from a single isolates of Klebsiella pneumoniae and Escherichia coli; both recovered from a patient repatriated to Sweden after treatment in New Delhi hospital, India [1]. NDM-1 is an enzyme that cleaves the amide bond of β-lactam ring and provides resistance against major classes of β-lactam antibiotics [2]. It has zinc ions at its active site that hydrolyses all beta lactam antibiotics excluding aztreonam [1,3].
After the initial report; the Health Protection Agency (HPA) in the United Kingdom (UK) concerned over the rapid increase in the number of cases of human colonization and infection with NDM-1 and other carbapenemases producing Enterobacteriaceae in hospitals across the country has raised a national alert in July 2009 [4]. Similarly, to the first case of NDM-1, the majority of patients with NDM-1 positive bacteria in the UK had a history of travel to India or Pakistan where many of them had been hospitalized with various indications including elective surgery and renal dialysis [5]. However, it is presumed that there are other reservoirs of infected patients in the Balkan countries and Middle East. Moreover, NDM-1-producing bacteria have been recovered from many infections such as urinary tract infections, pneumonia, septicaemia, wound and deviceassociated infections [3]. NDM is reported almost worldwide but did not successfully spread in most countries of Europe except the UK and recently France [6].
New Delhi Metallo-β-lactamase-1 gene (bla NDM-1 ) codes for NDM-1 [2]. An association with other resistance mechanisms makes majority of Enterobacteriaceae with bla NDM-1 gene extensively resistant to antibiotics and susceptible only to colistin and less consistently, tigecycline [1]. Dissemination of the plasmid borne bla NDM-1 through horizontal gene transfer is a potential threat to the society [2]. Therefore, this research aimed at detecting the presence of NDM-1 producers in clinical bacterial isolates in Kano-Nigeria.

METHODOLOGY
A total of 500 clinical bacterial isolates were collected from Microbiology Departments of Aminu Kano Teaching Hospital, Muhammad Abdullahi Wase Specialist Hospital and Murtala Muhammad specialists Hospital, Kano, Nigeria after obtaining an ethical clearance from the respective hospitals' ethical committees. Bacterial isolates were characterized using biochemical tests for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi and Salmonella paratyphi and screened for carbapenemase production according to the procedure described by Clinical and Laboratory Standard Institute guidelines using disc diffusion techniques with imipenem (10 µg) and meropenem (10 µg) obtained from oxoid UK. Any isolate that exhibited resistance or reduced susceptibility of 23 mm and 21 mm for meropenem and imipenem, respectively were subjected to further confirmatory tests [7].
Modified Hodge test was performed to confirm Carbapenemase production as described by the CLSI guidelines using Disc diffusion techniques with IPM (10 µg) and MEM (10 µg).
EDTA disc synergy test was performed as described by the CLSI guidelines [7] using Disc diffusion techniques with two meropenem discs; one with MEM (10 µg) and other containing 10 µl of 0.1 M anhydrous EDTA (292 µg). A strain producing a diameter of >4 mm around the disc with MEM-EDTA and not around the disc with MEM alone was considered phenotypically positive for NDM-1. Escherichia coli ATCC 25922 was used as the control strain.
Phenol chloroform method was used for DNA extraction according to manufacturer's instructions (ThermoFisher Scientific).The DNA was subjected to Polymerase Chain Reaction (PCR) with bla NDM-1 gene primers NDM-Fm (5′-GGTTTGGCGATCTGGTTTTC-3′,) and NDM-Rm (5′ CGGAATGGCTCATCACGATC-3′,), as designed by Nordmann et al. [8]. Using 50µl micro test tubes, 1.5 µl of NDM-1 primers each were pipetted and dispensed into the tubes; then 0.2 ml of dNTPS each, the cofactor (mgCl 2 ) 1.5 mM g M g Cl 2 , 14 mM tris-HCl Buffer (PH 8.2) and the Taq polymerase of 1.0 µl were added. Finally, 2 µl of the template DNA were also added to the reaction mixture. Klebsiella pneumoiae NCTC 13443 was used as the bla NDM-1 positive control. Then, the following conditions were used 94°C for 5 minutes, 94°C for 30 seconds, 43°C for 30 seconds, 72°C for 1 minute, and 72°C for 10 minutes for 35 cycles. The amplicons were run on 1.5% agarose gel in TAE buffer at 120 volt for 1 hour. The DNA bands were visualized using UV light box (Gel documentation Unit).
Four PCR positive products were sequenced by Sanger sequencing dye termination method using Beckman Coulter Kit and setup according to manufacturer's instructions. Finally the DNA sequence was compared using Basic Local Alignment Search Tool (BLAST).

RESULTS
Out of 500 clinical bacterial isolates screened, 162(32.4%) were found to produce carbapenemase. Frequency of phenotypically detected New Delhi Metallo Beta Lactamases (MBLs) in this study was found to be 43(8.6%) ( Table 1).
A representative result of Gel electrophoresis showing bla NDM-1 gene was given in Fig. 1.

DISCUSSION
The prevalence of carbapenemase producing bacterial pathogens (32.4%) was recorded in this study which is higher compared to that reported by Yusuf and Arzai [9] and Motayo et al. [10] with 14% in Kano, Northwest and 9.3% in Abeokuta, Southwest Nigeria respectively. However, it is lower to that of Yusuf et al. (34.5%). [11] According to the 2009 data from the European Antimicrobial Resistance Surveillance Network, the rates of carbapenem resistance were: 43.5% in Greece, 17.0% in Cyprus, 1.3% in Italy, 1.2% in Belgium and below 1% in other 23 reporting  [14]. The differences in prevalence rates may be due to the differences in sample size and study area.

CONCLUSION
A novel carbapenemase, New Delhi Metallo Beta Lactamase 1(NDM-1) was first detected among clinical bacterial isolates in Kano, Northwestern Nigeria. Prevalence of NDM-1 producers is highest among blood samples. Therefore its identification in clinical bacterial infections will be suspected on any decreased susceptibility to carbapenem in Enterobacteriaceae.