Emergence of New Delhi Metallo-β-Lactamase (NDM) Producing Multidrug-Resistant Escherichia Coli in Sudan

Emergence of New Delhi Metallo-β-Lactamase (NDM) Producing Multidrug-Resistant Escherichia Coli in Sudan This study investigated the prevalance of New Delhi Metallo-β-Lactamase (NDM) resistant gene in 57 E. coli collected clinical isolates from hospitals in Khartoum state, Sudan. An emerging carbapenemase, New Delhi metallo-lactamase (NDM), was first reported in a Swedish patient with a hospitalization history in India, it exhibited resistant to all β-lactams except for monobactams, and has great potential to cause global health crisis [1]. Initially, bla NDM gene was endemic to India subcontinent and NDM-producing isolates tested worldwide have geographical links with these high prevalence areas. Out of 200 Escherichia coli collected from clinical samples; 57 resistant strains were identified in different Khartoum state hospitals (n= 57). These resistance isolates were processed by bacteriological conventional methods and then were tested by antimicrobial profile and concentrated on Meropenem and Imipenem antibiotics and then RT-PCR with β-lactamase NDM primer were done. The NDM positive E.coli were 48 (80.7%) from total (n=57) resistant isolates. Within the positive blaNDM; 40.3% were resistant to Imipenem, 59.6% sensitive to Imipenem. bla NDM positive showed sensitivity profile to Meropenem; 38.5% of them were resistant to Meropenem, 61.5% were sensitive to Meropenem. The MHT showed that 56.2% of the bla NDM positive organisms were positive for Imipenem MHT and 64.9% were positive for Meropenem MHT. Our findings provides additional evidence that the majority of the circulating clinical isolates Escherichia coli in Khartoum hospitals in Sudan harbouring NDM antibiotic resistance gene.

he was hospitalized, initially in the Punjab area, but then in New Delhi, for the management of a gluteal abscess. In January 2008 he was admitted to a hospital in Orebro, Sweden, where, on the day after admission, a urine culture yielded an isolate of Klebsiella pneumoniae that was resistant to multiple antibiotics including Carbapenems (ertapenem, Imipenem and Meropenem) [7]. This strain was not isolated from any subsequent cultures, but stool samples tested following transfer of the patient to a nursing home in March 2008 yielded a Carbapenems resistant strain of Escherichia coli. Phenotypic testing of both isolates suggested that the Carbapenems resistance was due to the production of a Metalloβ-Lactamase (MBL), but PCR analysis failed to detect known MBL genes. The global dissemination of carbapenem resistance has recently received much attention especially following reports of the international spread of Enterobacteriaceae strains producing carbapenem mases such as KPC and NDM from endemic areas [8].
This dissemination is facilitated by increased international travel, which has been described as a risk factor for the acquisition of infections due to drug resistant organisms such as CTX-M-producing E. coli, as well as various carbapenemase-producing Gram-negative bacteria, including NDM and KPC producers [9].

Material and Methods
This study was conducted at the Khartoum teaching Hospital, Bahri, and Omdurman hospitals. Receiving referrals from 3 districts and serving a population of more than sex million) in the period between December 2016 to November 2018.

Antimicrobial Susceptibility Testing
The enzymatic tests of meropenem and imipenem were done after diffusion method that measured on Muller Hinton Agar plates (Oxoid, Basingstoke, UK) by the agar diffusion method and interpreted according to CLSI [10]. Meropenem and Imipenem discs were purchased from Himedia, Mumbai (India). Antimicrobial profile against Escherichia coli drugs was done for the carbapenemresistant isolates using disc-diffusion method according to CLSI [10].

DNA Extraction and Sample Preparation
DNA extraction was performed using iNtRON genomic DNA extraction kit. The isolates were cultivated in nutrient agar and incubated at 37 O C overnight aerobically. A 300 µl of heavy organism suspension was prepared in a 1.5ml tube to be extracted. The kit consists of washing buffer, lysis buffer, protein precipitation solution and rehydration solution as well as 70% Ethanol. All the materials were brought to room temperature before use.

Extraction Protocol
A volume 900µl of washing solution was added to the prepared sample to remove all media artifacts and metabolic products of the organism. Vortexed sufficiently the mixture was then incubated for 10min on ice and centrifuged at 13,000rpm for 30sec to obtain clean cell pellet. The supernatant solution was removed, and the cell pallet was suspended and 600µl of Lysis Buffer was added to the tube. To enhance the lysis, the lysate was incubated in a sonication water bath at 65c˚ and frequency of 50KHZ for 10 minutes. To prevent the protein contamination, 200µl of Protein PPT Buffer was added and vortexed for 10 secs, then incubated in ice bath for 5min.
After incubation, the samples were centrifuged at 13,000rpm for 5 min at room temperature. The supernatant was then transferred to a fresh 1.5 tube and 600µl of isopropanol was added and mixed well then, the mixture was centrifuged at 13,000rpm for 1minutes at room temperature. To precipitate the DNA, 600µl of 70% Ethanol was added and gently mixed, the tubes were then centrifuged at 13,000rpm for 1 min at room temperature. Then the Ethanol was removed, and the DNA pallet was dried. For DNA rehydration 100µl of DNA Rehydration Solution was added to the tubes. All samples were stored at -20c.

Real-Time PCR Conditions
Using Sacycler 96 instrument of Sacace biotechnology, real-time

Results
In the bla NDM ; 80.7%of the samples were positive (Figures 1 &   2). Strong positive samples were indicated by the concentration of the PCR product and fluorescent reading.
bla NDM positive showed sensitivity profile to Meropenem; 38.5% of them were resistant to Meropenem, 61.5% were sensitive to Meropenem ( Figure 3).

bla NDM and Enzyme Assay
Enzymes Assays reveled the following; 64.9% of the bla NDM positive organisms were positive in the Meropenem enzyme assay, and 56% were positive for the Imipenem enzyme assay (Figure 4).  Africa and Middle East countries such as Ethiopia [12], Kenya [13], Uganda [14], Egypt [15]. , South Africa [16], Saudi Arabia [17], Iraqi [18], Kuwait [19], and Lebanon [20], were agreed with our study and were found a high rate of bla-NDM gene. In the same our view many studies were found increased rate of NDM as Kumarasamy and colleagues [21], provide compelling evidence that NDMproducing Enterobacteriaceae (mostly K. pneumoniae and E. coli) are widespread in India and Pakistan. They also found that many isolates from different hospitals in Southern Vietnam [28].

Conclusion
The outcome of this work revealed that in Sudan must be take