The demographic characteristics of patients under study and the frequency of GNB isolates according to age groups are shown in (Figure.1). The most of the isolates were from neonates less than one year old in 42.5% followed by adult group between 13–80 years 38.3% and 19.5% for age group between 1–12 years. Males 53.4% (110/206) were predominant among admitted wards patients and females 46.6% (96/206).
The antibiotic resistance pattern is shown in (Fig. 2). Out of 206 isolates tested, the highest percentage of resistance 98%, 93.5% were found in ampicillin and cephalexin respectively, followed by amoxicillin clavulanic acid 90%, cefotaxime 89.7%, ceftriaxone 88.4%, ceftazidime 84.2% and aztreonam 66%, temocillin 64%, Sulfamethoxasole-trimethoprim 78.4% and nitrofurantoin 75.2%. The resistance rate was also higher in ciprofloxacin 83.1%, gentamicin 85% and amikacin 70%. High resistant rate associated with meropenem 63.1% and imipenem 61.6%.
Prevalence of carbapenemase producing Gram-negative bacilli based on phenotypic tests
Carbapenemase activity was detected in 171 (83%) of the 206 clinical isolates, which were positive for the production of one or more carbapenemase enzymes by phenotypic methods as the following 24 (14%) by MHT method and Boronic acid screen, 105 (61.5%) by the EDTA test and 42 (24.5%) of the isolates were positive for both EDTA and Boronic acid methods. Details of the carbapenemase activity among different isolates by phenotypic tests are shown in Table 3. This suggests that the MBL type is the most prevalent type of carbapenemase hydrolysis enzyme among Gram-negative bacilli in Khartoum state, OXA and KPC types are present at a low level.
Prevalence and distribution of Carbapenemase genes among Gram negative bacilli
Carbapenemase genes were detected in 121 (58.7%) of the 206 study isolates using PCR, one or more carbapenemase genes were detected in the isolates. blaNDM was the most commonly detected among the isolates, mainly in K. pneumonia, which was the species with the highest number of these genes. blaNDM was also detected more often in A. baumannii, P. aeruginosa and E. coli. The most prevalent gene was blaNDM 107(88.4%), followed by blaIMP 7 (5.7%), blaOXA-48 5(4.1%), blaVIM 2 (1.6%) and blaKPC 0 (0%). ESBL were detected among these isolates, it showed a high prevalence in 183 isolates (88.8%) as the following blaCTXM 127(61.6%), blaSHV 84(40.7%) and blaTEM 80(38.8). The genes were unevenly distributed among the different study isolates. For more details, see Table 4.
Co resistance genes carried with NDM gene among Gram-negative bacilli
Many isolates carried more than one gene with blaNDM gene. Co-resistance carbapenemase genes were observed in a small number of isolates, blaNDM + blaOXA-48 were detected in three isolates, while blaNDM + blaVIM and blaNDM + blaIMP were detected in two different isolates. On the other hand, ESBL were often observed together with blaNDM in 87 (81.3%) of blaNDM positive isolates (107). Most of the isolates carried blaNDM with one ESBL gene in (43.5%) as the following; blaNDM + blaCTXM in (24 isolates, 27.6%), blaNDM + blaTEM (8 isolates, 9.1%), and blaNDM + blaSHV (6 isolates, 6.8%). Isolates carried blaNDM with two ESBL genes in (39.2%) as the following: blaNDM + blaCTXM + blaSHV (10 isolates, 11.5%), blaNDM + blaCTXM + blaTEM (10 isolates, 11.5%), blaNDM + blaSHV + blaTEM (14 isolates, 16.2%). Isolates carried blaNDM with three ESBL genes, blaNDM + blaCTXM + blaSHV + blaTEM (15 isolates, 17.3%). The distribution of co resistance genes among different Gram negative bacilli is shown in Table 5.
The frequency of carbapenemase producer Gram-negative bacilli by type of specimens, hospital units and bacteria species isolates.
Carbapenemase producing isolates were more frequently distributed among clinical specimens including blood (36%) and then followed by wounds (24%), urine (21%), body fluids (7%), catheter tips (6%) and sputum samples were (6%).
With regard to the distribution of Carbapenemase producer among hospital units, the most carbapenemase producing isolates associated with patients were found in the neonatal intensive care unit NICU 32(26%), followed by medicine wards 26(22%) and pediatric wards 22 (18%), Surgery 18(15%), ICU 15(12%) and Renal Unit 8(7%). Carbapenemase genes were predominant in Klebsiella pneumoniae isolates: 70(42.6%), followed by Pseudomonas aeruginosa: 33(20%); Acinetobacter baumannii: 30(18.2%) and Escherichia coli 18(10.9%)
Molecular characterization of NDM genes
Out of 107 blaNDM genes detected 75 (70%) were blaNDM-1. Other subtypes of blaNDM were identified by sequencing including blaNDM- 5, and blaNDM-6. (Fig. 3)
Bioinformatics analysis of blaNDM genes
Fourteen samples were sequenced by and all showed 97–100% similarity with blaNDM genes from the NCBI database with accession number MF379688 and MG764089.
Multiple sequence alignment:
The Nucleotide sequences of NDM Deoxyribonucleic acid (DNA) sequences were compared against the DNA databank using BLASTp. Fourteen NDM beta-lactamases genes were compared against the NDM in the database, (http://blast.ncbi.nlm.nih.gov/Blast.cgi), they have produced a significant alignment to NDM-1 beta-lactamase of Klebsiella pneumoniae (gb|MK425054|), and the isolates were shown to have 97–100% identity.
When multiple sequence alignment of NDM proteins was undertaken using MEGA7 software version 18.104.22.168 against similar proteins that obtained from BLASTp, NDM-1 from Sudan were similar to |KX100583.1| Escherichia coli NDM-1 (blaNDM) gene from India and | MH891562| Klebsiella pneumoniae NDM-1 from Bangladesh. NDM-5 from Sudan were similar to | MH991817 |, Escherichia coli NDM-5 from India and | MH168510| Klebsiella pneumoniae NDM-5 from Bangladesh while NDM-6 from Sudan were similar to | MH683607 | Escherichia coli from India, | JN967644 | Escherichia coli from the United States and | JQ235754 | Escherichia coli from New Zealand.
Nucleotide sequence accession number
The sequence of the 14 NDM genes have been deposited in the GenBank database under accession numbers MK033562, MK033563, MK033564, MK363705, MK363706, MK363707, MK363708, MK363709, MK363710, MK371542, MK371543, MK371544, MK37154 5, and MK371546.
The phylogenetic analysis of the NDM proteins sequences revealed that the NDM-1 and NDM-5 were related to the same NDM lineage as the Indian and Bangladeshi isolates. The NDM-6 gene was found to be close to NDM-6 from India, New Zealand, and the United States as shown in Fig. 3.