Demographic distribution
The demographic characteristics of the inpatients and the frequency of GNB isolates according to age groups are shown in Figure 1. Most of the isolates were from pediatric patients less than one year old (42.5%), followed by age group 13-80 years (38%) and the remainder of paediatric patients age group 1-12 years (19.5%). Males 53.4% (110/206) were predominant among inpatients with females at 46.6% (96/206).
Antimicrobial susceptibility
The antibiotic resistance pattern is shown in Figure 2. Out of 206 isolates tested, the highest percentage resistance was 98% and 93.5%, for ampicillin and cephalexin respectively, followed by amoxicillin clavulanic acid 90%, cefotaxime 89.7%, ceftriaxone 88.4%, ceftazidime 84.2%, aztreonam 66%, temocillin 64%, sulfamethoxazole-trimethoprim 78.4%, nitrofurantoin 75.2%. The resistance rate was also high in ciprofloxacin 83.1%, gentamicin 85% and amikacin 70%. The resistance rate for meropenem and imipenem was 63.1% and 61.6%, respectively.
Prevalence of carbapenemase producing Gram-negative bacilli based on phenotypic tests
Carbapenemase activity was detected in 171 (83%) of the 206 clinical isolates. These isolates were positive for the production of one or more carbapenemase enzymes by phenotypic methods as the following; 24 (11.7%) by Modified Hodge test (MHT) method and Boronic acid screen, 105 (51%) by the EDTA test and 27 (13.1%) of the isolates were positive for both EDTA and Boronic acid methods while 15 (7.2%) were positive with all methods. Carbapenemase enzymes were predominant in Klebsiella pneumoniae isolates: 71(41.5%), followed by Pseudomonas aeruginosa: 33(19.3%); Acinetobacter baumannii: 30(17.6%) and Escherichia coli 26(15.2%). Details of the carbapenemase activity among different isolates by phenotypic tests are given in Table 1. This suggests that the MBL type is the most prevalent type of carbapenemase hydrolysis enzyme among Gram-negative bacilli, 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(52%), followed by blaIMP 7 (3.4%), blaOXA-48 5(2.4%), blaVIM 2 (0.9%) and blaKPC 0 (0%). ESBL were detected among these isolates with high prevalence in 183 isolates (88.8%) as the following; blaCTXM 126(61.6%), blaSHV 84(40.7%) and blaTEM 78(37.8). The genes were unevenly distributed among the different study isolates and more details are given in Table 2.
Co-resistance genes carried with NDM gene among Gram-negative bacilli
Several 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 38(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 in 15 isolates (17.3%). The distribution of co-resistance genes among different Gram negative bacilli is shown in Table 3.
The frequency of carbapenemase producer Gram-negative bacilli by type of specimens and hospital units
Carbapenemase producing isolates were more frequently distributed among the following clinical specimens; blood (36%) followed by wound samples (24%), urine (21%), body fluids (7%), catheter tips (6%) and sputum samples (6%).
With regard to the distribution of carbapenemase producers among hospital location, the most carbapenemase producing isolates were found in the neonatal intensive care unit 32(26%), followed by medicine wards 26(22%), pediatric wards 22 (18%), surgery 18(15%), ICU 15(12%) and renal unit 8(7%).
Molecular characterisation 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 Figure 3.
Bioinformatics analysis of blaNDM genes
A subset samples were analysed to confirm the presumed most prevalent (NDM) gene type. Fourteen samples were sequenced and all showed 97-100% similarity with blaNDM genes from the NCBI database with accession number MF379688 and MG764089.
Multiple sequence alignment:
The nucleotide sequence of NDM Deoxyribonucleic acid (DNA) sequences were compared against the DNA databank using BLASTp. Fourteen NDM beta-lactamase genes were compared against those NDM genes recorded in the database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The alignment of the NDM genes in the isolates were shown to have identical similarities within range of 97-100% with those available in the database.When multiple sequence alignment of NDM proteins was undertaken using MEGA7 software version 7.0.9.0 against similar proteins obtained from BLASTp, NDM-1 from Sudan was similar to the |KX100583.1| Escherichia coli NDM-1 (blaNDM) gene from India and the | MH891562| Klebsiella pneumoniae NDM-1 from Bangladesh. NDM-5 from Sudan was similar to | MH991817 |, Escherichia coli NDM-5 from India and | MH168510| Klebsiella pneumoniae NDM-5 from Bangladesh while NDM-6 from Sudan was 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 the following accession numbers: MK033562, MK033563, MK033564, MK363705, MK363706, MK363707, MK363708, MK363709, MK363710, MK371542, MK371543, MK371544, MK37154 5, and MK371546.
Phylogenetic tree
The phylogenetic analysis of the NDM protein sequences revealed that 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 Figure 3.