Doctoral Degree. University of KwaZulu-Natal, Durban.Gram-negative ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp) pathogens are a major healthcare concern globally due to their increasing multidrug resistance and ability to cause debilitating infections. Phenotypic and genotypic characteristics of multidrug resistant Gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana were investigated. Two hundred (200) clinical, non-duplicate Gram-negative bacterial pathogens were randomly selected from various human specimens routinely processed by the diagnostic microbiological laboratory in the hospital. Multidrug resistant (isolates resistant to at least one agent in three or more antibiotic class) isolates selected from each group of Gram-negative ESKAPE pathogens constituted the final sample. Identification and antibiotic susceptibility profiles were carried out using Vitek-2. Identity of isolates for whole genome sequencing was further confirmed by MALDI-TOF MS. Four P. aeruginosa and 10 K. pneumoniae were subjected to whole genome sequencing based on their extensively drug resistant profiles and resistance to third-generation cephalosporins respectively using Illumina MiSeq, after genomic DNA extraction using the NucliSens easyMAG®. Antibiotic resistance genes and plasmids were identified by mapping the sequence data to an online database using ResFinder and plasmidFinder respectively. MLST was also determined from the WGS data. The raw read sequences and assembled whole genome contigs have been deposited in GenBank under project number PRJNA411997. An average multidrug resistance of 89.5% was observed, ranging from 53.8% in Enterobacter spp to 100.0% in Acinetobacter spp and P. aeruginosa. Gram-negative ESKAPE bacteria constituted 48.5% (97) of the 200 isolates. P. aeruginosa (n=4) belonging to ST234 harboured blaDIM-1, blaIMP-34, blaOXA-10, blaOXA-129, blaOXA-50, blaPAO aadA1, aac4 aph(3’)-IIb, fosA, sul1, dfrB5, catB7, arr-2 conferring resistance to β-lactams, aminoglycosides, fosfomycin, sulphonamides, trimethoprim phenicols and rifampin respectively. qnrVC was detected in two of the four isolates
. Both blaDIM-1 and blaIMP-34-like positive contigs showed identical DNA sequences and were linked to type 1 integron structures. BlaDIM-1 was 100% identical to the blaDIM-1 prototype gene, while blaIMP-34-like had two base pair (bp) differences T190C and C314G respectively compared to blaIMP-34, leading to one amino acid substitution in IMP-34-like indicating that, the gene may have independently evolved, perhaps due to selection pressure. Blast analysis did not reveal identical genetic structures deposited in NCBI, neither among the nucleotide collection, completed genomes nor among the completed plasmids. β-lactamases (blaCTX-M-15, blaSHV-11, blaTEM-1B) and resistance genes for aminoglycosides (aac(3)-IIa-like,aph(3')-Ia) quinolones/fluoroquinolones (oqxA-like,oqxB-like,qnrB10-like,qnrB2) and others including fosfomycin (fosA), trimethoprim (dfrA14), and sulphonamide (sul2) were found in the K. pneumoniae (n=10). Multiple and diverse mutations of the quinolone resistance-determining regions gyrA, gyrB and parC genes were detected in the K. pneumoniae (n=4), which were clonally distinct. The diversity of resistance genes expressed by Gram-negative ESKAPE pathogens conferring resistance to multiple antibiotics is problematic in a resource-constrained country like Ghana, necessitating urgent antibiotic stewardship and infection prevention and control interventions
