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Browsing by Author "Agyepong, Nicholas."

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    Molecular characterization of multi-drug resistant (MDR) gram-negative bacterial pathogens from environments, patients and staff in a teaching hospital in Ghana.
    (2023) Yeboah, Esther Eyram Asare.; Essack, Sabiha Yusuf.; Owusu-Ofori, Alexander.; Agyepong, Nicholas.; Abia, Akebe Luther King.; Amoako, Daniel Gyamfi.; Mbanga, Joshua.
    Multidrug resistant Gram-negative bacteria (MDR GNB) are implicated in serious infections both of community and nosocomial origin and may be disseminated in the hospital in the absence of efficient infection prevention and control (IPC) practices. The prevalence and risk factors for rectal colonization of MDR GNB among patients, the carriage of MDR GNB on healthcare workers’ (HCWs’) hands and the contamination patients’ environments with MDR GNB were investigated in a teaching hospital in Ghana. In this prospective study, conducted between April 2021 to July 2021, the phenotypic profiles of the MDR GNB isolates were determined using the VITEK 2 system. Risk factors for colonization with MDR GNB were assessed using univariate and multivariate analysis of associated data. The resistome, virulome, mobilome and genetic relatedness of MDR extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and ESBL-producing or carbapenem resistant Klebsiella pneumoniae isolates from patients and their environment were also determined using whole genome sequencing performed on the Nextseq 550 (2 x 150 bp) and bioinformatics analysis. A total of 585 samples were collected from patients, HCWs’ hands and the hospital environment within the study period. The prevalence of MDR GNB rectal colonization among patients was 50.62% on admission and 44.44% after 48 hours. MDR GNB, frequently E. coli and K. pneumoniae were isolated from 6 (5.26%) and 24 (11.54%) of HCW’s hand swabs and environmental swabs, respectively. Previous hospitalization (p-value = 0.021, OR,95% CI= 7.170 (1.345-38.214) was significantly associated with colonization by MDR GNB after 48 hours of admission while age (21-30 years) (p-value =0.022, OR, 95% CI =0.103(0.015-0.716) was significantly identified as a protective factor associated with a reduced risk of rectal MDR GNB colonization. Rectal carriage and acquisition of ESBL-producing E. coli among patients was 13.65% and 11.32% respectively. blaTEM-1B and blaCTX-M-15 were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Multiple virulence genes, predominantly, terC were detected in the ESBL E. coli isolates. Sequence types (STs) were diverse and included one novel ST (ST13846) present in two isolates. Phylogenetic analysis grouped the ESBL E. coli isolates into four main clusters. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities. Of the ten selected MDR K. pneumoniae isolates, the β-lactamase gene, blaCTX-M-15 was observed in six isolates. Mutations were found in both ompK36 and ompK37 in all isolates (both carriage isolates and isolates from hospital environments). Genes encoding resistance to fluoroquinolone (qnrB), aminoglycosides (aadA1, aadA2, aac(3)-IIa, aac(6')-Ib-cr,aph(3'')-Ib , aph(6)-Id) sulphamethoxazole/trimethoprim (sul1, sul2, dfrA14, dfrA15) were also detected. The K. pneumoniae isolates belonged to seventeen different STs with ST39 most commonly observed and common to both carriage isolates and isolates from hospital environments. A myriad of virulence genes, including irp1, irp2, iutA, gndA, ompA, fes, fep, mrkD and fimH were detected in both carriage and isolates from the hospital environment. IncFIB was the most abundant plasmid replicon occurring in nine (four carriage isolates and five isolates from hospital environments). ESBL-producing K. pneumoniae isolates appeared to be introduced into the hospital from the community. The high colonization of MDR GNB in patients, the carriage of MDR GNB on HCW’s hands, the contamination of hospital environments and the circulation of ESBL-producing E. coli and K. pneumoniae isolates with diverse genomic characteristics, highlights the need for patient screening, and stringent infection prevention and control practices to prevent the spread of MDR GNB in hospitals. The observed clonal relatedness among isolates from patients and the hospital environment, as well as between different patients, suggests a possible transmission within and between sources, hence infection prevention and control practices need to be enhanced to prevent the dissemination and transmission of these resistant strains in the hospital. This study further highlights the usefulness of whole genome sequencing as an effective tool in AMR surveillance.
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    Molecular profile of gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana.
    (2017) Agyepong, Nicholas.; Essack, Sabiha Yusuf.; Owusu-Ofori, Alex.; Govinden, Usha.
    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.