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Browsing School of Health Sciences by Author "Amoako, Daniel Gyamfi."

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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 epidemiology of antibiotic-resistant Enterococcus spp. from farm-to-fork in intensive pig production in KwaZulu-Natal, South Africa.
    (2021) Badul, Sasha.; Essack, Sabiha Yusuf.; Bester, Linda Antionette.; Amoako, Daniel Gyamfi.; Akebe, Luther King Abia.
    Background: Substantial antibiotic use and high population densities in intensive farming systems results in the emergence and spread of antibiotic-resistant commensals and pathogens. This study investigated the molecular epidemiology of antibiotic resistance (ABR) and virulence in Enterococcus spp. from pigs in an intensive food production continuum from farm-to-fork in the uMgungundlovu district, Kwa-Zulu Natal. Methods: A total of 174 samples obtained along the pig farm-to-fork continuum (farm, transport, abattoir, and retail meat) were subjected to the quantification and putative identification of Enterococcus spp. using the IDEXX Enterolert® method and selective media, respectively. Up to three presumptive enterococcal colonies were picked per sampling point for molecular confirmation by real-time PCR, targeting the genus- and species-specific (tuf and sodA) genes, respectively. Antibiotic resistance profiles were determined by the Kirby-Bauer disk diffusion method against a panel of antibiotics for Enterococcus spp. recommended by the WHO-AGISAR using EUCAST guidelines. Selected antibiotic resistance and virulence genes were detected by real-time PCR. Clonal relatedness between isolates across the continuum was evaluated by REP-PCR. Results: A total of 284 isolates constituted the final sample. Real-time PCR confirmed 79.2% of the isolates as E. faecalis, 6.7% as E. faecium, 2.5% as E. casseliflavus, 0.4% as E. gallinarum, and 11.2% as other Enterococcus spp. Antibiotic susceptibility testing revealed resistance to sulfamethoxazole-trimethoprim (78.8%), tetracycline (76.9%), erythromycin (68.1%), streptomycin (62.6%), chloramphenicol (27.0%), ciprofloxacin (8.5%), gentamicin (8.1%), and levofloxacin (5.6%) but no vancomycin, teicoplanin, tigecycline or linezolid resistance was detected. E. faecium displayed 44.4% resistance to quinupristin-dalfopristin. A total of 78% of enterococcal isolates were MDR. Phenotypic resistance to tetracycline, aminoglycosides, and macrolides was corroborated by the presence of the tetM, aph(3’)-IIIa, and ermB genes in 99.1%, 96.1%, and 88.3% of the isolates, respectively. The most commonly detected virulence genes were: gelE, efaAfs, and cpd in 89.1%, 78.5%, and 77.1% of isolates conferring autolysin and biofilm formation capabilities, cell adhesion, and conjugative plasmid accumulation, respectively. Clonality evaluated by REP-PCR revealed that E. faecalis isolates belonged to diverse clones along the continuum with major REP-types, largely consisting of isolates from the same sampling source but different sampling rounds (on the farm). E. faecium isolates revealed a less diverse profile. There was minimal evidence of clonal transmission across the continuum. Conclusion: Multi-drug resistant Enterococcus spp. were isolated along the farm-to-fork continuum. Isolates harboured a diversity of antibiotic resistance and virulence genes in different combinations forming reservoirs for the potential transfer of these genes from pigs to occupationally exposed workers and consumers via direct contact with animals and animal products/food, respectively. The results highlight the need for more robust guidelines for antibiotic use in intensive farming practices and the necessity of including Enterococcus spp. as an indicator in antibiotic resistance surveillance systems in food animals.
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    Molecular epidemiology of carbapenem-resistant Enterobacterales colonization in an intensive care unit.
    (2021) Madni, Osama.; Essack, Sabiha Yusuf.; Rout, Joan Allison.; Amoako, Daniel Gyamfi.; Akebe, Luther King Abia.
    Background: Due to the high association with mortality and morbidity, carbapenem-resistant Enterobacterales (CRE) in general, and carbapenem-resistant Klebsiella pneumoniae, in particular, have been listed as high-priority pathogens by the World Health Organization (WHO) for the research and development of new antibiotics. Concomitant resistance to multiple antibiotics of different classes, impedes efficient clinical management of CRE infections. We characterized carbapenemase-producing K. pneumoniae (CPKP) isolates from sequential rectal screening of patients in a single intensive care unit (ICU) in a public hospital in the uMgungundlovu District of Kwazulu-Natal, South Africa, collected over one month. Method: Ninety-seven rectal swabs collected from consenting adult patients (n=31) on day 1, 3, 7 and weekly thereafter were screened for carbapenemase-production using Chrome-ID selective media. Fourteen CPKP were subjected to speciation and antibiotic susceptibility testing using the VITEK 2® automated system and their clonality was ascertained by ERIC/PCR. A sub-sample of eight isolates from five patients underwent whole genome sequencing (WGS) on the Illumina MiSeq platform followed by bioinformatics analysis to delineate the resistome, virulome, mobilome, clonality and phylogeography. Results: All isolates (100%) were resistant to ertapenem and meropenem and 71.4% (n=10) were resistant to imipenem. All isolates harbored the blaOXA-181 carbapenemase (100%, n=8) and also carried other β-lactamase genes such as OXA-1, CTX-M-15, TEM-1B and SHV-1. IncF, IncX3, and Col plasmid replicons groups and class I integrons (ln191 and ln27) were detected. All isolates belonged to the same sequence type ST307 and capsular serotypes (K102, O2v2) and several were associated with a single bed located in the ICU. All but one isolate carried the same plasmid multilocus sequence type [K7:A-:B-] and the same virulence repertoire was identified reflecting the epidemiological relationships between isolates. BlaOXA-181 were presumably located on a multi-replicon plasmid similar to that of E. coli p010_B-OXA181, and isolates were aligned with several South African and international clades, demonstrating horizontal and vertical transboundary distribution. Conclusion: OXA-181-producing K. pneumoniae belonging to ST307 was found to be potentially endemic in the hospital ICU environment of a public hospital in KwaZulu-Natal South Africa. The presence of a myriad of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in different permutations and combinations presents challenges to clinical management and infection prevention and control measures. This necessitates a CRE screening programme and strict infection prevention and control measures to detect and eliminate this endemic clone.