Browsing by Author "Amoako, D.G."

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    Genomic Analysis of Enterococcus spp. Isolated From a Wastewater Treatment Plant and Its Associated Waters in Umgungundlovu District, South Africa
    (Frontiers in Microbiology, 2021-06-14) Mbanga, J.; Amoako, D.G.; Abia, A.L.; Allam, M.; Ismail, A.; Essack, S.Y.
    We investigated the antibiotic resistome, mobilome, virulome, and phylogenomic lineages of Enterococcus spp. obtained from a wastewater treatment plant and its associated waters using whole-genome sequencing (WGS) and bioinformatics tools. The whole genomes of Enterococcus isolates including Enterococcus faecalis (n = 4), Enterococcus faecium (n = 5), Enterococcus hirae (n = 2), and Enterococcus durans (n = 1) with similar resistance patterns from different sampling sites and time points were sequenced on an Illumina MiSeq machine. Multilocus sequence typing (MLST) analysis revealed two E. faecalis isolates that had a common sequence type ST179; the rest had unique sequence types ST841, and ST300. The E. faecium genomes belonged to 3 sequence types, ST94 (n = 2), ST361 (n = 2), and ST1096 (n = 1). Detected resistance genes included those encoding tetracycline [tet(S), tet(M), and tet(L)], and macrolides [msr(C), msr(D), erm(B), and mef(A)] resistance. Antibiotic resistance genes were associated with insertion sequences (IS6, ISL3, and IS982), and transposons (Tn3 and Tn6000). The tet(M) resistance gene was consistently found associated with a conjugative transposon protein (TcpC). A total of 20 different virulence genes were identified in E. faecalis and E. faecium including those encoding for sex pheromones (cCF10, cOB1, cad, and came), adhesion (ace, SrtA, ebpA, ebpC, and efaAfs), and cell invasion (hylA and hylB). Several virulence genes were associated with the insertion sequence IS256. No virulence genes were detected in E. hirae and E. durans. Phylogenetic analysis revealed that all Enterococcus spp. isolates were more closely related to animal and environmental isolates than clinical isolates. Enterococcus spp. with a diverse range of resistance and virulence genes as well as associated mobile genetic elements (MGEs) exist in the wastewater environment in South Africa.
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    Genomic Insights of Multidrug-Resistant Escherichia coli From Wastewater Sources and Their Association With Clinical Pathogens in South Africa
    (Frontiers in Veterinary Science, 2021-02-26) Mbanga, J.; Amoako, D.G.; Abia, A.L.; Allam, M.; Ismail, A.; Essack, S. Y.
    There is limited information on the comparative genomic diversity of antibiotic-resistant Escherichia coli from wastewater. We sought to characterize environmental E. coli isolates belonging to various pathotypes obtained from a wastewater treatment plant (WWTP) and its receiving waters using whole-genome sequencing (WGS) and an array of bioinformatics tools to elucidate the resistomes, virulomes, mobilomes, clonality, and phylogenies. Twelve multidrug-resistant (MDR) diarrheagenic E. coli isolates were obtained from the final effluent of a WWTP, and the receiving river upstream and downstream of the WWTP were sequenced on an Illumina MiSeq machine. The multilocus sequence typing (MLST) analysis revealed that multiple sequence types (STs), the most common of which was ST69 (n = 4) and ST10 (n = 2), followed by singletons belonging to ST372, ST101, ST569, ST218, and ST200. One isolate was assigned to a novel ST ST11351. A total of 66.7% isolates were positive for β-lactamase genes with 58.3% harboring the blaTEM1B gene and a single isolate the blaCTX−M−14 and blaCTX−M−55 extended-spectrum β-lactamase (ESBL) genes. One isolate was positive for the mcr-9 mobilized colistin resistance gene. Most antibiotic resistance genes (ARGs) were associated with mobile genetic support: class 1 integrons (In22, In54, In191, and In369), insertion sequences (ISs), and/or transposons (Tn402 or Tn21). A total of 31 virulence genes were identified across the study isolates, including those responsible for adhesion (lpfA, iha, and aggR), immunity (air, gad, and iss), and toxins (senB, vat, astA, and sat). The virulence genes were mostly associated with IS (IS1, IS3, IS91, IS66, IS630, and IS481) or prophages. Co-resistance to heavy metal/biocide, antibiotics were evident in several isolates. The phylogenomic analysis with South African E. coli isolates from different sources (animals, birds, and humans) revealed that isolates from this study mostly clustered with clinical isolates. Phylogenetics linked with metadata revealed that isolates did not cluster according to source but according to ST. The occurrence of pathogenic and MDR isolates in the WWTP effluent and the associated river is a public health concern.
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    Mbanga, J., Abia, A.L.K., Amoako, D.G. and Essack, S.Y., 2021. Longitudinal surveillance of antibiotic resistance in Escherichia coli and Enterococcus spp. from a wastewater treatment plant and its associated waters in KwaZulu-Natal, South Africa. Microbial Drug Resistance, 27(7), pp.904-918.
    (Microbial Drug Resistance, 2021) Mbanga, J.; Abia, A.L.K.; Amoako, D.G.; Essack, S.Y.
    We assessed the prevalence, distribution, and antibiotic resistance patterns of Escherichia coli and Enterococcus spp. isolated from raw and treated wastewater of a major wastewater treatment plant (WWTP) in KwaZulu-Natal, South Africa and the receiving river water upstream and downstream from the WWTP discharge point. Escherichia coli and enterococci were isolated and counted using the Colilert®-18 Quanti-Tray® 2000 and Enterolert®-18 Quanti-Tray 2000 systems, respectively. A total of 580 quantitative PCR-confirmed E. coli and 579 enterococci were randomly chosen from positive samples and tested for in vitro antibiotic susceptibility using the disk diffusion assay against 20 and 16 antibiotics, respectively. The removal success of the bacterial species through the treatment procedure at the WWTP was expressed as log removal values (LRVs). Most E. coli were susceptible to meropenem (94.8%) and piperacillin-tazobactam (92.9%), with most Enterococcus susceptible to ampicillin (97.8%) and vancomycin (96.7%). In total, 376 (64.8%) E. coli and 468 (80.8%) Enterococcus isolates showed multidrug resistance (MDR). A total of 42.4% (246/580) E. coli and 65.1% (377/579) enterococci isolates had multiple antibiotic resistance indices >0.2. The LRV for E. coli ranged from 2.97 to 3.99, and for enterococci the range was observed from 1.83 to 3.98. A high proportion of MDR E. coli and enterococci were present at all sampled sites, indicating insufficient removal during wastewater treatment. There is a need to appraise the public health risks associated with bacterial contamination of environmental waters arising from such WWTPs to protect the health of users of the receiving water bodies.
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    Mobile genetic elements-mediated Enterobacterales-associated carbapenemase antibiotic resistance genes propagation between the environment and humans: A One Health South African study
    (Elsevier, 2021-09-23) Ramsamy, Y.; Mlisana, K.P.; Amoako, D.G.; Abia, A.L.K.; Ismail, A.; Allam, M.; Mbanga, J.; Singh, R.; Essack, S.Y.
    We, (1) studied carbapenem-resistant Enterobacterales (CRE) in the environment, humans, and animals, within the same geographical area and, (2) delineated the isolates' resistome, mobilome, virulome, and phylogeny. Following ethical approval, 587 samples (humans = 230, pigs = 345, and water = 12) were collected and cultured on CRE selective media. Confirmatory identification and antibiotic susceptibility testing were performed using the VITEK 2 automated platform. The resistomes, virulomes, mobilomes, and phylogenies were ascertained by whole genome sequencing. Nineteen (3.2%), i.e., 15/19 humans and 4/19 environmental, but no pig, CRE were obtained. CREs included Klebsiella pneumoniae 9/19 (47%), Enterobacter hormaechei 6/19 (32%), Klebsiella quasipneumoniae 2/19 (11%), a novel ST498 Citrobacter freundii 1/19 (5%) and Serratia marcescens 1/19 (5%). Eleven isolates were extensively drug-resistant; eight were multidrug-resistant. Sixteen CRE harbored the blaOXA-181, blaOXA-48, blaOXA-484, blaNDM-1, and blaGES-5 genes. Multiple species/clones carried blaOXA-48 and blaNDM-1 carbapenemase-encoding genes with respective mobile genetic elements (MGEs). The IncFIB(K) plasmid replicon was found in most human K. pneumoniae strains (7/9) and all environmental K. quasipneumoniae isolates; most K. pneumoniae produced OXA-181 (5/9). The (Col440I) plasmid replicon, identified in 11 (26.82%) isolates, mainly E. hormaechei (n = 6), predominated both sectors. Most β-lactamase-encoding genes were associated with class 1 integrons IntI1, insertion sequences (IS) (IS91, IS5075, IS30, IS3000, IS3, IS19, ISKpn19, IS5075) and transposons (Tn3). The IncL/M(pMU407) and IncL/M(pOXA48) plasmid replicons were found exclusively in K. pneumoniae; all but one of these strains produced OXA-181. Also, the Klebsiella spp. harbored 80 virulence genes. Phylogenomic clustered identified isolates with other carbapenemase-producing K. pneumoniae, E. hormaechei, S. marcescens, and C. freundii from different South African sources (animals, environment, and humans). We delineated the resistome, mobilome, virulome, and phylogeny of carbapenemase-producing Enterobacterales in humans and environment, highlighting antibiotic resistance genes propagation via MGEs across sectors, emphasizing a One Health approach to AMR.
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    Whole genome sequencing reveals insights into antibiotic resistant Klebsiella grimontii novel sequence type ST350 isolated from a wastewater source in South Africa
    (Journal of Biotech Research, 2022) Mbanga, J.; Amoako, D.G.; Abia, A.L.; Fatoba, D.; Essack, S.
    Klebsiella grimontii is a recently identified species that has been implicated in clinical infections. Few or no reports on environmental K. grimontii exist from Africa. This study was part of a broader longitudinal research that aimed to assess the distribution, antibiotic resistance patterns, and genomics of Enterobacterales obtained from raw and treated wastewater and the associated river water of a wastewater treatment plant (WWTP) in KwaZulu-Natal, South Africa. We reported the genomics of an antibiotic resistant Klebsiella grimontii isolate obtained from the raw influent of the WWTP. Following phenotypic characterisation and antibiotic susceptibility testing, the isolate was sequenced on an Illumina MiSeq machine. Raw reads were assembled de novo by using SPAdes (v3. 6.2) prior to bioinformatics analysis. The assembled K. grimontii INF139 genome was 6,369,878 bp, with 113 contiguous sequences (> 200 bp) and 55.4% GC content. The isolate was assigned a novel sequence type ST350. Genomic analysis revealed the presence of chromosomally encoded β-lactamase (blaOXY-6-1) and fluoroquinolone (oqxB) resistance genes. Virulence factors encoding antiphagocytic, iron acquisition, and Intracellular survival properties were identified. Mobilome analysis revealed the presence of plasmid replicons (ColRNAI, FIA (pBK30683), IncFII, IncFII (Yp)) and insertion sequences (ISIX2, ISSen9, ISIS, IS1A). To our knowledge, this is the first description of antibiotic resistant K. grimontii from the water environment from Africa. The presence of this antibiotic resistant and potentially pathogenic isolate in the water environment is worrying as it may be disseminated into river systems used by informal settlements.