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- ItemMbanga, 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.
- ItemGenomic 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.
- ItemBiodiversity of Aflatoxigenic Aspergillus Species in Dairy Feeds in Bulawayo, Zimbabwe(Frontiers in Microbiology, 2021-01-21) Nleya, N.; Ngoma, L.; Adetunji, M.C.; Mwanza, M.The presence of molds, especially certain species of Aspergillus, in food commodities may contribute to aflatoxin contamination. The aim of this study was to determine the biodiversity of Aspergillus species in dairy feeds from farms in select locations in Zimbabwe and assess their aflatoxin production potential using a polyphasic approach. A total of 96 feed samples were collected, which consisted of dairy feed concentrate, mixed ration, brewers’ spent grain, and grass from 13 farms during the dry season (August–October, 2016) and the following rainy season (January–March, 2017). A total of 199 presumptive isolates representing four sections from genus Aspergillus (Nigri, Fumigati, Flavi, and Circumdati) were recovered from the feeds. Section Flavi, which includes several aflatoxin producers, constituted 23% (n = 46) of the isolates. Species from this section were A. flavus, A. nomius, A. oryzae, A. parasiticus, and A. parvisclerotigenus, and 39 (84.4%) of these showed evidence of aflatoxin production in plate assays. Of the 46 section Flavi isolates examined, some lacked one or more of the five targeted aflatoxin cluster genes (aflD, aflR, aflS, aflM, and aflP). The presence of the five genes was as follows: aflD (76.9%), aflR (48.7%), aflS (74.4%), aflM (64.1%), and aflP (79.5%). This study highlights the species diversity of aflatoxigenic fungi that have the potential to contaminate different types of feed for dairy cows. Our findings underscore the importance of preventing contamination of feedstuffs by these fungi so that aflatoxins do not end up in the diets of consumers.
- ItemAnalysis of β-amylase gene (Amyβ) variation reveals allele association with low enzyme activity and increased firmness in cooked sweetpotato (Ipomoea batatas) from East Africa(Elsevier, 2021-02-15) Banda, L.; Kyallo, M.; Entfellner, J.B.D.; Moyo, M.; Swanckaert, J.; Mwanga, R.O.; Onyango, A.; Magiri, E.; Gemenet, D.C.; Yao, N.; Pelle, R.β-amylase is a thermostable enzyme that hydrolyses starch during cooking of sweetpotato (Ipomoea batatas) storage roots, thereby influencing eating quality. Its activity is known to vary amongst genotypes but the genetic diversity of the beta-amylase gene (Amyβ) is not well studied. Amyβ has a highly conserved region between exon V and VI, forming part of the enzyme's active site. To determine the gene diversity, a 2.3 kb fragment, including the conserved region of the Amyβ gene was sequenced from 25 sweetpotato genotypes. The effect of sequence variation on gene expression, enzyme activity, and firmness in cooked roots was determined. Six genotypes carrying several SNPs within exon V, linked with an AT or ATGATA insertion in intron V were unique and clustered together. The genotypes also shared an A336E substitution in the amino acid sequence, eight residues upstream of a substrate-binding Thr344. The genotypes carrying this allele exhibited low gene expression and low enzyme activity. Enzyme activity was negatively correlated with firmness (R = −0.42) in cooked roots. This is the first report of such an allele, associated with low enzyme activity. These results suggest that genetic variation within the AmyB locus can be utilized to develop markers for firmness in sweetpotato breeding.
- ItemMobile 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.