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There is a rapid spread of antibiotic resistance in the environment. However, the impact of antibiotic resistance in drinking water is relatively underexplored. Thus, this study aimed to quantify antibiotic resistance genes (ARGs) and antibiotic residues in two drinking water production facilities (NW-E and NW-C) in North West Province, South Africa and link these parameters to bacterial communities. Physicochemical and ARG levels were determined using standard procedures. Residues (antibiotics and fluconazole) and ARGs were quantified using ultra-high performance liquid chromatography (UHPLC) chemical analysis and real-time PCR, respectively. Bacterial community compositions were determined by high-throughput 16S rRNA sequencing. Data were analysed using redundancy analysis and pairwise correlation. Although some physicochemical levels were higher in treated than in raw water, drinking water in NW-E and NW-C was safe for human consumption using the South African Water Quality Guideline (SAWQG). ARGs were detected in raw and treated water. In NW-E, the concentrations of ARGs (sul1, intl1, EBC, FOX, ACC and DHA) were higher in treated water than in raw water. Regarding antimicrobial agents, antibiotic and fluconazole concentrations were higher in raw than in treated water. However, in NW-C, trimethoprim concentrations were higher in raw than in treated water. Redundancy analysis showed that bacterial communities were not significantly correlated (Monte Carlo simulations, p-value >0.05) with environmental factors. However, pairwise correlation showed significant differences (p-value <0.05) for Armatimonas, CL500-29 marine group, Clade III, Dickeya and Zymomonas genera with environmental factors. The presence of ARGs and antibiotic residues in the current study indicated that antibiotic resistance is not only a clinical phenomenon but also in environmental settings, particularly in drinking water niches. Consumption of NW-E and NW-C treated water may facilitate the spread of antibiotic resistance among consumers. Thus, regulating and monitoring ARGs and antibiotic residues in drinking water production facilities should be regarded as paramount. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli, a ubiquitous inhabitant of the gut microbiota, has been recognized as an indicator of fecal contamination and a potential reservoir for antibiotic resistance genes. Its prevalence in drinking water sources raises concerns about the potential dissemination of antibiotic resistance within aquatic ecosystems and the subsequent impact on public health. The ability of E. coli to acquire and transfer resistance genes, coupled with the constant exposure to low levels of antibiotics in the environment, underscores the need for comprehensive surveillance and rigorous antimicrobial stewardship strategies to safeguard the quality and safety of drinking water supplies, ultimately mitigating the escalation of antibiotic resistance and its implications for human well-being. Methods: WG5D strain, isolated from a drinking water distribution source in North-West Province, South Africa, underwent genomic analysis following isolation on nutrient agar, anaerobic cultivation, and DNA extraction. Paired-end Illumina sequencing with a Nextera XT Library Preparation kit was performed. The assembly, annotation, and subsequent genomic analyses, including phylogenetic analysis using TYGS, pairwise comparisons, and determination of genes related to antimicrobial resistance and virulence, were carried out following standard protocols and tools, ensuring comprehensive insights into the strain's genomic features. Results: This study explores the notable characteristics of E. coli strain WG5D. This strain stands out because it possesses multiple antibiotic resistance genes, encompassing tetracycline, cephalosporin, vancomycin, and aminoglycoside resistances. Additionally, virulence-associated genes indicate potential heightened pathogenicity, complemented by the identification of mobile genetic elements that underscore its adaptability. The intriguing possibility of bacteriophage involvement and factors contributing to pathogenicity further enriches our understanding. We identified E. coli WG5D as a potential human pathogen associated with a drinking water source in South Africa. The analysis provided several antibiotic resistance-associated genes/mutations and mobile genetic elements. It further identified WG5D as a potential human pathogen. The occurrence of E. coli WG5D raised the awareness of the potential pathogens and the carrying of antibiotic resistance in drinking water. Conclusions: The findings of this study have highlighted the advantages of the genomic approach in identifying the bacterial species and antibiotic resistance genes of E. coli and its potential as a human pathogen. [ABSTRACT FROM AUTHOR]

Practices in intensive animal farming such as the extensive use of antimicrobials have significant impacts on the genetic make-up of bacterial communities, especially on that of human/animal commensals. In this report, whole genome sequencing of two vancomycin-resistant enterococci (VRE) isolates from a cattle feedlot in the North West Province, South Africa, was used to highlight the threats that extensive antimicrobial usage in intensive animal rearing represents for environmental microbiomes and the food chain. The genomic DNA of the studied strains was extracted using a DNA extraction kit. Whole-genome sequencing was performed through next-generation sequencing. The genomes of Enterococcus durans strain NWUTAL1 and Enterococcus gallinarum strain S52016 consisted of 3,279,618 and 2,374,946 bp, respectively with G + C contents of 40.76 and 43.13%, respectively. Antibiotic resistance genes (ARG), plasmids and virulence factors (involved in biofilm formation, colonization and copper/silver efflux system), were detected in the genomes of both strains. The presence of these genetic determinants in the studied strains is a cause for concern as they may disseminate and find their way into the food chain via horizontal gene transfer amongst bacteria of the different ecological niches. Issues of this nature cannot be undermined and are relevant as far as food safety is concerned. [ABSTRACT FROM AUTHOR]

Endophytic fungi have the ability to live inside the host plant tissues without causing neither symptoms of diseases/or harm. Opportunistic infections are accountable for majority of the outbreaks, thereby putting a burden on the health system. To investigate and characterize the bioactive compounds for the control of bacteria of clinical importance, extracts from endophytic fungi were isolated from indigenous South African medicinal plants. Extracts from endophytic fungi were isolated from 133 fungal strains and screened against Gram positive and negative bacteria namely Bacillus cereus, Escherichia coli, Enterococcus faecium, and E. gallinarum using disk diffusion. Furthermore, gas chromatography–mass spectrometry was performed to identify the bioactive compounds. Sixteen out of one hundred and thirty-three (12%) fungi extracts exhibited antibacterial properties against some of the selected bacteria. E. coli was found to be the most susceptible in contrast to E. faecium and E. gallinarum which were the most resistant. The isolate MHE 68, identified as Alternaria sp. displayed the greater spectrum of antibacterial activities by controlling selected clinical bacteria strains including resistant E. faecium and E. gallinarum. The chemical analysis of the extract from MHE 68 indicated that linoleic acid (9,12-octadecadienoic acid (Z,Z)) and cyclodecasiloxane could be accountable for the antibacterial activity. This is the first study conducted on the secondary metabolites produced by endophytic fungal strains isolated from the Pelargonium sidoides DC. possessing antibacterial properties. [ABSTRACT FROM AUTHOR]

Endophytic fungi have the ability to co-exist with their host plants without causing any harm and are beneficial to both the plant and the fungi. The current study determined the antimicrobial properties and identify the chemical compounds of secondary metabolites produced by endophytic fungi isolated from Sceletium tortuosum L. A total of 60 endophytic fungi produced secondary metabolites that were detected after fermentation and extraction. Antibacterial properties of the secondary metabolites were determined using the disc diffusion assay against pathogenic environmental Gram-positive and Gram-negative bacteria as well as control stains. The chemical compounds were characterized by GC-MS. Overall, 15% of fungal extracts displayed narrow spectrum of activity against the bacteria strains. Despite this, none of the fungal extracts inhibited growth of Enterococcus faecalis (ATCC S1299) and Enterococcus gallinarum (ATCC 700425) while Bacillus cereus (ATCC 10876) was the most susceptible against the fungal extracts. Fusarium oxysporum (GG 008) with accession no. KJ774041.1 displayed significant antibacterial activity that was linked to high levels of 5-hydroxymethylfurfural (HMF) and octadecanoic acid as revealed by GC-MS. This study revealed the presence of bioactive secondary metabolites with antibacterial activities from fungi isolated from Sceletium tortuosum L. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • First report of 60 endophytic fungi from Sceletium. • Fusarium , Aspergillus and Penicillium were the most dominant. • Three novel endophytic fungi. Abstract Throughout history, mankind has used plants as their primary source of sustainability, in agricultural commodities, clothing, fragrances, fertilizers, flavours, and providing shelter. There is a strong symbiotic relationship between the plant and its endophytes. Endophytes are harboured within the living plant tissues without causing neither diseases nor symptoms. They produce bioactive compounds that protect the host plants against attack of insects, pathogens and herbivores. The bioactive compounds might be utilized for pharmaceutical, agricultural, or biotechnological applications.This paper reported on the various endophytic fungi strains that were isolated from isolated from a medicinal plant, Sceletium tortuosum. Fifty Sceletium tortuosum plants were collected from three different provinces in South Africa and leaves and roots used to isolate culturable endophytes. Morphological characteristics and a genus specific PCR designed to amplify fungal internal transcribe spacer (ITS) region (ITS1 and ITS4) and elongation factor (EF 1 and 2) was used for identification. A total of 60 fungal isolates belonging to 16 genera were identified and classified. Isolates were identified to species level based on similarities with known sequences in GenBank and a large proportion of the fungi were Fusarium species (37%) followed Aspergillus (25%) and Penicillium (7%) species. Phylogenetic analysis was performed using nuclear ribosomal DNA sequences and three potentially new isolates (DR 019 Fusarium penzigii , DR 010 Phomopsis columnaris , DR 007 Fusarium oxysporum f. sp. lycopersici) were identified in the phylogenic tree that was constructed. Our results offers basic data on the symbiotic/or mutualistic relationship between the medicinal plant Sceletium tortuosum and its endophytic fungi, as well as novel species. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the presence and diversity of AmpC β-lactamase and integrase genes among DNA (genomic and plasmid) from bacterial populations in selected aquatic systems. Following an enrichment step, DNA was isolated and subjected to polymerase chain reaction (PCR) and digital droplet PCR. The intI1 gene and AmpC β-lactamase genes were present in genomic and plasmid DNA from all sites in the Mooi, Crocodile and Marico Rivers, with the exception of intI1 in the Marico River. Digital droplet PCR demonstrated that copy numbers varied considerably (0.0 to 29.38 copies per picogram of DNA). Some samples in which ampC was not detected, intI1 was present. Amplicons of ampC genes were subjected to restriction digest using HindIII. Samples where the restriction markers were absent were purified by cloning followed by plasmid extraction, PCR amplification, and sequencing of individual AmpC gene fragments. Phylogenetic analysis identified all positive AmpC genes as Class C β-lactamases, comprising of ampC, CMY- and ACT-families. Detecting AmpC and intl1 genes on plasmids suggests a high risk of horizontal gene transfer and potential dissemination of these and other antibiotic resistance genes surrounding immediate aquatic environments. Consequences of β-lactamase diversity in aquatic ecosystems are relatively unexplored in South African aquatic ecosystems. [ABSTRACT FROM AUTHOR]

Increased heavy metal pollution generated through anthropogenic activities into the environment has necessitated the need for eco-friendly remediation strategies such as mycoremediation. With a view to prospecting for fungi with heavy metal remediation potentials, the tolerance of fiveAspergillusspecies isolated from soils of three active gold and gemstone mining sites in southwestern Nigeria to varied heavy metal concentrations was investigated. IsolatedAspergillusstrains were identified based on the internal transcribed spacer 1 and 2 (ITS 1 and ITS 2) regions. Growth ofAspergillusstrains were challenged with a range of varied concentrations of heavy metals: cadmium (Cd) (0–100), copper (Cu) (0–1000), lead (Pb) (0–400), arsenic (As) (0–500), and iron (Fe) (0–800) concentrations (ppm) incorporated into Malt Extract Agar (MEA) in triplicates. Mycelial radial growths were recorded at intervals of 3 days during a 13-day incubation period.Aspergillusstrains were identified asA. tubingensis, A. fumigatus, A. terreus, A. nidulans, andA. nomius. A. tubingensistolerated Cd, Cu, Pb, As, and Fe at all test concentrations (100–1000 ppm), showing no significant (p> .05) difference compared with the control. Similarly,A. nomiustolerated all concentrations of Cu, Pb, As, and Fe and only 50 ppm Cd concentrations.A. nidulans, A. terreus, andA. fumigatus, on the other hand, tolerated all concentrations of Cu, Pb, and Fe with no statistical significance (p> .05) difference from the controls. Overall, theAspergillusspecies showed tolerance to heavy metal concentrations above permissible limits for contaminated soils globally. These heavy metal tolerance traits exhibited by theAspergillusisolates may suggest that they are potential candidates for bioremediation of heavy metal–polluted environments. [ABSTRACT FROM AUTHOR]

Virulence factors in Enterococcus may be indicative of potential pathogenicity. The aim of this study was to determine the relationship between the presence of clinically relevant virulence genes, in Enterococcus spp. from environmental water, and their in vitro expression. One hundred and twenty-four Enterococcus isolates (seven species), from five surface water systems in the North West Province, South Africa, were screened for the presence of asa1, cylA, esp, gelE and hyl using polymerase chain reaction. The expression of cylA, hyl and gelE was determined by phenotypic assessments. Sixty-five percent of the isolates were positive for one virulence gene and 13% for two or more. Most frequently detected genes were gelE (32%) and cylA (28%). Enterococcal surface protein was absent in all isolates screened. The presence of virulence genes was correlated with their extracellular enzyme production. The results show that a large percentage of these environmental Enterococcus spp. possess virulence factors that could be expressed in vitro. This is a cause for concern and could have implications for individuals using this water for recreational and cultural purposes. Further investigation is required into the sources of these potential pathogenic Enterococcus isolates and measures to minimize their presence in water sources. [ABSTRACT FROM AUTHOR]

This study aimed to identify bacterial community structures in the Vaal River using PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) and high-throughput sequencing. The impact of physico-chemical characteristics on bacterial structures was investigated through multivariate analysis. Samples were collected from 4 sampling stations along the Upper Vaal River during winter (June 2009) and summer (December 2010). Physico-chemical analysis was conducted on-site. Additional physico-chemical data were obtained from statutory bodies. DNA was directly isolated from water samples and PCR amplified using universal bacterial primer pairs. PCR products were subjected to DGGE fingerprinting and high-throughput sequencing, followed by Shannon-Weaver diversity calculations, cluster analysis and multivariate analysis. Physico-chemical parameters did not exceed the prescribed South African water quality standards for domestic use, aquatic ecosystems, livestock watering and irrigation. DGGE banding patterns revealed similar bacterial community structures for 3 of the 4 sampling stations. PCA and RDA indicated that pH, water temperature and inorganic nutrient concentrations could be used to explain changes in bacterial community structures. High-throughput sequencing data showed that bacterial assemblages were dominated by common freshwater groups: Cyanobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria. Other freshwater phyla such as Deltaproteobacteria, Epsilonbacteria, Acidobacteria, Verrucomicrobia, Firmicutes, Fusobacteria, Flavobacteria and Fibrobacteres were found in low proportions. This study provides an overview of the dominant bacterial groups in the Upper Vaal River and the impact of environmental changes on bacterial diversity. [ABSTRACT FROM AUTHOR]

Culture-independent methods employed in fungal genetic studies using in vitro amplification (PCR) and analysis of specific genes or gene fragments have proved to be useful for detection, identification, and molecular taxonomy of various plant pathogens including Fusarium spp. This approach may be faster than culture-dependent methods, and could especially be of value for the rapid detection of slow-growing toxin-producing species in food samples. The present study was aimed at the development and evaluation of multiplex PCR-based methods for the detection and identification of potential fumonisin-producing Fusarium spp. in traditional morogo—leafy vegetables supplementing the maize-based staple diet of rural communities in South Africa. In these rural subsistence settings, some morogo plants grow as weeds in maize fields where they might become contaminated with potential fumonisin-producing Fusarium strains before being collected for consumption. Substances released by senescent vegetables could induce toxin production during storage. Using fumonisin-positive MRC Fusarium verticillioides strains as reference cultures, the following primer sets for the detection of specific gene fragments in fumonisin-positive Fusarium spp. were evaluated: (i) the conserved transcription elongation factor gene (EF-1), (ii) the FUM1 gene encoding polyketide synthase for fumonisin B1 production, and (iii) 18S rRNA gene. Preliminary results indicated that, these DNA fragments were amplified from MRC reference strains as well as Fusarium spp. isolated from morogo. The annealing temperature for the multiplex PCR was 55°C and each reaction contained 25 pmol of each of the primer sets EF and FUM1 and 12.5 pmol of the 18S primer set. The detection limit of the individual primers was up to 1 ng and for the multiplex up to 10 ng. This demonstrates the potential of this method for the detection of potential fumonisin-positive strains. [ABSTRACT FROM AUTHOR]

Sceletium tortuosum (L.) N.E.Br. (Mesembryanthemaceae), commonly known as kanna or kougoed, is an effective indigenous medicinal plant in South Africa, specifically to the native San and Khoikhoi tribes. Today, the plant has gained strong global attraction and reputation due to its capabilities to promote a sense of well-being by relieving stress with calming effects. Historically, the plant was used by native San hunter-gatherers and Khoi people to quench their thirst, fight fatigue and for healing, social, and spiritual purposes. Various studies have revealed that extracts of the plant have numerous biological properties and isolated alkaloids of Sceletium tortuosum are currently being used as dietary supplements for medicinal purposes and food. Furthermore, current research has focused on the commercialization of the plant because of its treatment in clinical anxiety and depression, psychological and psychiatric disorders, improving mood, promoting relaxation and happiness. In addition, several studies have focused on the isolation and characterization of various beneficial bioactive compounds including alkaloids from the Sceletium tortuosum plant. Sceletium was reviewed more than a decade ago and new evidence has been published since 2008, substantiating an update on this South African botanical asset. Thus, this review provides an extensive overview of the biological and pharmaceutical properties of Sceletium tortuosum as well as the bioactive compounds with an emphasis on antimicrobial, anti-inflammatory, anti-oxidant, antidepressant, anxiolytic, and other significant biological effects. There is a need to critically evaluate the bioactivities and responsible bioactive compounds, which might assist in reinforcing and confirming the significant role of kanna in the promotion of healthy well-being in these stressful times. [ABSTRACT FROM AUTHOR]

With the increasing spread of antimicrobial resistance, there is growing attention to the contribution made by drinking water systems. The potential health impact of two drinking water treatment and distribution systems (A and B) in the North-West Province of South Africa was determined by investigating the water quality and occurrence of antimicrobial-resistant heterotrophic bacteria and genes in the raw and treated water over four seasons. Most of the physicochemical parameters except for electrical conductivity were within permissible limits. Coliform bacteria reduced from raw to potable water except for counts higher than the threshold recorded in Summer and Winter. A total of 203 heterotrophic bacterial isolates were recovered on chromogenic R2A medium and subjected to susceptibility testing to twelve antibiotics. Most of the isolates were resistant to β-lactam antibiotics and Trimethoprim, whereas they were susceptible to Ciprofloxacin, Erythromycin, and Neomycin. The proportions of Cephalothin and Kanamycin-resistant isolates were significantly higher (p < 0.05) after treatment for site A, compared to significantly lower β-lactam, Oxytetracycline, and Trimethoprim-resistant isolates for B. Over 50% of isolates were of high risk, indicating their origin from high antibiotic-use sources. Seventy-one (35%) isolates were multidrug-resistant, out of which the majority (53.5%, n = 38) possessed the strA gene, followed by strB 21 (29.6%), dfrB 13 (18.3%), aadA 11 (15.5%), blaCTX-M 5 (7.0%), and tetA 3 (4.2%). The 16S rRNA gene sequences of the isolates revealed strains belonging to eight bacterial families, some of which are clinically important. [ABSTRACT FROM AUTHOR]

Busseola fusca (Fuller) (Lepidoptera: Noctuidae), a major insect pest of maize in sub-Saharan Africa, has developed high levels of non-recessive resistance to Cry1Ab toxin expressed in genetically modified Bt maize. Multiple resistance mechanisms to various Cry toxins have been identified in Lepidoptera, but no study has yet been done to determine the mechanism of Cry1Ab resistance in B. fusca. Therefore, the larval transcriptome of B. fusca was sequenced, de novo assembled and characterized. Differential expression analysis was performed to compare gene expression profiles of Cry toxin challenged and unchallenged neonate larvae to assess the molecular basis of the defence mechanism employed by this insect. Several genes associated with Cry toxin resistance in other lepidopteran pests were detected in B. fusca. Results suggest that differential expression of metabolic and immune-related genes might explain Cry1Ab toxin defence in this pest (supplemental file). Transcript expression profiles of neonates demonstrated that 33.59% and 60.31% of the 131 differentially expressed genes were upregulated and downregulated in the toxin-challenged neonate larvae, respectively. Transcripts were grouped into two subclusters according to the similarity of their expression patterns. Transcripts in subcluster 1 were moderately upregulated in the toxin-challenged neonate larvae, and, conversely, downregulated in the unchallenged neonate larvae. The solute carrier organic anion transporter, which is involved in insecticide detoxification, was upregulated in the toxin-challenged neonate larvae. Conversely, most of the transcripts in subcluster 2 were moderately downregulated in the toxin-challenged neonate larvae, and upregulated for neonates feeding on non-challenged maize. Four unidentified transcripts were extremely down-regulated in the toxin-challenged neonate larvae, and upregulated in the unchallenged neonate larvae. Further studies are recommended to establish if there is a direct correlation between these differentially expressed genes and the observed resistance. Elucidation of such defence mechanisms is crucial for developing insect resistance management strategies to ensure sustainable use of genetically modified maize in Africa. Nevertheless, this is the first study on gene expression profiles of B. fusca strains challenged with Cry toxin. The transcriptome characterized in this study provides a significant resource base for future studies on B. fusca and contributes to understanding some of the gene regulation and signalling networks involved in the defence of B. fusca against Cry1Ab toxin. • First whole transcriptome analysis of Busseola fusca • Busseola fusca defence against Cry1Ab toxin. • Differential expression of immune genes in response to toxin challenge. • Identification of potential Bt resistance genes • Signal transduction and immune system pathways identified for Busseola fusca. [ABSTRACT FROM AUTHOR]