Cornelius Carlos Bezuidenhout

Characterisation of Escherichia coli O157 strains from humans, cattle and pigs in the North-West Province, South Africa

Escherichia coli O157 strains cause diseases in humans that result from the consumption of food and water contaminated with faeces of infected animals and/or individuals. The objectives of this study were to isolate and characterise E. coli O157 strains from humans, cattle and pigs and to determine their antibiotic resistant profiles as well as detection of virulence genes by PCR. Eight hundred faecal samples were analysed for typical E. coli O157 and 76 isolates were positively identified as E. coli O157 strains. 16S rRNA sequence data were used to confirm the identity of the isolates. Susceptibility profiles to 9 antibiotics were determined and the multiple antibiotic resistant (MAR) patterns were compiled. A large proportion (52.6%-92.1%) of the isolates from pigs, cattle and humans were resistant to tetracycline, sulphamethoxazole and erythromycin. Thus the phenotype Smx-T-E (sulphamethozaxole-tetracycline-erythromycin) was present in most of the predominant MAR phenotypes obtained. Cluster analysis of antibiotic resistances revealed a closer relationship between isolates from pig and human faeces than cattle and humans. PCR were performed to amplify STEC virulence and tetracycline resistance gene fragments. A tetB gene fragment was amplified among the isolates. Eighteen (60%) of the isolates possessed the hlyA gene and 7(23.3%) the eae gene while only 5(16.7%) possessed both genes. Although shiga toxin genes were detected in the E. coli O157:H7 positive control strain none of the isolates that were screened possessed these genes. In a related study we reported that the prevalence of E. coli O157 was higher in pigs than cattle and humans. A high market demand for pork and beef in South Africa amplifies the risk that diseased animals pose to human health. This highlighted the need for proper hygiene management to reduce the prevalence of E. coli O157 in farm animals and prevent the spread from animals to humans.

Bacterial community composition of an urban river in the North West Province, South Africa, in relation to physico-chemical water quality

The aim of this study was to determine the impacts of anthropogenic disturbances on bacterial community composition in an urban river (Mooi River). Physico-chemical analysis, bacterial enumeration and 454-pyrosequencing were conducted on the Mooi River system upstream and downstream of an urban settlement in the North West Province, South Africa. Pyrosequencing and multivariate analysis showed that nutrient inputs and faecal pollution strongly impacted the physico-chemical and microbiological quality at the downstream sites. Also, bacterial communities showed higher richness and evenness at the downstream sites. Multivariate analysis suggested that the abundances of Betaproteobacteria, Epsilonproteobacteria, Acidobacteria, Bacteroidetes and Verrucomicrobia are related to temperature, pH, dissolved oxygen (DO), sulphate and chlorophyll-a levels. These results suggest that urbanisation caused the overall water quality of this river to deteriorate, which in turn affected the bacterial community composition. In addition, our work identified potential indicator groups that may be used to track faecal and organic pollution in freshwater systems.

Linolenic acid and folate in wild-growing African dark leafy vegetables (morogo)

BACKGROUND Transition from a low-fat vegetable-rich rural diet to a high-fat Westernised diet is considered a factor in the escalating occurrence of vascular-related diseases and type 2 diabetes in urban black South Africans. Consumption of morogo is a distinguishing feature of rural African diets. OBJECTIVE To determine fatty acid profiles and folate contents of three widely consumed, wild-growing, African dark green leafy vegetables (morogo). DESIGN GC-MS was applied for analysis of fatty acid composition and a validated microbiological assay conducted to determine folic acid contents of wild-growing morogo sampled from deep rural villages in three different geographical regions of South Africa. RESULTS Measured fatty acids ranged from 1610.2 to 2941.6 mg/100 g dry mass, with PUFA concentrations 1.4 to 2.8 times those of SFA. Calculated from the relative percentages of linoleic acid (18:2n-6) and linolenic acid (18:3n-3), the ratio of 18:2n-6 to 18:3n-3 PUFA was 1.0:3.4 to 1.0:8.9. The only MUFA was palmitoleic acid (16:1), measured at 34.7 (sd 0.3) to 79.0 (sd 9.3) mg/100 g dry mass, and the predominant SFA was palmitic acid (16:0), measured at 420.6 (sd 83.3) to 662.0 (sd 21.2) mg/100 g dry mass. Folic acid concentration varied from 72 to 217 microg/100 g fresh sample. CONCLUSION Morogo is low-fat food item high in folate and with 18:3n-3 in excess of 18:2n-6, the proposed anti-inflammatory effects of which may lower risks of vascular-related chronic diseases and type 2 diabetes.

Heavy metal tolerance traits of filamentous fungi isolated from gold and gemstone mining sites

Increased environmental pollution has necessitated the need for eco-friendly clean-up strategies. Filamentous fungal species from gold and gemstone mine site soils were isolated, identified and assessed for their tolerance to varied heavy metal concentrations of cadmium (Cd), copper (Cu), lead (Pb), arsenic (As) and iron (Fe). The identities of the fungal strains were determined based on the internal transcribed spacer 1 and 2 (ITS 1 and ITS 2) regions. Mycelia growth of the fungal strains were subjected to a range of (0–100 Cd), (0–1000 Cu), (0–400 Pb), (0–500 As) and (0–800 Fe) concentrations (mgkg−1) incorporated into malt extract agar (MEA) in triplicates. Fungal radial growths were recorded every three days over a 13-days’ incubation period. Fungal strains were identified as Fomitopsis meliae, Trichoderma ghanense and Rhizopus microsporus. All test fungal exhibited tolerance to Cu, Pb, and Fe at all test concentrations (400–1000 mgkg−1), not differing significantly (p > 0.05) from the controls and with tolerance index >1. T. ghanense and R. microsporus demonstrated exceptional capacity for Cd and As concentrations, while showing no significant (p > 0.05) difference compared to the controls and with a tolerance index >1 at 25 mgkg−1 Cd and 125 mgkg−1 As. Remarkably, these fungal strains showed tolerance to metal concentrations exceeding globally permissible limits for contaminated soils. It is envisaged that this metal tolerance trait exhibited by these fungal strains may indicate their potentials as effective agents for bioremediative clean-up of heavy metal polluted environments.

PCR-denaturing gradient gel electrophoresis analysis of microbial community in soy-daddawa, a Nigerian fermented soybean (Glycine max (L.) Merr.) condiment.

Soy-daddawa, a fermented soybean (Glycine max (L.) Merr.) condiment, plays a significant role in the culinary practice of West Africa. It is essential to understand the microbial community of soy-daddawa for a successful starter culture application. This study investigated the microbial community structure of soy-daddawa samples collected from Nigerian markets, by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the V3-V5 region of the 16S rRNA gene of bacteria and internal transcribed spacer 2 (ITS2) region of fungi. Six bacterial and 16 fungal (nine yeasts and seven molds) operational taxonomic units (OTUs)/species were obtained at 97% sequence similarity. Taxonomic assignments revealed that bacterial OTUs belonged to the phyla Firmicutes and Actinobacteria, and included species from the genera Atopostipes, Bacillus, Brevibacterium and Nosocomiicoccus. Densitometric analysis of DGGE image/bands revealed that Bacillus spp. were the dominant OTU/species in terms of population numbers. Fungal OTUs belonged to the phyla Ascomycota and Zygomycota, and included species from the genera, Alternaria, Aspergillus, Candida, Cladosporium, Dokmaia, Issatchenkia, Kodamaea, Lecythophora, Phoma, Pichia, Rhizopus, Saccharomyces and Starmerella. The majority of fungal species have not been previously reported in soy-daddawa. Potential opportunistic human pathogens such as Atopostipes suicloacalis, Candida rugosa, Candida tropicalis, and Kodamaea ohmeri were detected. Variation in soy-daddawa microbial communities amongst samples and presence of potential opportunistic pathogens emphasises the need for starter culture employment and good handling practices in soy-daddawa processing.

Gut microbiota of Busseola fusca (Lepidoptera: Noctuidae)

Busseola fusca (Fuller) (Lepidoptera: Noctuidae) is a stemborer pest that attacks maize (Zea mays) throughout sub-Saharan Africa. Genetically modified maize has been shown to be effective against B. fusca. However, resistance of B. fusca against Bt-maize has developed and spread throughout South Africa. Previous studies suggested that gut microbiota contribute to mortality across a range of Lepidoptera. To fully assess the role of microbiota within the gut, it is essential to understand the microbiota harboured by natural B. fusca populations. This study aimed to identify the gut-associated bacteria by 16S rRNA gene sequencing. A total of 78 bacterial strains were characterised from the midgut of B. fusca larvae that were collected from 30 sites across the maize producing region of South Africa. Molecular phylogenetic analyses revealed bacteria affiliated to Proteobacteria, Actinobacteria, and Firmicutes. Taxonomic distribution placed these isolates into 15 different genera representing 20 species. The majority of bacteria identified belong to the genera Bacillus, Enterococcus, and Klebsiella. The B. fusca gut represents an intriguing and unexplored niche for analysing microbial ecology. The study could provide opportunities for developing new targets for pest management and contribute to understanding the phenomenon of resistance evolution of this species.Graphical Abstract

Heavy metal tolerance potential of Aspergillus strains isolated from mining sites

ABSTRACT 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 five Aspergillus species isolated from soils of three active gold and gemstone mining sites in southwestern Nigeria to varied heavy metal concentrations was investigated. Isolated Aspergillus strains were identified based on the internal transcribed spacer 1 and 2 (ITS 1 and ITS 2) regions. Growth of Aspergillus strains 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. Aspergillus strains were identified as A. tubingensis, A. fumigatus, A. terreus, A. nidulans, and A. nomius. A. tubingensis tolerated 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. nomius tolerated all concentrations of Cu, Pb, As, and Fe and only 50 ppm Cd concentrations. A. nidulans, A. terreus, and A. fumigatus, on the other hand, tolerated all concentrations of Cu, Pb, and Fe with no statistical significance (p > .05) difference from the controls. Overall, the Aspergillus species showed tolerance to heavy metal concentrations above permissible limits for contaminated soils globally. These heavy metal tolerance traits exhibited by the Aspergillus isolates may suggest that they are potential candidates for bioremediation of heavy metal–polluted environments.

Genetic diversity in four populations of Nguni (Zulu) sheep assessed by microsatellite analysis

Zulu sheep are found mainly in the rural KwaZulu-Natal province and the numbers are declining due to indiscriminate inbreeding. There is thus a need for phenotypic and genetic characterisation as a first phase for planning conservation strategies. Zulu sheep populations sampled were from Makhathini research station (MS) (n=33), University of Zululand (UZ) (n=21), a community at KwaMthethwa (KM) (n=32) and from Msinga (EM) (n=33). One European breed Appenninica (AP) was used as out group. Microsatellite analysis using 29 microsatellite loci was used in this study. Among the Zulu sheep, the mean number of alleles per locus was the lowest (3.86) in UZ and the highest (6.24) was realised in EM. The mean values of observed and expected heterozygosity were 0.57 and 0.61, respectively. Neighbour-joining tree showed two main Zulu sheep clusters: the UZ, KM and MS sheep populations clustered together and the second cluster included only representatives from the EM population. The STRUCTURE analysis showed that KM, AP and EM were founded in separate clusters, whereas UZ and MS clustered together. The study demonstrated that there was a common origin of the population from the research stations (MS and UZ populations). It also demonstrated that the EM had a different history for the other three populations. This work suggests that exchange of rams could be useful in reducing inbreeding when considering conservation breeding programmes.

Characterisation of Xanthomonas campestris pv. campestris isolates from South Africa using genomic DNA fingerprinting and pathogenicity tests

The genetic diversity of Xanthomonas campestris pv. campestris isolates from South Africa was evaluated using 28 isolates obtained from the Johannesburg Fresh Produce Market. Samples were collected from cabbage supplies from farms in Gauteng, Mpumalanga and North West Provinces. Strains were isolated from small sections of infected cabbage leaf samples and cultured on Yeast Dextrose Agar. Isolates identity was confirmed by ELISA and Pathogenicity test. Pathogenicity tests were performed by inoculating leaves of known susceptible cabbage seedlings. Infection symptoms induced could be categorized into three groups, ranging from typical to non-typical black rot symptoms. Four differential Brassica cultivars with known avirulence genes were used for race typing done by spray inoculation. Four races, namely 1, 3, 4 and 6, were identified. Of the 28 isolates, four were identified as race 1, two as race 3, 19 as race 4 and three as race 6. Repetitive DNA polymerase chain reaction-based fingerprinting using Eric- and Box-primers was used to assess the genetic diversity. Generated fingerprints of X. c pv. campestris were relatively similar. Cluster analysis could not strictly group isolates by their geographical origin, suggesting limited diversity of Xanthomonas campestris pv. campestris strains within cabbage producing regions in South Africa.

Molecular analysis of bacterial community dynamics during the fermentation of soy-daddawa condiment

Bacterial community dynamics during soy-daddawa fermentation was investigated using culture-dependent and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) molecular methods. The total titratable acidity (TTA), pH, and bacterial counts (BCs) were monitored daily during a 72-h fermentation period. Bacteria were characterized based on 16S rRNA gene sequencing. TTA ranged from 0.08 to 0.26 mg lactic acid/g, whereas pH ranged from 7.01 to 8.19. BCs increased from 3.9 to 10.61 log CFU/g. Fifty-eight isolates were obtained by culture method and clustered into seven operational taxonomic units (OTUs) at 97% sequence similarity, whereas four OTUs were obtained from the PCR-DGGE method. Taxonomic identification revealed that bacteria belonged to the genera Bacillus, Enterobacter, Enterococcus, and Staphylococcus with B. subtilis being present throughout fermentation. Medically significant isolates, including B. anthracis, Enterococcus casseliflavus, and Enterobacter hormaechei were detected. These results emphasize the need for starter culture utilization and offer a platform for starter culture screening and selection.