Analysis of bacterial communities of three cassava-based traditionally fermented Nigerian foods (abacha, fufu and garri).

Globally, cassava is an important food crop that contributes significantly to food security. In Nigeria, cassava can be traditionally processed into abacha (fermented strips), fufu (submerged-fermented porridge) and garri (solid-state fermented farinated granules) for human consumption. Despite the widespread consumption of these foods, there is a major knowledge gap in understanding their core bacterial diversity. This study, therefore, applied next-generation sequencing of 16S rRNA gene to delineate the bacterial diversity in abacha, fufu and garri. Amplicon sequence variants belonging to nine phyla were present in the three foods. Firmicutes dominated the bacterial community of abacha and fufu, whereas, Proteobacteria was the dominant phylum in garri. At genus level taxa, Lactococcus, Lysinibacillus and Pseudomonas dominated the bacterial community in abacha, fufu and garri, respectively. Other dominant phylotypes reported in the foods belonged to Bacillus, Clostridium sensu stricto (cluster 1), Cupriavidus, Enterobacter, Sphingomonas and Staphylococcus. To the best of our knowledge, Clostridium sensu stricto cluster 1 and Lysinibacillus in fufu, and Brevundimonas, Cupriavidus, Sphingomonas and Strenotrophomomas in garri are reported for the first time. Although some potential pathogenic genera were recorded, the foods contained potentially functional species that could be explored to improve artisanal food production, food security and safeguard consumer health.

Fusarium: more than a node or a foot-shaped basal cell.

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae. Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae. Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium. Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae. Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae (e.g., Cosmosporella, Macroconia, Microcera). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium. To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa (act1, CaM, his3, rpb1, rpb2, tef1, tub2, ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [F. dimerum species complex (SC)], Cyanonectria (F. buxicola SC), Geejayessia (F. staphyleae SC), Neocosmospora (F. solani SC) or Rectifusarium (F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org).

Re-examination of species limits in Aspergillus section Flavipedes using advanced species delimitation methods and description of four new species.

Since the last revision in 2015, the taxonomy of section Flavipedes evolved rapidly along with the availability of new species delimitation techniques. This study aims to re-evaluate the species boundaries of section Flavipedes members using modern delimitation methods applied to an extended set of strains (n = 90) collected from various environments. The analysis used DNA sequences of three house-keeping genes (benA, CaM, RPB2) and consisted of two steps: application of several single-locus (GMYC, bGMYC, PTP, bPTP) and multi-locus (STACEY) species delimitation methods to sort the isolates into putative species, which were subsequently validated using DELINEATE software that was applied for the first time in fungal taxonomy. As a result, four new species are introduced, i.e. A. alboluteus, A. alboviridis, A. inusitatus and A. lanuginosus, and A. capensis is synonymized with A. iizukae. Phenotypic analyses were performed for the new species and their relatives, and the results showed that the growth parameters at different temperatures and colonies characteristics were useful for differentiation of these taxa. The revised section harbors 18 species, most of them are known from soil. However, the most common species from the section are ecologically diverse, occurring in the indoor environment (six species), clinical samples (five species), food and feed (four species), droppings (four species) and other less common substrates/environments. Due to the occurrence of section Flavipedes species in the clinical material/hospital environment, we also evaluated the susceptibility of 67 strains to six antifungals (amphotericin B, itraconazole, posaconazole, voriconazole, isavuconazole, terbinafine) using the reference EUCAST method. These results showed some potentially clinically relevant differences in susceptibility between species. For example, MICs higher than those observed for A. fumigatus wild-type were found for both triazoles and amphotericin B for A. ardalensis, A. iizukae, and A. spelaeus whereas A. lanuginosus, A. luppiae, A. movilensis, A. neoflavipes, A. olivimuriae and A. suttoniae were comparable to or more susceptible as A. fumigatus. Finally, terbinafine was in vitro active against all species except A. alboviridis.

Bacterial contaminants and their antibiotic susceptibility patterns in ready-to-eat foods vended in Ogun state, Nigeria.

Contamination of ready-to-eat (RTE) foods by pathogenic bacteria may predispose consumers to foodborne diseases. This study investigated the presence of bacterial contaminants and their antibiotic susceptibility patterns in three locally processed RTE foods (eko, fufu and zobo) vended in urban markets in Ogun state, Nigeria. Bacteria isolated from a total of 120 RTE food samples were identified by 16S rRNA gene phylogeny while susceptibility patterns to eight classes of antibiotics were determined by the disc diffusion method. Species belonging to the genera Acinetobacter and Enterobacter were recovered from all RTE food types investigated, Klebsiella and Staphylococcus were recovered from eko and fufu samples, while those of Shigella were recovered from eko samples. Enterobacter hormaechei was the most prevalent species in all three RTE food types. Precisely 99% of 149 isolates were multidrug-resistant, suggesting a high risk for RTE food handlers and consumers. Co-resistance to ampicillin and cephalothin was the most frequently observed resistance phenotype. Results demonstrate that improved hygiene practices by food processors and vendors are urgently required during RTE processing and retail. Also, adequate food safety guidelines, regulation and enforcement by relevant government agencies are needed to improve the safety of RTE foods and ensure the protection of consumer health.

Mycotoxin contamination of foods in Southern Africa: A 10-year review (2007-2016).

Major staple foods in Southern Africa are prone to mycotoxin contamination, posing health risks to consumers and consequent economic losses. Regional climatic zones favor the growth of one or more main mycotoxin producing fungi, Aspergillus, Fusarium and Penicillium. Aflatoxin contamination is mainly reported in maize, peanuts and their products, fumonisin contamination in maize and maize products and patulin in apple juice. Lack of awareness of occurrence and risks of mycotoxins, poor agricultural practices and undiversified diets predispose populations to dietary mycotoxin exposure. Due to a scarcity of reports in Southern Africa, reviews on mycotoxin contamination of foods in Africa have mainly focused on Central, Eastern and Western Africa. However, over the last decade, a substantial number of reports of dietary mycotoxins in South Africa have been documented, with fewer reports documented in Botswana, Lesotho, Malawi, Mozambique, Zambia and Zimbabwe. Despite the reported high dietary levels of mycotoxins, legislation for their control is absent in most countries in the region. This review presents an up-to-date documentation of the epidemiology of mycotoxins in agricultural food commodities and discusses the implications on public health, current and recommended mitigation strategies, legislation, and challenges of mycotoxin research in Southern Africa.

Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins and other mycotoxins.

Aflatoxins and ochratoxins are among the most important mycotoxins of all and producers of both types of mycotoxins are present in Aspergillus section Flavi, albeit never in the same species. Some of the most efficient producers of aflatoxins and ochratoxins have not been described yet. Using a polyphasic approach combining phenotype, physiology, sequence and extrolite data, we describe here eight new species in section Flavi. Phylogenetically, section Flavi is split in eight clades and the section currently contains 33 species. Two species only produce aflatoxin B1 and B2 (A. pseudotamarii and A. togoensis), and 14 species are able to produce aflatoxin B1, B2, G1 and G2: three newly described species A. aflatoxiformans, A. austwickii and A. cerealis in addition to A. arachidicola, A. minisclerotigenes, A. mottae, A. luteovirescens (formerly A. bombycis), A. nomius, A. novoparasiticus, A. parasiticus, A. pseudocaelatus, A. pseudonomius, A. sergii and A. transmontanensis. It is generally accepted that A. flavus is unable to produce type G aflatoxins, but here we report on Korean strains that also produce aflatoxin G1 and G2. One strain of A. bertholletius can produce the immediate aflatoxin precursor 3-O-methylsterigmatocystin, and one strain of Aspergillus sojae and two strains of Aspergillus alliaceus produced versicolorins. Strains of the domesticated forms of A. flavus and A. parasiticus, A. oryzae and A. sojae, respectively, lost their ability to produce aflatoxins, and from the remaining phylogenetically closely related species (belonging to the A. flavus-, A. tamarii-, A. bertholletius- and A. nomius-clades), only A. caelatus, A. subflavus and A. tamarii are unable to produce aflatoxins. With exception of A. togoensis in the A. coremiiformis-clade, all species in the phylogenetically more distant clades (A. alliaceus-, A. coremiiformis-, A. leporis- and A. avenaceus-clade) are unable to produce aflatoxins. Three out of the four species in the A. alliaceus-clade can produce the mycotoxin ochratoxin A: A. alliaceus s. str. and two new species described here as A. neoalliaceus and A. vandermerwei. Eight species produced the mycotoxin tenuazonic acid: A. bertholletius, A. caelatus, A. luteovirescens, A. nomius, A. pseudocaelatus, A. pseudonomius, A. pseudotamarii and A. tamarii while the related mycotoxin cyclopiazonic acid was produced by 13 species: A. aflatoxiformans, A. austwickii, A. bertholletius, A. cerealis, A. flavus, A. minisclerotigenes, A. mottae, A. oryzae, A. pipericola, A. pseudocaelatus, A. pseudotamarii, A. sergii and A. tamarii. Furthermore, A. hancockii produced speradine A, a compound related to cyclopiazonic acid. Selected A. aflatoxiformans, A. austwickii, A. cerealis, A. flavus, A. minisclerotigenes, A. pipericola and A. sergii strains produced small sclerotia containing the mycotoxin aflatrem. Kojic acid has been found in all species in section Flavi, except A. avenaceus and A. coremiiformis. Only six species in the section did not produce any known mycotoxins: A. aspearensis, A. coremiiformis, A. lanosus, A. leporis, A. sojae and A. subflavus. An overview of other small molecule extrolites produced in Aspergillus section Flavi is given.

Phylogenetic analyses of bacteria associated with the processing of iru and ogiri condiments.

Analysis of the bacterial community dynamics during the production of traditional fermented condiments is important for food safety assessment, quality control and development of starter culture technology. In this study, bacteria isolated during the processing of iru and ogiri, two commonly consumed condiments in Nigeria, were characterized based on phylogenetic analyses of the bacterial 16S rRNA gene. A total of 227 isolates were obtained and clustered into 12 operational taxonomic units (OTUs) based on 97% 16S rRNA gene similarity. The OTUs spanned three phyla (Firmicutes, Actinobacteria and Proteobacteria), and nine genera: Acinetobacter, Aerococcus, Bacillus, Enterococcus, Enterobacter, Lysinibacillus, Micrococcus, Proteus and Staphylococcus. OTUs closely related to species of Bacillus dominated the processing stages of both condiments. Although no single OTU occurred throughout iru processing stages, an OTU (mostly related to B. safensis) dominated the ogiri processing stages indicating potentials for the development of starter culture. However, other isolates such as those of Enterococcus spp. and Lysinibacillus spp. may be potential starters for iru fermentation. Presumptive food-borne pathogens were also detected at some stages of the condiments' processing, possibly due to poor hygienic practices. SIGNIFICANCE AND IMPACT OF THE STUDY: Iru and ogiri are important condiments used for flavour enhancement in foods and serve as protein substitutes in diets among rural populations across West Africa. Consumption of these condiments is growing, reinforcing the need to scale up their production. Production of these condiments includes spontaneous fermentation, which often leads to inconsistent product quality and unguaranteed safety. This study has demonstrated the bacterial succession in iru and ogiri processing and highlights species that could be selected and exploited for starter culture development. This study provides a starting point to produce quality and microbiologically safe iru and ogiri condiments.

Environmental impacts of cage culture in Lake Victoria: the case of Shirati Bay-Sota, Tanzania.

The experimental cage culture was conducted at Shirati bay, Lake Victoria from February to August 2013, to investigate the impacts of the small scale cage culture on the environment. Three locations along the cages, at the intermediate and one in the offshore (control) were sampled for water quality parameters, phytoplankton and macro invertebrates. A notable increase in nutrient concentration was observed after the set of cages among the stations. However DO, pH, and water transparency showed no major changes and was within the recommended ranges. Cyanophytes an indicator of inorganic pollution dominated before and after the set of cages, an increase in phytoplankton numerical abundance was observed after stocking of fish in cages. In addition there was an increase in the invertebrate community especially bivalves and gastropods. In conclusion we found no consistent environmental change caused by cage culture, and therefore it can be allowed in Lake Victoria, Tanzania part, with close monitoring of its impacts.

Distribution of aflatoxigenic Aspergillus section Flavi in commercial poultry feed in Nigeria.

The distribution and aflatoxigenicity of Aspergillus section Flavi isolates in 58 commercial poultry feed samples obtained from 17 states in five agro-ecological zones (AEZs) in Nigeria were determined in order to assess the safety of the feeds with respect to aflatoxin-producing fungi. Correlation was also performed for incidence of species, aflatoxin-producing ability of isolates in vitro, and aflatoxin (AFB1) concentrations in the feed. A total of 1006 Aspergillus section Flavi isolates were obtained from 87.9% of the feed samples and identified as Aspergillus flavus, unnamed taxon SBG, Aspergillus parasiticus and Aspergillus tamarii. A. flavus was the most prevalent (91.8%) of the isolates obtained from the feed in the AEZs while A. parasiticus had the lowest incidence (0.1%) and was isolated only from a layer mash sample collected from the DS zone. About 29% of the Aspergillus isolates produced aflatoxins in maize grains at concentrations up to 440,500µg/kg B and 341,000µg/kgG aflatoxins. The incidence of toxigenic isolates was highest (44.4%) in chick mash and lowest (19.9%) in grower mash. The population of A. flavus in the feed had positive (r=0.50) but non significant (p>0.05) correlations with proportion of toxigenic isolates obtained from the feed while SBG had significant (p<0.001) positive (r=0.99) influence on AFB1 concentrations in the feed. Poultry feed in Nigerian markets are therefore highly contaminated with aflatoxigenic Aspergillus species and consequently, aflatoxins. This is a potential threat to the poultry industry and requires urgent intervention.

Assessment of aflatoxigenic Aspergillus and other fungi in millet and sesame from Plateau State, Nigeria.

Sixteen fonio millet and 17 sesame samples were analysed for incidence of moulds, especially aflatoxigenic Aspergillus species, in order to determine the safety of both crops to consumers, and to correlate aflatoxin levels in the crops with levels produced by toxigenic isolates on laboratory medium. Diverse moulds including Alternaria, Aspergillus, Cercospora, Fusarium, Mucor, Penicillium, Rhizopus and Trichoderma were isolated. Aspergillus was predominantly present in both crops (46-48%), and amongst the potentially aflatoxigenic Aspergillus species, A. flavus recorded the highest incidence (68% in fonio millet; 86% in sesame kernels). All A. parvisclerotigenus isolates produced B and G aflatoxins in culture while B aflatoxins were produced by only 39% and 20% of A. flavus strains isolated from the fonio millet and sesame kernels, respectively. Aflatoxin concentrations in fonio millet correlated inversely (r = -0.55; p = 0.02) with aflatoxin levels produced by toxigenic isolates on laboratory medium, but no correlation was observed in the case of the sesame samples. Both crops, especially sesame, may not be suitable substrates for aflatoxin biosynthesis. This is the first report on A. parvisclerotigenus in sesame.

Fungal and mycotoxin assessment of dried edible mushroom in Nigeria.

In order to determine whether dried mushrooms are a foodstuff that may be less susceptible to infection by toxigenic molds and consequently to mycotoxin contamination, 34 dried market samples were analyzed. Fungal population was determined in the samples by conventional mycological techniques and molecular studies, while the spectrum of microbial metabolites including mycotoxins was analyzed by a liquid chromatography tandem mass spectrometric method covering 320 metabolites. Molds such as Fusarium, Penicillium, Trichoderma and aflatoxigenic species of Aspergillus (Aspergillus flavus and Aspergillus parvisclerotigenus) were recovered from all samples at varying levels. None of the mycotoxins addressed by regulatory limits in the EU was positively identified in the samples. However, 26 other fungal metabolites occurred at sub- to medium µg/kg levels in the samples, including aflatoxin/sterigmatocystin bio-precursors, bis-anthraquinone derivatives from Talaromyces islandicus, emerging toxins (e.g. enniatins) and other Fusarium metabolites, and clavine alkaloids. Although little is known on the toxicology of these substances, the absence of aflatoxins and other primary mycotoxins suggests that dried mushrooms may represent a relatively safe type of food in view of mycotoxin contamination.

Mycotoxins and fungal metabolites in groundnut- and maize-based snacks from Nigeria.

This exploratory study was aimed at investigating the spectrum of fungal metabolites in the processed food and snacks. Twenty types of snacks made separately from groundnut (n = 10), maize (n = 8) and a combination of groundnut and maize (n = 2) were analysed for naturally occurring mycotoxins and other fungal metabolites by a liquid chromatography-tandem mass spectrometric multi-mycotoxin method. A total of 18, 21 and 32 metabolites were detected and quantified in the groundnut-, groundnut/maize- and maize-based snacks, respectively. Aflatoxins contaminated 2, 3 and 5 of the groundnut/maize-, groundnut- and maize-based snacks at concentrations up to 14, 1041 and 74 µg kg(-1), respectively. Thus, the National Agency for Food and Drug Administration and Control (NAFDAC) recommended limit of 20 µg kg(-1) for aflatoxins was exceeded in 6 of the 20 snacks. Fumonisins contaminated all the maize- and groundnut/maize-based snacks with higher concentrations in the maize-based snacks (mean = 218.7 µg kg(-1)) compared with the groundnut/maize-based snacks (mean = 178.5 µg kg(-1)). Up to 26 different metabolites were found to co-occur in the same samples, thus posing an additional threat to the consumers due to possible additive and/or synergistic effects.

Fungal and bacterial metabolites in commercial poultry feed from Nigeria.

Metabolites of toxigenic fungi and bacteria occur as natural contaminants (e.g. mycotoxins) in feedstuffs making them unsafe to animals. The multi-toxin profiles in 58 commercial poultry feed samples collected from 19 districts in 17 states of Nigeria were determined by LC/ESI-MS/MS with a single extraction step and no clean-up. Sixty-three (56 fungal and seven bacterial) metabolites were detected with concentrations ranging up to 10,200 µg kg⁻¹ in the case of aurofusarin. Fusarium toxins were the most prevalent group of fungal metabolites, whereas valinomycin occurred in more than 50% of the samples. Twelve non-regulatory fungal and seven bacterial metabolites detected and quantified in this study have never been reported previously in naturally contaminated stored grains or finished feed. Among the regulatory toxins in poultry feed, aflatoxin concentrations in 62% of samples were above 20 µg kg⁻¹, demonstrating high prevalence of unsafe levels of aflatoxins in Nigeria. Deoxynivalenol concentrations exceeded 1000 µg kg⁻¹ in 10.3% of samples. Actions are required to reduce the consequences from regulatory mycotoxins and understand the risks of the single or co-occurrence of non-regulatory metabolites for the benefit of the poultry industry.

Aflatoxin-mediated Sperm and Blood Cell Abnormalities in Mice Fed with Contaminated Corn.

The effect of aflatoxin-contaminated corn on albino mice was investigated using the sperm morphology assay. Blood parameter levels including; total white blood cells (WBC), total red blood cells (RBC), packed cell volume (PCV), serum bilirubin (SB) and fasting blood sugar (FBS) were also determined in the tested mice. Test mice were exposed to aflatoxin-contaminated corn (contamination level of 100 ppb) for 1~4 weeks while aflatoxin-free corn and cyclophosphamide were used as negative and positive controls, respectively. Sperm cells showed varieties of morphological abnormality when assessed after 5 weeks. The percentage frequencies of the negative and positive controls were 18.8% and 48.87%, respectively, while the percentage abnormalities for the 1, 2, 3 and 4 weeks exposures were 41.38%, 48.17%, 57.13% and 61.67%, respectively. PCV, WBC, total bilirubin and glucose level values of mice in all concentrations were higher and statistically significant as compared to the negative control values using Dunnett's test. Therefore, abnormal sperm cell induction is concentration-dependent such that continuous consumption of aflatoxin-contaminated corn is capable of negatively affecting spermatogenesis by inducing or increasing the frequency of morphologically abnormal sperm cells produced.