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This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR, gel electrophoresis, and Sanger sequencing. Students retrieved their sequences and identified their fish using the NCBI BLAST nucleotide database. Slightly more than half of the samples yielded sequences identical or close to expected (based on the identification of the fish on the packaging); some other samples matched unanticipated fish or other organisms, due to an incomplete database, minor sequencing errors, or laboratory contamination (human and fungal sequences); 1 canned tuna sample identified as hake could represent food fraud.

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The biosynthetic pathway for amanitins and related cyclic peptides in deadly Amanita (Amanitaceae) mushrooms represents the first known ribosomal cyclic peptide pathway in the Fungi. Amanitins are found outside of the genus in distantly related agarics Galerina (Strophariaceae) and Lepiota (Agaricaceae). A long-standing question in the field persists: why is this pathway present in these phylogenetically disjunct agarics? Two deadly mushrooms, A. pallidorosea and A. subjunquillea, were deep sequenced, and sequences of biosynthetic genes encoding MSDINs (cyclic peptide precursor) and prolyl oligopeptidases (POPA and POPB) were obtained. The two Amanita species yielded 29 and 18 MSDINs, respectively. In addition, two MSDIN sequences were cloned from L. brunneoincarnata basidiomes. The toxin MSDIN genes encoding amatoxins or phallotoxins from the three genera were compared, and a phylogenetic tree constructed. Prolyl oligopeptidase B (POPB), a key enzyme in the biosynthetic pathway, was used in phylogenetic reconstruction to infer the evolutionary history of the genes. Phylogenies of POPB and POPA based on both coding and amino acid sequences showed very different results: while POPA genes clearly reflected the phylogeny of the host species, POPB did not; strikingly, it formed a well-supported monophyletic clade, despite that the species belong to different genera in disjunct families. POPA, a known house-keeping gene, was shown to be restricted in a branch containing only Amanita species and the phylogeny resembled that of those Amanita species. Phylogenetic analyses of MSDIN and POPB genes showed tight coordination and disjunct distribution. A POPB gene tree was compared with a corresponding species tree, and distances and substitution rates were compared. The result suggested POPB genes have significant smaller distances and rates than the house-keeping rpb2, discounting massive gene loss. Under this assumption, the incongruency between the gene tree and species tree was shown with strong support. Additionally, k-mer analyses consistently cluster Galerina and Amanita POPB genes, while Lepiota POPB is distinct. Our result suggests that horizontal gene transfer (HGT), at least between Amanita and Galerina, was involved in the acquisition of POPB genes, which may shed light on the evolution of the α-amanitin biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Maize ( Zea mays) is a staple in many developing countries but is known to be prone to pest (insects, birds, and rodents) and fungal infestation. In Guatemala, mycotoxin contamination of cultivated products may occur owing to such factors as environmental conditions and the use of traditional agriculture operations. To assess the current maize conditions in Guatemala, a small-scale study was performed. Mold and insect counts and mycotoxin (aflatoxin and fumonisin) concentrations were determined on 25 farms in two townships (Chiantla and Todos Santos) of the Huehuetenango Department. Total fungal counts were 3.6 to 6.83 log CFU/g with no significant differences ( P > 0.05) across farms at different altitudes. Farms where maize was not produced but was purchased were at higher risk of fumonisin contamination, whereas local producers were mostly affected by aflatoxins. Aflatoxin was present in maize from 100% of farms at 1.0 to 85.3 ppb, and fumonisin was detected on 52% of farms at 0.4 to 31.0 ppm. Average mycotoxin consumption amounts were above the recommended maximum intake for aflatoxin in both produced and purchased maize and above the provisional maximum tolerable daily intake for fumonisin in purchased maize. Estimated daily intake was 0.01 to 0.85 μg/kg of body weight per day for aflatoxin and 2.9 to 310.0 μg/kg of body weight per day for fumonisin. An entomological analysis revealed overall 32% prevalence of Ephestia kuehniella (flour moth), 16% prevalence of Sitophilus zeamais (maize weevil), and 8% prevalence of Tribolium sp. (flour beetle) on the analyzed farms. This study highlighted poor agricultural practices used in the highlands of Guatemala. Current practices should be revised for the production of maize that is safe for consumption by the population in this region. [ABSTRACT FROM AUTHOR]

Maize is a staple crop in Guatemala, especially in the rural regions where it is consumed in high amounts. Given that traditional pre- and post-harvest practices lead to exposure to the environmental surroundings where pests and microorganisms may be present, maize quality and safety can be compromised severely. In order to assess the potential degree of risk, an exploratory study involving maize mycobiota from six farms from Huehuetenango, Guatemala was conducted. DNA was extracted from the maize samples, and the ITS1 region was subjected to Illumina sequencing. This survey identified 52 fungal taxa in the 90-day maize storage period. For the samples where the maize moisture content exceeded 20%, a high yeast content was observed which can reflect spoilage during storage. A significant amount of Fusarium and Aspergillus – mycotoxin-producing molds – was found, representing a potential for mycotoxin contamination. This indicates a plausible health risk in a region where maize represents a significant portion of the diet. Potential maize pathogens in the genera Acremonium and Cladosproium, and Stenocarpella maydis, were also common. Results from this study can help better understand the potential health-risk scenario in the Highlands of Guatemala if poor grain handling practices are adopted. [ABSTRACT FROM AUTHOR]

Small bowel transplantation (SBT) can be a life-saving medical procedure. However, these recipients experience high risk of bloodstream infections caused by Candida. This research aims to characterise the SBT recipient gut microbiota over time following transplantation and investigate the epidemiology of candidaemia in seven paediatric patients. Candida species from the recipients' ileum and bloodstream were identified by internal transcribed spacer sequence and distinguished to strain by multilocus sequence typing and randomly amplified polymorphic DNA. Antifungal susceptibility of bloodstream isolates was determined against nine antifungals. Twenty-two ileostomy samples harboured at least one Candida species. Fungaemia were caused by Candida parapsilosis, Candida albicans, Candida glabrata, Candida orthopsilosis and Candida pelliculosa. All but three bloodstream isolates showed susceptibility to all the antifungals tested. One C. glabrata isolate showed multidrug resistance to itraconazole, amphotericin B and posaconazole and intermediate resistance to caspofungin. Results are congruent with both endogenous ( C. albicans, C. glabrata) and exogenous ( C. parapsilosis) infections; results also suggest two patients were infected by the same strain of C. parapsilosis. Continuing to work towards a better understanding of sources of infection-particularly the exogenous sources-would lead to targeted prevention strategies. [ABSTRACT FROM AUTHOR]

Many species of fungi have been detected in the healthy human gut; however, nearly half of all taxa reported have only been found in one sample or one study. Fungi capable of growing in and colonizing the gut are limited to a small number of species, mostlyCandidayeasts and yeasts in the family Dipodascaceae (Galactomyces, Geotrichum, Saprochaete).Malasseziaand the filamentous fungusCladosporiumare potential colonizers; more work is needed to clarify their role. Other commonly-detected fungi come from the diet or environment but either cannot or do not colonize (PenicilliumandDebaryomycesspecies, which are common on fermented foods but cannot grow at human body temperature), while still others have dietary or environmental sources (Saccharomyces cerevisiae, a fermentation agent and sometime probiotic;Aspergillusspecies, ubiquitous molds) yet are likely to impact gut ecology. The gut mycobiome appears less stable than the bacterial microbiome, and is likely subject to environmental factors. [ABSTRACT FROM PUBLISHER]

This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR, gel electrophoresis, and Sanger sequencing. Students retrieved their sequences and identified their fish using the NCBI BLAST nucleotide database. Slightly more than half of the samples yielded sequences identical or close to expected (based on the identification of the fish on the packaging); some other samples matched unanticipated fish or other organisms, due to an incomplete database, minor sequencing errors, or laboratory contamination (human and fungal sequences); 1 canned tuna sample identified as hake could represent food fraud. [ABSTRACT FROM AUTHOR]

Fungal DNA was selectively amplified, and the ITS region sequenced, from fecal samples taken from 45 healthy human volunteers at one (21 volunteers) or two (24 volunteers) time points. Seventy-two operational taxonomic units, representing two phyla and ten classes of fungi, were recovered. Candida yeasts, notably Candida tropicalis (present in 51 samples), and yeasts in the Dipodascaceae (39 samples), dominated, while 38 OTUs were detected in a single sample each. Fungi included known human symbionts ( Candida , Cryptococcus , Malassezia and Trichosporon spp.), common airborne fungi ( Cladosporium sp.) and fungi known to be associated with food ( Debaryomyces hansenii and high salt fermented foods; Penicillium roqueforti and blue cheese). In contrast with gut-associated bacteria, fungi occurred in much lower abundance and diversity, and fungal composition appeared unstable over time. [ABSTRACT FROM AUTHOR]

Candida yeasts are the dominant fungi in the healthy human microbiome, but are well-known for causing disease following a variety of perturbations. Evaluation of fungal populations from the healthy human gut revealed a significant negative correlation between the foodborne yeast, Debaryomyces hansenii , and Candida species. D. hansenii is reported to produce killer toxins (mycocins) effective against other yeast species. In order to better understand this phenomenon, a collection of 42 D. hansenii isolates was obtained from 22 cheeses and evaluated for killer activity against Candida albicans and Candida tropicalis over a range of temperatures and pH values. Twenty three strains demonstrated killer activity against both C. albicans and C. tropicalis , which was pH- and temperature-dependent, with no killer activity observed for any strain at pH 6.5 or higher, or at ≥ 35 °C (physiological conditions in the human gastrointestinal tract). A cell-free mycocin preparation showed transient killer activity against C. albicans at 35 °C and a cheese sample containing a killer D. hansenii strain demonstrated sustained killer activity against both C. albicans and C. tropicalis . Together, these observations raise the possibility that D. hansenii could influence Candida populations in the gut. [ABSTRACT FROM AUTHOR]

Introduction: Kombucha is a popular fermented tea that has attracted considerable attention due, in part, to its suggested health benefits. Previous results from animal models led us to hypothesize kombucha may reduce blood sugar levels in humans with diabetes. The objective of this pilot clinical study was to evaluate kombucha for its anti-hyperglycemic activities in adults with diabetes mellitus type II., Methods: The study was organized as a prospective randomized double-blinded crossover study at a single-center urban hospital system. Participants ( n  = 12) were instructed to consume either a kombucha product or a placebo control (each 240 mL) for 4 weeks. After an 8-week washout period, participants consumed the alternate product. Fasting blood glucose levels were self-determined at baseline and at 1 and 4 weeks during each treatment period. Secondary health outcomes, including overall health, insulin requirement, gut health, skin health, mental health, and vulvovaginal health were measured by questionnaire at the same time points. The kombucha microbiota was assessed by selective culturing and 16S rRNA gene (bacteria) and ITS (fungi) sequencing. Fermentation end products were assessed by HPLC. Statistical significance of changes in fasting blood glucose was determined using paired, two-tailed student's t -tests., Results: Kombucha lowered average fasting blood glucose levels at 4 weeks compared to baseline (164 vs. 116 mg/dL, p  = 0.035), whereas the placebo did not (162 vs. 141 mg/dL, p  = 0.078). The kombucha microbiota, as assessed by cultural enumeration, was mainly comprised of lactic acid bacteria, acetic acid bacteria, and yeast, with each group present at about 10 6 colony forming units (CFU)/mL. Likewise, 16S rRNA gene sequencing confirmed that lactic acid and acetic acid bacteria were the most abundant bacteria, and ITS sequencing showed Dekkera was the most abundant yeast. The primary fermentation end products were lactic and acetic acids, both less than 1%. Ethanol was present at 1.5%., Discussion: Although this pilot study was limited by a small sample size, kombucha was associated with reduced blood glucose levels in humans with diabetes. Larger follow-up studies are warranted., Clinical Trial Registration: ClinicalTrials.gov, identifier NCT04107207., Competing Interests: RH is a co-founder of Synbiotic Health; JA has a financial interest in Synbiotic Health. DM serves as President of the Board of Directors of the International Scientific Association for Probiotics and Prebiotics, a non-paid position. All kombucha and placebo drinks were donated by Craft Kombucha. Craft Kombucha did not have any access to data reported in this study. No author has any financial ties with Craft Kombucha. SD was employed by MedStar Health. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mendelson, Sparkes, Merenstein, Christensen, Sharma, Desale, Auchtung, Kok, Hallen-Adams and Hutkins.)

The deadly toxin α-amanitin is a bicyclic octapeptide biosynthesized on ribosomes. A phylogenetically disjunct group of mushrooms in Agaricales (Amanita, Lepiota, and Galerina) synthesizes α-amanitin. This distribution of the toxin biosynthetic pathway is possibly related to the horizontal transfer of metabolic gene clusters among taxonomically unrelated mushrooms with overlapping habitats. Here, our work confirms that two biosynthetic genes, P450-29 and FMO1, are oxygenases important for amanitin biosynthesis. Phylogenetic and genetic analyses of these genes strongly support their origin through horizontal transfer, as is the case for the previously characterized biosynthetic genes MSDIN and POPB. Our analysis of multiple genomes showed that the evolution of the α-amanitin biosynthetic pathways in the poisonous agarics in the Amanita, Lepiota, and Galerina clades entailed distinct evolutionary pathways including gene family expansion, biosynthetic genes, and genomic rearrangements. Unrelated poisonous fungi produce the same deadly amanitin toxins using variations of the same pathway. Furthermore, the evolution of the amanitin biosynthetic pathway(s) in Amanita species generates a much wider range of toxic cyclic peptides. The results reported here expand our understanding of the genetics, diversity, and evolution of the toxin biosynthetic pathway in fungi.

Maize ( Zea mays) is a staple in many developing countries but is known to be prone to pest (insects, birds, and rodents) and fungal infestation. In Guatemala, mycotoxin contamination of cultivated products may occur owing to such factors as environmental conditions and the use of traditional agriculture operations. To assess the current maize conditions in Guatemala, a small-scale study was performed. Mold and insect counts and mycotoxin (aflatoxin and fumonisin) concentrations were determined on 25 farms in two townships (Chiantla and Todos Santos) of the Huehuetenango Department. Total fungal counts were 3.6 to 6.83 log CFU/g with no significant differences ( P > 0.05) across farms at different altitudes. Farms where maize was not produced but was purchased were at higher risk of fumonisin contamination, whereas local producers were mostly affected by aflatoxins. Aflatoxin was present in maize from 100% of farms at 1.0 to 85.3 ppb, and fumonisin was detected on 52% of farms at 0.4 to 31.0 ppm. Average mycotoxin consumption amounts were above the recommended maximum intake for aflatoxin in both produced and purchased maize and above the provisional maximum tolerable daily intake for fumonisin in purchased maize. Estimated daily intake was 0.01 to 0.85 μg/kg of body weight per day for aflatoxin and 2.9 to 310.0 μg/kg of body weight per day for fumonisin. An entomological analysis revealed overall 32% prevalence of Ephestia kuehniella (flour moth), 16% prevalence of Sitophilus zeamais (maize weevil), and 8% prevalence of Tribolium sp. (flour beetle) on the analyzed farms. This study highlighted poor agricultural practices used in the highlands of Guatemala. Current practices should be revised for the production of maize that is safe for consumption by the population in this region.