Sijmen E. Schoustra

Background: Resistance to medical azoles has been well investigated in Aspergillus fumigatus, which is primarily driven by exposure to azole fungicides. Azole resistance selection in other Aspergillus species remains understudied. We investigated azole resistance selection in Aspergillus series Nigri.Methods: 165 clinical series Nigri isolates were collected from Dutch and Belgian hospitals and 77 environmental isolates were obtained from air and other environmental sources, including known hotspots for azole resistance selection. Isolates were screened for resistance to medical azoles and selected fungicides using the EUCAST microbroth dilution reference method. Epidemiological cut-off values (ECOFFs) were used to identify non-wildtype isolates. All isolates were subjected to whole-genome sequencing (WGS), and global sequence databases were used for population genomic analyses. Clinical information was collected for azole-resistant isolates.Findings: Aspergillus tubingensis was the dominant species lineage among the clinical isolates (120/165, 72·7 %), whereas species of the A. niger lineage were more frequently identified in environmental samples (55/78, 70·5 %). Overall A. tubingensis isolates showed higher MICs than A. niger isolates; 27 clinical isolates (17·0 %) showed a non-wildtype phenotype for medical azoles as well as 24 of 78 (30·8 %) environmental isolates. WGS revealed a worldwide distribution of multiple clonal groups. Cross resistance between medical azoles and azole fungicides was observed in A. tubingensis lineage (ATL) isolates, which were recovered from hotspots, air and patient samples. Pan-azole resistant A. tubingensis was recovered from four mould-active azole-naïve patients, including a patient with proven invasive aspergillosis.Interpretation: Our study shows azole resistance in ATL isolates and evidence that supports environmental resistance selection. Population genomics indicate a risk of global migration of resistant clones in A. tubingensis. Next to confirmative studies regarding black aspergilli, mycobiome analysis of hotspots are needed to identify potential selection of intrinsic or acquired resistant fungal species that may cause infections.

Chepa shutki is a fermented fish product from Bangladesh that is produced traditionally by spontaneous fermentation. Although it is very popular, there is limited understanding of how processing variations and bacterial communities affect fermentation outcomes and final product quality. In this study, controlled fish fermentations mimicking chepa shutki production were performed to investigate the effect of fish soaking time on water activity, pH, microbial growth, aroma compounds and organic acids. Additionally, the impact of adding mixtures of endogenous Tetragenococcus halophilus and coagulase-negative Staphylococcus isolates as adjunct starter cultures was assessed under controlled laboratory conditions. Water activity increased with longer soaking times and remained constant during fermentation. Bacteria grew faster and reached higher concentrations in longer-soaked fish, while pH remained stable in all fermentations. The adjunct starter cultures had a limited effect on fermentation outcome and the process relied mainly on microorganisms present in the dried fish. Aroma production was influenced by fermentation time and fish soaking time, together explaining more than 60% of the variation observed across samples. These results suggest that high-quality chepa shutki can be consistently produced by standardizing processing conditions, particularly soaking and fermentation time, while reliably relying on the natural microbial community for fermentation.

Species sorting underlies changes in microbial community composition under varying environments, yet predicting the species diversity and their functional outcomes when exposed to novel conditions remains challenging. We explored this using mabisi, a Zambian traditional fermented milk, by propagating a shared starting mabisi microbial community across five novel substrates - raw bovine milk (control), low-fat milk, full-cream milk, and the infant formulas F100 and S26 – under static conditions and fixed dilution for ~ 33 generations, at three rural farm sites. The microbial community composition was profiled through 16 S rDNA sequencing, and community-level functioning through volatile organic compounds, pH, and consistency. We observed a substrate-driven divergence of microbial communities, with low-fat milk enriched with Acetobacter , full-cream milk enriched with Lactococcus , and S26 enriched with Lactiplantibacillus and Leuconostoc . This pattern exhibited a temporal community succession but remained consistent between sites. Similarly, the community-level functionality diverged but remained largely repeatable to properties known for mabisi. Our findings suggest that novel environments drive a species sorting process in natural microbial communities, and this process persists over time. Additionally, the repeatability of its community-level functionality points to underlying functional redundancy within diverse and successive microbial communities. Our study not only advances the understanding of how microbial communities adapt to environmental changes but also provides a basis for harnessing the mabisi-derived microbial community for broad biotechnological applications, particularly supporting the feasibility and effectiveness of its use in an in-house formulated F100 infant formula for treating malnourished children in developing regions.

Societal Impact Statement: Traditional fermented foods worldwide depend on naturally occurring microbes, yet the environmental sources shaping these microbes and their effects on food quality remain poorly understood. We investigated bacteria living inside the roots of wild plants used to produce Munkoyo, a widely consumed nonalcoholic fermented cereal beverage in Zambia, to determine how plant species and local growing conditions influence these microbial communities. We found that both plant species identity and soil environment shape the root microbes that seed fermentation. These findings can improve beverage quality, support the cultivation of threatened species, and promote sustainable access to ingredients, helping protect nutrition, livelihoods and cultural heritage. Summary: Plants interact with diverse soil microbes through their roots, and these bacterial communities play vital roles in plant health and nutrient acquisition. In some traditional foods, root microbiomes of specific wild plant species act as natural starter cultures for fermentation. Yet, it remains unclear how distinct these root bacterial communities are among plant species and how much variation reflects plant identity versus growing environment. This study examined three wild plant species used as inoculum for producing Munkoyo, a traditional, nonalcoholic fermented cereal beverage in Zambia. Root endosphere, rhizosphere and bulk soil samples were collected from five districts, and bacterial communities were characterised using 16S rRNA gene amplicon sequencing. Bulk soil bacterial communities varied strongly across field sites and were structured by soil physicochemical properties, whereas root endosphere microbiomes were more distinct and differed among plant species and locations. The findings contribute to a better understanding of the bacterial communities involved in traditional fermented beverage production. They highlight the importance of plant species and soil environment in structuring root microbiomes, with implications for fermentation quality, sustainable cultivation of fermentation plants, and cultural heritage preservation.

Chepa shutki is a spontaneously fermented fish product appreciated for its rich flavour and widely consumed in Bangladesh. However, it is unknown how processing influences the flavour and microbial community. This study investigates the effects of processing variations on volatile organic compounds (VOCs) and bacterial communities in chepa shutki. 120 samples were collected cross-sectionally from different regions of Bangladesh, while recording fish type, district, fermentation time, container type, water activity and pH. Substantial variation in VOC profiles was observed, with phenols, pyrazines, alcohols, alkanes and acids as the predominant VOC groups. Most processing parameters were associated with one or more VOC groups, with fish type showing the strongest effect. Puma chepa shutki exhibited a higher VOC abundance, particularly pyrazines, whereas punti chepa shutki lacked phenols and showed higher levels of furanones. Tetragenococcus and Staphylococcus were detected in all samples, although overall bacterial community composition varied. Fish type and district significantly influenced bacterial communities, jointly explaining 13% of the variation. Limited correlation between bacterial communities and VOCs suggest functional redundancy among bacterial species. These findings highlight key processing parameters shaping functional properties of chepa shutki and inform future mechanistic studies to standardize traditional chepa shutki processing.

Species sorting underlies changes in microbial community composition under varying environments, yet predicting the species diversity and their functional outcomes when exposed to novel conditions remains challenging. We explored this using mabisi, a Zambian traditional fermented milk, by propagating a shared starting mabisi microbial community across five novel substrates - raw bovine milk (control), low-fat milk, full-cream milk, and the infant formulas F100 and S26 – under static conditions and fixed dilution for ~ 66 generations, at three rural farm sites. The microbial community composition was profiled through 16S rDNA sequencing, and community-level functioning through volatile organic compounds, pH, and consistency. We observed a substrate-driven divergence of microbial communities, with early-phase (~ 20 generations) communities enriched with Lactococcus , and transitioning to late-phase (~ 66 generations) communities enriched with Acetobacter , Lactiplantibacillus , Paucilactobacillus , Leuconostoc , Lacticaseibacillus , and Lactobacillus . This succession pattern remained consistent between sites. Despite community diversity shifts, community-level functionality remained largely repeatable. Our findings suggest that novel environments drive a species sorting process in natural microbial communities, and this process persists over time. Additionally, the maintenance of its community-level functionality despite community turnover points to underlying functional redundancy within diverse and successive microbial communities. Our study not only advances the understanding of how microbial community adapts to environmental changes but also provides a basis for harnessing the mabisi-derived microbial community for broad biotechnological applications, particularly supporting the feasibility and effectiveness of its use in an in-house formulated F100 infant formula for treating malnourished children in developing regions.

Sorting of maize ears based on husk cover at harvest may be considered as an approach to minimise the aflatoxin (AF) levels in maize grain intended for consumption. Aflatoxins are known natural human carcinogens produced by certain species of Aspergillus section (sect.) Flavi and impair livestock productivity. We investigated the influence of maize ear husk cover inadequacy on the natural infection of preharvest maize with Aspergillus sect. Flavi and contamination with AFs thereof. Additional investigated factors that may influence maize Aspergillus sect. Flavi infection and AF contamination were rainfall intensity, field burning, insect pest infestation, seed maturity and crop rotation. Maize and soil samples were collected over two seasons from two climatically contrasting regions of Zambia. One region was in the wetter Agro-Climatic Zone (ACZ) and another in the drier ACZ. Plate count technique on modified rose Bengal agar was used to quantify Aspergillus sect. Flavi densities in maize and soil. AF levels in maize were determined by High-Performance Liquid Chromatography with immunoaffinity column clean-up. Aspergillus sect. Flavi was present in soils. On maize, Aspergillus sect. Flavi was mainly detected under dry spell conditions despite being detected in all soils. Similarly, AF in maize was only detected under dry spell conditions. Findings showed that inadequate husk cover alone did not influence Aspergillus sect. Flavi infection of maize and subsequent AF contamination. Rather, dry spell with late rains predisposed the Aspergillus sect. Flavi infection and AF contamination. Insect pest incidence was the second most important factor aggravating the Aspergillus sect. Flavi infection.

The human gut contains a complex and highly variable microbial ecosystem of which the composition is affected by the health condition, lifestyle and diet of the host. Fermented dairy products harbour microorganisms favourable to the human gut microbial community. Mabisi, a spontaneous fermented local dairy product from Zambia, carries a variety of potentially beneficial microorganisms. Using the gastrointestinal tract (GI-tract) model system, SHIME^®, we tested how the administration of mabisi affects the composition and functionality of the human colon gut microbiota. After ten days of feeding mabisi into the GI-tract model system, the composition of the gut microbial community shifted towards a more even distribution of genera was similar to the community composition obtained by intervention with a standard prebiotic, fructooligosaccharide (FOS). This effect remained even when mabisi was heat-treated and all bacteria there were killed prior to the administration. Comparably to FOS, the microbial shift after mabisi treatment coincides with an increase in concentration of short chain fatty acids. Our findings suggest that mabisi carries important bioactive compounds with a prebiotic potential and might support and stabilize the gut microbial community.

The gut microbiota consisting of bacteria, fungi, archea, and viruses and plays a role as a barrier against pathogen invasion to promote intestinal health and preventing organismal infections. This study examined the potential protective properties of Mabisi, a widely consumed traditional fermented dairy product in Zambia, against pathogen invasion in the gut. Using the in-vitro static INFOGEST model, we challenged the gut microbiota from a pooled stool sample pf six Zambian children) and previously exposed to Mabisi by adding model pathogens. We hypothesized that pathogen invasion is suppressed in in-vitro gut microbiota treated with Mabisi due to its ability to modulate the gut microbiota composition. Our microbiota challenge treatments were Mabisi, fructooligosaccharides (FOS) as a positive control, sterile water as a negative control, and with and without pathogens. Out of the two strains of pathogens added to the gut microbiota, Escherichia coli established in FOS treated gut microbiota, however not in Mabisi and sterile water treatments. Furthermore, we show that Mabisi modulated gut microbiota by increasing Pediococcus , a commensal bacteria and increased production of short chain fatty acids including acetate, propionate, lactate, isobutyrate, formate, and succinate in Mabisi treated gut microbiota compared to both FOS and sterile water treated gut microbiota. This study contributes to evidence of the beneficial role of fermented foods such as Mabisi through the prevention of pathogen invasion of the gut microbiota in children. Importance: This study provides evidence supporting the protective role of Mabisi, a traditional fermented dairy product, in preventing pathogen invasion in the gut microbiota of children. By using an in-vitro approach, we demonstrate that Mabisi treatment suppresses the establishment of Escherichia coli, a key pathogen, unlike fructooligosaccharides (FOS), a well-known prebiotic. Additionally, Mabisi modulates gut microbiota composition by increasing beneficial bacteria such as Pediococcus and enhancing short-chain fatty acid (SCFA) production, which is essential for gut health. These findings highlight the potential of traditional fermented foods as affordable, accessible dietary interventions for improving gut health and preventing infections, particularly in low-resource settings like Zambia.

Integrating fermented cereals like mahewu into school meals is a cost-effective strategy to enhance nutrition and health outcomes, particularly in resource-constrained settings.