Antimicrobial plant secondary metabolites, MDR transporters and antimicrobial resistance in cereal-associated lactobacilli: is there a connection?

Cereal-associated lactobacilli resist antimicrobial plant secondary metabolites. This study aimed to identify multi-drug-resistance (MDR) transporters in isolates from mahewu, a Zimbabwean fermented cereal beverage, and to determine whether these MDR-transporters relate to resistance against phenolic compounds and antibiotics. Comparative genomic analyses indicated that all seven mahewu isolates harbored multiple MATE and MFS MDR proteins. Strains of Lactiplantibacillus plantarum and Limosilactobacillus fermentum encoded for the same gene, termed mahewu phenolics resistance gene mprA, with more than 99% nucleotide identity, suggesting horizontal gene transfer. Strains of Lp. plantarum were more resistant than strains of Lm. fermentum to phenolic acids, other antimicrobials and antibiotics but the origins of strains were not related to resistance. The resistance of several strains exceeded EFSA thresholds for several antibiotics. Analysis of gene expression in one strain each of Lp. plantarum and Lm. fermentum revealed that at least one MDR gene in each strain was over-expressed during growth in wheat, sorghum and millet relative to growth in MRS5 broth. In addition, both strains over-expressed a phenolic acid reductase. The results suggest that diverse lactobacilli in mahewu share MDR transporters acquired by lateral gene transfer, and that these transporters mediate resistance to secondary plant metabolites and antibiotics.

Composition and Origin of the Fermentation Microbiota of Mahewu, a Zimbabwean Fermented Cereal Beverage.

Mahewu is a fermented cereal beverage produced in Zimbabwe. This study determined the composition and origin of mahewu microbiota. The microbiota of mahewu samples consisted of 3 to 7 dominant strains of lactobacilli and two strains of yeasts. Enterobacteriaceae were not detected. Candida glabrata was present in high cell counts from samples collected in summer but not from samples collected in winter. Millet malt is the only raw ingredient used in the production of mahewu and is a likely source of fermentation microbiota; therefore, malt microbiota was also analyzed by culture-dependent and high-throughput 16S rRNA gene sequencing methodologies. Millet malt contained 8 to 19 strains of Enterobacteriaceae, lactobacilli, bacilli, and very few yeasts. Strain-specific quantitative PCR assays were established on the basis of the genome sequences of Lactobacillus fermentum FUA3588 and FUA3589 and Lactobacillus plantarum FUA3590 to obtain a direct assessment of the identity of strains from malt and mahewu. L. fermentum FUA3588 and FUA3589 were detected in millet malt, demonstrating that millet malt is a main source of mahewu microbiota. Strains which were detected in summer were not detected in samples produced at the same site in winter. Model mahewu fermentations conducted with a 5-strain inoculum consisting of lactobacilli, Klebsiella pneumoniae, and Cronobacter sakazakii demonstrated that lactobacilli outcompete Enterobacteriaceae, which sharply decreased in the first 24 h. In conclusion, mahewu microbiota is mainly derived from millet malt microbiota, but minor components of malt microbiota rapidly outcompete Enterobacteriaceae and Bacillus species during fermentation.IMPORTANCE This study provides insight into the composition and origin of the microbiota of mahewu and the composition of millet malt microbiota. Fermentation microbiota are often hypothesized to be derived from the environment, but the evidence remains inconclusive. Our findings confirm that millet malt is the major source of mahewu microbiota. By complementing culture methods with high-throughput sequencing of 16S rRNA amplicons and strain-specific quantitative PCR, this study provides evidence about the source of mahewu microbiota, which can inform the development of starter cultures for mahewu production. The study also documents the fate of Enterobacteriaceae during the fermentation of mahewu. There are concerns regarding the safety of traditionally prepared mahewu, and this requires in-depth knowledge of the fermentation process. Therefore, this study elucidated millet malt microbiota and identified cultures that are able to control the high numbers of Enterobacteriaceae that are initially present in mahewu fermentations.

African cereal fermentations: A review on fermentation processes and microbial composition of non-alcoholic fermented cereal foods and beverages.

Africa has a rich tradition of cereal fermentations to produce diverse products including baked goods, porridges, non-alcoholic beverages and alcoholic beverages. Diversity also relates to the choice of the fermentation substrates, which include wheat, maize, teff, sorghum and millet, and the fermentation processes that are used in food production. For fermentation processes that are used in baking and brewing, it is well established that the composition of fermentation microbiota and thus the impact of fermentation on product quality is determined by the choice of fermentation conditions. This link has not been systematically explored for African cereal fermentations. This review therefore aims to provide an overview on the diversity of African fermented cereal products, and to interrogate currently available literature data with respect to the impact of fermentation substrate and fermentation processes on the assembly of fermentation microorganisms and product quality.