High fat intake sustains sorbitol intolerance after antibiotic-mediated Clostridia depletion from the gut microbiota.

Carbohydrate intolerance, commonly linked to the consumption of lactose, fructose, or sorbitol, affects up to 30% of the population in high-income countries. Although sorbitol intolerance is attributed to malabsorption, the underlying mechanism remains unresolved. Here, we show that a history of antibiotic exposure combined with high fat intake triggered long-lasting sorbitol intolerance in mice by reducing Clostridia abundance, which impaired microbial sorbitol catabolism. The restoration of sorbitol catabolism by inoculation with probiotic Escherichia coli protected mice against sorbitol intolerance but did not restore Clostridia abundance. Inoculation with the butyrate producer Anaerostipes caccae restored a normal Clostridia abundance, which protected mice against sorbitol-induced diarrhea even when the probiotic was cleared. Butyrate restored Clostridia abundance by stimulating epithelial peroxisome proliferator-activated receptor-gamma (PPAR-γ) signaling to restore epithelial hypoxia in the colon. Collectively, these mechanistic insights identify microbial sorbitol catabolism as a potential target for approaches for the diagnosis, treatment, and prevention of sorbitol intolerance.

A chemically-defined growth medium to support Lactobacillus-Acetobacter sp. community analysis.

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli or Acetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe's vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli and Acetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions.

Lactiplantibacillus plantarum uses ecologically relevant, exogenous quinones for extracellular electron transfer.

IMPORTANCE: While quinones are essential for respiratory microorganisms, their importance for microbes that rely on fermentation metabolism is not understood. This gap in knowledge hinders our understanding of anaerobic microbial habitats, such in mammalian digestive tracts and fermented foods. We show that Lactiplantibacillus plantarum, a model fermentative lactic acid bacteria species abundant in human, animal, and insect microbiomes and fermented foods, uses multiple exogenous, environmental quinones as electron shuttles for a hybrid metabolism involving EET. Interestingly, quinones both stimulate this metabolism as well as cause oxidative stress when extracellular electron acceptors are absent. We also found that quinone-producing, lactic acid bacteria species commonly enriched together with L. plantarum in food fermentations accelerate L. plantarum growth and medium acidification through a mainly quinone- and EET-dependent mechanism. Thus, our work provides evidence of quinone cross-feeding as a key ecological feature of anaerobic microbial habitats.

Extracellular electron transfer increases fermentation in lactic acid bacteria via a hybrid metabolism.

Energy conservation in microorganisms is classically categorized into respiration and fermentation; however, recent work shows some species can use mixed or alternative bioenergetic strategies. We explored the use of extracellular electron transfer for energy conservation in diverse lactic acid bacteria (LAB), microorganisms that mainly rely on fermentative metabolism and are important in food fermentations. The LAB Lactiplantibacillus plantarum uses extracellular electron transfer to increase its NAD+/NADH ratio, generate more ATP through substrate-level phosphorylation, and accumulate biomass more rapidly. This novel, hybrid metabolism is dependent on a type-II NADH dehydrogenase (Ndh2) and conditionally requires a flavin-binding extracellular lipoprotein (PplA) under laboratory conditions. It confers increased fermentation product yield, metabolic flux, and environmental acidification in laboratory media and during kale juice fermentation. The discovery of a single pathway that simultaneously blends features of fermentation and respiration in a primarily fermentative microorganism expands our knowledge of energy conservation and provides immediate biotechnology applications.

Bacteria produce the energy they need to live through two processes, respiration and fermentation. While respiration is often more energetically efficient, many bacteria rely on fermentation as their sole means of energy production. Respiration normally depends on the presence of small soluble molecules, such as oxygen, that can diffuse inside the cell, but some bacteria can use metals or other insoluble compounds found outside the cell to perform 'extracellular electron transfer'. Lactic acid bacteria are a large group of bacteria that have several industrial uses and live in many natural environments. These bacteria survive using fermentation, but they also carry a group of genes needed for extracellular electron transfer. It is unclear whether they use these genes for respiration or if they have a different purpose. Tejedor-Sanz, Stevens et al. used a lactic acid bacterium called Lactiplantibacillus plantarum to study whether and how this group of bacteria use extracellular electron transfer. Analysis of L. plantarum and its effect on its surroundings showed that these bacteria use a hybrid process to produce energy: the cells use aspects of extracellular respiration to increase the yield and efficiency of fermentation. Combining these two approaches may allow L. plantarum to adapt to different environments and grow faster, allowing it to compete against other species. Tejedor-Sanz, Stevens et al. provide new information on a widespread group of bacteria that are often used in food production and industry. The next step will be to understand how the hybrid system is controlled and how it varies among species. Understanding this process could result in new biotechnologies and foods that are healthier, produce less waste, or have different tastes and textures.

Microbiota Assessments for the Identification and Confirmation of Slit Defect-Causing Bacteria in Milk and Cheddar Cheese.

Validated methods are needed to detect spoilage microbes present in low numbers in foods and ingredients prior to defect onset. We applied propidium monoazide combined with 16S rRNA gene sequencing, qPCR, isolate identification, and pilot-scale cheese making to identify the microorganisms that cause slit defects in industrially produced Cheddar cheese. To investigate milk as the source of spoilage microbes, bacterial composition in milk was measured immediately before and after high-temperature, short-time (HTST) pasteurization over 10-h periods on 10 days and in the resulting cheese blocks. Besides HTST pasteurization-induced changes to milk microbiota composition, a significant increase in numbers of viable bacteria was observed over the 10-h run times of the pasteurizer, including 68-fold-higher numbers of the genus Thermus However, Thermus was not associated with slit development. Milk used to make cheese which developed slits instead contained a lower number of total bacteria, higher alpha diversity, and higher proportions of Lactobacillus, Bacillus, Brevibacillus, and Clostridium Only Lactobacillus proportions were significantly increased during cheese aging, and Limosilactobacillus (Lactobacillus) fermentum, in particular, was enriched in slit-containing cheeses and the pre- and post-HTST-pasteurization milk used to make them. Pilot-scale cheeses developed slits when inoculated with strains of L. fermentum, other heterofermentative lactic acid bacteria, or uncultured bacterial consortia from slit-associated pasteurized milk, thereby confirming that low-abundance taxa in milk can negatively affect cheese quality. The likelihood that certain microorganisms in milk cause slit defects can be predicted based on comparisons of the bacteria present in the milk used for cheese manufacture.IMPORTANCE Food production involves numerous control points for microorganisms to ensure quality and safety. These control points (e.g., pasteurization) are difficult to develop for fermented foods wherein some microbial contaminants are also expected to provide positive contributions to the final product and spoilage microbes may constitute only a small proportion of all microorganisms present. We showed that microbial composition assessments with 16S rRNA marker gene DNA sequencing are sufficiently robust to detect very-low-abundance bacterial taxa responsible for a major but sporadic Cheddar cheese spoilage defect. Bacterial composition in the (pasteurized) milk and cheese was associated with slit defect development. The application of Koch's postulates showed that individual bacterial isolates as well as uncultured bacterial consortia were sufficient to cause slits, even when present in very low numbers. This approach may be useful for detection and control of low-abundance spoilage microorganisms present in other foods.