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.

A citizen-science-enabled catalogue of the vaginal microbiome and associated factors.

Understanding the composition and function of the vaginal microbiome is crucial for reproductive and overall health. Here we established the Isala citizen-science project to analyse the vaginal microbiomes of 3,345 women in Belgium (18-98 years) through self-sampling, 16S amplicon sequencing and extensive questionnaires. The overall vaginal microbiome composition was strongly tied to age, childbirth and menstrual cycle phase. Lactobacillus species dominated 78% of the vaginal samples. Specific bacterial taxa also showed to co-occur in modules based on network correlation analysis. Notably, the module containing Lactobacillus crispatus, Lactobacillus jensenii and Limosilactobacillus taxa was positively linked to oestrogen levels and contraceptive use and negatively linked to childbirth and breastfeeding. Other modules, named after abundant taxa (Gardnerella, Prevotella and Bacteroides), correlated with multiple partners, menopause, menstrual hygiene and contraceptive use. With this resource-rich vaginal microbiome map and associated health, life-course, lifestyle and dietary factors, we provide unique data and insights for follow-up clinical and mechanistic research.

Characterization of potential probiotic starter cultures of lactic acid bacteria isolated from Ethiopian fermented cereal beverages, Naaqe, and Cheka.

AIMS: To test the in vitro probiotic potential and starter culture capacity of lactic acid bacteria (LAB) isolated from Naaqe and Cheka, cereal-based Ethiopian traditional fermented beverages. METHODS AND RESULTS: A total of 44 strains were isolated from spontaneously fermented Ethiopian cereal-based beverages, Naaqe and Cheka with 24 putatively identified as LAB and 14 identified up to the species level. The species Limosilactobacillus fermentum (6/12; 50%) and Weissella confusa (5/12, 41.67%) were the predominant species identified from Naaqe, while the two Cheka isolates were L. fermentum and Pediococcus pentosaceus. Six LAB strains inhibited eight of the nine gastrointestinal indicator key pathogens in Ethiopia, including Escherichia coli, Salmonella enterica subsp. enterica var. Typhimurium, Staphylococcus aureus, Shigella flexneri, and Listeria monocytogenes. Three of the LAB isolates exhibited strain-specific immunostimulation in human monocytes. Based on these probiotic properties and growth, six strains were selected for in situ evaluation in a mock fermentation of Naaqe and Cheka. During primary fermentations, L. fermentum 73B, P. pentosaceus 74D, L. fermentum 44B, W. confusa 44D, L. fermentum 82C, and Weissella cibaria 83E and their combinations demonstrated higher pH-lowering properties and colony-forming unit counts compared to the control spontaneous fermentation. The same pattern was also observed in the secondary mock fermentation by the Naaqe LAB isolates. CONCLUSIONS: In this study, we selected six LAB strains with antipathogenic, immunostimulatory, and starter culture potentials that can be used as autochthonous probiotic starters for Naaqe and Cheka fermentations once their health benefit is ascertained in a clinical trial as a next step.

Boosting aerobic microbial protein productivity and quality on brewery wastewater: Impact of anaerobic acidification, high-rate process and biomass age.

Consortia of aerobic heterotrophic bacteria (AHB) are appealing as sustainable alternative protein ingredient for aquaculture given their high nutritional qualities, and their production potential on feed-grade industrial wastewater. Today, the impacts of pre-treatment, bioprocess choice and key parameter settings on AHB productivity and nutritional properties are unknown. This study investigated for the first time AHB microbial protein production effects based on (i) raw vs anaerobically fermented brewery wastewater, (ii) high-rate activated sludge (HRAS) without vs with feast-famine conditions, and (iii) three short solid retention time (SRT): 0.25, 0.50 and 1.00 d. High biomass (4.4-8.0 g TSS/L/d) and protein productivities (1.9-3.2 g protein/L/d) were obtained while achieving COD removal efficiencies up to 98 % at SRT 0.50 d. The AHB essential amino acid (EAA) profiles were above rainbow trout requirements, excluding the S-containing EAA, highlighting the AHB biomass replacement potential for unsustainable fishmeal in salmonid diets.

Purple bacteria screening for photoautohydrogenotrophic food production: Are new H2-fed isolates faster and nutritionally better than photoheterotrophically obtained reference species?

Photoautohydrogenotrophic enrichments of wastewater treatment microbiomes were performed to obtain hypothetically high-potential specialist species for biotechnological applications. From these enrichment cultures, ten photoautohydrogenotrophic species were isolated: six Rhodopseudomonas species, three Rubrivivax members and Rhodobacter blasticus. The performance of these isolates was compared to three commonly studied, and originally photoheterotrophically enriched species (Rhodopseudomonas palustris, Rhodobacter capsulatus and Rhodobacter sphaeroides), designated as reference species. Repeated subcultivations were applied to improve the initial poor performance of the isolates (acclimation effect), which resulted in increases in both maximum growth rate and protein productivity. However, the maximum growth rate of the reference species remained 3-7 times higher compared to the isolates (0.42-0.84 d-1 at 28 °C), while protein productivities remained 1.5-1.7 times higher. This indicated that H2-based enrichment did not result in photoautohydrogenotrophic specialists, suggesting that the reference species are more suitable for intensified biomass and protein production. On the other hand, the isolates were able to provide equally high protein quality profiles as the references species, providing full dietary essential amino acid matches for human food. Lastly, the effect of metabolic carbon/electron switching (back and forth between auto- to heterotrophic conditions) initially boosted µmax when returning to photoautohydrogenotrophic conditions. However, the switch negatively impacted lag phase, protein productivities and pigment contents. In the case of protein productivity, the acquired acclimation was partially lost with decreases of up to 44 % and 40 % respectively for isolates and reference species. Finally, the three reference species, and specifically Rh. capsulatus, remained the most suitable candidate(s) for further biotechnological development.

Spontaneous Riboflavin-Overproducing Limosilactobacillus reuteri for Biofortification of Fermented Foods.

Riboflavin-producing lactic acid bacteria represent a promising and cost-effective strategy for food biofortification, but production levels are typically insufficient to support daily human requirements. In this study, we describe the novel human isolate Limosilactobacillus reuteri AMBV339 as a strong food biofortification candidate. This strain shows a high natural riboflavin (vitamin B2) overproduction of 18.36 µg/ml, biomass production up to 6 × 1010 colony-forming units/ml (in the typical range of model lactobacilli), and pH-lowering capacities to a pH as low as 4.03 in common plant-based (coconut, soy, and oat) and cow milk beverages when cultured up to 72 h at 37°C. These properties were especially pronounced in coconut beverage and butter milk fermentations, and were sustained in co-culture with the model starter Streptococcus thermophilus. Furthermore, L. reuteri AMBV339 grown in laboratory media or in a coconut beverage survived in gastric juice and in a simulated gastrointestinal dialysis model with colon phase (GIDM-colon system) inoculated with fecal material from a healthy volunteer. Passive transport of L. reuteri AMBV339-produced riboflavin occurred in the small intestinal and colon stage of the GIDM system, and active transport via intestinal epithelial Caco-2 monolayers was also demonstrated. L. reuteri AMBV339 did not cause fecal microbiome perturbations in the GIDM-colon system and inhibited enteric bacterial pathogens in vitro. Taken together, our data suggests that L. reuteri AMBV339 represents a promising candidate to provide riboflavin fortification of plant-based and dairy foods, and has a high application potential in the human gastrointestinal tract.

Microbial enrichment and storage for metagenomics of vaginal, skin, and saliva samples.

Few validated protocols are available for large-scale collection, storage, and analysis of microbiome samples from the vagina, skin, and mouth. To prepare for a large-scale study on the female microbiome by remote self-sampling, we investigated the impact of sample collection, storage, and host DNA depletion on microbiome profiling. Vaginal, skin, and saliva samples were analyzed using 16S rRNA gene amplicon and metagenomic shotgun sequencing, and qPCR. Of the two tested storage buffers, the eNAT buffer could keep the microbial composition stable during various conditions. All three tested host DNA-depletion approaches showed a bias against Gram-negative taxa. However, using the HostZERO Microbial DNA and QIAamp DNA Microbiome kits, samples still clustered according to body site and not by depletion approach. Therefore, our study showed the effectiveness of these methods in depleting host DNA. Yet, a suitable approach is recommended for each habitat studied based on microbial composition.

Oxygen control and stressor treatments for complete and long-term suppression of nitrite-oxidizing bacteria in biofilm-based partial nitritation/anammox.

Mainstream nitrogen removal by partial nitritation/anammox (PN/A) can realize energy and cost savings for sewage treatment. Selective suppression of nitrite oxidizing bacteria (NOB) remains a key bottleneck for PN/A implementation. A rotating biological contactor was studied with an overhead cover and controlled air/N2 inflow to regulate oxygen availability at 20 °C. Biofilm exposure to dissolved oxygen concentrations < 0.51 ± 0.04 mg O2 L-1 when submerged in the water and < 1.41 ± 0.31 mg O2 L-1 when emerged in the headspace (estimated), resulted in complete and long-term NOB suppression with a low relative nitrate production ratio of 10 ± 4%. Additionally, weekly biofilm stressor treatments with free ammonia (FA) (29 ± 1 mg NH3-N L-1 for 3 h) could improve the NOB suppression while free nitrous acid treatments had insufficient effect. This study demonstrated the potential of managing NOB suppression in biofilm-based systems by oxygen control and recurrent FA exposure, opening opportunities for resource efficient nitrogen removal.

The nasal mutualist Dolosigranulum pigrum AMBR11 supports homeostasis via multiple mechanisms.

Comparing the nasal microbiome of healthy individuals and chronic rhinosinusitis (CRS) patients revealed Dolosigranulum pigrum as a species clearly associated with nasal health, although isolates obtained from healthy individuals are scarce. In this study, we explored the properties of this understudied lactic acid bacterium by integrating comparative genomics, habitat mining, cultivation, and functional characterization of interaction capacities. Mining 10.000 samples from the Earth Microbiome Project of 17 habitat types revealed that Dolosigranulum is mainly associated with the human nasal cavity. D. pigrum AMBR11 isolated from the nose of a healthy individual exerted antimicrobial activity against Staphylococcus aureus, decreased proinflammatory cytokine production in airway epithelial cells, and Galleria mellonella larvae mortality induced by this important nasal pathobiont. Furthermore, the strain protected the nasal barrier function in a mouse model using interleukin-4 as disruptive cytokine. Hence, D. pigrum AMBR11 is a mutualist with high potential as topical live biotherapeutic product.

Cotton and Surgical Face Masks in Community Settings: Bacterial Contamination and Face Mask Hygiene.

During the current COVID-19 pandemic, the use of face masks has become increasingly recommended and even mandatory in community settings. To evaluate the risk of bacterial cross-contamination, this study analyzed the bacterial bioburden of disposable surgical masks and homemade cotton masks, and surveyed the habits and face mask preferences of the Flemish population. Using culture approaches and 16S rRNA gene amplicon sequencing, we analyzed the microbial community on surgical and/or cotton face masks of 13 healthy volunteers after 4 h of wearing. Cotton and surgical masks contained on average 1.46 × 105 CFU/mask and 1.32 × 104 CFU/mask, respectively. Bacillus, Staphylococcus, and Acinetobacter spp. were mostly cultured from the masks and 43% of these isolates were resistant to ampicillin or erythromycin. Microbial profiling demonstrated a consistent difference between mask types. Cotton masks mainly contained Roseomonas, Paracoccus, and Enhydrobacter taxa and surgical masks Streptococcus and Staphylococcus. After 4 h of mask wearing, the microbiome of the anterior nares and the cheek showed a trend toward an altered beta-diversity. According to dedicated questions in the large-scale Corona survey of the University of Antwerp with almost 25,000 participants, only 21% of responders reported to clean their cotton face mask daily. Laboratory results indicated that the best mask cleaning methods were boiling at 100°C, washing at 60°C with detergent or ironing with a steam iron. Taken together, this study suggests that a considerable number of bacteria, including pathobionts and antibiotic resistant bacteria, accumulate on surgical and even more on cotton face masks after use. Based on our results, face masks should be properly disposed of or sterilized after intensive use. Clear guidelines for the general population are crucial to reduce the bacteria-related biosafety risk of face masks, and measures such as physical distancing and increased ventilation should not be neglected when promoting face mask use.

Case-Control Microbiome Study of Chronic Otitis Media with Effusion in Children Points at Streptococcus salivarius as a Pathobiont-Inhibiting Species.

Chronic otitis media with effusion (OME) has been associated with a shift in microbiome composition and microbial interaction in the upper respiratory tract (URT). While most studies have focused on potential pathogens, this study aimed to find bacteria that could be protective against OME through a case-control microbiome study and characterization of isolates from healthy subjects. The URT and ear microbiome profiles of 70 chronic OME patients and 53 controls were compared by 16S rRNA amplicon sequencing. Haemophilus influenzae was the most frequent classic middle ear pathobiont. However, other taxa, especially Alloiococcus otitis, were also frequently detected in the ear canal of OME patients. Streptococci of the salivarius group and Acinetobacter lwoffii were more abundant in the nasopharynx of healthy controls than in OME patients. In addition to the microbiome analysis, 142 taxa were isolated from healthy individuals, and 79 isolates of 13 different Streptococcus species were tested for their pathobiont-inhibiting potential. Of these, Streptococcus salivarius isolates showed a superior capacity to inhibit the growth of H. influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, A. otitis, and Corynebacterium otitidis S. salivarius strains thus show potential as a probiotic for prevention or treatment of OME based on their overrepresentation in the healthy nasopharynx and their ability to inhibit the growth of respiratory pathobionts. (This study has been registered at ClinicalTrials.gov under registration no. NCT03109496.)IMPORTANCE The majority of probiotics marketed today target gastrointestinal health. This study searched for bacteria native to the human upper respiratory tract, with a beneficial potential for respiratory and middle ear health. Comparison of the microbiomes of children with chronic otitis media with effusion (OME) and of healthy controls identified Streptococcus salivarius as a health-associated and prevalent inhabitant of the human nasopharynx. However, beneficial potential should be assessed at strain level. Here, we also isolated specific S. salivarius strains from the healthy individuals in our study. These isolates showed a beneficial safety profile and efficacy potential to inhibit OME pathogens in vitro These properties will now have to be evaluated and confirmed in human clinical studies.

Robustness of fermented carrot juice against Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7.

Artisanal vegetable fermentations are regaining popularity in industrialized countries, but they could be prone to contamination with foodborne pathogens. By simulating home or small-scale restaurant fermentations, we evaluated the microbiological safety of spontaneous carrot juice fermentations. Raw carrot juice was spiked with Listeria monocytogenes, Salmonella enterica subsp. enterica Typhimurium and Escherichia coli O157:H7, and the microbial dynamics were followed throughout the entire fermentation process by cultivation and amplicon sequencing. In addition, the behavior of these pathogens was also monitored after addition of raw cucumber juice and storage under refrigerated conditions to mimic post-contamination issues. Although the numbers of the pathogens increased during the first phase of the fermentation, the pathogens were not able to persist throughout the fermentation. Their numbers fell below the detection limit after 8 days of fermentation at 20 °C. Further investigation using amplicon sequencing also showed that there was no major impact on the general microbial dynamics of the spontaneous carrot juice fermentation. This indicates that the artisanal carrot juice fermentation is a robust process which resists the persistence of pathogens. More caution is needed however when mixing the final fermented product with a raw juice. When simulating pathogen post-contamination, both Salmonella enterica and Escherichia coli were able to survive in the refrigerated fermented juice up to 10 days after the fermentation. Listeria monocytogenes was detected up to 8 days in the refrigerated juice. Pasteurization of the raw juice before adding it to the fermented product is thus recommended.

Lactobacillus rhamnosus probiotic prevents airway function deterioration and promotes gut microbiome resilience in a murine asthma model.

Allergic asthma is a highly prevalent inflammatory disease of the lower airways, clinically characterized by airway hyperreactivity and deterioration of airway function. Immunomodulatory probiotic bacteria are increasingly being explored to prevent asthma development, alone or in combination with other treatments. In this study, wild-type and recombinant probiotic Lactobacillus rhamnosus GR-1 were tested as preventive treatment of experimental allergic asthma in mice. Recombinant L. rhamnosus GR-1 was designed to produce the major birch pollen allergen Bet v 1, to promote allergen-specific immunomodulation. Administration of wild-type and recombinant L. rhamnosus GR-1 prevented the development of airway hyperreactivity. Recombinant L. rhamnosus GR-1 also prevented elevation of airway total cell counts, lymphocyte counts and lung IL-1ß levels, while wild-type L. rhamnosus GR-1 inhibited airway eosinophilia. Of note, a shift in gut microbiome composition was observed after asthma development, which correlated with the severity of airway inflammation and airway hyperreactivity. In the groups that received L. rhamnosus GR-1, this asthma-associated shift in gut microbiome composition was not observed, indicating microbiome-modulating effects of this probiotic. These data demonstrate that L. rhamnosus GR-1 can prevent airway function deterioration in allergic asthma. Bet v 1 expression by L. rhamnosus GR-1 further contributed to lower airway inflammation, although not solely through the expected reduction in T helper 2-associated responses, suggesting involvement of additional mechanisms. The beneficial effects of L. rhamnosus GR-1 correlate with increased gut microbiome resilience, which in turn is linked to protection of airway function, and thus further adds support to the existence of a gut-lung axis.

Return-Sludge Treatment with Endogenous Free Nitrous Acid Limits Nitrate Production and N2O Emission for Mainstream Partial Nitritation/Anammox.

Nitrite oxidizing bacteria (NOB) and nitrous oxide (N2O) hinder the development of mainstream partial nitritation/anammox. To overcome these, endogenous free ammonia (FA) and free nitrous acid (FNA), which can be produced in the sidestream, were used for return-sludge treatment for two integrated-film activated sludge reactors containing biomass in flocs and on carriers. The repeated exposure of biomass from one reactor to FA shocks had a limited impact on NOB suppression but inhibited anammox bacteria (AnAOB). In the other reactor, repeated FNA shocks to the separated flocs failed to limit the system's nitrate production since NOB activity was still high on the biofilms attached to the unexposed carriers. In contrast, the repeated FNA treatment of flocs and carriers favored aerobic ammonium-oxidizing bacteria (AerAOB) over NOB activity with AnAOB negligibly affected. It was further revealed that return-sludge treatment with higher FNA levels led to lower N2O emissions under similar effluent nitrite concentrations. On this basis, weekly 4 h FNA shocks of 2.0 mg of HNO2-N/L were identified as an optimal and realistic treatment, which not only enabled nitrogen removal efficiencies of ∼65% at nitrogen removal rates of ∼130 mg of N/L/d (20 °C) but also yielded the lowest cost and carbon footprint.

Applications of plant-based fermented foods and their microbes.

Plant-based fermentations and their microbes provide an underexplored source for novel biotechnological applications. Recent advances in DNA sequencing technologies and analyses of sequencing data highlight that a diverse array of lactic acid bacteria (LAB) frequently dominate these plant fermentations. Because of the long history of safe LAB use in fermented foods, we argue here that various novel probiotic, synbiotic and a range of other industrial applications can be produced based on new insights in the functional and genetic potential of these LAB. To aid in this quest, comparative genomics tools are increasingly available enabling a more rational design of wet-lab experiments to screen for the most relevant properties. This is also true for the exploration of useful enzymatic and (secondary) metabolic production capacities of the LAB that can be isolated from these plant-based fermentations, such as the recent discovery of a cellulase enzyme in specific Lactobacillus plantarum group members.

The microbiome of the invertebrate model host Galleria mellonella is dominated by Enterococcus.

BACKGROUND: The popularity of Galleria mellonella as invertebrate model is increasing rapidly, because it forms an attractive alternative to study bacterial, fungal and viral infections, toxin biology, and to screen antimicrobial drugs. For a number of vertebrate and invertebrate animal and plant models, it has been established that the commensals present within the microbial communities on various host surfaces will influence the host's immune and growth development state and the colonization capacity of newly introduced micro-organisms. The microbial communities of Galleria mellonella larvae have, however, not yet been well characterized. RESULTS: In this study, we present the bacterial communities that were found by 16S rRNA amplicon sequencing on different body sites of G. mellonella larvae. These communities showed very little diversity and were mostly dominated by one Enterococcus taxon. In addition, we found that the production conditions (as 'bait' for fishing or under more controlled 'research grade' conditions - with or without hormones and antibiotics) appear to have little impact on the microbiota of the larvae. CONCLUSIONS: Establishment of the simplicity of the microbiota of G. mellonella larvae underlines the potential of the larvae as a model host system for microbiome-host interactions.

Carrot Juice Fermentations as Man-Made Microbial Ecosystems Dominated by Lactic Acid Bacteria.

Spontaneous vegetable fermentations, with their rich flavors and postulated health benefits, are regaining popularity. However, their microbiology is still poorly understood, therefore raising concerns about food safety. In addition, such spontaneous fermentations form interesting cases of man-made microbial ecosystems. Here, samples from 38 carrot juice fermentations were collected through a citizen science initiative, in addition to three laboratory fermentations. Culturing showed that Enterobacteriaceae were outcompeted by lactic acid bacteria (LAB) between 3 and 13 days of fermentation. Metabolite-target analysis showed that lactic acid and mannitol were highly produced, as well as the biogenic amine cadaverine. High-throughput 16S rRNA gene sequencing revealed that mainly species of Leuconostoc and Lactobacillus (as identified by 8 and 20 amplicon sequence variants [ASVs], respectively) mediated the fermentations in subsequent order. The analyses at the DNA level still detected a high number of Enterobacteriaceae, but their relative abundance was low when RNA-based sequencing was performed to detect presumptive metabolically active bacterial cells. In addition, this method greatly reduced host read contamination. Phylogenetic placement indicated a high LAB diversity, with ASVs from nine different phylogenetic groups of the Lactobacillus genus complex. However, fermentation experiments with isolates showed that only strains belonging to the most prevalent phylogenetic groups preserved the fermentation dynamics. The carrot juice fermentation thus forms a robust man-made microbial ecosystem suitable for studies on LAB diversity and niche specificity.IMPORTANCE The usage of fermented food products by professional chefs is steadily growing worldwide. Meanwhile, this interest has also increased at the household level. However, many of these artisanal food products remain understudied. Here, an extensive microbial analysis was performed of spontaneous fermented carrot juices which are used as nonalcoholic alternatives for wine in a Belgian Michelin star restaurant. Samples were collected through an active citizen science approach with 38 participants, in addition to three laboratory fermentations. Identification of the main microbial players revealed that mainly species of Leuconostoc and Lactobacillus mediated the fermentations in subsequent order. In addition, a high diversity of lactic acid bacteria was found; however, fermentation experiments with isolates showed that only strains belonging to the most prevalent lactic acid bacteria preserved the fermentation dynamics. Finally, this study showed that the usage of RNA-based 16S rRNA amplicon sequencing greatly reduces host read contamination.