Detection and classification of the integrative conjugative elements of Lactococcus lactis.

Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.

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.

Salivary microbiome of healthy women of reproductive age.

IMPORTANCE: The salivary microbiome has been proven to play a crucial role in local and systemic diseases. Moreover, the effects of biological and lifestyle factors such as oral hygiene and smoking on this microbial community have already been explored. However, what was not yet well understood was the natural variation of the saliva microbiome in healthy women and how this is associated with specific use of hormonal contraception and with the number of different sexual partners with whom microbiome exchange is expected regularly. In this paper, we characterized the salivary microbiome of 255 healthy women of reproductive age using an in-depth questionnaire and self-sampling kits. Using the large metadata set, we were able to investigate the associations of several host-related and lifestyle variables with the salivary microbiome profiles. Our study shows a high preservation between individuals.

Capacity of a Microbial Synbiotic To Rescue the In Vitro Metabolic Activity of the Gut Microbiome following Perturbation with Alcohol or Antibiotics.

The human gut microbiome contributes crucial bioactive metabolites that support human health and is sensitive to perturbations from the ingestion of alcohol and antibiotics. We interrogated the response and recovery of human gut microbes after acute alcohol or broad-spectrum antibiotic administration in a gut model simulating the luminal and mucosal colonic environment with an inoculated human microbiome. Both alcohol and antibiotic treatments reduced the production of major short-chain fatty acids (SCFAs) (acetate, propionate, and butyrate), which are established modulators of human health. Treatment with a microbial synbiotic restored and enhanced gut function. Butyrate and acetate production increased by up to 29.7% and 18.6%, respectively, relative to untreated, dysbiotic samples. In parallel, treatment led to increases in the relative abundances of beneficial commensal organisms not found in the synbiotic (e.g., Faecalibacterium prausnitzii and the urolithin-producing organism Gordonibacter pamelaeae) as well as species present in the synbiotic (e.g., Bifidobacterium infantis), suggesting synergistic interactions between supplemented and native microorganisms. These results lead us to conclude that functional shifts in the microbiome, evaluated by both metabolite production and specific taxonomic compositional changes, are an appropriate metric to assess microbiome "recovery" following a dysbiosis-inducing disruption. Overall, these findings support the execution of randomized clinical studies to determine whether a microbial synbiotic can help restore microbiome function after a disruption. IMPORTANCE The human gut microbiome is sensitive to disruptions by common stressors such as alcohol consumption and antibiotic treatment. In this study, we used an in vitro system modeling the gut microbiome to investigate whether treatment with a microbial synbiotic can help restore microbiome function after stress. We find that a complex gut community treated with alcohol or antibiotics showed reduced levels of production of short-chain fatty acids, which are critical beneficial molecules produced by a healthy gut microbiota. Treatment of stressed communities with a microbial synbiotic resulted in the recovery of SCFA production as well as an increase in the abundance of beneficial commensal organisms. Our results suggest that treatment with a microbial synbiotic has the potential to restore healthy gut microbiome function after stress and merits further investigation in clinical studies.

Development of a live biotherapeutic throat spray with lactobacilli targeting respiratory viral infections.

Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the formulation of a throat spray with live lactobacilli exhibiting several in vitro mechanisms of action against respiratory viral infections, including induction of interferon regulatory pathways and direct inhibition of respiratory viruses. Rational selection of Lactobacillaceae strains was based on previously documented beneficial properties, up-scaling and industrial production characteristics, clinical safety parameters, and potential antiviral and immunostimulatory efficacy in the URT demonstrated in this study. Using a three-step selection strategy, three strains were selected and further tested in vitro antiviral assays and in formulations: Lacticaseibacillus casei AMBR2 as a promising endogenous candidate URT probiotic with previously reported barrier-enhancing and anti-pathogenic properties and the two well-studied model strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 that display immunomodulatory capacities. The three strains and their combination significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus, and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form. Thus, the developed spray with live lactobacilli will be further explored in the clinic as a potential broad-acting live biotherapeutic strategy against respiratory viral diseases.

Functional response to a microbial synbiotic in the gastrointestinal system of children: a randomized clinical trial.

BACKGROUND: Oral microbial therapy has been studied as an intervention for a range of gastrointestinal disorders. Though research suggests that microbial exposure may affect the gastrointestinal system, motility, and host immunity in a pediatric population, data have been inconsistent, with most prior studies being in neither a randomized nor placebo-controlled setting. The aim of this randomized, placebo-controlled study was to evaluate the efficacy of a synbiotic on increasing weekly bowel movements (WBMs) in constipated children. METHODS: Sixty-four children (3-17 years of age) were randomized to receive a synbiotic (n = 33) comprising mixed-chain length oligosaccharides and nine microbial strains, or placebo (n = 31) for 84 days. Stool microbiota was analyzed on samples collected at baseline and completion. The primary outcome was a change from baseline of WBMs in the treatment group compared to placebo. RESULTS: Treatment increased (p < 0.05) the number of WBMs in children with low baseline WBMs, despite broadly distinctive baseline microbiome signatures. Sequencing revealed that low baseline microbial richness in the treatment group significantly anticipated improvements in constipation (p = 0.00074). CONCLUSIONS: These findings suggest the potential for (i) multi-species-synbiotic interventions to improve digestive health in a pediatric population and (ii) bioinformatics-based methods to predict response to microbial interventions in children. IMPACT: Synbiotic microbial treatment improved the number of spontaneous weekly bowel movements in children compared to placebo. Intervention induced an increased abundance of bifidobacteria in children, compared to placebo. All administered probiotic species were enriched in the gut microbiome of the intervention group compared to placebo. Baseline microbial richness demonstrated potential as a predictive biomarker for response to intervention.

Assessment of shelf-life and metabolic viability of a multi-strain synbiotic using standard and innovative enumeration technologies.

The number of live bacterial cells is the most used parameter to assess the quality of finished probiotic products. Plate counting (PC) is the standard method in industry to enumerate cells. Application of PC implies critical aspects related to the selection of optimal nutrient media and growth conditions and underestimation of viable but not cultivable (VBNC) cells. Flow-cytometry (FC) is a culture-independent methodology having the potential to selectively enumerate live, damaged, and dead cells representing a powerful tool for in-depth monitoring of probiotic products. We monitored the shelf life of a clinical batch of a synbiotic composition PDS-08 targeting the pediatric population by means of PC and FC according to International Conference on Harmonization (ICH) pharma guidelines testing the Arrhenius model as predictive tool; PC enumeration revealed higher destruction rate than FC suggesting a faster reduction in cultivability than membrane integrity and thus a possible shift of the bacteria into a VBNC status. PDS-08 maintained acidification capability over time, when re-suspended in nutrient medium, even in samples tested sub-optimally for CFU detection (below 1 billion cells/dose). Due to similar kinetics described by the study of metabolic activity and membrane integrity, FC might be suggested as a valid tool for the study of functional stability of a probiotic product.

Randomized, Double-Blind, Placebo-Controlled Trial of a Throat Spray with Selected Lactobacilli in COVID-19 Outpatients.

Primary care urgently needs treatments for coronavirus disease 2019 (COVID-19) patients because current options are limited, while these patients who do not require hospitalization encompass more than 90% of the people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a throat spray containing three Lactobacillaceae strains with broad antiviral properties in a randomized, double-blind, placebo-controlled trial. Before the availability of vaccines, 78 eligible COVID-19 patients were randomized to verum (n = 41) and placebo (n = 37) within 96 h of a positive PCR-based SARS-CoV-2 diagnosis, and a per-protocol analysis was performed. Symptoms and severity were reported daily via an online diary. Combined nose-throat swabs and dried blood spots were collected at regular time points in the study for microbiome, viral load, and antibody analyses. The daily reported symptoms were highly variable, with no added benefit for symptom resolution in the verum group. However, based on 16S V4 amplicon sequencing, the acute symptom score (fever, diarrhea, chills, and muscle pain) was significantly negatively associated with the relative abundance of amplicon sequence variants (ASVs) that included the applied lactobacilli (P < 0.05). Furthermore, specific monitoring of these applied lactobacilli strains showed that they were detectable via quantitative PCR (qPCR) analysis in 82% of the patients in the verum group. At the end of the trial, a trend toward lower test positivity for SARS-CoV-2 was observed for the verum group (2/30; 6.7% positive) than for the placebo group (7/27; 26% positive) (P = 0.07). These data indicate that the throat spray with selected antiviral lactobacilli could have the potential to reduce nasopharyngeal viral loads and acute symptoms but should be applied earlier in the viral infection process and substantiated in larger trials. IMPORTANCE Viral respiratory tract infections result in significant health and economic burdens, as highlighted by the COVID-19 pandemic. Primary care patients represent 90% of those infected with SARS-CoV-2, yet their treatment options are limited to analgesics and antiphlogistics, and few broadly acting antiviral strategies are available. Microbiome or probiotic therapy is a promising emerging treatment option because it is based on the multifactorial action of beneficial bacteria against respiratory viral disease. In this study, an innovative topical throat spray with select beneficial lactobacilli was administered to primary COVID-19 patients. A remote study setup (reducing the burden on hospitals and general practitioners) was successfully implemented using online questionnaires and longitudinal self-sampling. Our results point toward the potential mechanisms of action associated with spray administration at the levels of viral loads and microbiome modulation in the upper respiratory tract and pave the way for future clinical applications of beneficial bacteria against viral diseases.

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.

Why Are Bifidobacteria Important for Infants?

The presence of Bifidobacterium species in the maternal vaginal and fecal microbiota is arguably an evolutionary trait that allows these organisms to be primary colonizers of the newborn intestinal tract. Their ability to utilize human milk oligosaccharides fosters their establishment as core health-promoting organisms throughout life. A reduction in their abundance in infants has been shown to increase the prevalence of obesity, diabetes, metabolic disorder, and all-cause mortality later in life. Probiotic strains have been developed as supplements for premature babies and to counter some of these ailments as well as to confer a range of health benefits. The ability to modulate the immune response and produce short-chain fatty acids, particularly acetate and butyrate, that strengthen the gut barrier and regulate the gut microbiome, makes Bifidobacterium a core component of a healthy infant through adulthood.

Delivery of Metabolically Neuroactive Probiotics to the Human Gut.

The human microbiome is a rich factory for metabolite production and emerging data has led to the concept that orally administered microbial strains can synthesize metabolites with neuroactive potential. Recent research from ex vivo and murine models suggests translational potential for microbes to regulate anxiety and depression through the gut-brain axis. However, so far, less emphasis has been placed on the selection of specific microbial strains known to produce the required key metabolites and the formulation in which microbial compositions are delivered to the gut. Here, we describe a double-capsule technology to deliver high numbers of metabolically active cells derived from the 24-strain probiotic product SH-DS01 to the gastrointestinal tract, including the small intestine, where immune responses and adsorption of metabolites into the bloodstream occur. Based on its genome sequence, Limosilactobacillus reuteri SD-LRE2-IT was predicted to have the genetic capacity to de novo produce a specific metabolite of interest to brain health, vitamin B12, which could be confirmed in vitro. Taken together, our data conceptualizes the importance of rationally defined microbial strain characterization based on genomics and metabolomics data, combined with carefully designed capsule technology for delivery of live cells and concomitant functionality in and beyond the gut ecosystem.

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.

Topical Microbial Therapeutics against Respiratory Viral Infections.

Emerging evidence suggests that microbial therapeutics can prevent and treat respiratory viral diseases, especially when applied directly to the airways. This review presents established beneficial effects of locally administered microbial therapeutics against respiratory viral diseases and the inferred related molecular mechanisms. Several mechanisms established in the intestinal probiotics field as well as novel, niche-specific insights are relevant in the airways. Studies at cellular and organism levels highlight biologically plausible but strain-specific and host and virus context-dependent mechanisms, underlying the potential of beneficial bacteria. Large-scale clinical studies can now be rationally designed to provide a bench-to-bedside translation of the multifactorial bacterial mechanisms within the host respiratory tract, to diminish the incidence and severity of viral infections and the concomitant complications.

Lipoproteins Contribute to the Anti-inflammatory Capacity of Lactobacillus plantarum WCFS1.

Bacterial lipoproteins are well-recognized microorganism-associated molecular patterns, which interact with Toll-like receptor (TLR) 2, an important pattern recognition receptor of the host innate immune system. Lipoproteins are conjugated with two- or three-acyl chains (di- or tri-acyl), which is essential for appropriate anchoring in the cell membrane as well as for the interaction with TLR2. Lipoproteins have mostly been studied in pathogens and have established roles in various biological processes, such as nutrient import, cell wall cross-linking and remodeling, and host-cell interaction. By contrast, information on the role of lipoproteins in the physiology and host interaction of probiotic bacteria is scarce. By deletion of lgt, encoding prolipoprotein diacylglyceryl transferase, responsible for lipidation of lipoprotein precursors, we investigated the roles of the collective group of lipoproteins in the physiology of the probiotic model strain Lactobacillus plantarum WCFS1 using proteomic analysis of secreted proteins. To investigate the consequences of the lgt mutation in host-cell interaction, the capacity of mutant and wild-type bacteria to stimulate TLR2 signaling and inflammatory responses was compared using (reporter-) cell-based models. These experiments exemplified the critical contribution of the acyl chains of lipoproteins in immunomodulation. To the best of our knowledge, this is the first study that investigated collective lipoprotein functions in a model strain for probiotic lactobacilli, and we show that the lipoproteins in L. plantarum WCFS1 are critical drivers of anti-inflammatory host responses toward this strain.

Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis.

Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays. Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.

Understanding mode of action can drive the translational pipeline towards more reliable health benefits for probiotics.

The different levels of knowledge described in a translational pipeline (the connection of molecular mechanisms with pre-clinical physiological and human health effects) are not complete for many probiotics. At present, we are not in a position to fully understand the mechanistic basis of many well established probiotic health benefits which, in turn, limits our ability to use mechanisms to predict which probiotics are likely to be effective in any given population. Here we suggest that this concept of a translation pipeline connecting mechanistic insights to probiotic efficacy can support the selection and production of improved probiotic products. Such a conceptual pipeline would also provide a framework for the design of clinical trials to convincingly demonstrate the benefit of probiotics to human health in well-defined subpopulations.

Renaissance of traditional DNA transfer strategies for improvement of industrial lactic acid bacteria.

The ever-expanding genomic insight in natural diversity of lactic acid bacteria (LAB) has revived the industrial interest in traditional and natural genetic mobilization methodologies. Here, we review recent advances in horizontal gene transfer processes in LAB, including natural competence, conjugation, and phage transduction. In addition, we envision the possibilities for industrial strain improvement arising from the recent discoveries of molecular exchanges between bacteria through nanotubes and extracellular vesicles, as well as the constantly expanding genome editing possibilities using the CRISPR-Cas technology.

Transcriptome Analysis of a Spray Drying-Resistant Subpopulation Reveals a Zinc-Dependent Mechanism for Robustness in L. lactis SK11.

The viability of starter cultures is essential for an adequate contribution to the fermentation process and end-product. Therefore, robustness during processing and storage is an important characteristic of starter culture strains. For instance, during spray drying cells are exposed to heat and oxidative stress, generally resulting in loss of viability. In this study, we exposed the industrially relevant but stress-sensitive Lactococcus lactis strain SK11 to two cycles of heat stress, with intermediate recovery and cultivation at moderate temperatures. After these two cycles of heat exposure, the abundance of robust derivatives was increased as compared with the original culture, which enabled isolation of heat-resistant subpopulations displaying up to 1,000-fold enhanced heat stress survival. Moreover, this heat-resistant subpopulation demonstrated an increased survival during spray drying. Derivatives from two independent lineages displayed different transcriptome changes as compared with the wild type strain, indicating that the increased robustness within these lineages was established by different adaptive strategies. Nevertheless, an overlap in differential gene expression in all five derivatives tested in both lineages included three genes in an operon involved in zinc transport. The link between zinc homeostasis and heat stress survival in L. lactis was experimentally established by culturing of the wild type strain SK11 in medium with various levels of zinc ions, which resulted in alterations in heat stress survival phenotypes. This study demonstrates that robust derivatives of a relatively sensitive L. lactis strain can be isolated by repeated exposure to heat stress. Moreover, this work demonstrates that transcriptome analysis of these robust derivatives can provide clues for improvement of the robustness of the original strain. This could boost the industrial application of strains with specific desirable traits but inadequate robustness characteristics.

Lactic Acid Bacteria for Delivery of Endogenous or Engineered Therapeutic Molecules.

Food-grade lactic acid bacteria (LAB) are considered suitable vehicles for the production and/or delivery of health promoting or therapeutic, bioactive molecules. The molecules considered for health-beneficial use include the endogenous effector molecules produced by probiotics (mostly lactobacilli), as well as heterologous bioactives that can be produced in LAB by genetic engineering (mostly using lactococci). Both strategies aim to deliver appropriate dosages of specific bioactive molecules to the site of action. This review uses specific examples of both strategies to illustrate the different avenues of research involved in these applications as well as their translation to human health-promoting applications. These examples pinpoint that despite the promising perspectives of these approaches, the evidence for their effective applications in human populations is lagging behind.