Ethanolamine enhances adhesion, promotes microcompartment formation, and modulates gene expression in Levilactobacillus brevis ATCC 14869.

Ethanolamine is an abundant compound in the gastrointestinal tract and a valuable source of carbon and nitrogen for pathogenic bacteria harboring ethanolamine utilization (eut) genes. Eut-positive pathogens can consume free ethanolamine to outcompete commensal microbes, which often lack eut genes, and establish infection. Ethanolamine can also act as a host recognition signal for eut-positive pathogens to upregulate virulence genes during colonization. Therefore, reducing free ethanolamine titers may represent a novel approach to preventing infection by eut-positive pathogens. Interestingly, the commensal microorganism Levilactobacillus brevis ATCC 14869 was found to encode over 18 eut genes within its genome. This led us to hypothesize that L. brevis can compete with eut-positive pathogens by clearing free ethanolamine from the environment. Our results demonstrate that despite being unable to metabolize ethanolamine under most conditions, L. brevis ATCC 14869 responds to the compound by increasing the expression of genes encoding proteins involved in microcompartment formation and adhesion to the intestinal epithelial barrier. The improved intestinal adhesion of L. brevis in the presence of ethanolamine also enhanced the exclusion of eut-positive pathogens from adhering to intestinal epithelial cells. These findings support further studies to test whether L. brevis ATCC 14869 can counter enteric pathogens and prevent or reduce the severity of infections. Overall, the metabolic capabilities of L. brevis ATCC 14869 offer a unique opportunity to add to the armamentarium of antimicrobial therapies as well as our understanding of the mechanisms used by beneficial microbes to sense and adapt to host microenvironments.

Dissecting mechanisms of fecal microbiota transplantation efficacy in disease.

Fecal microbiota transplantation (FMT) has emerged as an alternative or adjunct experimental therapy for microbiome-associated diseases following its success in the treatment of recurrent Clostridioides difficile infections (rCDIs). However, the mechanisms of action involved remain relatively unknown. The term 'dysbiosis' has been used to describe microbial imbalances in relation to disease, but this traditional definition fails to consider the complex cross-feeding networks that define the stability of the microbiome. Emerging research transitions toward the targeted restoration of microbial functional networks in treating different diseases. In this review, we explore potential mechanisms responsible for the efficacy of FMT and future therapeutic applications, while revisiting definitions of 'dysbiosis' in favor of functional network restoration in rCDI, inflammatory bowel diseases (IBDs), metabolic diseases, and cancer.

Multi-site microbiota alteration is a hallmark of kidney stone formation.

BACKGROUND: Inquiry of microbiota involvement in kidney stone disease (KSD) has largely focussed on potential oxalate handling abilities by gut bacteria and the increased association with antibiotic exposure. By systematically comparing the gut, urinary, and oral microbiota of 83 stone formers (SF) and 30 healthy controls (HC), we provide a unified assessment of the bacterial contribution to KSD. RESULTS: Amplicon and shotgun metagenomic sequencing approaches were consistent in identifying multi-site microbiota disturbances in SF relative to HC. Biomarker taxa, reduced taxonomic and functional diversity, functional replacement of core bioenergetic pathways with virulence-associated gene markers, and community network collapse defined SF, but differences between cohorts did not extend to oxalate metabolism. CONCLUSIONS: We conclude that multi-site microbiota alteration is a hallmark of SF, and KSD treatment should consider microbial functional restoration and the avoidance of aberrant modulators such as poor diet and antibiotics where applicable to prevent stone recurrence. Video Abstract.

Corrigendum: New perspectives on an old grouping: the genomic and phenotypic variability of Oxalobacter formigenes and the implications for calcium oxalate stone prevention.

[This corrects the article DOI: 10.3389/fmicb.2022.1011102.].

Vitamins as regulators of calcium-containing kidney stones - new perspectives on the role of the gut microbiome.

Calcium-based kidney stone disease is a highly prevalent and morbid condition, with an often complicated and multifactorial aetiology. An abundance of research on the role of specific vitamins (B6, C and D) in stone formation exists, but no consensus has been reached on how these vitamins influence stone disease. As a consequence of emerging research on the role of the gut microbiota in urolithiasis, previous notions on the contribution of these vitamins to urolithiasis are being reconsidered in the field, and investigation into previously overlooked vitamins (A, E and K) was expanded. Understanding how the microbiota influences host vitamin regulation could help to determine the role of vitamins in stone disease.

The Role of Microbiota-Derived Vitamins in Immune Homeostasis and Enhancing Cancer Immunotherapy.

Not all cancer patients who receive immunotherapy respond positively and emerging evidence suggests that the gut microbiota may be linked to treatment efficacy. Though mechanisms of microbial contributions to the immune response have been postulated, one likely function is the supply of basic co-factors to the host including selected vitamins. Bacteria, fungi, and plants can produce their own vitamins, whereas humans primarily obtain vitamins from exogenous sources, yet despite the significance of microbial-derived vitamins as crucial immune system modulators, the microbiota is an overlooked source of these nutrients in humans. Microbial-derived vitamins are often shared by gut bacteria, stabilizing bioenergetic pathways amongst microbial communities. Compositional changes in gut microbiota can affect metabolic pathways that alter immune function. Similarly, the immune system plays a pivotal role in maintaining the gut microbiota, which parenthetically affects vitamin biosynthesis. Here we elucidate the immune-interactive mechanisms underlying the effects of these microbially derived vitamins and how they can potentially enhance the activity of immunotherapies in cancer.

The Role of Urinary Modulators in the Development of Infectious Kidney Stones.

Introduction: The pathogenesis of infectious kidney stones is poorly understood. Bacteria have been implicated in promoting infectious stones via urease production; however, there is mounting evidence indicating the relationship is more complex. The aim of our study was to characterize suspected biotic and abiotic extrinsic factors that may modulate the formation of infectious stones. Materials and Methods: A high-throughput experimental model with Griffith's artificial urine was used to test a wide variety of urinary modulators and cytoplasmic enzymes present in crude cell-free extracts (CFEs) from bacterial strains to investigate how they impact struvite and calcium (Ca) phosphate crystal production. Crystal formation was evaluated with spectrophotometry and growth curve analysis. Light microscopy and scanning electron microscopy/X-ray diffraction was used for crystal structure and composition identification. Results: The acidic urinary modulators used in this study prevented crystal formation, whereas osteopontin had a significant inhibitory effect. Addition of CFEs from Proteus mirabilis 175A and 177A resulted in Ca phosphate and struvite crystals. Of interest, Klebsiella pneumoniae and Klebsiella oxytoca produced crystals including Ca phosphate and Ca oxalate, respectively. Pseudomonas aeruginosa had no urease production detected and produced Ca phosphate crystals. Discussion: Urinary modulators can have a wide variety of effects on infectious stone formation and the role of pH is important but does not guarantee robust crystal formation. Bacterial strains can produce Ca oxalate, Ca phosphate, and struvite stones with and without urease activity. Conclusion: Various urinary modulators appear to influence the process and are worthy of further evaluation as a potential therapeutic strategy to prevent infection-related urinary stone formation. Stones formed from urinary tract infections may be a result of multiple encoded metabolic pathways and discovering these would improve our understanding of the stone-bacterial relationship.

Osteopontin phosphopeptide mitigates calcium oxalate stone formation in a Drosophila melanogaster model.

Kidney stone disease affects nearly one in ten individuals and places a significant economic strain on global healthcare systems. Despite the high frequency of stones within the population, effective preventative strategies are lacking and disease prevalence continues to rise. Osteopontin (OPN) is a urinary protein that can inhibit the formation of renal calculi in vitro. However, the efficacy of OPN in vivo has yet to be determined. Using an established Drosophila melanogaster model of calcium oxalate urolithiasis, we demonstrated that a 16-residue synthetic OPN phosphopeptide effectively reduced stone burden in vivo. Oral supplementation with this peptide altered crystal morphology of calcium oxalate monohydrate (COM) in a similar manner to previous in vitro studies, and the presence of the OPN phosphopeptide during COM formation and adhesion significantly reduced crystal attachment to mammalian kidney cells. Altogether, this study is the first to show that an OPN phosphopeptide can directly mitigate calcium oxalate urolithiasis formation in vivo by modulating crystal morphology. These findings suggest that OPN supplementation is a promising therapeutic approach and may be clinically useful in the management of urolithiasis in humans.

Probiotic Bifidobacteria Mitigate the Deleterious Effects of para-Cresol in a Drosophila melanogaster Toxicity Model.

Renal impairment associated with chronic kidney disease (CKD) causes the buildup of uremic toxins that are deleterious to patient health. Current therapies that manage toxin accumulation in CKD offer an incomplete therapeutic effect against toxins such as para-cresol (p-cresol) and p-cresyl sulfate. Probiotic therapies can exploit the wealth of microbial diversity to reduce toxin accumulation. Using in vitro culture techniques, strains of lactobacilli and bifidobacteria from a 24-strain synbiotic were investigated for their ability to remove p-cresol. Four strains of bifidobacteria internalized p-cresol from the extracellular environment. The oral supplementation of these toxin-clearing probiotics was more protective than control strains in a Drosophila melanogaster toxicity model. Bifidobacterial supplementation was also associated with higher abundance of lactobacilli in the gut microbiota of p-cresol-exposed flies. The present findings suggest that these strains might reduce p-cresol in the gut in addition to increasing the prevalence of other beneficial bacteria, such as lactobacilli, and should be tested clinically to normalize the dysbiotic gut microbiota observed in CKD patients. IMPORTANCE Chronic kidney disease (CKD) affects approximately 10% of the global population and has limited treatment options. The accumulation of gut microbiota-derived uremic toxins, such as para-cresol (p-cresol) and p-cresyl sulfate, is associated with the onset of comorbidities (i.e., atherosclerosis and cognitive disorders) in CKD. Unfortunately, dialysis, the gold standard therapy is unable to remove these toxins from the bloodstream due to their highly protein-bound nature. Some strains of Bifidobacterium have metabolic properties that may be useful in managing uremic toxicity. Using a Drosophila model, the present work highlights why dosing with certain probiotic strains may be clinically useful in CKD management.

Long-Duration Space Travel Support Must Consider Wider Influences to Conserve Microbiota Composition and Function.

The microbiota is important for immune modulation, nutrient acquisition, vitamin production, and other aspects for long-term human health. Isolated model organisms can lose microbial diversity over time and humans are likely the same. Decreasing microbial diversity and the subsequent loss of function may accelerate disease progression on Earth, and to an even greater degree in space. For this reason, maintaining a healthy microbiome during spaceflight has recently garnered consideration. Diet, lifestyle, and consumption of beneficial microbes can shape the microbiota, but the replenishment we attain from environmental exposure to microbes is important too. Probiotics, prebiotics, fermented foods, fecal microbiota transplantation (FMT), and other methods of microbiota modulation currently available may be of benefit for shorter trips, but may not be viable options to overcome the unique challenges faced in long-term space travel. Novel fermented food products with particular impact on gut health, immune modulation, and other space-targeted health outcomes are worthy of exploration. Further consideration of potential microbial replenishment to humans, including from environmental sources to maintain a healthy microbiome, may also be required.

New perspectives on an old grouping: The genomic and phenotypic variability of Oxalobacter formigenes and the implications for calcium oxalate stone prevention.

Oxalobacter formigenes is a unique bacterium with the ability to metabolize oxalate as a primary carbon source. Most kidney stones in humans are composed of calcium and oxalate. Therefore, supplementation with an oxalate-degrading bacterium may reduce stone burden in patients suffering from recurrent calcium oxalate-based urolithiasis. Strains of O. formigenes are divided into two groups: group I and group II. However, the differences between strains from each group remain unclear and elucidating these distinctions will provide a better understanding of their physiology and potential clinical applications. Here, genomes from multiple O. formigenes strains underwent whole genome sequencing followed by phylogenetic and functional analyses. Genetic differences suggest that the O. formigenes taxon should be divided into an additional three species: Oxalobacter aliiformigenes sp. nov, Oxalobacter paeniformigenes sp. nov, and Oxalobacter paraformigenes sp. nov. Despite the similarities in the oxalyl-CoA gene (oxc), which is essential for oxalate degradation, these strains have multiple unique genetic features that may be potential exploited for clinical use. Further investigation into the growth of these strains in a simulated fecal environment revealed that O. aliiformigenes strains are capable of thriving within the human gut microbiota. O. aliiformigenes may be a better therapeutic candidate than current group I strains (retaining the name O. formigenes), which have been previously tested and shown to be ineffective as an oral supplement to mitigate stone disease. By performing genomic analyses and identifying these novel characteristics, Oxalobacter strains better suited to mitigation of calcium oxalate-based urolithiasis may be identified in the future.

High-Throughput in vitro Gel-Based Plate Assay to Screen for Calcium Oxalate Stone Inhibitors.

OBJECTIVE: Kidney stones are a common medical condition that is increasing in prevalence worldwide. Approximately, ∼80% of urinary calculi are composed of calcium oxalate (CaOx). There is a growing interest toward identifying therapeutic compounds that can inhibit the formation of CaOx crystals. However, some chemicals (e.g., antibiotics and bacterial metabolites) may directly promote crystallization. Current knowledge is limited regarding crystal promoters and inhibitors. Thus, we have developed an in vitro gel-based diffusion model to screen for substances that directly influence CaOx crystal formation. MATERIALS AND METHODS: We used double diffusion of sodium oxalate and calcium chloride-loaded paper disks along an agar medium to facilitate the controlled formation of monohydrate and dihydrate CaOx crystals. A third disk was used for the perpendicular diffusion of a test substance to assess its influence on CaOx crystal formation. RESULTS: We confirmed that citrates and magnesium are effective inhibitors of CaOx crystals. We also demonstrated that 2 strains of uropathogenic Escherichia coli are able to promote crystal formation. While the other tested uropathogens and most antibiotics did not change crystal formation, ampicillin was able to reduce crystallization. CONCLUSION: We have developed an inexpensive and high-throughput model to evaluate substances that influence CaOx crystallization.

Interstrain Variability of Human Vaginal Lactobacillus crispatus for Metabolism of Biogenic Amines and Antimicrobial Activity against Urogenital Pathogens.

Lactobacillus crispatus is the dominant species in the vagina of many women. With the potential for strains of this species to be used as a probiotic to help prevent and treat dysbiosis, we investigated isolates from vaginal swabs with Lactobacillus-dominated and a dysbiotic microbiota. A comparative genome analysis led to the identification of metabolic pathways for synthesis and degradation of three major biogenic amines in most strains. However, targeted metabolomic analysis of the production and degradation of biogenic amines showed that certain strains have either the ability to produce or to degrade these compounds. Notably, six strains produced cadaverine, one produced putrescine, and two produced tyramine. These biogenic amines are known to raise vaginal pH, cause malodour, and make the environment more favourable to vaginal pathogens. In vitro experiments confirmed that strains isolated from women with a dysbiotic vaginal microbiota have higher antimicrobial effects against the common urogenital pathogens Escherichia coli and Enterococcus faecium. The results indicate that not all L. crispatus vaginal strains appear suitable for probiotic application and the basis for selection should not be only the overall composition of the vaginal microbiota of the host from which they came, but specific biochemical and genetic traits.

Abiraterone acetate preferentially enriches for the gut commensal Akkermansia muciniphila in castrate-resistant prostate cancer patients.

Abiraterone acetate (AA) is an inhibitor of androgen biosynthesis, though this cannot fully explain its efficacy against androgen-independent prostate cancer. Here, we demonstrate that androgen deprivation therapy depletes androgen-utilizing Corynebacterium spp. in prostate cancer patients and that oral AA further enriches for the health-associated commensal, Akkermansia muciniphila. Functional inferencing elucidates a coinciding increase in bacterial biosynthesis of vitamin K2 (an inhibitor of androgen dependent and independent tumor growth). These results are highly reproducible in a host-free gut model, excluding the possibility of immune involvement. Further investigation reveals that AA is metabolized by bacteria in vitro and that breakdown components selectively impact growth. We conclude that A. muciniphila is a key regulator of AA-mediated restructuring of microbial communities, and that this species may affect treatment response in castrate-resistant cohorts. Ongoing initiatives aimed at modulating the colonic microbiota of cancer patients may consider targeted delivery of poorly absorbed selective bacterial growth agents.

Lactobacillus spp. attenuate antibiotic-induced immune and microbiota dysregulation in honey bees.

Widespread antibiotic usage in apiculture contributes substantially to the global dissemination of antimicrobial resistance and has the potential to negatively influence bacterial symbionts of honey bees (Apis mellifera). Here, we show that routine antibiotic administration with oxytetracycline selectively increased tetB (efflux pump resistance gene) abundance in the gut microbiota of adult workers while concurrently depleting several key symbionts known to regulate immune function and nutrient metabolism such as Frischella perrera and Lactobacillus Firm-5 strains. These microbial changes were functionally characterized by decreased capped brood counts (marker of hive nutritional status and productivity) and reduced antimicrobial capacity of adult hemolymph (indicator of immune competence). Importantly, combination therapy with three immunostimulatory Lactobacillus strains could mitigate antibiotic-associated microbiota dysbiosis and immune deficits in adult workers, as well as maximize the intended benefit of oxytetracycline by suppressing larval pathogen loads to near-undetectable levels. We conclude that microbial-based therapeutics may offer a simple but effective solution to reduce honey bee disease burden, environmental xenobiotic exposure, and spread of antimicrobial resistance.

Missing Microbes in Bees: How Systematic Depletion of Key Symbionts Erodes Immunity.

Pesticide exposure, infectious disease, and nutritional stress contribute to honey bee mortality and a high rate of colony loss. This realization has fueled a decades-long investigation into the single and combined effects of each stressor and their overall bearing on insect physiology. However, one element largely missing from this research effort has been the evaluation of underlying microbial communities in resisting environmental stressors and their influence on host immunity and disease tolerance. In humans, multigenerational bombardment by antibiotics is linked with many contemporary diseases. Here, we draw a parallel conclusion for the case in honey bees and suggest that chronic exposure to antimicrobial xenobiotics can systematically deplete honey bees of their microbes and hamper cross-generational preservation of host-adapted symbionts that are crucial to health.

Novel probiotic approach to counter Paenibacillus larvae infection in honey bees.

American foulbrood (AFB) is a highly virulent disease afflicting honey bees (Apis mellifera). The causative organism, Paenibacillus larvae, attacks honey bee brood and renders entire hives dysfunctional during active disease states, but more commonly resides in hives asymptomatically as inactive spores that elude even vigilant beekeepers. The mechanism of this pathogenic transition is not fully understood, and no cure exists for AFB. Here, we evaluated how hive supplementation with probiotic lactobacilli (delivered through a nutrient patty; BioPatty) affected colony resistance towards a naturally occurring AFB outbreak. Results demonstrated a significantly lower pathogen load and proteolytic activity of honey bee larvae from BioPatty-treated hives. Interestingly, a distinctive shift in the microbiota composition of adult nurse bees occurred irrespective of treatment group during the monitoring period, but only vehicle-supplemented nurse bees exhibited higher P. larvae loads. In vitro experiments utilizing laboratory-reared honey bee larvae showed Lactobacillus plantarum Lp39, Lactobacillus rhamnosus GR-1, and Lactobacillus kunkeei BR-1 (contained in the BioPatty) could reduce pathogen load, upregulate expression of key immune genes, and improve survival during P. larvae infection. These findings suggest the usage of a lactobacilli-containing hive supplement, which is practical and affordable for beekeepers, may be effective for reducing enzootic pathogen-related hive losses.

Deleterious Effects of Neonicotinoid Pesticides on Drosophila melanogaster Immune Pathways.

Neonicotinoid insecticides are common agrochemicals that are used to kill pest insects and improve crop yield. However, sublethal exposure can exert unintentional toxicity to honey bees and other beneficial pollinators by dysregulating innate immunity. Generation of hydrogen peroxide (H2O2) by the dual oxidase (Duox) pathway is a critical component of the innate immune response, which functions to impede infection and maintain homeostatic regulation of the gut microbiota. Despite the importance of this pathway in gut immunity, the consequences of neonicotinoid exposure on Duox signaling have yet to be studied. Here, we use a Drosophila melanogaster model to investigate the hypothesis that imidacloprid (a common neonicotinoid) can affect the Duox pathway. The results demonstrated that exposure to sublethal imidacloprid reduced H2O2 production by inhibiting transcription of the Duox gene. Furthermore, the reduction in Duox expression was found to be a result of imidacloprid interacting with the midgut portion of the immune deficiency pathway. This impairment led to a loss of microbial regulation, as exemplified by a compositional shift and increased total abundance of Lactobacillus and Acetobacter spp. (dominant microbiota members) found in the gut. In addition, we demonstrated that certain probiotic lactobacilli could ameliorate Duox pathway impairment caused by imidacloprid, but this effect was not directly dependent on the Duox pathway itself. This study is the first to demonstrate the deleterious effects that neonicotinoids can have on Duox-mediated generation of H2O2 and highlights a novel coordination between two important innate immune pathways present in insects.IMPORTANCE Sublethal exposure to certain pesticides (e.g., neonicotinoid insecticides) is suspected to contribute to honey bee (Apis mellifera) population decline in North America. Neonicotinoids are known to interfere with immune pathways in the gut of insects, but the underlying mechanisms remain elusive. We used a Drosophila melanogaster model to understand how imidacloprid (a common neonicotinoid) interferes with two innate immune pathways-Duox and Imd. We found that imidacloprid dysregulates these pathways to reduce hydrogen peroxide production, ultimately leading to a dysbiotic shift in the gut microbiota. Intriguingly, we found that presupplementation with probiotic bacteria could mitigate the harmful effects of imidacloprid. Thus, these observations uncover a novel mechanism of pesticide-induced immunosuppression that exploits the interconnectedness of two important insect immune pathways.

Immobilization of cadmium and lead by Lactobacillus rhamnosus GR-1 mitigates apical-to-basolateral heavy metal translocation in a Caco-2 model of the intestinal epithelium.

Heavy metals are highly toxic elements that contaminate the global food supply and affect human and wildlife health. Purification technologies are often too expensive or not practically applicable for large-scale implementation, especially in impoverished nations where heavy metal contamination is widespread. Lactobacillus rhamnosus GR-1 (LGR-1) was shown in previous work to reduce heavy metal bioaccumulation in a Tanzanian cohort of women and children through indeterminant mechanisms. Here, it was hypothesized that LGR-1 could sequester the heavy metals lead (Pb) and cadmium (Cd), thereby reducing their absorption across intestinal epithelium. LGR-1 and other lactobacilli significantly reduced the amount of Pb and Cd in solution at all concentrations tested (0.5 mg/L - 50 mg/L) and exhibited sustained binding profiles over a 48-hour period. Relative binding efficiency of LGR-1 decreased as Pb concentration increased, with an absolute minimum binding threshold apparent at concentrations of 2 mg/L and above. Electron microscopy revealed that Pb formed irregular cell-surface clusters on LGR-1, while Cd appeared to form intracellular polymeric clusters. Additionally, LGR-1 was able to significantly reduce apical-to-basolateral translocation of Pb and Cd in a Caco-2 model of the intestinal epithelium. These findings demonstrate the absorbent properties of LGR-1 can immobilize Pb and Cd, effectively reducing their translocation across the intestinal epithelium in vitro. Oral administration of heavy metal-binding Lactobacillus spp. (many of which are known human symbionts and strains of established probiotics) may offer a simple and effective means to reduce the amount of heavy metals absorbed from foods in contaminated regions of the world.