The Role of Prebiotics in Modulating Gut Microbiota: Implications for Human Health.

The human gut microbiota, an intricate ecosystem within the gastrointestinal tract, plays a pivotal role in health and disease. Prebiotics, non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of beneficial microorganisms, have emerged as a key modulator of this complex microbial community. This review article explores the evolution of the prebiotic concept, delineates various types of prebiotics, including fructans, galactooligosaccharides, xylooligosaccharides, chitooligosaccharides, lactulose, resistant starch, and polyphenols, and elucidates their impact on the gut microbiota composition. We delve into the mechanisms through which prebiotics exert their effects, particularly focusing on producing short-chain fatty acids and modulating the gut microbiota towards a health-promoting composition. The implications of prebiotics on human health are extensively reviewed, focusing on conditions such as obesity, inflammatory bowel disease, immune function, and mental health. The review further discusses the emerging concept of synbiotics-combinations of prebiotics and probiotics that synergistically enhance gut health-and highlights the market potential of prebiotics in response to a growing demand for functional foods. By consolidating current knowledge and identifying areas for future research, this review aims to enhance understanding of prebiotics' role in health and disease, underscoring their importance in maintaining a healthy gut microbiome and overall well-being.

Exploitation of a novel adjuvant for polymyxin B against multidrug-resistant Acinetobacter baumannii.

BACKGROUND: Although polymyxin has been used as a last-resort antibiotic against resistant bacteria, its use is restricted due to nephrotoxicity and neurotoxicity. While the present antibiotic resistance issue compels clinicians to reconsider polymyxin use in severe illness cases, polymyxin-resistant microorganisms exert an effect. OBJECTIVES: To address the issue of antibiotic resistance, the cycle of developing new antibiotics to counteract emerging resistance must be discontinued. Here we tried to develop novel therapies that do not rely on direct antimicrobial activity and thus do not promote antibiotic resistance. METHODS: By a high-throughout screening system based on bacterial respiration, chemical compounds accelerating the antimicrobial effects of polymyxin B were screened. In vitro and in vivo tests were performed to validate adjuvanticity. In addition, membrane depolarization and total transcriptome analysis were used to determine molecular mechanisms. RESULTS: PA108, a newly discovered chemical compound, was used to eradicate polymyxin-resistant A. baumannii and three other species in the presence of polymyxin B at concentrations less than the MIC. Since this molecule lacks self-bactericidal action, we hypothesized that PA108 acts as an antibiotic adjuvant, enhancing the antimicrobial activity of polymyxin B against resistant bacteria. At working concentrations, no toxicity was observed in cell lines or mice, although co-treatment with PA108 and polymyxin B increased survival of infected mouse and decreased bacterial loads in organs. CONCLUSIONS: Boosting antibiotic efficiency through the use of antibiotic adjuvants holds significant promise for tackling the rise in bacterial antibiotic resistance.

Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation.

Cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as an active stress response. How these phage fossils affect persister cell resuscitation has, however, not been explored. Persister cells form as a result of stresses such as starvation, antibiotics and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis and Lyme disease. Deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system resulting from inactivating the transcriptional regulator AlpA of the CP4-57 cryptic prophage. Deletion of alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Both phosphate regulators PhoP and PhoB stimulate resuscitation. This suggests a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.

Crossing the kingdom border: Human diseases caused by plant pathogens.

Interactions between pathogenic microorganisms and their hosts are varied and complex, encompassing open-field scale interactions to interactions at the molecular level. The capacity of plant pathogenic bacteria and fungi to cause diseases in human and animal systems was, until recently, considered of minor importance. However, recent evidence suggests that animal and human infections caused by plant pathogenic fungi, bacteria and viruses may have critical impacts on human and animal health and safety. This review analyses previous research on plant pathogens as causal factors of animal illness. In addition, a case study involving disruption of type III effector-mediated phagocytosis in a human cell line upon infection with an opportunistic phytopathogen, Pseudomonas syringae pv. tomato, is discussed. Further knowledge regarding the molecular interactions between plant pathogens and human and animal hosts is needed to understand the extent of disease incidence and determine mechanisms for disease prevention.

Identification of a potent indigoid persister antimicrobial by screening dormant cells.

The subpopulation of bacterial cells that survive myriad stress conditions (e.g., nutrient deprivation and antimicrobials) by ceasing metabolism, revive by activating ribosomes. These resuscitated cells can reconstitute infections; hence, it is imperative to discover compounds which eradicate persister cells. By screening 10,000 compounds directly for persister cell killing, we identified 5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride (NPIMA) kills Escherichia coli persister cells more effectively than the best indigoid found to date, 5-iodoindole, and better than the DNA-crosslinker cisplatin. In addition, NPIMA eradicated Pseudomonas aeruginosa persister cells in a manner comparable to cisplatin. NPIMA also eradicated Staphylococcus aureus persister cells but was less effective than cisplatin. Critically, NPIMA kills Gram-positive and Gram-negative bacteria by damaging membranes and causing lysis as demonstrated by microscopy and release of extracellular DNA and protein. Furthermore, NPIMA was effective in reducing P. aeruginosa and S. aureus cell numbers in a wound model, and no resistance was found after 1 week. Hence, we identified a potent indigoid that kills persister cells by damaging their membranes.

Viable but non-culturable and persistence describe the same bacterial stress state.

Bacteria are often thought of as having two dormant phenotypes: the viable but non-culturable (VBNC) state and the persister state. Here we investigate the relatedness of the two stress-induced phenotypes at the single-cell level and examine cell morphology and quantify cell resuscitation. Using the classic starvation conditions to create VBNC cells, we found that the majority of the remaining Escherichia coli population are spherical, have empty cytosol and fail to resuscitate; however, some of the spherical cells resuscitate immediately (most probably those with dense cytosol). Critically, all the culturable cells in this starved population became persister cells within 14 days of starvation. We found that the persister cells initially are rod-like, have clear but limited membrane damage, can resuscitate immediately and gradually become spherical by aging. After 24 h, only rod-shaped persister cells survive, and all the spherical cells lyse. Both cell populations formed under the VBNC-inducing conditions and the persister conditions are metabolically inactive. Therefore, the bacterial population consists of dead cells and persister cells in the VBNC-inducing conditions; that is, the non-lysed particles that do not resuscitate are dead, and the dormant cells that resuscitate are persister cells. Hence, 'VBNC' and 'persister' describe the same dormant phenotype.

Single cell observations show persister cells wake based on ribosome content.

Since persister cells survive antibiotic treatments through dormancy and resuscitate to reconstitute infections, it is imperative to determine the rate at which these cells revive. Using two sets of Escherichia coli persister cells, those arising after antibiotic treatment at low levels and those generated at high levels by ceasing transcription via rifampicin pretreatment (shown to be bona fide persisters through eight sets of experiments), we used microscopy of single cells to determine that the resuscitation of dormant persisters is heterogeneous and includes cells that grow immediately. In all, five phenotypes were found during the observation of persister cells when fresh nutrients were added: (i) immediate division, (ii) immediate elongation followed by division, (iii) immediate elongation but no division, (iv) delayed elongation/division and (v) no growth. In addition, once cell division begins, the growth rate is that of exponential cells. Critically, the greater the ribosome content, the faster the persister cells resuscitate.

Pseudomonas syringae evades phagocytosis by animal cells via type III effector-mediated regulation of actin filament plasticity.

Certain animal and plant pathogenic bacteria have developed virulence factors including effector proteins that enable them to overcome host immunity. A plant pathogen, Pseudomonas syringae pv. tomato (Pto) secretes a large repertoire of effectors via a type III secretory apparatus, thereby suppressing plant immunity. Here, we show that Pto causes sepsis in mice. Surprisingly, the effector HopQ1 disrupted animal phagocytosis by inhibiting actin rearrangement via direct interaction with the LIM domain of the animal target protein LIM kinase, a key regulator of actin polymerization. The results provide novel insight into animal host-plant pathogen interactions. In addition, the current study firstly demonstrates that certain plant pathogenic bacteria such as Pto evade phagocytosis by animal cells due to cross-kingdom suppression of host immunity.

GhoT of the GhoT/GhoS toxin/antitoxin system damages lipid membranes by forming transient pores.

GhoT is a bacterial toxin of the type V toxin/antitoxin system that allows Escherichia coli to reduce its metabolism in response to oxidative and bile stress. GhoT functions by increasing membrane permeability and reducing both ATP levels and the proton motive force. However, how GhoT damages the inner membrane has not been elucidated. Here we investigated how GhoT damages membranes by studying its interaction with lipid bilayers and determined that GhoT does not cause macroscopic disruption of the lipid bilayer to increase membrane permeability to the dye carboxyfluorescein. Using circular dichroism, we found that GhoT forms an alpha helical structure in lipid bilayers that agrees with the structure predicted by the I-TASSER protein structure prediction program. The structure generated using I-TASSER was used to conduct coarse-grained molecular dynamics simulations, which indicate that GhoT damages the cell membrane, as a multimer, by forming transient transmembrane pores.

Fumarate-Mediated Persistence of Escherichia coli against Antibiotics.

Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) inEscherichia coliled to a higher frequency of persister formation. The persister frequency ofE. coliwas increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-relatedhipA7mutation indicated that surplus fumarate markedly elevated theE. colipersister frequency. AnE. colistrain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears thatSDHandFRDrepresent a paired system that gives rise to and maintainsE. colipersisters by producing and utilizing fumarate, respectively.

Complementary Therapeutic Effect of Fecal Microbiota Transplantation in Ulcerative Colitis after the Response to Anti-Tumor Necrosis Factor Alpha Agent Was Lost: A Case Report.

In patients with ulcerative colitis (UC), the development of an antidrug antibody (ADA) to anti-tumor necrosis factor (TNF)α agent is a crucial problem which aggravates the clinical course of the disease, being cited as one of the most common causes for discontinuing anti-TNFα treatment. This is due to ADA eventually causing secondary LOR, leading to discontinuation of anti-TNFα treatment. Recently, research on the microbiome and relationship between worsening UC and dysbiosis has been conducted. Further, investigations on the association between the microbiome and secondary LOR are increasing. Here, we present the therapeutic effect of fecal microbiota transplantation (FMT) on a 42-year-old man with secondary LOR and high ADA levels. FMT has recently been used for the treatment of, and for overcoming, drug resistance through microbiome modification. Stool samples were collected from the patient before and 4 weeks after FMT. Symptoms, including hematochezia and Mayo endoscopy sub-scores, improved after FMT, while ADA levels decreased by one-third to less than half the value (29 ng/mL) compared to before FMT (79 ng/mL). Additionally, the trough level of infliximab became measurable, which reflects the improvement in the area under the concentration (AUC). Butyricicoccus, Faecalibacterium, Bifidobacterium, Ligilactobacillus, Alistipes, and Odoribacter, which regulate immune responses and alleviate inflammation, also increased after FMT. We report a case in which microbiome modification by FMT increased the AUC of anti-TNFα in a patient who developed secondary LOR during anti-TNFα treatment, thereby improving symptoms and mucosal inflammation.

Facemask acne attenuation through modulation of indirect microbiome interactions.

During the COVID-19 pandemic, facemasks played a pivotal role in preventing person-person droplet transmission of viral particles. However, prolonged facemask wearing causes skin irritations colloquially referred to as 'maskne' (mask + acne), which manifests as acne and contact dermatitis and is mostly caused by pathogenic skin microbes. Previous studies revealed that the putative causal microbes were anaerobic bacteria, but the pathogenesis of facemask-associated skin conditions remains poorly defined. We therefore characterized the role of the facemask-associated skin microbiota in the development of maskne using culture-dependent and -independent methodologies. Metagenomic analysis revealed that the majority of the facemask microbiota were anaerobic bacteria that originated from the skin rather than saliva. Previous work demonstrated direct interaction between pathogenic bacteria and antagonistic strains in the microbiome. We expanded this analysis to include indirect interaction between pathogenic bacteria and other indigenous bacteria classified as either 'pathogen helper (PH)' or 'pathogen inhibitor (PIn)' strains. In vitro screening of bacteria isolated from facemasks identified both strains that antagonized and promoted pathogen growth. These data were validated using a mouse skin infection model, where we observed attenuation of symptoms following pathogen infection. Moreover, the inhibitor of pathogen helper (IPH) strain, which did not directly attenuate pathogen growth in vitro and in vivo, functioned to suppress symptom development and pathogen growth indirectly through PH inhibitory antibacterial products such as phenyl lactic acid. Taken together, our study is the first to define a mechanism by which indirect microbiota interactions under facemasks can control symptoms of maskne by suppressing a skin pathogen.

Mucoadhesive Mesalamine Prodrug Nanoassemblies to Target Intestinal Macrophages for the Treatment of Inflammatory Bowel Disease.

While mesalamine, a 5-aminosalicylic acid (5-ASA), is pivotal in the management of inflammatory bowel disease (IBD) through both step-up and top-down approaches in clinical settings, its widespread utilization is limited by low bioavailability at the desired site of action due to rapid and extensive absorption in the upper gastrointestinal (GI) tract. Addressing mesalamine's pharmacokinetic challenges, here, we introduce nanoassemblies composed exclusively of a mesalamine prodrug that pairs 5-ASA with a mucoadhesive and cathepsin B-cleavable peptide. In an IBD model, orally administered nanoassemblies demonstrate enhanced accumulation and sustained retention in the GI tract due to their mucoadhesive properties and the epithelial enhanced permeability and retention (eEPR) effect. This retention enables the efficient uptake by intestinal pro-inflammatory macrophages expressing high cathepsin B, triggering a burst release of the 5-ASA. This cascade fosters the polarization toward an M2 macrophage phenotype, diminishes inflammatory responses, and simultaneously facilitates the delivery of active agents to adjacent epithelial cells. Therefore, the nanoassemblies show outstanding therapeutic efficacy in inhibiting local inflammation and contribute to suppressing systemic inflammation by restoring damaged intestinal barriers. Collectively, this study highlights the promising role of the prodrug nanoassemblies in enhancing targeted drug delivery, potentially broadening the use of mesalamine in managing IBD.

3-Fucosyllactose-mediated modulation of immune response against virus infection.

Viral pathogens, particularly influenza and SARS-CoV-2, pose a significant global health challenge. Given the immunomodulatory properties of human milk oligosaccharides, in particular 2'-fucosyllactose and 3-fucosyllactose (3-FL), we investigated their dietary supplementation effects on antiviral responses in mouse models. This study revealed distinct immune modulations induced by 3-FL. RNA-sequencing data showed that 3-FL increased the expression of interferon receptors, such as Interferon Alpha and Beta Receptor (IFNAR) and Interferon Gamma Receptor (IFNGR), while simultaneously downregulating interferons and interferon-stimulated genes, an effect not observed with 2'-fucosyllactose supplementation. Such modulation enhanced antiviral responses in both cell culture and animal models while attenuating pre-emptive inflammatory responses. Nitric oxide concentrations in 3-FL-supplemented A549 cells and mouse lung tissues were elevated exclusively upon infection, reaching 5.8- and 1.9-fold increases over control groups, respectively. In addition, 3-FL promoted leukocyte infiltration into the site of infection upon viral challenge. 3-FL supplementation provided protective efficacy against lethal influenza challenge in mice. The demonstrated antiviral efficacy spanned multiple influenza strains and extended to SARS-CoV-2. In conclusion, 3-FL is a unique immunomodulator that helps protect the host from viral infection while suppressing inflammation prior to infection.

Effect of altered gene expression in lipid metabolism on cognitive improvement in patients with Alzheimer's dementia following fecal microbiota transplantation: a preliminary study.

Background: The brain-gut axis has emerged as a potential target in neurodegenerative diseases, including dementia, as individuals with dementia exhibit distinct gut microbiota compositions. Fecal microbiota transplantation (FMT), the transfer of fecal solution from a healthy donor to a patient, has shown promise in restoring homeostasis and cognitive enhancement. Objective: This study aimed to explore the effects of FMT on specific cognitive performance measures in Alzheimer's dementia (AD) patients and investigate the relationship between cognition and the gut microbiota by evaluating changes in gene expression following FMT. Methods: Five AD patients underwent FMT, and their cognitive function [Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and Clinical Dementia Rating Scale Sum of Boxes (CDR-SOB)] was assessed before and after FMT. The patients' fecal samples were analyzed with 16S rRNA to compare the composition of their gut microbiota. We also assessed modifications in the serum mRNA expression of patients' genes related to lipid metabolism using serum RNA sequencing and quantitative real-time polymerase chain reaction. Results: Significant improvements in cognitive function, as measured by the MMSE (pre- and post-FMT was 13.00 and 18.00) and MoCA were seen. The MoCA scores at 3 months post-FMT (21.0) were the highest (12.0). The CDR-SOB scores at pre- and post-FMT were 10.00 and 5.50, respectively. Analysis of the gut microbiome composition revealed changes via 16S rRNA sequencing with an increase in Bacteroidaceae and a decrease in Enterococcaceae. Gene expression analysis identified alterations in lipid metabolism-related genes after FMT. Conclusion: These findings suggest a link between alterations in the gut microbiome, gene expression related to lipid metabolism, and cognitive function. The study highlights the importance of gut microbiota in cognitive function and provides insights into potential biomarkers for cognitive decline progression. FMT could complement existing therapies and show potential as a therapeutic intervention to mitigate cognitive decline in AD.

Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus.

Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) >> P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the cassettes were integrated into the chromosome, the stochastic behavior was markedly reduced. These combined results imply that the gene expression stochasticity should be overcome in order to use this strain for delicate metabolic engineering, which would require the co-expression of several genes.

Dissection of SNARE-driven membrane fusion and neuroexocytosis by wedging small hydrophobic molecules into the SNARE zipper.

Neuronal SNARE proteins mediate neurotransmitter release at the synapse by facilitating the fusion of vesicles to the presynaptic plasma membrane. Cognate v-SNAREs and t-SNAREs from the vesicle and the plasma membrane, respectively, zip up and bring about the apposition of two membranes attached at the C-terminal ends. Here, we demonstrate that SNARE zippering can be modulated in the midways by wedging with small hydrophobic molecules. Myricetin, which intercalated into the hydrophobic inner core near the middle of the SNARE complex, stopped SNARE zippering in motion and accumulated the trans-complex, where the N-terminal region of v-SNARE VAMP2 is in the coiled coil with the frayed C-terminal region. Delphinidin and cyanidin inhibited N-terminal nucleation of SNARE zippering. Neuronal SNARE complex in PC12 cells showed the same pattern of vulnerability to small hydrophobic molecules. We propose that the half-zipped trans-SNARE complex is a crucial intermediate waiting for a calcium trigger that leads to fusion pore opening.

Collagen Peptide Exerts an Anti-Obesity Effect by Influencing the Firmicutes/Bacteroidetes Ratio in the Gut.

Alterations in the intestinal microbial flora are known to cause various diseases, and many people routinely consume probiotics or prebiotics to balance intestinal microorganisms and the growth of beneficial bacteria. In this study, we selected a peptide from fish (tilapia) skin that induces significant changes in the intestinal microflora of mice and reduces the Firmicutes/Bacteroidetes ratio, which is linked to obesity. We attempted to verify the anti-obesity effect of selected fish collagen peptides in a high-fat-diet-based obese mouse model. As anticipated, the collagen peptide co-administered with a high-fat diet significantly inhibited the increase in the Firmicutes/Bacteroidetes ratio. It increased specific bacterial taxa, including Clostridium_sensu_stricto_1, Faecalibaculum, Bacteroides, and Streptococcus, known for their anti-obesity effects. Consequently, alterations in the gut microbiota resulted in the activation of metabolic pathways, such as polysaccharide degradation and essential amino acid synthesis, which are associated with obesity inhibition. In addition, collagen peptide also effectively reduced all obesity signs caused by a high-fat diet, such as abdominal fat accumulation, high blood glucose levels, and weight gain. Ingestion of collagen peptides derived from fish skin induced significant changes in the intestinal microflora and is a potential auxiliary therapeutic agent to suppress the onset of obesity.

Population Dynamics of Intestinal Enterococcus Modulate Galleria mellonella Metamorphosis.

Microbes found in the digestive tracts of insects are known to play an important role in their host's behavior. Although Lepidoptera is one of the most varied insect orders, the link between microbial symbiosis and host development is still poorly understood. In particular, little is known about the role of gut bacteria in metamorphosis. Here, we explored gut microbial biodiversity throughout the life cycle of Galleria mellonella, using amplicon pyrosequencing with the V1 to V3 regions, and found that Enterococcus spp. were abundant in larvae, while Enterobacter spp. were predominant in pupae. Interestingly, eradication of Enterococcus spp. from the digestive system accelerated the larval-to-pupal transition. Furthermore, host transcriptome analysis demonstrated that immune response genes were upregulated in pupae, whereas hormone genes were upregulated in larvae. In particular, regulation of antimicrobial peptide production in the host gut correlated with developmental stage. Certain antimicrobial peptides inhibited the growth of Enterococcus innesii, a dominant bacterial species in the gut of G. mellonella larvae. Our study highlights the importance of gut microbiota dynamics on metamorphosis as a consequence of the active secretion of antimicrobial peptides in the G. mellonella gut. IMPORTANCE First, we demonstrated that the presence of Enterococcus spp. is a driving force for insect metamorphosis. RNA sequencing and peptide production subsequently revealed that antimicrobial peptides targeted against microorganisms in the gut of Galleria mellonella (wax moth) did not kill Enterobacteria species, but did kill Enterococcus species, when the moth was at a certain stage of growth, and this promoted moth pupation.