The impact of vegan production on the kimchi microbiome.

Despite previous inquiry into the fermentative bacterial community of kimchi, there has been little insight into the impacts of starting ingredients on the establishment and dynamics of the microbial community. Recently some industrial producers have begun to utilize vegan production methods that omit fermented seafood ingredients. The community-level impacts of this change are unknown. In this study, we investigated the differences in the taxonomic composition of the microbial communities of non-vegan kimchi and vegan kimchi prepared through quick fermentation at room temperature. In addition to tracking the community dynamics over the fermentation process, we looked at the impact of the constituent ingredients and the production facility environment on the microbial community of fermenting kimchi. Our results indicate that the bacterial community of the prepared vegan product closely mirrors the progression and final structure of the non-vegan final product. We also found that room temperature-fermented kimchi differs minimally from more traditional cold-fermented kimchi. Finally, we found that the bacterial community of the starting ingredients show a low relative abundance of the lactic acid bacteria in fermented kimchi, whereas the production facility is dominated by these bacteria.

Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector.

Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.

Immunomodulation by the Commensal Microbiome During Immune-Targeted Interventions: Focus on Cancer Immune Checkpoint Inhibitor Therapy and Vaccination.

Emerging evidence in clinical and preclinical studies indicates that success of immunotherapies can be impacted by the state of the microbiome. Understanding the role of the microbiome during immune-targeted interventions could help us understand heterogeneity of treatment success, predict outcomes, and develop additional strategies to improve efficacy. In this review, we discuss key studies that reveal reciprocal interactions between the microbiome, the immune system, and the outcome of immune interventions. We focus on cancer immune checkpoint inhibitor treatment and vaccination as two crucial therapeutic areas with strong potential for immunomodulation by the microbiota. By juxtaposing studies across both therapeutic areas, we highlight three factors prominently involved in microbial immunomodulation: short-chain fatty acids, microbe-associate molecular patterns (MAMPs), and inflammatory cytokines. Continued interrogation of these models and pathways may reveal critical mechanistic synergies between the microbiome and the immune system, resulting in novel approaches designed to influence the efficacy of immune-targeted interventions.

Metatranscriptomics Reveals Antibiotic-Induced Resistance Gene Expression in the Murine Gut Microbiota.

Antibiotic resistance is a current and expanding threat to the practice of modern medicine. Antibiotic therapy has been shown to perturb the composition of the host microbiome with significant health consequences. In addition, the gut microbiome is known to be a reservoir of antibiotic resistance genes. Work has demonstrated that antibiotics can alter the collection of antibiotic resistance genes within the microbiome through selection and horizontal gene transfer. While antibiotics also have the potential to impact the expression of resistance genes, metagenomic-based pipelines currently lack the ability to detect these shifts. Here, we utilized a dual sequencing approach combining shotgun metagenomics and metatranscriptomics to profile how three antibiotics, amoxicillin, doxycycline, and ciprofloxacin, impact the murine gut resistome at the DNA and RNA level. We found that each antibiotic induced broad, but untargeted impacts on the gene content of the resistome. In contrast, changes in ARG transcript abundance were more targeted to the antibiotic treatment. Doxycycline and amoxicillin induced the expression of tetracycline and beta-lactamase resistance genes, respectively. Furthermore, the increased beta-lactamase resistance gene transcripts could contribute to an observed bloom of Bacteroides thetaiotaomicron during amoxicillin treatment. Based on these findings, we propose that the utilization of a dual sequencing methodology provides a unique capacity to fully understand the response of the resistome to antibiotic perturbation. In particular, the analysis of transcripts reveals that the expression and utilization of resistance genes is far narrower than their abundance at the genomic level would suggest.

Consumption of a Western-Style Diet Modulates the Response of the Murine Gut Microbiome to Ciprofloxacin.

Dietary composition and antibiotic use have major impacts on the structure and function of the gut microbiome, often resulting in dysbiosis. Despite this, little research has been done to explore the role of host diet as a determinant of antibiotic-induced microbiome disruption. Here, we utilize a multi-omic approach to characterize the impact of Western-style diet consumption on ciprofloxacin-induced changes to gut microbiome structure and transcriptional activity. We found that Western diet consumption dramatically increased Bacteroides abundances and shifted the community toward the metabolism of simple sugars and mucus glycoproteins. Mice consuming a Western-style diet experienced a greater expansion of Firmicutes following ciprofloxacin treatment than those eating a control diet. Transcriptionally, we found that ciprofloxacin reduced the abundance of tricarboxylic acid (TCA) cycle transcripts on both diets, suggesting that carbon metabolism plays a key role in the response of the gut microbiome to this antibiotic. Despite this, we observed extensive diet-dependent differences in the impact of ciprofloxacin on microbiota function. In particular, at the whole-community level we detected an increase in starch degradation, glycolysis, and pyruvate fermentation following antibiotic treatment in mice on the Western diet, which we did not observe in mice on the control diet. Similarly, we observed diet-specific changes in the transcriptional activity of two important commensal bacteria, Akkermansia muciniphila and Bacteroides thetaiotaomicron, involving diverse cellular processes such as nutrient acquisition, stress responses, and capsular polysaccharide (CPS) biosynthesis. These findings demonstrate that host diet plays a role in determining the impacts of ciprofloxacin on microbiome composition and microbiome function.IMPORTANCE Due to the growing incidence of disorders related to antibiotic-induced dysbiosis, it is essential to determine how our "Western"-style diet impacts the response of the microbiome to antibiotics. While diet and antibiotics have profound impacts on gut microbiome composition, little work has been done to examine their combined effects. Previous work has shown that nutrient availability, influenced by diet, plays an important role in determining the extent of antibiotic-induced disruption to the gut microbiome. Thus, we hypothesize that the Western diet will shift microbiota metabolism toward simple sugar and mucus degradation and away from polysaccharide utilization. Because of bacterial metabolism's critical role in antibiotic susceptibility, this change in baseline metabolism will impact how the structure and function of the microbiome are impacted by ciprofloxacin exposure. Understanding how diet modulates antibiotic-induced microbiome disruption will allow for the development of dietary interventions that can alleviate many of the microbiome-dependent complications of antibiotic treatment.

Microbial Metabolism Modulates Antibiotic Susceptibility within the Murine Gut Microbiome.

Although antibiotics disturb the structure of the gut microbiota, factors that modulate these perturbations are poorly understood. Bacterial metabolism is an important regulator of susceptibility in vitro and likely plays a large role within the host. We applied a metagenomic and metatranscriptomic approach to link antibiotic-induced taxonomic and transcriptional responses within the murine microbiome. We found that antibiotics significantly alter the expression of key metabolic pathways at the whole-community and single-species levels. Notably, Bacteroides thetaiotaomicron, which blooms in response to amoxicillin, upregulated polysaccharide utilization. In vitro, we found that the sensitivity of this bacterium to amoxicillin was elevated by glucose and reduced by polysaccharides. Accordingly, we observed that dietary composition affected the abundance and expansion of B. thetaiotaomicron, as well as the extent of microbiome disruption with amoxicillin. Our work indicates that the metabolic environment of the microbiome plays a role in the response of this community to antibiotics.

Urogenital schistosomiasis is associated with signatures of microbiome dysbiosis in Nigerian adolescents.

Urogenital schistosomiasis is a neglected tropical disease caused by the parasite Schistosoma haematobium, which resides in the vasculature surrounding the urogenital system. Previous work has suggested that helminthic infections can affect the intestinal microbiome, and we hypothesized that S. haematobium infection could result in an alteration of immune system-microbiota homeostasis and impact the composition of the gut microbiota. To address this question, we compared the fecal microbiomes of infected and uninfected schoolchildren from the Argungu Local Government Area of Kebbi State, Nigeria, detecting significant differences in community composition between the two groups. Most remarkably, we observed a decreased abundance of Firmicutes and increased abundance of Proteobacteria - a shift in community structure which has been previously associated with dysbiosis. More specifically, we detected a number of changes in lower taxa reminiscent of inflammation-associated dysbiosis, including decreases in Clostridiales and increases in Moraxellaceae, Veillonellaceae, Pasteurellaceae, and Desulfovibrionaceae. Functional potential analysis also revealed an enrichment in orthologs of urease, which has been linked to dysbiosis and inflammation. Overall, our analysis indicates that S. haematobium infection is associated with perturbations in the gut microbiota and may point to microbiome disruption as an additional consequence of schistosome infection.

Antibiotic Persistence as a Metabolic Adaptation: Stress, Metabolism, the Host, and New Directions.

Persistence is a phenomenon during which a small fraction of a total bacterial population survives treatment with high concentrations of antibiotics for an extended period of time. In conjunction with biofilms, antibiotic persisters represent a major cause of recalcitrant and recurring infections, resulting in significant morbidity and mortality. In this review, we discuss the clinical significance of persister cells and the central role of bacterial metabolism in their formation, specifically with respect to carbon catabolite repression, sugar metabolism, and growth regulation. Additionally, we will examine persister formation as an evolutionary strategy used to tolerate extended periods of stress and discuss some of the response mechanisms implicated in their formation. To date, the vast majority of the mechanistic research examining persistence has been conducted in artificial in vitro environments that are unlikely to be representative of host conditions. Throughout this review, we contextualize the existing body of literature by discussing how in vivo conditions may create ecological niches that facilitate the development of persistence. Lastly, we identify how the development of next-generation sequencing and other "big data" tools may enable researchers to examine persistence mechanisms within the host to expand our understanding of their clinical importance.

Microbial competition between Escherichia coli and Candida albicans reveals a soluble fungicidal factor.

Localized and systemic fungal infections caused by Candida albicans can lead to significant mortality and morbidity. However, severe C. albicans infections are relatively rare, occurring mostly in the very young, the very old, and immunocompromised individuals. The fact that these infections are rare is interesting because as much as 80 percent of the population is asymptomatically colonized with C. albicans. It is thought that members of the human microbiota and the immune system work in concert to reduce C. albicans overgrowth through competition and modification of the growth environment. Here, we report that Escherichia coli (strain MG1655) outcompetes and kills C. albicans (strain SC5314) in vitro. We find that E. coli produces a soluble factor that kills C. albicans in a magnesium-dependent fashion such that depletion of available magnesium is essential for toxicity.

Coinfection With Influenza A Virus and Klebsiella oxytoca: An Underrecognized Impact on Host Resistance and Tolerance to Pulmonary Infections.

Pneumonia is a world health problem and a leading cause of death, particularly affecting children and the elderly (1, 2). Bacterial pneumonia following infection with influenza A virus (IAV) is associated with increased morbidity and mortality but the mechanisms behind this phenomenon are not yet well-defined (3). Host resistance and tolerance are two processes essential for host survival during infection. Resistance is the host's ability to clear a pathogen while tolerance is the host's ability to overcome the impact of the pathogen as well as the host response to infection (4-8). Some studies have shown that IAV infection suppresses the immune response, leading to overwhelming bacterial loads (9-13). Other studies have shown that some IAV/bacterial coinfections cause alterations in tolerance mechanisms such as tissue resilience (14-16). In a recent analysis of nasopharyngeal swabs from patients hospitalized during the 2013-2014 influenza season, we have found that a significant proportion of IAV-infected patients were also colonized with Klebsiella oxytoca, a gram-negative bacteria known to be an opportunistic pathogen in a variety of diseases (17). Mice that were infected with K. oxytoca following IAV infection demonstrated decreased survival and significant weight loss when compared to mice infected with either single pathogen. Using this model, we found that IAV/K. oxytoca coinfection of the lung is characterized by an exaggerated inflammatory immune response. We observed early inflammatory cytokine and chemokine production, which in turn resulted in massive infiltration of neutrophils and inflammatory monocytes. Despite this swift response, the pulmonary pathogen burden in coinfected mice was similar to singly-infected animals, albeit with a slight delay in bacterial clearance. In addition, during coinfection we observed a shift in pulmonary macrophages toward an inflammatory and away from a tissue reparative phenotype. Interestingly, there was only a small increase in tissue damage in coinfected lungs as compared to either single infection. Our results indicate that during pulmonary coinfection a combination of seemingly modest defects in both host resistance and tolerance may act synergistically to cause worsened outcomes for the host. Given the prevalence of K. oxytoca detected in human IAV patients, these dysfunctional tolerance and resistance mechanisms may play an important role in the response of patients to IAV.

Microbial Community Analysis of Sauerkraut Fermentation Reveals a Stable and Rapidly Established Community.

Despite recent interest in microbial communities of fermented foods, there has been little inquiry into the bacterial community dynamics of sauerkraut, one of the world’s oldest and most prevalent fermented foods. In this study, we utilize 16S rRNA amplicon sequencing to profile the microbial community of naturally fermented sauerkraut throughout the fermentation process while also analyzing the bacterial communities of the starting ingredients and the production environment. Our results indicate that the sauerkraut microbiome is rapidly established after fermentation begins and that the community is stable through fermentation and packaging for commercial sale. Our high-throughput analysis is in agreement with previous studies that utilized traditional microbiological assessments but expands the identified taxonomy. Additionally, we find that the microbial communities of the starting ingredients and the production facility environment exhibit low relative abundance of the lactic acid bacteria that dominate fermented sauerkraut.

The salivary microbiome is consistent between subjects and resistant to impacts of short-term hospitalization.

In recent years, a growing amount of research has begun to focus on the oral microbiome due to its links with health and systemic disease. The oral microbiome has numerous advantages that make it particularly useful for clinical studies, including non-invasive collection, temporal stability, and lower complexity relative to other niches, such as the gut. Despite recent discoveries made in this area, it is unknown how the oral microbiome responds to short-term hospitalization. Previous studies have demonstrated that the gut microbiome is extremely sensitive to short-term hospitalization and that these changes are associated with significant morbidity and mortality. Here, we present a comprehensive pipeline for reliable bedside collection, sequencing, and analysis of the human salivary microbiome. We also develop a novel oral-specific mock community for pipeline validation. Using our methodology, we analyzed the salivary microbiomes of patients before and during hospitalization or azithromycin treatment to profile impacts on this community. Our findings indicate that azithromycin alters the diversity and taxonomic composition of the salivary microbiome; however, we also found that short-term hospitalization does not impact the richness or structure of this community, suggesting that the oral cavity may be less susceptible to dysbiosis during short-term hospitalization.

Bactericidal antibiotics induce programmed metabolic toxicity.

The misuse of antibiotics has led to the development and spread of antibiotic resistance in clinically important pathogens. These resistant infections are having a significant impact on treatment outcomes and contribute to approximately 25,000 deaths in the U.S. annually. If additional therapeutic options are not identified, the number of annual deaths is predicted to rise to 317,000 in North America and 10,000,000 worldwide by 2050. Identifying therapeutic methodologies that utilize our antibiotic arsenal more effectively is one potential way to extend the useful lifespan of our current antibiotics. Recent studies have indicated that modulating metabolic activity is one possible strategy that can impact the efficacy of antibiotic therapy. In this review, we will address recent advances in our knowledge about the impacts of bacterial metabolism on antibiotic effectiveness and the impacts of antibiotics on bacterial metabolism. We will particularly focus on two studies, Lobritz, et al. (PNAS, 112(27): 8173-8180) and Belenky et al. (Cell Reports, 13(5): 968-980) that together demonstrate that bactericidal antibiotics induce metabolic perturbations that are linked to and required for bactericidal antibiotic toxicity.