Fecal microbiota spores, live-brpk (VOWST™/VOS) for prevention of recurrent Clostridioides difficile infection.

Clostridioides difficile infection (CDI) is a health crisis comprising a majority of healthcare-associated infections and is now being seen in the community. Persistent dysbiosis despite treatment with standard-of-care antibiotics increases risk of recurrent infections. Fecal microbiota transplantation has been an effective way of addressing dysbiosis, but the studies have lacked standardization, which makes outcome and safety data difficult to interpret. Standardized microbiome therapies have demonstrated efficacy and safety for recurrent CDI and have been approved to prevent recurrent infection. In this review, we discuss the data behind and the practice use of fecal microbiota spores, live-brpk (VOWST™ / VOS), a US FDA approved live biotherapeutic for the prevention of recurrent CDI.

Clostridioides difficile infection (CDI) is a serious infection of the gut. It often happens in hospitals and can come back after treatment. Antibiotics are usually used to treat CDI, but they do not always stop it from returning. Fecal microbiota transplantation (FMT), where healthy bacteria from the stool of a healthy donor are transferred to a patient, can work better than antibiotics alone. FMT is not yet approved for use because of the differences in donor screening, fecal material preparation and storage. Because of this, new treatments called standardized microbiome therapies are being developed. One such treatment, VOS, comes as an ingestible capsule containing live bacteria to colonize the gut with healthy bacteria that stop Clostridioides difficile from growing. It is a simple and easy treatment, though may have some mild side effects like gas or stomach pain. This treatment offers a new way to prevent CDI, especially for people at high risk. More research is needed to see how safe VOS is for pregnant women and people with weakened immune systems.

Next generation probiotics for human health: An emerging perspective.

Over recent years, the scientific community has acknowledged the crucial role of certain microbial strains inhabiting the intestinal ecosystem in promoting human health, and participating in various beneficial functions for the host. These microorganisms are now referred to as next-generation probiotics and are currently considered as biotherapeutic products and food or nutraceutical supplements. However, the majority of next-generation probiotic candidates pose nutritional demands and exhibit high sensitivity towards aerobic conditions, leading to numerous technological hurdles in large-scale production. This underscores the need for the development of suitable delivery systems capable of enhancing the viability and functionality of these probiotic strains. Currently, potential candidates for next generation probiotics (NGP) are being sought among gut bacteria linked to health, which include strains from the genera Bacteroids, Faecalibacterium, Akkermansia and Clostridium. In contrast to Lactobacillus spp. and Bifidobacterium spp., NGP, particularly Bacteroids spp. and Clostridium spp., appear to exhibit greater ambiguity regarding their potential to induce infectious diseases. The present review provides a comprehensive overview of NGPs in terms of their health beneficial effects, regulation framework and risk assessment targeting relevant criteria for commercialization in food and pharmaceutical markets.

Microbiota during pregnancy and early life: role in maternal-neonatal outcomes based on human evidence.

Here, we explored the vast potential of microbiome-based interventions in preventing and managing non-communicable diseases including obesity, diabetes, allergies, celiac disease, inflammatory bowel diseases, malnutrition, and cardiovascular diseases across different life stages. We discuss the intricate relationship between microbiome and non-communicable diseases, emphasizing on the "window of opportunity" for microbe-host interactions during the first years after birth. Specific biotics and also live biotherapeutics including fecal microbiota transplantation emerge as pivotal tools for precision medicine, acknowledging the "one size doesn't' fit all" aspect. Challenges in implementation underscore the need for advanced technologies, scientific transparency, and public engagement. Future perspectives advocate for understanding maternal-neonatal microbiome, exploring the maternal exposome and delving into human milk's role in the establishment and restoration of the infant microbiome and its influence over health and disease. An integrated scientific approach, employing multi-omics and accounting for inter-individual variance in microbiome composition and function appears central to unleash the full potential of early-life microbiome interventions in revolutionizing healthcare.

Feasibility of engineered Bacillus subtilis for use as a microbiome-based topical drug delivery platform.

Non-adherence to medication is a major challenge in healthcare that results in worsened treatment outcomes for patients. Reducing the frequency of required administrations could improve adherence but is challenging for topical drug delivery due to the generally short residence time of topical formulations on the skin. In this study, we sought to determine the feasibility of developing a microbiome-based, long-acting, topical delivery platform using Bacillus subtilis for drug production and delivery on the skin, which was assessed using green fluorescent protein as a model heterologous protein for delivery. We developed a computational model of bacteria population dynamics on the skin and used its qualitative predictions to guide experimental design choices. Using an ex vivo pig skin model and a human skin tissue culture model, we saw persistence of delivered bacteria for multiple days and observed little evidence of cytotoxicity to human keratinocyte cells in vitro. Finally, using an in vivo mouse model, we found that the delivered bacteria persisted on the skin for at least 1 day during every-other-day application and did not appear to present safety concerns. Taken together, our results support the feasibility of using engineered B. subtilis for topical drug delivery.

Next generation probiotics: Engineering live biotherapeutics.

The population dynamics of the human microbiome have been associated with inflammatory bowel disease, cancer, obesity, autoimmune diseases, and many other human disease states. An emerging paradigm in treatment is the administration of live engineered organisms, also called next-generation probiotics. However, the efficacy of these microbial therapies can be limited by the organism's overall performance in the harsh and nutrient-limited environment of the gut. In this review, we summarize the current state of the art use of bacterial and yeast strains as probiotics, highlight the recent development of genetic tools for engineering new therapeutic functions in these organisms, and report on the latest therapeutic applications of engineered probiotics, including recent clinical trials. We also discuss the supplementation of prebiotics as a method of manipulating the microbiome and improving the overall performance of engineered live biotherapeutics.

Positioning the preventive potential of microbiome treatments for cystic fibrosis in the context of current therapies.

Antibiotics and cystic fibrosis transmembrane conductance regulator (CFTR) modulators play a pivotal role in cystic fibrosis (CF) treatment, but both have limitations. Antibiotics are linked to antibiotic resistance and disruption of the airway microbiome, while CFTR modulators are not widely accessible, and structural lung damage and pathogen overgrowth still occur. Complementary strategies that can beneficially modulate the airway microbiome in a preventive way are highly needed. This could be mediated via oral probiotics, which have shown some improvement of lung function and reduction of airway infections and exacerbations, as a cost-effective approach. However, recent data suggest that specific and locally administered probiotics in the respiratory tract might be a more targeted approach to prevent pathogen outgrowth in the lower airways. This review aims to summarize the current knowledge on the CF airway microbiome and possibilities of microbiome treatments to prevent bacterial and/or viral infections and position them in the context of current CF therapies.

Novel Signal Peptides and Episomal Plasmid System for Enhanced Protein Secretion in Engineered Bacteroides Species.

The genus Bacteroides, a predominant group in the human gut microbiome, presents significant potential for microbiome engineering and the development of live biotherapeutics aimed at treating gut diseases. Despite its promising capabilities, tools for effectively engineering Bacteroides species have been limited. In our study, we have made a breakthrough by identifying novel signal peptides in Bacteroides thetaiotaomicron and Akkermansia muciniphila. These peptides facilitate efficient protein transport across cellular membranes in Bacteroides, a critical step for therapeutic applications. Additionally, we have developed an advanced episomal plasmid system. This system demonstrates superior protein secretion capabilities compared to traditional chromosomal integration plasmids, making it a vital tool for enhancing the delivery of therapeutic proteins in Bacteroides species. Initially, the stability of this episomal plasmid posed a challenge; however, we have overcome this by incorporating an essential gene-based selection system. This novel strategy not only ensures plasmid stability but also aligns with the growing need for antibiotic-free selection methods in clinical settings. Our work, therefore, not only provides a more robust secretion system for Bacteroides but also sets a new standard for the development of live biotherapeutics.

Synthetic Communities of Gut Microbes for Basic Research and Translational Approaches in Animal Health and Nutrition.

Microbes and animals have a symbiotic relationship that greatly influences nutrient uptake and animal health. This relationship can be studied using selections of microbes termed synthetic communities, or SynComs. SynComs are used in many different animal hosts, including agricultural animals, to investigate microbial interactions with nutrients and how these affect animal health. The most common host focuses for SynComs are currently mouse and human, from basic mechanistic research through to translational disease models and live biotherapeutic products (LBPs) as treatments. We discuss SynComs used in basic research models and findings that relate to human and animal health and nutrition. Translational use cases of SynComs are discussed, followed by LBPs, especially within the context of agriculture. SynComs still face challenges, such as standardization for reproducibility and contamination risks. However, the future of SynComs is hopeful, especially in the areas of genome-guided SynCom design and custom SynCom-based treatments.

Bifidobacterium mechanisms of immune modulation and tolerance.

Bifidobacterium is a widely distributed commensal bacterial genus that displays beneficial pro-homeostatic and anti-inflammatory immunomodulatory properties. Depletion or absence of Bifidobacterium in humans and model organisms is associated with autoimmune responses and impaired immune homeostasis. At the cellular level, Bifidobacterium upregulates suppressive regulatory T cells, maintains intestinal barrier function, modulates dendritic cell and macrophage activity, and dampens intestinal Th2 and Th17 programs. While there has been a large volume of literature characterizing the probiotic properties of various Bifidobacterial species, the likely multifactorial mechanisms underlying these effects remain elusive, in particular, its immune tolerogenic effect. However, recent work has shed light on Bifidobacterium surface structural polysaccharide and protein elements, as well as its metabolic products, as commensal mediators of immune homeostasis. This review aims to discuss several mechanisms Bifidobacterium utilizes for immune modulation as well as their indirect impact on the regulation of gut microbiome structure and function, from structural molecules to produced metabolites. These mechanisms are pertinent to an increasingly networked understanding of immune tolerance and homeostasis in health and disease.

Systems and synthetic biology-driven engineering of live bacterial therapeutics.

The past decade has seen growing interest in bacterial engineering for therapeutically relevant applications. While early efforts focused on repurposing genetically tractable model strains, such as Escherichia coli, engineering gut commensals is gaining traction owing to their innate capacity to survive and stably propagate in the intestine for an extended duration. Although limited genetic tractability has been a major roadblock, recent advances in systems and synthetic biology have unlocked our ability to effectively harness native gut commensals for therapeutic and diagnostic purposes, ranging from the rational design of synthetic microbial consortia to the construction of synthetic cells that execute "sense-and-respond" logic operations that allow real-time detection and therapeutic payload delivery in response to specific signals in the intestine. In this review, we outline the current progress and latest updates on microbial therapeutics, with particular emphasis on gut commensal engineering driven by synthetic biology and systems understanding of their molecular phenotypes. Finally, the challenges and prospects of engineering gut commensals for therapeutic applications are discussed.

Drug and gut microbe relationships: Moving beyond antibiotics.

Our understanding of drug-microbe relationships has evolved from viewing microbes as mere drug producers to a dynamic, modifiable system where they can serve as drugs or targets of precision pharmacology. This review highlights recent findings on the gut microbiome, particularly focusing on four aspects of research: (i) drugs for bugs, covering recent strategies for targeting gut pathogens; (ii) bugs as drugs, including probiotics; (iii) drugs from bugs, including postbiotics; and (iv) bugs and drugs, discussing additional types of drug-microbe interactions. This review provides a perspective on future translational research, including efficient companion diagnostics in pharmaceutical interventions.

Targeting microbial quorum sensing: the next frontier to hinder bacterial driven gastrointestinal infections.

Bacteria synchronize social behaviors via a cell-cell communication and interaction mechanism termed as quorum sensing (QS). QS has been extensively studied in monocultures and proved to be intensively involved in bacterial virulence and infection. Despite the role QS plays in pathogens during laboratory engineered infections has been proved, the potential functions of QS related to pathogenesis in context of microbial consortia remain poorly understood. In this review, we summarize the basic molecular mechanisms of QS, primarily focusing on pathogenic microbes driving gastrointestinal (GI) infections. We further discuss how GI pathogens disequilibrate the homeostasis of the indigenous microbial consortia, rebuild a realm dominated by pathogens, and interact with host under worsening infectious conditions via pathogen-biased QS signaling. Additionally, we present recent applications and main challenges of manipulating QS network in microbial consortia with the goal of better understanding GI bacterial sociality and facilitating novel therapies targeting bacterial infections.

Clostridioides difficile Infection: Landscape and Microbiome Therapeutics.

Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and is common in the community. Both younger individuals who may be healthy otherwise and older individuals with comorbid conditions are at risk for developing CDI, with the predominant risk factor being antibiotic use. Unlike other gastrointestinal infections, CDI is not self-limited, requires antimicrobial therapy, and tends to recur at high rates even without additional risk factor exposure. The goals of CDI management include controlling active symptoms and using a recurrence prevention strategy such as a narrow-spectrum antibiotic, tapered and pulsed regimens, antibody- based therapies (directed against toxin B), or microbiome restoration. In recent years, fecal microbiota transplantation (FMT) has been the most used modality to prevent recurrent CDI with high cure rates. Heterogeneity, lack of scalability, and serious adverse events from FMT have led to development of standardized microbiota restoration therapies (MRTs). The US Food and Drug Administration has approved 2 stool-derived MRTs for prevention of recurrent CDI: fecal microbiota, live-jslm, an enema-based therapy; and fecal microbiota spores, live-brpk, an oral therapy. A phase 3 trial for a synthetic oral MRT is underway. This article outlines the pathophysiology and treatment of CDI, focusing primarily on the gut microbiome and standardized MRTs.

Recombinant Lactococcus lactis Expressing Human LL-37 Prevents Deaths from Viral Infections in Piglets and Chicken.

Novel antibiotic substitutes are increasingly in demand in the animal husbandry industry. An oral recombinant Lactococcus lactis (L. lactis) expressing human LL-37 (oral LL-37) was developed and its safety and antiviral effectiveness in vivo was tested. In addition to impairing liposome integrity, LL-37 polypeptide from recombinant L. lactis could prevent the host cell infection by a variety of viruses, including recombinant SARS, SARS-CoV-2, Ebola virus, and vesicular stomatitis virus G. Subchronic toxicity studies performed on Sprague-Dawley rats showed that no cumulative toxicity was found during short-term intervention. Oral LL-37 treatment after the onset of fever could reduce mortality in piglets infected with porcine reproductive and respiratory syndrome virus. Moreover, body weight gain of piglets receiving treatment was progressively restored, and nucleic acid positive rebound was not undetected after discontinuation. Oral LL-37 consistently increased the lifespan of chickens infected with Newcastle viruses. These findings suggested a potential use of recombinantly modified microorganisms in veterinary medicine.

Engineered probiotics as live biotherapeutics for diagnosis and treatment of human diseases.

The use of probiotics to regulate the intestinal microbiota to prevent and treat a large number of disorders and diseases has been an international research hotspot. Although conventional probiotics have a certain regulatory role in nutrient metabolism, inhibiting pathogens, inducing immune regulation, and maintaining intestinal epithelial barrier function, they are unable to treat certain diseases. In recent years, aided by the continuous development of synthetic biology, engineering probiotics with desired characteristics and functionalities to benefit human health has made significant progress. In this article, we summarise the mechanism of action of conventional probiotics and their limitations and highlight the latest developments in the design and construction of probiotics as living diagnostics and therapeutics for the detection and treatment of a series of diseases, including pathogen infections, cancer, intestinal inflammation, metabolic disorders, vaccine delivery, cognitive health, and fatty liver. Besides we discuss the concerns regarding engineered probiotics and corresponding countermeasures and outline the desired features in the future development of engineered live biotherapeutics.

Bacterial expression of a designed single-chain IL-10 prevents severe lung inflammation.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is active as a swapped domain dimer and is used in bacterial therapy of gut inflammation. IL-10 can be used as treatment of a wide range of pulmonary diseases. Here we have developed a non-pathogenic chassis (CV8) of the human lung bacterium Mycoplasma pneumoniae (MPN) to treat lung diseases. We find that IL-10 expression by MPN has a limited impact on the lung inflammatory response in mice. To solve these issues, we rationally designed a single-chain IL-10 (SC-IL10) with or without surface mutations, using our protein design software (ModelX and FoldX). As compared to the IL-10 WT, the designed SC-IL10 molecules increase the effective expression in MPN four-fold, and the activity in mouse and human cell lines between 10 and 60 times, depending on the cell line. The SC-IL10 molecules expressed in the mouse lung by CV8 in vivo have a powerful anti-inflammatory effect on Pseudomonas aeruginosa lung infection. This rational design strategy could be used to other molecules with immunomodulatory properties used in bacterial therapy.

Lachnospiraceae are emerging industrial biocatalysts and biotherapeutics.

The Lachnospiraceae is a family of anaerobic bacteria in the class Clostridia with potential to advance the bio-economy and intestinal therapeutics. Some species of Lachnospiraceae metabolize abundant, low-cost feedstocks such as lignocellulose and carbon dioxide into value-added chemicals. Others are among the dominant species of the human colon and animal rumen, where they ferment dietary fiber to promote healthy gut and immune function. Here, we summarize recent studies of the physiology, cultivation, and genetics of Lachnospiraceae, highlighting their wide substrate utilization and metabolic products with industrial applications. We examine studies of these bacteria as Live Biotherapeutic Products (LBPs), focusing on in vivo disease models and clinical studies using them to treat infection, inflammation, metabolic syndrome, and cancer. We discuss key research areas including elucidation of intra-specific diversity and genetic modification of candidate strains that will facilitate the exploitation of Lachnospiraceae in industry and medicine.

Considerations for Choosing Soluble Immune Markers to Determine Safety of Novel Vaginal Products.

Several soluble cytokines have been associated with microbicide-induced cervicovaginal inflammation, non-optimal vaginal microbiota, and risk of HIV acquisition. Many of these biomarkers are used in preclinical assays to estimate the safety of vaginally applied products. However, there are currently no validated biomarkers to evaluate the safety of novel vaginal products in clinical trials. This hinders the rapid and rational selection of novel products being tested in first-in-human trials. We reviewed available literature to assess how best to select and measure soluble immune markers to determine product safety in first in human clinical trials of novel vaginal products.

Microcin MccI47 selectively inhibits enteric bacteria and reduces carbapenem-resistant Klebsiella pneumoniae colonization in vivo when administered via an engineered live biotherapeutic.

The gastrointestinal (GI) tract is the reservoir for multidrug resistant (MDR) pathogens, specifically carbapenem-resistant (CR) Klebsiella pneumoniae and other Enterobacteriaceae, which often lead to the spread of antimicrobial resistance genes, severe extraintestinal infections, and lethal outcomes. Selective GI decolonization has been proposed as a new strategy for preventing transmission to other body sites and minimizing spreading to susceptible individuals. Here, we purify the to-date uncharacterized class IIb microcin I47 (MccI47) and demonstrate potent inhibition of numerous Enterobacteriaceae, including multidrug-resistant clinical isolates, in vitro at concentrations resembling those of commonly prescribed antibiotics. We then genetically modify the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to produce MccI47 from a stable multicopy plasmid by using MccI47 toxin production in a counterselection mechanism to engineer one of the native EcN plasmids, which renders provisions for inducible expression and plasmid selection unnecessary. We then test the clinical relevance of the MccI47-producing engineered EcN in a murine CR K. pneumoniae colonization model and demonstrate significant MccI47-dependent reduction of CR K. pneumoniae abundance after seven days of daily oral live biotherapeutic administration without disruption of the resident microbiota. This study provides the first demonstration of MccI47 as a potent antimicrobial against certain Enterobacteriaceae, and its ability to significantly reduce the abundance of CR K. pneumoniae in a preclinical animal model, when delivered from an engineered live biotherapeutic product. This study serves as the foundational step toward the use of engineered live biotherapeutic products aimed at the selective removal of MDR pathogens from the GI tract.