Modulating the Microbiome for Disease Treatment

Abstract

The central role of gut microbiota in the regulation of health and disease has been convincingly demonstrated. Polymicrobial inter-kingdom interactions between bacteria (the bacteriome) and fungal (the mycobiome) communities of the gut have become a prominent focus for development of potential therapeutic approaches. In addition to polymicrobial interactions, the complex gut ecosystem also mediates interactions between host and the microbiota. These interactions are complex and bidirectional, microbiota composition can be influenced by host immune response, disease-specific therapeutics, antimicrobial drugs, and overall ecosystems. However, the gut microbiota also influences host immune response to a drug or therapy by potentially transforming the drug’s structure and altering bioavailability, activity or toxicity, this is especially true in cases where the gut microbiota has produced a biofilm. The negative ramifications of biofilm formation include alteration of gut permeability, enhanced antimicrobial resistance, and alteration of host immune response effectiveness. Natural modulation of the gut microbiota, using pro- and pre-biotic approaches may also be used to affect the host microbiome, a type of “natural” modulation of the host microbiota composition. In this review we discuss potential bidirectional interactions between microbes and host, describe the changes in gut microbiota induced by probiotic and prebiotic approaches, and their potential clinical consequences and summarize how to develop a systematic approach to designing probiotics capable of altering the host microbiota in disease states, using Crohn’s Disease (CD) as a model chronic disease. Understanding how the effective changes in the microbiome may enhance treatment efficacy may unlock the possibility of modulating the gut microbiome to improve treatment using a natural approach.

A. Introduction:

Microbiome research examines the genetic material from all microorganisms (microbiota) within a specific niche, such as the human gut. This research area has massively expanded over the past decade empowered by the development of new analytic methodologies and bioinformatics which led to a better understanding of the association between pathogenesis of various diseases with changes in the composition of the microbiome, especially where an imbalance (dysbiosis) from healthy microbiome composition is evident. Several studies have illustrated the important role the microbiome plays in the development of diseases such as inflammatory bowel diseases, rheumatoid arthritis, depression, and cancer. ^1–10 Early in microbiome research, it was thought that the bacteriome (bacterial community), which represent a significant portion of the human microbiome, was the main (only) culprit behind physiological changes associated with the microbiota. However, researchers recently started to appreciate that effective management of microbiome-related diseases calls for a more comprehensive understanding of the impact of the total microbiota on host health and disease, which requires insight into intra- and cross-kingdom interactions among major members of the microbiome community including bacteria (bacteriome), fungal (mycobiome), and viral (virome) constituents, as well as potential microbial-host interactions ^11–13.

Defining detailed microbiome profiles in various health conditions ushers a path toward a new phase of microbiome research, focused on developing new therapeutic strategies to manage these conditions. Several pathways may be targeted to achieve this objective including developing nutritional supplements or biotherapeutic agents, both of which aim to address the underlying dysbiosis in a disease process with an ultimate goal of restoring and maintaining the microbial balance. Creating targeted therapeutics requires a carefully designed scientific approach to ensure the development of safe and effective new agents.

In this review, we will discuss potential tactics that can be used to develop probiotics to modulate the microbiome as a potential tool in the management of diseases/conditions. To illustrate these tactics, we will use Crohn’s disease (CD) as an example.

A.1. Crohn’s Disease

Crohn’s Disease (CD) is an autoinflammatory disorder of the digestive tract that currently affects approximately 500,000 Americans, with a prevalence of 100 to 300 per 100,000 individuals. Symptoms may include chronic diarrhea, abdominal pain, fever, weight loss, anal fissures, anal fistulas, and rectal bleeding. ¹⁴ CD can affect any part of the digestive tract with ileitis being the most common presentation. ¹⁴

Several factors may contribute to the development of CD including genetic predisposition. Between 5–20% of CD patients have a direct relative with the disorder, and an increased odds ratio is observed among Ashkenazi Jews wherein genome assessments have identified 71 loci on 17 chromosomes that contribute to disease manifestation. ^{15, 16} In addition to genetic contribution, other factors including host microbiome alteration, environmental influences, medications, and diet have all been reported to contribute significantly to disease development. ^17–21

Currently, the main CD course of treatment is based on modulating the production of local chemical mediators and the immune response to reduce inflammatory reactions. ^{22, 23} Treatment options include oral 5-aminosalicylates (e.g., sulfasalazine), glucocorticoids (e.g., prednisone), immunomodulators (e.g., azathioprine, 6-mercaptopurine, methotrexate), and biologic therapies (e.g., infliximab, adalimumab, certolizumab pegol, and ustekinumab). ²⁴ Although these treatments have been shown to effectively manage disease, they are associated with several side-effects such as nausea, headache, rash, male infertility, increased risk of infection, cytopenia, hepatitis, pneumonitis, neutropenia, pancreatitis, acute myocardial infarction, and agranulocytosis. ²⁵ Several studies have shown that among the reasons for medication non-adherence are the adverse side-effects caused by these drugs, and the need for more frequent agents or doses (>3–4 doses per day) in some cases. ^26–31 Initial analysis of early treatment regimens revealed that some of these treatments disrupted the host gut microbiome, an occurrence linked to increased pathogenesis of Crohn’s disease. ^{32, 33} Thus, introducing an adjuvant/supporting therapy that can modulate the gut microbiota and/or prevent gut microbiome dysbiosis may be a novel approach to supporting the management of CD. Not only does this approach have the potential of restoring the microbiome balance, but it may also help achieve better response to the currently utilized medications. ^34–36

A.2. Using the microbiome as a treatment target

Several studies have identified microbial influences on the development of Crohn’s Disease (CD). For example, Mycobacteria spp. appear to be elevated in those with CD compared to healthy controls, and viral infections have also been pinpointed as contributing to disease manifestation. ³⁰ Crohn’s patients also have elevated Bacteroidetes and Escherichia coli colonization and decreased Firmicutes spp. and Faecalibacterium prausnitzii (an anti-inflammatory bacteria) compared to the microbiome of healthy, non-CD subjects. ³¹ Causes of dysbiosis are not simply due to bacterial interspecies interactions, but rather a whole network of polymicrobial interactions, including fungal (mycobiome) interactions as well.

Due to the recently detailed characterization of a healthy microbiome profile, researchers have been able to identify differences in the microbiome of CD patients, and to develop new methods of disease identification based on microbiome profiling. For example, Saccharomyces cerevisiae has a relatively high abundance in healthy individuals, but may be severely decreased in CD patients. ³⁷ S. cerevisiae may also be used as a marker of inappropriate immune response. Anti-S. cerevisiae antibodies (ASCA) have been reported to be elevated in CD patients and used as a biomarker of Crohn’s disease. ^{31 38}

Recently, we demonstrated that compared to healthy relatives, CD patients had a higher abundance of the fungal pathogen Candida tropicalis, correlating with an increase of the bacterial species Serratia marcescens and Escherichia coli. Additionally, these three species were demonstrated to form a thick biofilm, with C. tropicalis interweaving its hyphal network of filaments, E. coli fusing to the fungal cells within the biofilm, and S. marcescens using its fimbriae to attach and aggregate the polymicrobial interaction (Figures 1–3). ³⁹ This study exhibits a clear, mutually cooperative relationship between pathogenic bacteria and fungi within the microbiome, indicating that various genera cross-talk within the host. To demonstrate this cross talk, we performed a metabolomic analysis of the single-species and triple-species cultures of the three pathogens and identified 15 metabolites that were highly increased in the triple-species culture, including the highly induced metabolite indole-3-acetic acid (IAA). This metabolite, that was previously shown to induce filamentation of certain fungi, promoted biofilm formation of C. tropicalis. ⁴⁰

Figure 1.

Confocal analysis of biofilms formed by C. tropicalis (CT) alone or in combination with E. coli (EC) and/or S. marcescens (SM). (A) Side view of biofilms formed by C. tropicalis plus E. coli plus S. marcescens, C. tropicalis plus S. marcescens, C. tropicalis plus E. coli, C. tropicalis alone, S. marcescens alone, or E. coli alone. (B) Mean thickness of biofilms. Reproduced from- Bacteriome and Mycobiome Interactions Underscore Microbial Dysbiosis in Familial Crohn’s Disease. mBio. 2016 Sep 20;7(5):e01250–16. doi: 10.1128/mBio.01250-16. PMID: 27651359; PMCID: PMC5030358.

Figure 3.

Transmission electron microscopy analyses of biofilms formed by C. tropicalis (CT) alone or in combination with E. coli (EC) and/or S. marcescens (SM). (A) C. tropicalis plus E. coli (bar, 0.5 μm); (B) C. tropicalis plus S. marcescens (bar, 500 nm); (C) C. tropicalis plus E. coli plus S. marcescens (bar, 0.5 μm); (D) C. tropicalis plus E. coli plus S. marcescens (bar, 200 nm). Reproduced from- Bacteriome and Mycobiome Interactions Underscore Microbial Dysbiosis in Familial Crohn’s Disease. mBio. 2016 Sep 20;7(5):e01250–16. doi: 10.1128/mBio.01250-16. PMID: 27651359; PMCID: PMC5030358.

Current research regarding CD treatment advocates for targeting the root microbial causes of the disorder, which may also help alleviate symptoms caused by a patient’s inflammatory response. This process involves modulating the microbial environment which can be done via administration of nutritional supplements (e.g., probiotics, prebiotics, or microbial metabolites), fecal transplants or biotherapeutics. ⁴¹ In this review, we focus on the use of probiotics as a potential mechanism for modulating the host gut microbiome, for a review of fecal microbiome transplant (FMT) or biotherapeutic treatment of CD the readers are referred to comprehensive reviews addressing these areas ^42–49, and ^{22, 50–53}, respectively.

B. The Biofilm Effect

Microbiota describes a complex society that includes bacteria, fungi, archaea and viruses, capable of forming polymicrobial or mixed-species biofilm communities comprised of the organisms themselves and their associated matrices. Formation of this matrix provides these communities with a distinct physiologic advantage during colonization and infection. Several investigators described mixed fungal–bacterial biofilms that often resulted in severe infections, especially on indwelling catheters, ^54–56 highlighting the coexistence of different microbial species, and their interdependence in biofilm formation. The formation of biofilms was not restricted to accumulation on foreign bodies, as numerous publications demonstrated the formation of naturally occurring biofilms in the mouth, in the gut, and on the skin. Indeed, the use of ‘biofilm’ to describe microbial aggregates on surfaces was reported by Costerton et al. over 50 years ago ⁵⁷, however, the term was also used in technical and environmental microbiology reports as early as 1935. ^{58, 59}

Although biofilms are complex microbial ecosystems, they can vary greatly in structure and composition from one micro-environmental niche to another. In addition to the organisms themselves, and their extracellular polymeric substances (EPS), primarily complex polysaccharide material, the matrix may also contain non-cellular materials such as mineral or organic particles. ⁶⁰ In terms of intestinal (gut) biofilms the main composition is aggregates of microorganisms that are embedded in a biopolymer matrix composed of host and microbial compounds that adhere to epithelial cells.

Abnormal and deleterious biofilms in contact with mucosal tissues have long been associated with human diseases, including some intestinal diseases. Despite these observations, biofilms also stabilize the gut by providing colonization resistance, phenotypic stability, elicitation of host defense and improved digestion of food and potential response to drug interactions. ⁶¹ The beneficial aspects of biofilms are still being explored and integrated into our current understanding of the gut microbiome.

Inflammatory Bowel Disease (IBD) patients exhibit microbiota growing in biofilms that adhere to the intestinal lining epithelium. ⁶² Cultivated bacteria obtained from CD or Ulcerative Colitis (UC) patients have been demonstrated to colonize and penetrate intestinal epithelia cells and elicit proinflammatory response signals. ⁶³ This response and the formation of the biofilm itself have been postulated to potentiate disease pathogenesis. ⁶⁴ Furthermore, organisms growing in a biofilm milieu are also protected from antimicrobials and attack from host immune response, thus resistance of endogenous organisms (even in dysbiosis) is enhanced by living within a biofilm microenvironment. ^65–68

In addition to bacteria, our work demonstrated an increase in fungal colonization of the gut of CD patients. ³⁹ Beyond increased fungal colonization, we further demonstrated that E. coli and S. marcesens exhibit increased abundance in CD patients, are positively correlated with C. tropicalis, and the combination of C. tropicalis, E. coli and S. marcesens were capable of forming robust biofilms in vitro and in vivo. ⁶⁹

One of the known bacterial pathogens associated with CD and biofilms is Adherent-invasive Escherichia coli (AIEC), a pathogen that has been implicated in the origin and exacerbation of CD. ⁷⁰ Recent reports have demonstrated the adaptation of AIEC to biofilm formation and the potential advantage of AIEC within biofilms of CD patients. ^{71, 72} Our in vivo modeling of polymicrobial biofilms using a dextran sulfate sodium (DSS) induced colitis model (Figure 4, 5) demonstrates that the combination of C. tropicalis, E. coli and S. marcesens results in the formation of a robust biofilm that show the intimate association of bacterial organisms with the host intestinal epithelium, mimicking what was observed in vitro with E. coli juxtaposed with fungal and host elements (Figure 3). This observation suggests that in addition to bacterial biofilms, the microbiota associated with the gut of CD patients may also thrive in a biofilm matrix produced by polymicrobial mixtures that include fungal microorganisms, and the bacterial-fungal association may be a key element in CD biofilms.

Figure 4.

Scanning electron microscopy analyses of biofilms formed in the intestinal epithelial of mice treated with either A: DSS alone or B: DSS in the presence of C. tropicalis combined with E. coli and S. marcescens introduced by gavage for 4 weeks. C: Transmission electron microscopy analyses of biofilms formed by C. tropicalis (CT) combined with E. coli and S. marcescens in the intestinal lumen of DSS treated mice.

Figure 5.

Biofilms formed by combining CT+EC+SM in vivo on murine intestine. Accumulation of the organisms of (A) Untreated and (B) treated with CT+EC+SM, demonstrate that biofilms could be formed by combining the organism.

Given this information, the next logical steps to alter the microbiota in CD patients are: 1. Modulate the microbiota, and 2. Interfere with biofilm formation. Thus, we have a two tier approach: 1) rebalance the microbiome and 2) eliminate the biofilm. This should result in rebalancing the microbiota, and reversing the formation of biofilms, with the ultimate aim of protecting the gut lining and reducing the associated gastrointestinal symptoms that exacerbate CD. We will discuss our approach in the following sections.

C. Pre- and Probiotics

Probiotics were originally defined in 2001 by a panel of experts assembled by the Food and Agricultural Organization of the United Nations and the World Health Organization (FAO/WHO) and published as guidelines for the Evaluation of Probiotics in Foods. ⁷³ In 2013, the International Scientific Association for Probiotics and Prebiotics (ISAPP) convened an Expert Panel to review the term probiotic, resulting in a publication that defined Probiotics as: “Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” ⁷⁴ Thus, probiotics are foods or supplements that contain live microorganisms intended to maintain or improve the “good/beneficial” bacteria (normal microflora) in the body.

In the same manner, the ISAPP also convened a panel of experts in microbiology, nutrition, and clinical research in 2016 to define prebiotics. The current consensus definition is: “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. ³⁷ Thus prebiotics are foods (typically high-fiber foods) that act as food for human microbiota.

Several studies have identified beneficial probiotic strains that are reduced in CD. Therefore, replenishing these organisms may help in reducing disease severity and mucosal damage. ^75–77 Replenishment could be achieved by direct administration of beneficial organisms (i.e., probiotics), administration of dietary fibers that enhance their growth (prebiotics), or a combination of both (synbiotics). ^78–84 Probiotics were shown to exhibit their effects in inflammatory bowel disease (IBD) animal models and in humans via several mechanisms including; downregulation of genes and interfering with signaling pathways responsible for production of proinflammatory cytokines, increased production of proteins necessary for epithelial integrity, and reduced pathogen growth and colonization. ^{76, 77, 85–88}

In terms of prebiotics, the benefit of using this option is that they are easily accessible, can be found in natural sources, and their use does not carry side effects and risks associated with antibiotics. ⁸⁹ Studies have shown that prebiotics increased the levels of Bifidobacterium and Lactobacillus, both of which are known to be beneficial organisms that are reduced in CD patients, using preclinical models. ^78–81 Administration of oligofructose-enriched inulin was shown to modulate the microbiome with increases in the level of Bifidobacterium longum, which positively correlated with reduction in disease severity, and increased butyrate and other short chain fatty acid (SFCA) levels which is reported to have anti-inflammatory effects. ^90–93

Furthermore, in a recent study, a prebiotic derived from grapes was shown to increase butyrate and SCFA-producing bacteria both of which are known for their mucosal protective roles by enhancing mucus production, promoting epithelial integrity, and regulating inflammatory activity. ^{94, 95} With that being said, one growing area of research is to analyze metabolites and biochemicals secreted by various members of the microbiome and determine the potential of these products as novel therapeutic options. ^{96, 97}

Using metabolomics and sequencing, nutrients and metabolites that are decreased or missing due to dysbiosis can be identified and provided to patients as supplements. ⁹⁸ For example, SCFAs, which have many beneficial effects, are produced by microbes that have been reported to be reduced in CD patients. ^{99, 100} Supplementing patients with prebiotics or probiotics may enhance the production of SCFAs; other options can be to directly administer the final product (i.e. a metabolite). Implementing the same concept to other microbial components or products, and conducting further analysis, may lead to the production of new drugs in addition to advanced nutritional supplements.

D. Modulating the Microbiome using Probiotics-An Example Approach

Microbiota dysbiosis has been implicated in a large number of diseases and conditions, therefore, the first step in modulating the microbiota profile is to identify a disease entity (e.g. colorectal cancer, irritable bowel disease, autism etc.) that is a viable candidate for supplementation. The next step is to use bioinformatic analyses to compare the microbiome profile of potential patients with matched (e.g., age, sex, race) healthy controls. This step helps to identify members of the microbial communities (both bacteria and fungi) that are statistically imbalanced (using abundance as an endpoint), and potentially responsible for dysbiosis. This analytic approach identifies whether the imbalance is due to a decrease in certain beneficial microorganism, an increase in pathogens, or combination thereof. Organisms that are either over- or under-represented comprise potential therapeutic target/s that can be the basis for microbiome-based therapy. The next step is to identify potential probiotic candidates that can restore microbiome balance with the ultimate goal of treating and ameliorating disease and associated symptoms. The final phase in this process is to conduct studies to evaluate the efficacy and safety of the potential therapeutic candidates using pre-clinical in vitro and in vivo models followed by clinical testing. Figure 6 summarizes the steps needed to drug the microbiome.

Figure 6.

Stages followed for developing a probiotic to drug the microbiome

E. Approach to Develop Microbiome-Based Modulation of the microbiota in Crohn’s Disease

Our strategy to modulating the microbiome in CD entailed following the general principles described above (Figure 6). Namely, 1. Use primary microbiome data to identify (by abundance) the microorganisms underlying dysbiosis, 2. Gain insight into the interactions between the identified pathogens, 3. Conduct correlation analysis to identify potential probiotic strains that antagonize these pathogens, as well as discover metabolites that can interrupt their interactions, 4. Validate the efficacy of the candidate formulation through preclinical in vitro and in vivo testing, and finally, clinical testing. Herein, we focus on the development of a probiotic to ameliorate CD, although the principles apply to the development of any targeted probiotic.

Phase 1: Identifying the dysbiosis

Several studies have reported that gut microbiome alteration plays an important role in the development of CD. ^{17, 18, 20, 21} Thus, following our approach, we sought to first identify the underlying dysbiosis in CD patients. To gain this insight, we used next generation sequencing (16S and ITS) to characterize the gut bacteriome (bacterial community) and the mycobiome (i.e. fungal community) in CD patients and their non-diseased first-degree relatives (NCDR) ³⁹. Fecal samples were collected from the participants, fungal and bacterial DNA were extracted, and next generation sequencing performed. ¹⁰¹ Principal component, diversity, and abundance analyses were conducted which revealed significant differences in the microbiome composition in CD patients compared to familial healthy controls. Assessing the abundance of different microorganisms in CD patients compared to the healthy controls revealed a reduction in Bacteroidetes phylum. Furthermore, Faecalibacterium prausnitzii, which has been reported to produce an anti-inflammatory effect, was also reduced. Additionally, we observed an increase in the levels of E. coli, S. marcescens and Ruminococcus gnavus, all of which have been reported to induce inflammation and mucus layer disruption. ^{100, 102–105} Importantly, an increase in the abundance of the fungus C. tropicalis in patients with CD compared to controls was also noted. Interestingly, further analysis revealed a positive correlation between the levels of C. tropicalis and E. coli and S. marcescens.

Phase 2: Understanding the microbial interaction between the identified dysbiotic microbes in CD patients

Investigations into the interactions of microorganisms within a given microbiome have shown that members of these communities interact and develop interactive microbial cooperative evolutionary strategies that often culminate in biofilm formation. In microbial dysbiosis, biofilms are beneficial to both bacterial and fungal communities, but detrimental to the host. Fungi benefit by a surge in their virulence factors, while both bacteria and fungi become tolerant to antimicrobial agents as a consequence of living in a biofilm setting in contrast to a planktonic one, where penetration of antimicrobials is blunted, and ability to circumvent immune cells also occurs ¹⁰⁶. The interkingdom cooperation observed in biofilms negatively impacts the host, as the fungi and bacteria produce extracellular enzymes and Candida undergoes morphological changes transforming from the yeast form to a hyphal one (hyphal structures are a known Candida virulence factor) that inflict epithelial tissue damage, leading to an increase in proinflammatory cytokines, which results in oxidative damage and apoptotic cell death.

Taking into consideration these strategies, a critical step in drugging the microbiome is to understand the relationship between these microbes and their role in the disease process. Because microbes in the intestine favor formation of biofilms ¹⁰⁷, we started our investigations by conducting in vitro testing of biofilm forming capacity of the three microorganisms identified in our analysis of CD patients versus healthy control family members. Furthermore, to test if these organisms interact with each other, we evaluated their ability to form biofilms alone and in combination with each other. In agreement with our hypothesis, using confocal and electron microscopy, we were able to confirm that the three dysbiotic microorganisms (C. tropicalis, E. coli and S. marcescens) interact and form biofilms that were higher in mass and thickness in combination polymicrobial biofilms (triplet compared to any of the species alone or in pairs). (Figure 1) Scanning electron microscopy and transmission electron microscopy analysis revealed that the two bacteria interact differently with C. tropicalis where E. coli appeared fused to the fungal cell wall, while S. marcescens interacted with E. coli and C. tropicalis using fimbria (Figures 2, 3). ³⁹ Further testing showed that this enhanced polymicrobial biofilm was Candida specific. Particularly, unlike C. tropicalis and C. albicans, the fungal species Trichosporon inkin and Saccharomyces fibuligera failed to form a thick polymicrobial biofilm with the two bacteria. ⁶⁹

Figure 2.

Scanning electron microscopy analyses of biofilms formed by C. tropicalis alone or in combination with E. coli and/or S. marcescens. (A) C. tropicalis plus E. coli (magnification, ×1,057); (B) C. tropicalis plus S. marcescens (magnification, ×1,000); (C) C. tropicalis plus E. coli plus S. marcescens (magnification, ×1,000); (D) C. tropicalis plus E. coli (magnification, ×5,000); (E) C. tropicalis plus S. marcescens (magnification, ×5,000); (F) C. tropicalis plus E. coli plus S. marcescens (magnification, ×5,000).

Reproduced from- Bacteriome and Mycobiome Interactions Underscore Microbial Dysbiosis in Familial Crohn’s Disease. mBio. 2016 Sep 20;7(5):e01250–16. doi: 10.1128/mBio.01250-16. PMID: 27651359; PMCID: PMC5030358.

The observation that C. tropicalis, E. coli and S. marcescens has the ability to form polymicrobial biofilms was confirmed using an in vivo preclinical model to mimic the human CD gut. In this experiment, biofilm formation was clearly evident in samples obtained from the colon of mice challenged with the combined three organisms compared to untreated controls (Figures 4, 5) (unpublished data). Based on the above in vitro and in vivo findings we hypothesized microbial dysbiosis in CD patients is caused by the increased abundance of C. tropicalis, E. coli and S. marcescens, and that biofilm formation is central to this process. The consequences of biofilms forming within the host gut has numerous implications for the host and the resident microbes. Biofilms change microbiota morphologically ^108–111, they produce metabolites ^{40, 112–115}, block nutrient absorption ^{60, 116}, enhance the resistance of the microorganisms, and damage the gut lining. ^{68, 117, 118} Consequences such as microbiota dysbiosis and biofilm formation need to be addressed in our modulation strategy for the microbiome.

Now that a target was identified, our objective was to discover beneficial microbial strains that could potentially interrupt and disrupt polymicrobial biofilms formed by these bacterial and fungal pathogens. To achieve this, we conducted bacterial-bacteria, fungal-fungal, and bacterial-fungal correlation analysis and selected four biotherapeutic strains: Bifidobacterium breve 19bx, Lactobacillus acidophilus 16axg, Lactobacillus rhamnosus 18fx, and Saccharomyces boulardii 16mxg. These strains were shown to have antibiofilm activity and to interfere with epithelial cell damage caused by C. tropicalis, E. coli and S. marcescens. ^{69, 119–125}. In addition to the selected microorganisms, we also added the enzyme amylase to the formulation to enhance biofilm abrogation. ^126–132

Phase 3: Validation of the selected therapeutic formulation

Modulation of the microbiome requires validation of the developed therapeutic formulation. This entails conducting preclinical (in vitro and in vivo testing) as well as clinical evaluation. The following is a description of the research and development process undertaken to validate the efficacy of the candidate formulation.

Phase 3a. Preclinical In vitro Evaluation

The first step in investigating any new therapies is to start with in vitro experiments in which the testing environment can be optimized and the activity and efficacy of the new agents can be evaluated accurately and rigorously. Moving forward, in vivo testing is the next stop, where the new agent can be tested in an environment similar to the human body. All of the accumulated preclinical data contributes to the design and conduct of clinical testing.

In our approach, we first tested the effect of the probiotic combination in prevention/treatment of polymicrobial biofilms using an in vitro model. Furthermore, since one of our targets is Candida, we also tested the effect of the probiotic combination on fungal cell germination. ⁶⁹ We also investigated whether addition of amylase, an enzyme that has been reported to disrupt biofilms, would improve the efficacy of the probiotic combination. ^{127, 130, 133} After growing the probiotic strains individually in broth, the cell pellets and cell supernatant of each organism was collected. Supernatants were filter sterilized and then combined in equal amounts. The probiotic supernatant combination was not only able to prevent the formation of biofilms but was also effective in disrupting mature biofilms. Addition of the probiotic filtrate as a preventive treatment resulted in a reduction in the biofilm matrix, thickness, and hyphal formation compared to untreated controls. Furthermore, examining the mature polymicrobial biofilm treatment with the filtrate showed complete absence of the extracellular matrix and lack of any structural biofilm matrix. Moreover, the filtrate caused a significant reduction in the percentage of C. albicans germ tube formation (an important Candida virulence factor) compared to untreated controls. ⁶⁹

Phase 3b. Preclinical Animal Models

Recently, we completed an in vivo pre-clinical study evaluating the effect of the probiotic-amylase combination (Bifidobacterium breve 19bx, Lactobacillus acidophilus 16axg, Lactobacillus rhamnosus 18fx, and Saccharomyces boulardii 16mxg plus amylase) using a spontaneous chronic CD like-Ileitis animal model (SAMP1/YitFc (SAMP)). Three groups of 7-week-old SAMP mice were compared using: 1) probiotic supplement (probiotic strains + amylase) diluted in sterile phosphate buffered saline (PBS) every day for 56 days through a gavage technique, 2) probiotic supplement (probiotic strains without amylase), and 3) control animals administered sterile phosphate buffered saline (PBS) alone. Following treatment, mice were sacrificed and ilea collected for histological scoring of ileitis and NanoString analysis. Stool samples were evaluated by 16S rRNA and gas chromatography/ mass spectrometry (GC/MS) analyses.

Histology scores showed that mice treated with probiotics + amylase had a significant decrease of ileitis severity compared to the other two groups. 16S rRNA and GC/MS analysis showed that abundance of species belonging to genus Lachnoclostridium and the species Mucispirillum schaedleri were significantly increased compared to the other two groups, and this increase was associated with augmented production of SCFAs. Furthermore, NanoString data showed that 27 genes involved in B memory cell development and T cell infiltration were significantly upregulated in probiotic+ amylase-treated mice and 17 genes were significantly down-regulated.

These findings suggest that administration of this novel probiotic preparation leads to functional changes that ameliorate the severity of CD-like ileitis. In addition, the hydrolytic activity of amylase appears to be essential for the anti-inflammatory effects of beneficial bacteria in the intestine. This article has been submitted and is currently under review.

Phase 4: Clinical testing

To this point, we accumulated a great amount of preclinical data that formed the basis for our next step, which were clinical (human) studies. After confirming the activity and efficacy of the probiotic combination in in vitro and in vivo experiments, we sought to examine its effect on the human microbiome. We established that the strains used in the combined probiotic were all classified as Generally Regarded as Safe (GRAS) organisms as under sections 201(s) and 409 of the Federal Food, Drug, and Cosmetic Act, whereby a substance is generally recognized, among qualified experts, as having been adequately shown to be safe under the conditions of its intended use, or unless the use of the substance is otherwise excepted from the definition of a food additive. Given the strains were GRAS, we started by testing the probiotic combination, which was also supplemented with amylase, on the human microbiome of healthy subjects. We enrolled and consented cohort of 49 volunteers to participate in 4 weeks of once-a-day probiotic consumption. The comprehensive intestinal microbiome (CIM, representing bacterial and fungal communities) profiles were assessed at baseline and following 4 weeks of probiotic consumption. We then compared the microbiome (both bacteria and fungi) of our subjects with those reported by the Human Microbiome Project (HMP) for healthy subjects as a control for bacterial abundance. Interestingly, the abundance of Candida spp. was significantly reduced compared to baseline. Furthermore, the treatment resulted in increased levels of Firmicutes and reduction in the Bacteroidetes. Based on our previous studies and data in literature, we reasoned these effects could be of great benefit to CD patients. For example, reduction in the levels of Candida will be helpful in preventing the formation of polymicrobial biofilms, a factor that has been linked to the inflammatory process in CD. ⁶⁴ Additionally, reduction in the Firmicutes/Bacteroidetes (F/B) ratio was shown to play a role in the disease process. ¹³⁴ Thus, using this probiotic and amylase combination may help in reversing the F/B ratio thereby restoring the gut balance. Not only this, but it will also help in replenishing some of the beneficial organisms, such as F. prausnitzii, that has been shown to exhibit an anti-inflammatory effect. ¹⁰²

Following this preliminary study, another clinical study (recently completed and submitted for publication) was performed to test the effect of the probiotic combination on individuals with self-reported gastrointestinal symptoms such as flatulence, bloating, and abdominal discomfort. Studies such as these and additional studies demonstrating that changes in the gut microbiome balance following probiotic modulation can be sustained by either diet modification or maintenance supplementation. ^135–137

Conclusions:

Gut microbiota in various hosts may influence disease. Numerous microbiota species have been reported to participate in degenerative diseases, including obesity, diabetes, cancer, cardiovascular disease, malignancy, liver diseases, and IBD. An imbalance of the gut microbiota composition (dysbiosis) may initiate or exacerbate several diseases, therefore, probiotics have been postulated as a mechanism to modulate gut microbiota composition imbalance thereby rebalancing the gut microbiome, increasing gut epithelium barrier function, and enhancing cytokine production. Thus, alteration of the gut microbiota composition via probiotic species designed to modulate dysbiosis is a viable therapeutic approach to allow the host microflora to treat diseases that manifest through dysbiosis. Implementing a probiotic capable of modulating the microbiome requires rationale design and appropriate experimental models (both in vitro, and in vivo) to provide insights into the gut microbiota composition, the microbial interplay expected during co-culture, and the potential effects on host commensal organisms. Identification of new probiotic formulations and key mixtures of probiotic polymicrobial species (that may contain additional supporting ingredients such enzymes/prebiotics) will help to shape future studies to promote host health through modulation of the microbiome.