Safety and Efficacy of an AIEC-targeted Bacteriophage Cocktail in a Mice Colitis Model.

BACKGROUND AND AIMS: Adherent invasive Escherichia coli [AIEC] are recovered with a high frequency from the gut mucosa of Crohn's disease patients and are believed to contribute to the dysbiosis and pathogenesis of this inflammatory bowel disease. In this context, bacteriophage therapy has been proposed for specifically targeting AIEC in the human gut with no deleterious impact on the commensal microbiota. METHODS: The in vitro efficacy and specificity of a seven lytic phage cocktail [EcoActive™] was assessed against [i] 210 clinical AIEC strains, and [ii] 43 non-E. coli strains belonging to the top 12 most common bacterial genera typically associated with a healthy human microbiome. These data were supported by in vivo safety and efficacy assays conducted on healthy and AIEC-colonized mice, respectively. RESULTS: The EcoActive cocktail was effective in vitro against 95% of the AIEC strains and did not lyse any of the 43 non-E. coli commensal strains, in contrast to conventional antibiotics. Long-term administration of the EcoActive cocktail to healthy mice was safe and did not induce dysbiosis according to metagenomic data. Using a murine model of induced colitis of animals infected with the AIEC strain LF82, we found that a single administration of the cocktail failed to alleviate inflammatory symptoms, while mice receiving the cocktail twice a day for 15 days were protected from clinical and microscopical manifestations of inflammation. CONCLUSIONS: Collectively, the data support the approach of AIEC-targeted phage therapy as safe and effective treatment for reducing AIEC levels in the gut of IBD patients.

The microbiome in reproductive health: protocol for a systems biology approach using a prospective, observational study design.

STUDY QUESTION: What is the microbiome profile across different body sites in relation to the normal menstrual cycle (with and without hormonal contraception), recurrent pregnancy loss (RPL) (before and during pregnancy, pregnancy loss or birth) and endometriosis (before, during and after surgery)? How do these profiles interact with genetics, environmental exposures, immunological and endocrine biomarkers? WHAT IS KNOWN ALREADY: The microbiome is a key factor influencing human health and disease in areas as diverse as immune functioning, gastrointestinal disease and mental and metabolic disorders. There is mounting evidence to suggest that the reproductive microbiome may be influential in general and reproductive health, fertility and pregnancy outcomes. STUDY DESIGN SIZE DURATION: This is a prospective, longitudinal, observational study using a systems biology approach in three cohorts totalling 920 participants. Since microbiome profiles by shot-gun sequencing have never been investigated in healthy controls during varying phases of the menstrual cycle, patients with RPL and patients with endometriosis, no formal sample size calculation can be performed. The study period is from 2017 to 2024 and allows for longitudinal profiling of study participants to enable deeper understanding of the role of the microbiome and of host-microbe interactions in reproductive health. PARTICIPANTS/MATERIALS SETTING METHODS: Participants in each cohort are as follows: Part 1 MiMens-150 healthy women with or without hormonal contraception; Part 2 MiRPL-200 couples with RPL, 50 healthy couples with prior uncomplicated pregnancy and 150 newborns; Part 3 MiEndo-120 patients with endometriosis requiring surgery with or without hormonal treatment. Microbiome profiles from saliva, faeces, rectal mucosa, vaginal fluid and endometrium will be studied, as well as the Omics profile, endocrine disrupting chemicals and endocrine and immune factors in blood, hair, saliva and urine. Pregnancy loss products, seminal microbiome, HLA types, endometriotic tissue and genetic risk and comprehensive questionnaire data will also be studied, where appropriate. Correlations with mental and physical health will be evaluated. STUDY FUNDING/COMPETING INTERESTS: This work is supported by funding from Ferring Pharmaceuticals ([#MiHSN01] to H.S.N., M.C.K., M.E.M., L.E.V., L.E., I.S.-K., F.B., L.W.H., E.F. and M.H.), Rigshospitalet's Research Funds ([#E-22614-01 and #E-22614-02] to M.C.K. and [#E-22222-06] to S.B.), Niels and Desiree Yde's Foundation (S.B., endocrine analyses [#2015-2784]), the Musikforlæggerne Agnes and Knut Mørk's Foundation (S.B., endocrine and immune analyses [#35108-001]) and Oda and Hans Svenningsen's Foundation ([#F-22614-08] to H.S.N.). Medical writing assistance with this manuscript was provided by Caroline Loat, PhD, and funded by Ferring Pharmaceuticals. H.S.N. reports personal fees from Ferring Pharmaceuticals, Merck Denmark A/S, Ibsa Nordic, Astra Zeneca and Cook Medical outside the submitted work. K.W. is a full-time employee of Ferring Pharmaceuticals. No other conflicts are reported. TRIAL REGISTRATION NUMBER: N/A. TRIAL REGISTRATION DATE: N/A. DATE OF FIRST PATIENT'S ENROLMENT: N/A.

Dysbiosis of the Human Oral Microbiome During the Menstrual Cycle and Vulnerability to the External Exposures of Smoking and Dietary Sugar.

Physiological hormonal fluctuations exert endogenous pressures on the structure and function of the human microbiome. As such, the menstrual cycle may selectively disrupt the homeostasis of the resident oral microbiome, thus compromising oral health. Hence, the aim of the present study was to structurally and functionally profile the salivary microbiome of 103 women in reproductive age with regular menstrual cycle, while evaluating the modifying influences of hormonal contraceptives, sex hormones, diet, and smoking. Whole saliva was sampled during the menstrual, follicular, and luteal phases (n = 309) of the cycle, and the participants reported questionnaire-based data concerning their life habits and oral or systemic health. No significant differences in alpha-diversity or phase-specific clustering of the overall microbiome were observed. Nevertheless, the salivary abundances of genera Campylobacter, Haemophilus, Prevotella, and Oribacterium varied throughout the cycle, and a higher species-richness was observed during the luteal phase. While the overall community structure maintained relatively intact, its functional properties were drastically affected. In particular, 11 functional modules were differentially abundant throughout the menstrual cycle, including pentose phosphate metabolism, and biosynthesis of cobalamin and neurotransmitter gamma-aminobutyric acid. The menstrual cycle phase, but not oral contraceptive usage, was accountable for greater variations in the metabolic pathways of the salivary microbiome. Further co-risk factor analysis demonstrated that Prevotella and Veillonella were increased in current smokers, whereas high dietary sugar consumption modified the richness and diversity of the microbiome during the cycle. This is the first large study to systematically address dysbiotic variations of the oral microbiome during the course of menstrual cycle, and document the additive effect of smoking and sugar consumption as environmental risk factors. It reveals the structural resilience and functional adaptability of the oral microbiome to the endogenous hormonal pressures of the menstrual cycle, while revealing its vulnerability to the exogenous exposures of diet and smoking.

Ex-Vivo Force Spectroscopy of Intestinal Mucosa Reveals the Mechanical Properties of Mucus Blankets.

Mucus is the viscous gel that protects mucosal surfaces. It also plays a crucial role in several diseases as well as in mucosal drug delivery. Because of technical limitations, mucus properties have mainly been addressed by in-vitro studies. However, this approach can lead to artifacts as mucus collection can alter its structure. Here we show that by using an implemented atomic force microscope it is possible to measure the interactions between micro-particles and mucus blankets ex-vivo i.e., on fresh excised mucus-covered tissues. By applying this method to study the small intestine, we were able to quantify the stiffness and adhesiveness of its mucus blanket at different pH values. We also demonstrate the ability of mucus blankets to bind and attract particles hundreds of µm away from their surface, and to trap and bury them even if their size is as big as 15 µm.

Bacteriophages Targeting Adherent Invasive Escherichia coli Strains as a Promising New Treatment for Crohn's Disease.

BACKGROUND AND AIMS: Adherent invasive Escherichia coli [AIEC] are abnormally predominant on the ileal mucosa of Crohn's disease [CD] patients. They bind to the CEACAM6 receptor expressed on the surface of epithelial cells. We aimed to assess the potential of bacteriophages, viruses infecting bacteria, to decrease the levels of AIEC bacteria associated with the intestinal mucosa. METHODS: We combined ex vivo and in vivo experiments with murine and human intestinal samples to quantify the ability of virulent bacteriophages to target the prototype AIEC strain LF82. RESULTS: We found that three virulent bacteriophages were able to replicate in ileal, caecal and colonic sections and faeces homogenates from murine gut samples colonised with the prototype AIEC strain LF82. A single day of per os treatment with the three bacteriophages cocktail given to LF82-colonised CEABAC10 transgenic mice, expressing the human CEACAM6 receptor for AIEC, decreased significantly the number of AIEC in faeces and in the adherent flora of intestinal sections. In addition, a single dose of the cocktail reduced dextran sodium sulphate-induced colitis symptoms on conventional mice colonised with the strain LF82 over a 2-week period. The cocktail targeted also LF82 bacteria in homogenates of ileal biopsies taken from CD patients. CONCLUSIONS: These findings demonstrate that bacteriophages are a new treatment option for targeting AIEC in CD patients and represent a strong basis for a clinical trial evaluation.