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Competition between the enteric pathogen Clostridium difficile and the commensal members of the gut microbiota for mucosal sugars

Periodic Reporting for period 1 - MUCDIFF (Competition between the enteric pathogen Clostridium difficile and the commensal members of the gut microbiota for mucosal sugars)

Reporting period: 2015-09-01 to 2017-08-31

The trillions of bacteria that live in the human gut form a complex ecological community that helps in food digestion, produces vitamins, and trains the developing immune system. The microbiota also protects against infection by enteropathogens, a phenomenon termed colonization resistance (CR). Perturbations of the gut microbiota, as occur upon antibiotic administration, lead to loss of CR and an increased susceptibility to infections by enteropathogens. Some of the mechanisms of CR are known and involve the production of antimicrobial compounds with narrow activity against pathogens, the production of microbial associated molecular patterns (MAMPs) that stimulate the production of adaptive or innate effectors by the host immune system, or by exhaustion of limited nutrient sources.
This project focuses on competition for host-derived nutrients as an important mechanism by which the colonic microbiota can suppress the growth of many gut pathogens. We are particularly interested in the pathogen Clostridium difficile, nowadays the most common cause of enteric infections associated with antibiotic therapy in developed countries. We seek to understand how C. difficile utilizes host mucosal sugars to establish and survive in the gut and how the presence of commensal bacteria competing for these mucosal sugars modify the outcome of C. difficile infections.
Using a combination of techniques such as genetic tools, in vivo colonization assays, and stable isotope probing (SIP) combined with fluorescence in situ hybridization (FISH) and high resolution secondary ion mass spectrometry (NanoSIMS), we aim to: 1) elucidate the role of mucosal sugars catabolism in C. difficile expansion in the gut; 2) identify commensal members of the gut microbiota that can efficiently catabolize these mucosal sugars in vivo; and 3) evaluate the ability of the identified organisms to outcompete C. difficile. Findings from this work will contribute to elucidate the mechanisms by which the gut microbiota prevents C. difficile colonization and to identify members of the gut microbiota that can be the basis for an effective, safe and standardized treatment to cure a C. difficile infection.
Clostridium difficile relies on the utilization of gut mucosal sugars to expand and cause infection and disease. One of the objectives of this work was to identify gut commensals that can efficiently use the same mucosal sugars as C. difficile and assess their ability to outcompete this pathogen. To address this question we have employed stable isotope probing to detect and identify specific members of the community that can be stimulated when ammended with these mucosal monossacharides. Since the microbial substrates we are interested in are very expensive as 13C or 15N isotopically labeled derivatives or are not even available commercially, we employed a recently developed method to measure the activity of individual microbes with Raman spectroscopy within complex samples. This method involves heavy water, were the hydrogen is replaced by a deuterium atom. Deuterium can still be used during lipid biosynthesis, leading to deuterated lipids of cells that are active in the community, which can be easily detected by non-destructive single cell Raman spectroscopy. Using this methodology we could observe stimulation of a significant part of the commensals from the gut mouse community, when these were incubated in vitro under anaerobic conditions and in the presence of D2O (Figure 1). Using Raman-based single cell sorting, these efficient mucosal sugar utilizers were sorted and collected. After cell lysis and multiple displacement amplification, these organisms are now being identified with basis on their 16S rRNA sequence.
Treatment of Clostridium difficile infection (CDI) includes stopping all previous antibiotic therapy, if possible, followed by the use of metronidazole or vancomycin. The main problem is that antibiotic treatment for a first infection eliminates many potentially protective commensal bacteria, facilitating reinfection. Thus, the gut microbiota needs to be restored to protect the intestinal epithelium and to prevent the recurrence of the disease. For this reason non-antibiotic based treatments, such as fecal microbiota transplantation (FMT), have been adopted to treat CDI. Despite the efficacy of FMT, which resolves recurrent CDI in more than 90% of the cases, this treatment has not been formally approved, which restricts its use. Moreover it requires time to identify a suitable donor and carries the risk of unintentional transmission of undetected pathogens, which together with general patient aversion represent major impediments to the broad application of this treatment. Thus, a major goal in the field is to identify specific members of the gut microbiota that can be the basis for an effective, safe and standardized treatment for CDI.
Clostridium difficile relies on the utilization of gut mucosal sugars to expand and cause infection and disease. Using stable isotope probing coupled to Raman Spectroscopy, we could observe stimulation of a significant part of the commensals from the gut mouse community when ammended with the same mucosal sugars that C.difficile utilizes during expansion in the gut. Using Raman-based single cell sorting, these efficient mucosal sugar utilizers were sorted and started to be identified. The ability of these commensal organisms (or closely related ones) to outcompete C. difficile will now be tested in vitro, and if successul results are achieved, then in vivo. If these organims reveal to be able to efficiently compete C. difficile, they can be used as the basis for an new bacterial-based treatment to erradicate C. difficile from the gut. C. difficile is currently a major concern is hospitals and health care institutions. In most European countries the number of deaths caused by CDI has more than quadrupled in the last decade, with medical costs associated with CDI also representing a major economic burden. Thus, the identification of organisms with potential to eradicate this pathogen will benefit the European economy and society in general.
Competition between Clostridium difficile and gut commensals for host-derived sugars in the gut