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Dissecting individual pathogen-commensal interactions within a complex gut microbiota community

Jack Hassall, Meera Unnikrishnan

Preprint posted on April 06, 2020 https://www.biorxiv.org/content/10.1101/2020.04.06.027128v1

Examination of how commensal bacteria interact with the pathogen C. difficile to prevent pathogenic infection within a biofilm

Selected by Josie Gibson

Categories: microbiology

Background:

The microbiota of the gut has multiple important roles, including metabolism, immune function and inhibition of pathogen colonisation. Commensals inhibit pathogen colonisation through resource competition, and directly through production of targeted antimicrobial molecules. In cases where alterations to the microbiota occur, for example following antibiotic treatment, disease can arise. This can lead to a higher risk of infection by pathogenic bacteria, for example Clostridioides difficile. This pre-print presents a new qPCR analysis of an in vitro multi-species gut community which is then used to examine how the microbiota interacts with C. difficile within this biofilm situation. This technique allows the authors to specifically examine individual bacterial interactions which is not possible in genomic approaches.

 

Key findings:

qPCR biofilm analysis

The authors first validate their qPCR biofilm analysis by comparing individual commensal bacterial growth within a biofilm, where all bacterial species are individually detected. This method also excludes dead bacterial cells allowing accurate examination of live bacteria. Currently the method cannot be used for long periods of time since bacterial survival begins to decline. Nevertheless, a useful experimental window of up to 72 hours is available and the method is employed to examine how microbiota interact with C. difficile.

Gut microbiota impede C. difficile colonisation

The ability of C. difficile to adhere and grow was reduced in the presence of the microbiota at the first stages of biofilm formation, whereas commensal bacterial species adherence was increased and their growth was similar or increased, compared to culture without C. difficile. This highlights how the microbiota can influence pathogenic species. The inhibitory effects upon C. difficile were enhanced when added to an existing biofilm, which better represents an established gut microbiota. Interestingly, individual commensals reacted differently when C. difficile was added to an existing biofilm, resulting in either a reduced, similar or increased bacterial number. A large increase in commensal growth was observed for Bacteroides dorei. Further experiments focussing on B. dorei alone, revealed that B. dorei bacterial numbers were increased in the presence of C. difficile, whilst also causing reduced C. difficile bacterial numbers. Therefore, this microbiota analysis enabled identification of key individual bacterial interactions within a complex microbial biofilm setting. Furthermore, pre-established microbiota was shown to be better at protecting against C. difficile, compared to addition of C. difficile at the stage of biofilm formation, signifying that alterations in the microbiota represent a foothold for C. difficile infection. Therefore, this methodology reflects real-life C. difficile infections, where infection likelihood is increased following changes in the microbiota, such as those caused by antibiotic treatment.

 

Why I chose this Preprint:

This pre-print is of interest for the study of host-pathogen interactions, especially how commensal bacteria regulate pathogenic infections. A new in vitro methodology for analysis of pathogen and commensal interactions within a microbial biofilm setting, more akin to the natural infection scenarios, is defined. The method described by the authors compares growth of individual bacterial species in co-culture conditions, which provides useful insights into infection dynamics and pathogen-commensal interactions. This pre-print demonstrates how this method can be applied, showing that C. difficile adherence in the biofilm setting is reduced in the presence of other commensal bacteria. The authors could then identify that out of nine commensal bacterial species used, Bacteroides dorei has a key role in reducing C. difficile growth.

 

Questions to the authors:

  1. In your assay, the trend of dying over time for bacterial species is shown. Therefore, could the biofilms be given fresh media/extra supplements to increase the timeframe of analysis? Do think improved fitness of all bacterial species would alter commensal-pathogen interactions?
  2. In the gut would the nine chosen commensal bacteria all be in equal number to start with? If not, could the numbers of bacteria present at the establishment of the biofilm be important for future analysis?
  3. You show that dorei negatively impacts C. difficile, and suggest these may be due to metabolic or soluble factors. Are there bacterial mutants available for these potential factors you could examine?
  4. C. difficile infection often occurs after alterations in the gut microbiome. If it is possible to determine the factor by which B. dorei negatively impacts C. difficile, do you envisage any therapeutic outcomes to influence C. difficile, during or after antibiotic treatment?

 

Posted on: 5th May 2020 , updated on: 25th March 2021

doi: https://doi.org/10.1242/prelights.20064

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Author's responses to questions

Meera Unnikrishnan shared

  1. We see bacteria declining in numbers only after 48 hours, hence we are confident that during this window we see an accurate representation of resource competition and interaction between the species up until this point. We agree, feeding the biofilm with fresh medium would extend this window, however it would have a big influence on the population as resources would suddenly become abundant from their limited state, making it difficult to differentiate between population dynamics caused by inter-species interactions and influx of resources. We believe the best way to study the biofilm over an extended period of time would be to have a constant influx of media using fluidics (which we are currently developing), avoiding the peak and trough dynamics made by adding fresh media periodically. Given the number of species in this community, any alteration in “fitness” or available medium would likely have an impact on population dynamics, and alter the commensal-pathogen interactions.
  2. The number of species present in the gut and their abundance have been shown to be highly variable from person to person. We do not know about absolute bacterial numbers in the gut as most numbers or proportions available are based on bacterial counts or sequencing data from faeces, which only include the luminal bacteria and not those attached to the mucosa. We chose to equalise bacterial inoculum by OD600 for practical reasons and to ensure repeatability. We could perform studies with equal numbers of all bacteria at the start, but given the small percentage of the initial inoculum that adhere, we believe we would see little change in the overall trends.
  3. Although we have some clues about the metabolic pathways changing in Bacteroides spp (Slater et al 2019), at present we do not know which specific factors impact C. difficile growth. Currently very few mutants of B. dorei have been constructed, as genetic manipulation tools are quite limited. We have ongoing research using functional genomic approaches to identify specific pathways between the two species.
  4. Yes indeed, if we identify an inhibitory small molecule secreted by B. dorei or a specific metabolite that allows B. dorei to outcompete C. difficile, it would be possible to design and develop new therapeutic or prophylactic strategies. It is also important to note that models like these also enable studies to understand the impact of current drugs on the microbiota; altering therapies would avoid the microbiota disturbances that leads to C. difficile colonisation in the first place.

 

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