Investigating the role of gut microbiota-mediated colonisation resistance against carbapenem-resistant Enterobacteriaceae

Carbapenem-resistant Enterobacteriaceae (CRE) are a group of Gram-negative bacteria which are highly resistant to antibiotic treatments and pose a great threat to human global health. These pathogens are responsible for many hospital-acquired invasive infections, such as pneumonia, urinary tract infections and blood stream infections. Intestinal colonisation of CRE is associated with high mortality rates, persistent infection and treatment resistance.

Antibiotic use has been shown to disrupt the natural colonisation resistance that the human gut microbiota can provide against multi-drug resistant organisms (MDROs). Currently the mechanism(s) of colonisation resistance provided by the gut microbiota are incompletely understood. A clearer understanding is required to develop microbiome therapeutics that can be used to restore colonisation resistance and thus combat intestinal colonisation of CRE.

The overall aim of this study is to identify mechanism(s) of gut microbiota-mediated colonisation resistance that are responsible for inhibiting CRE growth in healthy individuals. This will allow us to develop a therapeutic target that could be used as a treatment for CRE intestinal colonisation. We hypothesise that gut microbiota-mediated colonisation resistance prevents intestinal colonisation with CRE by restoring a microorganism, metabolite, or enzyme that inhibits CRE growth. The objectives of our study are as follows:

1) Screen the effects of antibiotics (known to promote CRE intestinal colonisation) on cultured gut microbiota and CRE growth using faecal batch cultures.
2) Measure the effects of antibiotics on CRE growth in an artificial gut model.
3) Measure the effects of FMT treatment on CRE growth in an artificial gut model.
4) Measure the direct effects of therapeutic targets (identified in objectives 2 and 3) on the growth of CRE isolates in batch cultures.

Faecal batch cultures will be used as a high-throughput method to measure the effects of a wide variety of antibiotics on gut microbiota-mediated colonisation resistance and their impact on CRE growth.

Results from faecal batch culture experiments will aid the design of longitudinal in vitro experiments using artificial gut ("chemostat") models. Firstly, we will compare the ability of CRE isolates to colonise a cultured gut microbiota in the presence and absence of antibiotic treatment.

Faecal microbiota transplants (FMT) have been shown to decolonise MDROs from the gut. FMT has been proposed to act by restoring gut microbiota colonisation resistance in patients with MDRO intestinal colonisation. Therefore, the next set of experiments will use artificial gut models to replicate FMT treatment in CRE colonised cultured gut microbiota.

Samples from faecal batch culture experiments and artificial gut model experiments will be analysed using various techniques. Changes in CRE growth will be monitored using selective plate counts, while changes in the composition of the cultured gut microbiota will be measured using 16S rRNA gene sequencing. Changes in microbial metabolites will be measured using 1H NMR spectroscopy and several mass spectrometry techniques.

Finally, to verify the direct action of therapeutic targets (e.g. microorganisms or metabolites) on reducing CRE growth, we will perform batch culture experiments; incubating CRE isolates with the therapeutic target identified in the artificial gut experiments. Changes in CRE growth will be measured to demonstrate the direct effects of the therapeutic target.

Together, these experiments will highlight key therapeutic targets that contribute to the restoration of colonisation resistance. These therapeutic targets will be further developed into treatments for CRE intestinal colonisation that could be used to treat or prevent intestinal CRE colonisation. Reduction or prevention of CRE growth in the intestine will prevent the development of invasive CRE infections.