Investigating the use of bacteriophages as a new decolonization strategy for intestinal carriage of CTX-M-15-producing ST131 Escherichia coli: An in vitro continuous culture system model

Objectives: We investigated the use of bacteriophages as a strategy to decolonize intestinal carriers of multidrug-resistant Escherichia coli. Methods: A fermentor was used as a continuous culture system for 48 h. Two different pools of faeces (studies I and II) obtained from volunteers were spiked with a CTX-M-15-producing ST131 E. coli (strain 4901.28) susceptible to bacteriophages and challenged with three doses of INTESTI Bacteriophage cocktail administered at 2, 6 and 10 h after the inoculum. Bacterial typing was performed by implementing microdilution panels, spot test, rep-PCR and whole-genome sequencing (including cgMLST and single-nucleotide variant analysis) obtained using Nanopore and Illumina platforms. Results: In study I, bacteriophages decreased the numbers of 4901.28 dramatically (≤101 CFU/mL after 6 h). In contrast, during study II, a phage-resistant mutant of 4901.28 persisted in the continuous culture (104 CFU/mL at 48 h). Whole-genome sequencing revealed the presence of two additional plasmids in the mutant as well as 11 single-nucleotide variants, including one chromosomal in a glycosyltransferase family 2 protein that is responsible for the transfer of sugars to polysaccharides and lipids. In both studies, the commensal E. coli population remained unchanged by the phage treatment maintaining itself at 108 CFU/mL. Conclusions: Our data indicates that bacteriophage cocktails may be implemented to decolonize some intestinal carriers. However, the individual microbiota composition may have an impact on the development of phage resistance. Mechanisms underlying this phenomenon are likely to be various and complex. Further in vivo studies and protein expression experiments are needed to confirm our observations and hypotheses.