Enterobacter-specific bacteriophages Entb₄3 and Entb₄5 interact with the intestinal epithelium to limit intracellular infection and may undergo partial translocation

Enterobacter cloacae complex (ECC) are opportunistic pathogens with an increasingly wide range of antibiotic resistance mechanisms described. Irrational antibiotic stewardship policy and their excessive use in various sectors have necessitated a return to therapeutic use of bacteriophages (phages) - viruses specific to bacteria. Bacteriophages are the most widespread biological entities in the environment and they can also interact with eukaryotic cells. In this study, we investigated the impact of Enterobacter-specific lytic bacteriophages, Entb₄3 and Entb₄5, on the intestinal epithelium using the immortalized human colorectal adenocarcinoma Caco-2 cell line. The tested phages do not negatively affect the intestinal epithelium integrity nor impair intestinal cells viability, which partially confirms their safety in the context of the gastrointestinal tract. Two multidrug-resistant clinical strains of E. hormaechei, isolated from patients undergoing experimental phage therapy and serving as hosts for the tested phages, were selected to assess their ability to develop intracellular infection. Intracellular bacterial invasion increased over time, but bacteria did not multiply within the cells. Importantly, Enterobacter-specific bacteriophages were able to protect epithelial cells from bacterial invasion and reduction of intracellular burden, which may indicate endocytosis of phages by epithelial cells or their interactions with epithelial cell membrane. The tested Enterobacter-specific phages were able to pass through the highly confluent intestinal epithelial monolayers cultured on the Transwell inserts after prolonged incubation time, confirming the potential for phage translocation across epithelia and the possible further exploration of this phenomenon.