ABSTRACT Specific elimination of cytolytic Enterococcus faecalis from the intestinal microbiota by bacteriophages (phages) attenuates ethanol-induced liver disease Clutch Solenoid in pre-clinical studies; however, other clinical phage therapy studies have reported the occurrence of phage-resistant variants.Here, we assessed phage resistance using a cytolytic E.faecalis clinical isolate, EF01.After infecting EF01 with ΦEf2.
1 (Myoviridae) or ΦEf2.2 (Podoviridae), four host variants (R-EF01ΦEf2.1-A and R-EF01ΦEf2.1-B from infection with ΦEf2.
1, and R-EF01ΦEf2.2-A and R-EF01ΦEf2.2-B from infection with ΦEf2.2) were isolated.
Although isolate R-EF01ΦEf2.2 exhibited resistance to both phages, isolate R-EF01ΦEf2.1 demonstrated partial resistance only to ΦEf2.1.
Whole-genome sequencing of these four isolates revealed an insertion sequence, IS256, -mediated disruption of xylA in R-EF01ΦEf2.1-A and R-EF01ΦEf2.1-B.In addition, a non-synonymous mutation in epaR, essential for the complete Enterococcus polysaccharide antigen (Epa), was identified in the R-EF01ΦEf2.
2-A isolate.Furthermore, R-EF01ΦEf2.2 isolates exhibited IS256-associated chromosomal deletions and lacked galE, a gene involved in Epa biosynthesis.After gavaging mice with EF01 WT, R-EF01ΦEf2.
1-A, R-EF01ΦEf2.2-A, and R-EF01ΦEf2.2-B isolates, colonization of R-EF01ΦEf2.2 isolates was significantly attenuated.
R-EF01ΦEf2.2 isolates exhibited less resistance to the bile salt sodium deoxycholate and showed reduced adherence to intestinal cell monolayers, suggesting that phage-resistant variants with alterations in bacterial surface molecules, potentially including those involved in Epa biosynthesis, reduced pathogen fitness by attenuating gut colonization.In summary, IS256 is involved in phage resistance of a cytolytic E.faecalis clinical isolate, and certain phage resistance mechanisms could contribute to favorable clinical outcomes by promoting the swift elimination of phage-resistant variants in the treatment of alcohol-associated hepatitis.
IMPORTANCEPhage therapy is a promising approach for precise editing of the gut microbiota.Notably, the specific elimination of cytolytic E.faecalis from the intestinal microbiota by phages attenuates ethanol-induced liver disease in pre-clinical studies.Despite the great promise RAW CACAO POWDER of phage therapy, the occurrence of phage-resistant variants represents a concern for the successful development of phage-based therapies.
In this context, we assessed phage resistance using a cytolytic E.faecalis clinical isolate.Isolated phage-resistant variants acquired mutations or deletions of Epa biosynthesis-related genes and exhibited attenuated colonization in the gut.These phage-resistant variants showed less resistance to bile salts and reduced adherence to intestinal cell monolayers.
These results suggest that even if phage-resistant variants arise during phage therapy, certain mechanisms of phage resistance may contribute to the rapid elimination of phage-resistant variants promoting favorable clinical outcomes in the treatment of alcohol-associated hepatitis.