Publications

Microbiology spectrum. 2025-04-15; e0330324.

Insertion sequence-mediated phage resistance contributes to attenuated colonization of cytolytic Enterococcus faecalis variants in the gut

Fujiki J, Nakamura T, Kreimeyer H, Llorente C, Fouts DE, Schnabl B

PMID: 40231830

Abstract

Specific elimination of cytolytic from the intestinal microbiota by bacteriophages (phages) attenuates ethanol-induced liver disease 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 clinical isolate, EF01. After infecting EF01 with ΦEf2.1 () or ΦEf2.2 (), four host variants (R-EF01-A and R-EF01-B from infection with ΦEf2.1, and R-EF01-A and R-EF01-B from infection with ΦEf2.2) were isolated. Although isolate R-EF01 exhibited resistance to both phages, isolate R-EF01 demonstrated partial resistance only to ΦEf2.1. Whole-genome sequencing of these four isolates revealed an insertion sequence, IS256, -mediated disruption of in R-EF01-A and R-EF01-B. In addition, a non-synonymous mutation in , essential for the complete polysaccharide antigen (Epa), was identified in the R-EF01-A isolate. Furthermore, R-EF01 isolates exhibited IS256-associated chromosomal deletions and lacked , a gene involved in Epa biosynthesis. After gavaging mice with EF01 WT, R-EF01-A, R-EF01-A, and R-EF01-B isolates, colonization of R-EF01 isolates was significantly attenuated. R-EF01 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 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.

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