Temporal shifts in antibiotic resistance elements govern phage-pathogen conflicts

Bacteriophage predation selects for diverse anti-phage systems that frequently cluster on mobilizable defense islands in bacterial genomes. However, there remains a lack of molecular insight into the reciprocal dynamics of phage-bacterial adaptations in nature, particularly in clinical contexts where there is need to inform phage therapy efforts and understand how phages drive pathogen evolution. Here,  we used time-shift assays to evaluate whether clinical Vibrio cholerae isolates were susceptible to infection by phage from past, future or contemporaneous patient samples. Across a 34-month sampling period, we discover that phage resistance is governed by fluctuations in SXT integrative and conjugative elements (ICEs), which notoriously also confer antibiotic resistance. We further demonstrate potential trade-offs for having SXT ICEs, as we show they can also can restrict beneficial mobile genetic elements. We discover phage counter-adaptation to SXT-mediated restriction in clinical samples, and show that flux of SXT ICEs in V. cholerae over time allows for re-emergence of phage resistance. We find that SXT ICEs, which are widespread in Gammaproteobacteria, invariably encode phage defense and function to protect other genera from phage attack following conjugation. Further, we find that phage infection stimulates high frequency SXT ICE conjugation, leading to the concurrent dissemination of phage and antibiotic resistance.

Methods
WYL-domain proteins and the downstream flanking gene encoding the conjugative relaxase TraI were extracted from all identifiable hotspot 5's encoded by SXT ICEs. MUSCLE alignments were performed on the nucleotide sequence for each unique gene, and a phylogenetic tree was constructed using PhyML with 100 bootstrap iteractions. Phylogenetic trees were visualized in FigTree v1.4.4.