Temporal shifts in antibiotic resistance elements govern phage-pathogen conflicts

Back to the future phage The interrelationships that prevail between bacteria and their phage parasites are subtle and evolutionarily dynamic. In Bangladesh, cholera remains endemic, and natural, clinically relevant infections have been monitored for decades. LeGault et al. investigated the relationship between antiphage defenses and phage counter-responses in human Vibrio cholerae cases. These bacteria have integrative and conjugative elements called SXT ICEs, which are notorious for carrying antibiotic resistance genes but also contain genes that defend bacteria from phage. Phage have their own counterdefense mechanisms. One constitutes a 44–amino acid peptide product in a phage lineage that inhibits the bacterium's SXT ICE defenses. In a further complication, SXT-ICEs also inhibit the lysogenic phage that transmit Vibrio virulence factors, including cholera toxin. Therefore, this process drives bacterial diversity as well as antibiotic resistance. Science, abg2166, this issue p. eabg2166 Vibrio cholerae susceptibility to bacteriophages is determined by mobile elements bearing phage defense as well as antibiotic resistance genes. INTRODUCTION In nature, bacteria contend with abundant and diverse viruses (phages), necessitating an extensive repertoire of defense systems. Antiphage systems cluster together with mobilome genes, which suggests that phage predation can drive bacterial evolution through mobile genetic element (MGE) flux. RATIONALE Despite the continued discovery of novel defense systems, we lack biologically relevant systems to study ongoing phage-host interactions, particularly in clinical settings. Approaches using heterologous hosts and model phages limit our understanding of fitness trade-offs and phage-encoded counteradaptations that drive diversification of phage defenses in nature. A promising model system to gain insight into phage-host coevolution lies in longitudinal sampling of the diarrheal pathogen Vibrio cholerae and its lytic phages, which are routinely co-isolated from clinical specimens in regions where cholera is endemic. RESULTS We performed time-shift assays that challenged V. cholerae isolates with phages directly from patient stool from the relative past, present, and future. All V. cholerae were susceptible to contemporaneous phages but restricted those from the past or future, indicative of a fluctuating resistance determinant. We sought to mechanistically uncover the driver of phage resistance in toxigenic V. cholerae and thereby understand how contemporaneous phages circumvented defenses. Combining comparative genomics with our time-shift results, we mapped resistances to fluctuations in integrative and conjugative elements (ICEs) of the SXT/R391 family. SXT ICEs are notorious for carrying antibiotic resistance genes in accessory “hotspot” regions. We found that SXT ICEs from clinical V. cholerae and other γ-proteobacteria carry identifiable defense systems (e.g., restriction modification systems and the recently discovered BREX system) in hotspot 5. An expanded analysis of 2600 toxigenic V. cholerae genomes revealed temporal dynamics of SXT ICEs and the unexpected persistence of strains lacking SXT ICEs. Genetic dissection and interrogation of the three dominant SXT ICEs in V. cholerae identified the defense systems responsible for restricting lytic phages. Rare clinical strains lacking an SXT ICE were highly susceptible to phage attack but did not incur the SXT ICE–mediated fitness cost associated with restricting beneficial MGEs. Phages in clinical samples were found to overcome cocirculating SXT ICEs through two distinct mechanisms: epigenetic escape from restriction modification, or a novel anti-BREX inhibitor protein, OrbA. After sequencing of clinical phages over a 34-month surveillance period, circulating phages with a deletion compromising expression of orbA were found only when the BREX-encoding SXT ICE was not circulating. SXT ICEs are self-transmissible to new taxa, conferring phage defense to new hosts; this is noteworthy because near-identical SXT ICEs are found in multiple taxa. Certain antibiotics stimulate high-frequency transfer of SXT ICEs, prompting us to test whether infection by phages that can overcome SXT ICE-mediated defense could similarly stimulate conjugation. We found that productive phage infection results in high-frequency transfer of SXT ICEs, leading to the concurrent dissemination of phage and antibiotic resistances. CONCLUSION SXT ICEs determine phage resistance in clinical V. cholerae, selecting for phage-encoded counteradaptations that can be lost as SXT ICEs fluctuate in dominance. A heterogeneous pool of SXT ICEs constitutes a “pan-immune” system that is able to restrict all phages and MGEs tested. SXT(–) V. cholerae may confer a population-wide benefit by limiting epigenetic escape, leading to subsequent bottlenecks that maintain the antiphage benefits afforded by SXT ICEs. Our work links phage and antibiotic resistances together on a single mobile genetic element, whose transfer is stimulated by phage infection. SXT ICEs determine phage resistance in toxigenic V. cholerae. SXT ICEs fluctuate in clinical V. cholerae (top left). SXT(–) isolates are sensitive to phages, and SXT ICEs restrict phages from the past or future but not contemporaneous phages with counteradaptations (below). Upon dissemination, which is stimulated by phage infection, SXT ICEs confer phage and antibiotic resistances to new hosts (right). Bacteriophage predation selects for diverse antiphage systems that frequently cluster on mobilizable defense islands in bacterial genomes. However, molecular insight into the reciprocal dynamics of phage-bacterial adaptations in nature is lacking, particularly in clinical contexts where there is need to inform phage therapy efforts and to understand how phages drive pathogen evolution. Using time-shift experiments, we uncovered fluctuations in Vibrio cholerae’s resistance to phages in clinical samples. We mapped phage resistance determinants to SXT integrative and conjugative elements (ICEs), which notoriously also confer antibiotic resistance. We found that SXT ICEs, which are widespread in γ-proteobacteria, invariably encode phage defense systems localized to a single hotspot of genetic exchange. We identified mechanisms that allow phage to counter SXT-mediated defense in clinical samples, and document the selection of a novel phage-encoded defense inhibitor. Phage infection stimulates high-frequency SXT ICE conjugation, leading to the concurrent dissemination of phage and antibiotic resistances.