Tracking the progeny of bacterial persisters using a CRISPR-based genomic recorder

• 成果类型: Article
• 署名作者: Rycroft, Julian Anthony; Giorgio, Rachel Teresa; Sargen, Molly; Helaine, Sophie
• 署名单位: Harvard University; Harvard Medical School
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13894
• DOI: 10.1073/pnas.2405983121
• 发表日期: 2024-10-15
• 关键词: antibiotic tolerance; mycobacterium-tuberculosis; spacer acquisition; cells; salmonella; resistance; inactivation; replication; EVOLUTION; DYNAMICS

摘要

The rise of antimicrobial failure is a global emergency, and causes beyond typical genetic resistance must be determined. One probable factor is the existence of sub- populations of transiently growth- arrested bacteria, persisters, that endure antibiotic treatment despite genetic susceptibility to the drug. The presence of persisters in infected hosts has been successfully established, notably through the development of fluorescent reporters. It is proposed that infection relapse is caused by persisters resuming growth after cessation of the antibiotic treatment, but to date, there is no direct evidence for this. This is because no tool or reporter currently exists to track the extent to which infection relapse is initiated by regrowth of persisters in the host. Indeed, once they have transitioned out of the persister state, the progeny of persisters are genetically and phenotypically identical to susceptible bacteria in the population, making it virtually impossible to ascertain the source of relapse. We designed pSCRATCH (plasmid for Selective CRISPR Array expansion To Check Heritage), a molecular tool that functions to record the state of antibiotic persistence in the genome of Salmonella persisters. We show that pSCRATCH successfully marks persisters by adding spacers in their CRISPR arrays and the genomic label is stable in persister progeny after exit from persistence. We further show that in a Salmonella infection model the system enables the discrimination of treatment failure originating from persistence versus resistance. Thus, pSCRATCH provides proof of principle for stable marking of persisters and a prototype for applications to more complex infection models and other pathogens.