From Tolerance to Resistance: Adaptive Pathways of Enterobacterales Persistence in the Gut Under Antibiotic Pressure

About This Grant

1 PROJECT SUMMARY and ABSTRACT 2 Hematopoietic stem cell transplantation (HCT) cures hematologic malignancies, but infection-related mortality 3 remains high, with bloodstream infections (BSIs) accounting for up to one-quarter of deaths in the first year. 4 Enterobacterales translocation from the gut is the primary source of these infections. While antibiotic (ABX) 5 prophylaxis reduces BSI risk, incomplete clearance of potential pathogens in the gut allows susceptible strains 6 to persist and acquire AMR under ongoing pressure, further limiting ABX effectiveness. Gut Enterobacterales 7 often persist despite in vitro susceptibility, suggesting a survival mechanism beyond resistance that remains 8 underexplored. 9 We hypothesize that antibiotic tolerance—the ability of bacteria to survive lethal antibiotic concentrations without 10 a change in minimum inhibitory concentration—is the key driver of Enterobacterales persistence in the gut and 11 a precursor to AMR. Preliminary data show that gut-resident E. coli and K. pneumoniae persist in almost two- 12 thirds of HCT patients despite antibiotic use and that tolerance levels rise during ABX and decrease after 13 withdrawal. We find that recurrent mutations in tolerance loci such as relA, hipA, and ptsI occur during ABX 14 treatment, and that tolerant strains acquire resistance more rapidly under antibiotic pressure in vitro. 15 To test this hypothesis, we will combine culture-based and genomic approaches across two large HCT cohorts. 16 In Aim 1 we will selectively culture E. coli and K. pneumoniae from stool samples and quantify tolerance using 17 high-throughput screening (TD test) and standardized time-kill assays with multiple antibiotics to measure both 18 isolate- and population-level survival. In Aim 2 we will identify genetic determinants of tolerance by sequencing 19 paired stool metagenomes and isolates, tracking the emergence of single-nucleotide variants in known 20 tolerance genes, and performing bacterial genome-wide association studies (GWAS) to discover novel loci. 21 Candidate genes will be validated through plasmid complementation and functional assays. In Aim 3 we will 22 link tolerance to clinical outcomes by integrating stool and bloodstream isolate sequencing with longitudinal 23 antibiotic exposure data in order to determine whether tolerant strains predict BSIs and accelerate acquisition 24 of phenotypic or genotypic AMR. 25 This project will be the first to investigate the reservoir of antibiotic tolerance in the human gut microbiome of 26 immunocompromised patients. Using complementary microbiology and isolate/stool genomics, we will directly 27 link in vivo tolerance phenotypes to genetic mechanisms and clinical outcomes. Establishing how tolerance 28 enables Enterobacterales to persist in the gut, seed bloodstream infections, and accelerate resistance in HCT 29 patients likely has impact for other vulnerable groups. By identifying tolerance as a critical determinant of infection 30 and AMR risk this work will identify novel strategies in overcoming tolerance to improve pathogen clearance, limit 31 multidrug-resistant transmission, and reduce infection-related mortality in immunocompromised patients.