Phenotypic and genotypic characterisation of thymine auxotrophy in Escherichia coli isolated from a patient with recurrent bloodstream infection

Introduction Thymine auxotrophic in vitro mutants of Escherichia coli were first reported in the mid-20th century. Later, thymine-dependent clinical strains of E. coli as well as other Enterobacterales, Enterococcus faecalis and Staphylococcus aureus have been recognized as the cause of persistent and recurrent infections. Objectives The aim of this study was to characterize the phenotype and investigate the molecular basis of thymine auxotrophy in ten E. coli isolates obtained at different time points from a patient with recurrent bloodstream infection (BSI) due to a chronic aortic graft infection treated with Trimethoprim/sulfamethoxazole (TMP-SMX). Methods Clinical data was obtained from hospital records. Growth characterization and antimicrobial susceptibility testing to TMP-SMX was performed on M9 agar and in MH broth with different thymine concentrations (0.5, 2, 5, 10 and 20 mu g/ mL), on Mueller-Hinton (MH) and blood agar. Whole genome sequencing (WGS) was performed on all E. coli isolates. Results E. coli were isolated from ten consecutive BSI episodes from a patient with chronic aortic graft infection. Six of these isolates were resistant to TMP-SMX when assayed on blood agar. Growth experiments with added thymine confirmed that these isolates were thyminedependent (thy-), and revealed growth defects (slower growth rate and smaller colony size) in these isolates relative to thy+ isolates (n = 4). WGS indicated that all isolates were of the same clonal lineage of sequence type 7358. Genomic analysis revealed a G172C substitution in thyA in all TMP-SMX resistant isolates, while mutations affecting genes involved in the deoxyribose salvage pathway (deoB and deoC) were identified in eight isolates. Conclusion This case highlights the risk of resistance development to TMP-SMX, especially for longterm treatment, and the possible pitfalls in detection of growth-deficient subpopulations from chronic infections, which could lead to treatment failure.

Automated summary

This study characterized the phenotype and molecular basis of thymine auxotrophy in E. coli isolates from a patient with recurrent bloodstream infection. The isolates showed resistance to TMP-SMX and growth defects. Genomic analysis identified gene mutations associated with resistance. This highlights the risk of resistance development and the challenges in detecting growth-deficient subpopulations in chronic infections.