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

Abstract

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 μg/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 thymine-dependent (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 long-term treatment, and the possible pitfalls in detection of growth-deficient subpopulations from chronic infections, which could lead to treatment failure.

Fig 1. Timeline of E. coli BSI episodes (above line) and TMP-SMX antimicrobial treatment (below line).

Isolated isolates are marked as susceptible (green) or resistant (red) to TMP-SMX. Mutations identified in thymidylate synthesis pathway genes include thyA G172C (black triangle), causing Glu58Gln in thymidylate synthase; deoB C801A (blue triangle), causing a premature stop codon in deoB; and a 515 bp deletion (Δ) in deoC (orange triangle).

Fig 2. Growth (OD₆₀₀) of E. coli BSI isolates at 37°C in liquid M9 medium with added thymine.

Fig 3. Classical auxotroph test of E. coli SO-ECO15-3 on M9 medium after 24 hours of incubation showing gradual increase in growth surrounding discs with increasing concentrations (10, 20 and 40 μg/mL as indicated) of thymine and no growth around the negative control (0 μg/mL).