Abstract
Abstract
Background:
Antimicrobial resistance is a major global health threat, with β-lactamases representing the most prevalent mechanism of resistance to β-lactam antibiotics in Gram-negative bacteria. Among these, TEM β-lactamases (
bla
TEM
) are the earliest identified and most widely disseminated resistance determinants in
Escherichia coli
. Given the established influence of canonical promoter variation on
bla
TEM
expression and β-lactam resistance, evolutionary diversification of the
bla
TEM
upstream regulatory region can affect the resistance phenotype. However, the extent to which upstream variation is associated with resistance beyond changes in the coding sequence remains unclear.
Results:
We analyzed 60,087
E. coli
genomes from BV-BRC to characterize
bla
TEM
upstream regulatory regions, focusing on -35 and -10 promoter motifs and their relationship with β-lactam resistance. P3 was the prevalent canonical promoter, while multiple non-canonical promoter types with distinct -10 and -35 motifs were identified. A causal-inference framework estimated promoter-associated differences in resistance probability while holding the underlying
bla
TEM
variant profile constant. Average treatment effect estimates showed that several P3 transitions were associated with increased predicted resistance probabilities across multiple β-lactam antibiotics. Novel motifs from single-
bla
TEM
isolates were further prioritized by occurrence frequency and evaluated structurally with σ⁷⁰ DNA-recognition domains using molecular dynamics simulations. Notably, the -35 TGCAAC motif maintained consistent σ⁷⁰ interactions, supporting its structural compatibility as a putative promoter motif potentially relevant to
bla
TEM
regulation and resistance-associated phenotypes.
Conclusion:
Overall, this study integrates large-scale genomics, causal inference, and structural analysis to investigate regulatory variation alongside coding-sequence changes, providing insight into the evolving mechanisms of antimicrobial resistance.