Functional characterization of the Est-type macrolide esterase ECO-1 from Escherichia coli

Abstract

Macrolides are widely used in both human and veterinary medicine, and enzymatic inactivation represents an important yet underexplored resistance mechanism beyond the well-studied Ere family. Here, we functionally characterized ECO-1, an Est-type macrolide esterase identified from Escherichia coli . Phylogenetic analysis placed ECO-1 as a distinct branch within the α/β-hydrolase superfamily, showing low sequence identity (37.8–46.7%) to previously characterized macrolide esterases. Heterologous expression of eco-1 in E. coli selectively increased resistance to 16-membered macrolides, with marked MIC elevation for tylosin (32-fold), tildipirosin (8-fold) and tilmicosin (4-fold), while showing minimal effects on 14- and 15-membered macrolides. Consistently, IC₅₀ values for five 16-membered macrolides (tylosin, tilmicosin, tildipirosin, spiramycin and kitasamycin) increased in ECO-1-expressing strains. Using purified recombinant ECO-1, agar diffusion assays demonstrated substantial loss of antibacterial activity after enzyme treatment. ESI-MS further confirmed hydrolysis products with a characteristic + 18 Da mass shift for all five substrates, supporting ester bond cleavage as the inactivation mechanism. Genomic context analysis revealed eco-1 embedded in a conserved multidrug resistance region associated with Tn3-family transposition and co-localized with clinically relevant resistance determinants including bla TEM-1 and tetracycline resistance genes ( tet(B)/tetR(B)/tet(C) ). A large-scale database search identified 136 ECO-1—positive genomes across 14 countries, predominantly in E. coli and Salmonella enterica , spanning animal, food, human, and environmental sources. Collectively, ECO-1 expands the repertoire of Est-type macrolide esterases with selective activity toward 16-membered macrolides and highlights the potential dissemination risk associated with mobile resistance gene clusters.