![]() It is an example of convergent evolution to exploit lactone hydrolysis for macrolide inactivation. The discovery of a novel macrolide esterase is remarkable. Robust characterization of EstT and homologs should be completed to better define the substrate specificities of these enzymes. ![]() The limited hydrolysis observed for S126A:macrolide reactions may have resulted from water acting as a nucleophile in the variant active site, calling for further investigation of the catalytic mechanism. In control experiments, no hydrolysis of tilmicosin was observed using cytosolic Escherichia coli proteins that were adventitiously enriched by IMAC, and the hydrolytic product appeared to be relatively stable ( Fig. 1 I). In contrast, the 14- and 15-atom–containing macrolides were not hydrolyzed by EstT. These macrolides were rapidly and completely converted to products showing mass shifts of 18 amu, consistent with the hydrolysis of the macrocyclic lactone. The 16-membered ring–containing macrolides tylosin, tilmicosin, and tildipirosin were EstT substrates ( Fig. 1 H). The products of EstT and EstT S126A-catalyzed reactions were analyzed by high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) and mass spectrometry (MS Fig. 1 G). Six macrolides-erythromycin, tulathromycin, gamithromycin, tylosin, tilmicosin, and tildipirosin-were tested as substrates based on variations in the size of their macrocycles from 14 to 16 atoms. Two forms were produced by Escherichia coli, and the lower molecular weight form (EstT 25–306) was characterized. Next, the α/β-hydrolase, which we named EstT, and the S126A variant were purified for a biochemical assay ( Fig. 1 F). Phenotypic screening of Escherichia coli transformed with the α/β-hydrolase showed a ≥32- or ≥4-fold increase in the minimum inhibitory concentration (MIC) of tildipirosin or tilmicosin compared to transformants carrying a catalytically inactive S126A variant ( Fig. 1 A). To evaluate the hypothetical role of the α/β-hydrolase in AMR, the gene and 194-bp upstream sequence were transformed into Escherichia coli DH5α using a high-copy plasmid. ( I) Chromatograms and MS showing control reactions: Escherichia coli IMAC impurities do not hydrolyze tilmicosin and the ring-opened product is relatively stable. ( H) Structure of EstT substrates and the proposed reaction. Representative results are shown for tylosin and tilmicosin after 30 min, whereas the other four reactions were observed after 4 h. ( G) HPLC/UV, HPLC/MS chromatograms, and mass spectra (MS) showing EstT and S126A reactions with six distinct macrolides: The 16-atom–containing macrolides are substrates of EstT. The purified form (red arrow) is EstT 25–306 (N-terminal sequence: MKEKI). Lanes are labeled for uninduced (UI) and induced (I) cultures, soluble (S), and insoluble (IS) fractions, IMAC flow-through (FT), IMAC elution (E), and pooled ~90% pure protein after SEC. ( F) SDS-PAGE analysis showing the purification of EstT and purified EstT S126A. ( E) Representative estT-containing loci with ARGs and transposases annotated. ( D) Prevalence of estT in bacterial pathogens: BRD pathogens ( Histophilus somni, Hs Mannheimia haemolytica, Mh Pasteurella multocida, Pm), the bird pathogen Riemerella anatipestifer ( Ra) Escherichia coli ( Ec) found in slaughterhouses where tet(X4) is prevalent, and Acinetobacter spp. ( C) Sample origins of bacteria with an EstT homolog (136 unique sequences >70% identity). faecium WB1 plasmid pWB1 map and ARG cluster showing the α/β-hydrolase (named estT, red). faecium WB1 and Escherichia coli transformants. Importantly, concordance between ARGs found in an organism recovered from watering bowls and relevant pathogens highlights the pragmatism of this approach.įig. The physical location of the α/β-hydrolase near ARGs, transposases, and/or on plasmids explains its cosmopolitan distribution and presence even in the human microbiome ( Fig. 1 E). Moreover, this α/β-hydrolase co-occurs with tet(X4), an emergent tigecycline resistance gene ( 4, 5). A retrospective analysis of relevant sequencing projects confirmed the high prevalence of the α/β-hydrolase in two known causative agents of bovine respiratory disease (BRD) ( 1) and bacteria infecting poultry and swine ( 2, 3). faecium WB1 genome includes a 17.5-kb plasmid with a cluster of three ARGs and an unannotated α/β-hydrolase ( Fig. 1 B) A search for homologs showed that this orphaned gene occurs in animal microbiomes and pathogen genomes ( Fig. 1 C and D). Selective cultivation of multidrug-resistant bacteria from watering bowls at a western Canadian feedlot resulted in the isolation of Sphingobacterium faecium WB1 ( Fig. 1 A).
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