Tetracycline-inactivating enzymes from environmental, human commensal, and pathogenic bacteria cause broad-spectrum tetracycline resistance

Hirdesh Kumar,Chanez T Symister,Carey-Ann D Burnham,Sidra Irum,Meghan Wallace,Bin Wang,Gautam Dantas,Jana L Markley,Timothy A Wencewicz,Saadia Andleeb,Niraj H Tolia,Luting Fang,Andrew J Gasparrini

doi:10.1038/s42003-020-0966-5

Abstract

Tetracycline resistance by antibiotic inactivation was first identified in commensal organisms but has since been reported in environmental and pathogenic microbes. Here, we identify and characterize an expanded pool of tet(X)-like genes in environmental and human commensal metagenomes via inactivation by antibiotic selection of metagenomic libraries. These genes formed two distinct clades according to habitat of origin, and resistance phenotypes were similarly correlated. Each gene isolated from the human gut encodes resistance to all tetracyclines tested, including eravacycline and omadacycline. We report a biochemical and structural characterization of one enzyme, Tet(X7). Further, we identify Tet(X7) in a clinical Pseudomonas aeruginosa isolate and demonstrate its contribution to tetracycline resistance. Lastly, we show anhydrotetracycline and semi-synthetic analogues inhibit Tet(X7) to prevent enzymatic tetracycline degradation and increase tetracycline efficacy against strains expressing tet(X7). This work improves our understanding of resistance by tetracycline-inactivation and provides the foundation for an inhibition-based strategy for countering resistance.

Highlights

Tetracycline resistance by antibiotic inactivation was first identified in commensal organisms but has since been reported in environmental and pathogenic microbes
These included libraries constructed from 53 soil samples, 176 human gut microbiota, 2 animal gut microbiota, and 13 latrine samples, and encompassed a total of 912 Gb of metagenomic DNA
We focused on putative tetracycline inactivators in these selections by searching for open-reading frames annotated as encoding an FADbinding domain

Summary

Introduction

Tetracycline resistance by antibiotic inactivation was first identified in commensal organisms but has since been reported in environmental and pathogenic microbes. We identify and characterize an expanded pool of tet(X)-like genes in environmental and human commensal metagenomes via inactivation by antibiotic selection of metagenomic libraries. While tetracycline resistance most frequently occurs via efflux or ribosomal protection[8], enzymatic detoxification of tetracycline was first reported in 19889,10 This mechanism of resistance, originally detected in the commensal Bacteroides fragilis, has since been identified in broader commensals, environmental microorganisms, and pathogens. Based on sequence homology to these soil-derived enzymes, we identified an additional enzyme, Tet(56), as a previously uncharacterized tetracycline resistance determinant in Legionella longbeachae These enzymes are structurally and functionally homologous to Tet(X)[12], the flavin-dependent monooxygenase (FMO) originally discovered in Bacteroides fragilis[13]. Tet(X3) and Tet(X4), were discovered in Enterobacteriaceae and Acinetobacter strains isolated from animal and human sources

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Conclusion