Enhanced resistance to macrolides and tetracyclines due to cooperation among plasmid-encoded genes associated with...

Antibiotic resistance genes associated with IS26-family mobile genetic elements are globally distributed in the Gram-negative plasmidome. In many cases, antibiotic resistance genes are co-located with genes of unknown function. Two particular IS26-associated gene clusters, mph(A)-mrx-mphR(A) and cysH-eamA-tet(A)-tetR(A), are prevalent on plasmids and chromosomes of common Gram-negative pathogens. While mph(A) encodes a macrolide phosphotransferase and tet(A) encodes a tetracycline efflux pump, the roles, if any, of mrx, cysH, and eamA remain poorly understood. To test the hypothesis that these genes consistently co-located with mph(A) and tet(A) act cooperatively to enhance antibiotic resistance, recombinant E. coli strains carrying various genetic combinations from each cluster were subjected to antimicrobial susceptibility testing. While the mph(A) gene alone provided minimal protection from azithromycin, inclusion of mrx increased resistance by at least 48-fold. Disruption of mrx by the insertion of a nonsense mutation or the addition of an efflux pump inhibitor abrogated this effect, indicating that the Mrx protein is a drug exporter that is required for maximal effect. In the absence of mph(A), mrx had no effect on azithromycin resistance, suggesting that Mrx only effluxes the phosphorylated form of azithromycin. Similarly, inclusion of cysH-eamA with tet(A) increased resistance to tetracycline antibiotics by at least 2-fold over tet(A) alone. Disruption of either cysH or eamA had no effect on this enhancement, but disruption of both genes eliminated it entirely. A more complete understanding of antibiotic resistance among Gram-negative bacteria requires elucidation of the roles of modulator genes co-located with primary resistance genes on mobile genetic elements.