Epidemiologic, Phenotypic, and Structural Characterization of Aminoglycoside-Resistance Gene aac(3)-IV

Abstract

Aminoglycoside antibiotics are powerful bactericidal therapeutics that are often used in the treatment of critical Gram-negative systemic infections. The emergence and global spread of antibiotic resistance, however, has compromised the clinical utility of aminoglycosides to an extent similar to that found for all other antibiotic-drug classes. Apramycin, a drug candidate currently in clinical development, was suggested as a next-generation aminoglycoside antibiotic with minimal cross-resistance to all other standard-of-care aminoglycosides. Here, we analyzed 591,140 pathogen genomes deposited in the NCBI National Database of Antibiotic Resistant Organisms (NDARO) for annotations of apramycin-resistance genes, and compared them to the genotypic prevalence of carbapenem resistance and 16S-rRNA methyltransferase (RMTase) genes. The 3-N-acetyltransferase gene aac(3)-IV was found to be the only apramycin-resistance gene of clinical relevance, at an average prevalence of 0.7%, which was four-fold lower than that of RMTase genes. In the important subpopulation of carbapenemase-positive isolates, aac(3)-IV was nine-fold less prevalent than RMTase genes. The phenotypic profiling of selected clinical isolates and recombinant strains expressing the aac(3)-IV gene confirmed resistance to not only apramycin, but also gentamicin, tobramycin, and paromomycin. Probing the structure-activity relationship of such substrate promiscuity by site-directed mutagenesis of the aminoglycoside-binding pocket in the acetyltransferase AAC(3)-IV revealed the molecular contacts to His124, Glu185, and Asp187 to be equally critical in binding to apramycin and gentamicin, whereas Asp67 was found to be a discriminating contact. Our findings suggest that aminoglycoside cross-resistance to apramycin in clinical isolates is limited to the substrate promiscuity of a single gene, rendering apramycin best-in-class for the coverage of carbapenem- and aminoglycoside-resistant bacterial infections.

Keywords: aac(3)-IV; acetyltransferase; aminoglycosides; antimicrobial resistance; apramycin.

Figure 1

Genomic prevalence of the aac(3)-IV gene in Gram-negative clinical isolates in comparison to RMTase resistance genes. (a) Gene-annotation analysis of 182,405 clinical-isolate genomes deposited in NCBI National Database of Antibiotic Resistant Organisms (NDARO) on 24 June 2020. (b) Gene-annotation analysis in a subpopulation of 21,195 carbapenemase-positive (CP) clinical isolates. Gene aac(3)-IV confers resistance to gentamicin, tobramycin, and apramycin, among others. RMTases confer pan-aminoglycoside resistance to all 4,6-disubstituted deoxystreptamines comprising all standard-of-care aminoglycoside antibiotics, but remain susceptible to apramycin.

Figure 2

Crystal structure of AAC(3)-IV in complex with apramycin (PDB ID: 6MN4) or gentamicin (PDB ID: 6MN3). (a) Structural overview of full protein showing superimposition of bound substrates apramycin (magenta) and gentamicin (orange). Detailed view of (b) apramycin and (c) gentamicin in the binding pocket. Amino acids modified in this study are highlighted in gray. Residues D67, D187, and E249 are differently rotated in the apramycin versus the gentamicin structure. (d) Shortest intermolecular distances between amino acid side chains and ligand, corresponding to dashed lines in (b,c).

Figure 3

Functional assessment of native and mutant protein AAC(3)-IV. Phenotypic susceptibility to apramycin and gentamicin in recombinant E. coli strains displayed as fold-changes in minimal-inhibitory-concentration (MIC) relative to a susceptible wild-type strain lacking aac(3)-IV (mean ± SE; n = 4).