Phenotypic Differentiation Within the aac(6' ) Aminoglycoside Resistance Gene Family Suggests a Novel Subtype IV of Contemporary Clinical Relevance

Abstract

Background: Whole genome sequencing of clinical bacterial isolates holds promise in predicting their susceptibility to antibiotic therapy, based on a detailed understanding of the phenotypic manifestation of genotypic variation. The aac(6') aminoglycoside acetyltransferase gene family is the most abundant aminoglycoside resistance determinant encountered in clinical practice. A variety of AAC(6') isozymes have been described, suggesting a phenotypic distinction between subtype I, conferring resistance to amikacin (AMK), and subtype II, conferring resistance to gentamicin (GEN) instead. However, the epidemiology and thus clinical relevance of the various and diverse isozymes and their phenotypic distinction demand systematic and contemporary re-assessment to reliably predict bacterial susceptibility to aminoglycoside antibiotics.

Methods: We analyzed the resistance gene annotations of 657,603 clinical bacterial isolates to assess the prevalence and diversity of aac(6') genes. Seventeen unique aac(6') amino acid sequences were cloned and expressed under defined promoter control in otherwise isogenic E. coli cells for phenotypic analysis with twenty distinct aminoglycoside antibiotics. A panel of clinical isolates was analyzed for the genotype-phenotype correlation of aac(6').

Results: An aac(6') resistance gene annotation was found in 139,236 (21.2%) of the clinical isolates analyzed. AMK resistance-conferring aac(6')-I genes dominated in Enterobacterales (28.5%). In Pseudomonas aeruginosa and Acinetobacter baumannii, a gene conferring the aac(6')-II phenotype but annotated as aac(6')-Ib₄ was the most prevalent. None of the aac(6') genes were annotated as subtype III, but gene aac(6')-Ii identified in Gram-positive isolates displayed a subtype III phenotype. Genes that were annotated as aac(6')-Ib₁₁ in Enterobacterales conferred resistance to both AMK and GEN, which we propose constitutes a novel subtype IV when applying established nomenclature. A phenotypic assessment facilitated structural re-assessment of the substrate promiscuity of AAC(6') enzymes.

Conclusions: Our study provides the most comprehensive analysis of clinically relevant aac(6') gene sequence variations to date, providing new insights into a differentiated substrate promiscuity across the genotypic spectrum of this gene family, thus translating into a critical contribution towards the development of amino acid sequence-based in silico antimicrobial susceptibility testing (AST).

Keywords: acetyltransferases; aminoglycoside antibiotics; in silico antimicrobial susceptibility testing; phenotype prediction; resistance gene annotation; surveillance; whole genome sequencing.

Figure 1

Clinical relevance of individual aac(6′) genes. Prevalence of aac(6′) gene annotations in 657,603 bacterial clinical isolates deposited in the NDARO on 1 July 2024. Gene and protein names were assigned according to the NDARO reference gene catalogue.

Figure 2

Amino acid sequence homology of individual aac(6′) genes. Unrooted phylogenetic tree of AAC(6′) amino acid sequences depicting the homology between clinically relevant isozymes and other subtypes previously described. Genes subcloned for phenotypic analysis are indicated in red font.

Figure 3

Phenotypic assessment of aac(6′) genes in engineered strains and clinical isolates. (a) Subtyping of representative aac(6′) genes by the changes in susceptibility to AMK and GEN relative to the wild-type (WT) laboratory strain DH5α. An interpretative threshold of > 2-fold increase in MIC is indicated by dotted lines; (b) manifestation of the corresponding phenotypes in clinical isolates not expressing aminoglycoside-modifying enzymes other than aac(6′). The size of the symbols corresponds to the number of isolates, and clinical breakpoints are indicated by dotted lines. (c,d) Heat map visualization of the differential activity of aac(6′) gene variants for individual gentamicin congeners (c) and fifteen additional aminoglycoside antibiotics (d) displayed as fold-increase over wild-type MIC.

Figure 4

Structural comparison of AAC(6′)-Ib₁₁ with AAC(6′)-Ib. (A) Ribbon structure of AAC(6′)-Ib with bound kanamycin B (yellow sticks) and co-enzyme A (PDB ID: 2qir); (B) ribbon structure of AAC(6′)-Ib₁₁ (PDB ID: 2pr8), showing the loop rearrangement (arrows) by amino acid substitutions Q101L, L102S relative to AAC(6′)-Ib, which results in an extended substrate promiscuity; (C) closeup view of kanamycin B (yellow sticks) and co-enzyme A (covered) in the substrate-binding pocket of AAC(6′)-Ib (represented as surface structure), highlighting the steric limitations for 1-N-substitutions superimposed onto kanamycin; (D,E) closeup views of two possible amikacin orientations (cyan sticks) modelled into the substrate-binding pocket of AAC(6′)-Ib₁₁ using AutoDock Vina. The 2-deoxystreptamine ring of amikacin needs to be rotated around the O4-C1′-glycosidic bond when compared to kanamycin to permit accommodation of its 1-N-LHABA substitution marked by the black circles. The loop rearrangement in AAC(6′)-Ib₁₁ results in a cleft (indicated by black arrows) in the binding pocket of co-enzyme A (sky blue), suggesting a wider space than in AAC(6′)-Ib available to better accommodate the 6′-disubstitutions of gentamicin C1 and C2a.