Phenotypic Differentiation Within the ' Aminoglycoside Resistance Gene Family Suggests a Novel Subtype IV of Contemporary Clinical Relevance.

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 ' 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 ' genes. Seventeen unique ' amino acid sequences were cloned and expressed under defined promoter control in otherwise isogenic cells for phenotypic analysis with twenty distinct aminoglycoside antibiotics. A panel of clinical isolates was analyzed for the genotype-phenotype correlation of '.

RESULTS

An ' resistance gene annotation was found in 139,236 (21.2%) of the clinical isolates analyzed. AMK resistance-conferring ' genes dominated in (28.5%). In and , a gene conferring the ' phenotype but annotated as ' was the most prevalent. None of the ' genes were annotated as subtype III, but gene ' identified in Gram-positive isolates displayed a subtype III phenotype. Genes that were annotated as ' in 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 ' 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).