Polymorphism of antibiotic-inactivating enzyme driven by ecology expands the environmental resistome

Abstract

The environmental resistome has been recognized as the origin and reservoir of antibiotic resistance genes and considered to be dynamic and ever expanding. In this study, a targeted gene sequencing approach revealed that the polymorphic diversity of the aminoglycoside-inactivating enzyme AAC(6')-Ib was ecological niche-specific. AAC(6')-Ib-cr, previously known as a clinical variant, was prevalent in various soils and the intestines of chickens and humans, suggesting that this variant might not have arisen from adaptive mutations in the clinic but instead originated from the environment. Furthermore, ecologically dominant polymorphic variants of AAC(6')-Ib were characterized and found to display different substrate specificities for quinolones and aminoglycosides, conferring the altered resistance spectra. Interestingly, a novel variant with the D179Y substitution showed an extended resistance spectrum to the recently developed fluoroquinolone gemifloxacin. Our results suggest that soil and animal microbiomes could be major reservoirs of antibiotic resistance; polymorphic diversity expands the antibiotic resistome in the environment, resulting in the potential emergence of novel resistance.

Revisiting Polymorphic Diversity of Aminoglycoside N-Acetyltransferase AAC(6′)-Ib Based on Bacterial Genomes of Human, Animal, and Environmental Origins

“…Among >130,000 bacterial genomes available in March 2018 from the NCBI genome database, including both complete and draft genomes, a total of 3,964 aac(6′)-Ib gene sequences (370 and 3,594 from complete and draft genome databases, respectively) were found to be present in the bacterial genomes. The relative abundance and Shannon entropy analysis of polymorphism in the AAC(6′)-Ib proteins revealed that three residues (102, 117, and 179) were prominent polymorphic residues ( Figure 1 ), as previously shown by the metagenomic studies conducted at various environmental sites ( Kim et al, 2018 ). Composition of AAC(6′)-Ib variants (Ib, IbL, and cr) were highly similar in the complete and draft genome databases ( Supplementary Figure 1 ).…”

“…Moreover, in this study, the presence of the cr variant was found to be correlated with the presence of IbL variant ( R 2 = 0.88) ( Supplementary Figure 2 ), suggesting that the mutation at L117 might be a prerequisite for the emergence of the cr variant, and evolution of these variants might be associated with each other. Other polymorphic variants of aac(6 ′ )-Ib such as R102/L117 and L117/Y179 variants, which were previously characterized to confer altered resistance spectra ( Kim et al, 2018 ), were detected mainly in the genera Klebsiella and Enterobacter ( Supplementary Table 1 ), indicating that these novel variants discovered in various environments were indeed present in clinical isolates.…”

“…aac(6′)-Ib gene sequences, searched from databases, were aligned with the reference gene and translated. After translation, Shannon entropy values ( H′ ) and the relative abundance of polymorphic amino acids from residues 54 to 186 of AAC(6′)-Ib proteins were calculated as described previously ( Iwai et al, 2010 ; Kim et al, 2018 ). Based on the major polymorphic residues 102, 117, and 179, all sequences were classified as follows: Ib (W102, S117, and D179), IbL (W102, L117, and D179), cr (R102, L117, and Y179), and others (other polymorphic variants) ( Kim et al, 2018 ).…”

“…A variant harboring the mutation S117L conferred increased resistance to amikacin and reduced resistance to gentamicin, as shown by changes in the minimal inhibitory concentration values, from 2 to 16 mg/L for amikacin and from 4 to 0.25 mg/L for gentamicin ( Rather et al, 1992 ). Recently, this mutation was found to be conserved in the cr variants of environmental origin, indicating the presence of three mutated residues in the cr variant ( Kim et al, 2018 ). The cr variant is known to be usually located in a cassette as part of an integron in a multi-resistance plasmid, and considered as plasmid-mediated quinolone resistance (PMQR) ( Jacoby et al, 2014 ).…”

“…In our recent study, targeted gene sequencing analysis of aac(6′)-Ib gene from various DNA samples, including river, wastewater, chicken and human intestines, and soils, revealed that prominent polymorphisms occurred at the three mutated residues in the cr variant, and that this variant was dominant in soil and intestine samples, suggesting that the distribution of polymorphic variants was ecological niche-specific ( Kim et al, 2018 ). Although this study was based on metagenomic DNA sequences and hence taxonomic information was absent, the results provided an overview of the prevalence of polymorphic variants at various ecological origins.…”