Work

Cloning and molecular characterization of ant(9)-If: a novel aminoglycoside adenylyltransferase gene conferring resistance to spectinomycin

Year: 2026

Type: article

Abstract: Background Enterococcus is an opportunistic pathogen with the potential to induce infections in both animals and humans. The antimicrobial resistance mechanism of Enterococcus is highly complex, leading to challenges in the clinical treatment of infectious diseases caused by this pathogen. Consequently, a better understanding of antibiotic resistance mechanisms is urgently needed. Methods Sewage samples were obtained from animal farms, and the streaking plate technique was used to isolate single colonies. The whole-genome sequence of the bacterium was obtained using next-generation sequencing platforms. Antimicrobial minimum inhibitory concentrations (MICs) were determined using the standard agar dilution method. Molecular cloning was performed to explore the function of the antibiotic resistance gene. The protein was expressed, and its enzyme kinetic parameters were analyzed. Bioinformatic methods were used to examine the sequence structure and evolutionary dynamics of the resistance gene. Results In this study, a novel plasmid-encoded aminoglycoside resistance gene, ant(9)-If , was discovered in Enterococcus faecium 520 isolated from animal farm sewage. Compared with the control strain JH2-2/pAM401, the recombinant strain JH2-2/pAM401- ant(9)-If demonstrated a 32-fold increase in the MIC of spectinomycin. ANT(9)-If shares 64.70% amino acid identity with the drug resistance enzyme ANT(9)-Ia and demonstrated high affinity and catalytic efficiency for spectinomycin, characterized by a Michaelis constant ( K m ) of 4.599 ± 0.198 μM and a k cat / K m value of (8.78 ± 0.39) × 10 4 M –1 ⋅s –1 . Sequence analysis revealed that the novel resistance gene ant(9)-If is associated with a mobile genetic element (MGE) encoded on the plasmid. The ant(9)-If ( - like) genes are broadly distributed across plasmids and chromosomes in various Gram-positive cocci and bacilli, including those of clinically significant species. Conclusion In contrast to ant(9)-Ic and ant(9)-Id , which were identified from the chromosomes of Gram-negative bacilli, the novel aminoglycoside resistance gene ant(9)-If , associated with the MGE encoded on the plasmid, was identified in a Gram-positive coccus strain isolated from animal farm sewage. Deeper exploration of the antimicrobial resistance mechanisms of bacteria from various sources can aid in the development of effective treatments for infections in both animal husbandry and human healthcare. These studies could expedite the development of strategies to prevent the spread of resistance among bacteria from various sources.

Source: Frontiers in Microbiology

DOI: 10.3389/fmicb.2026.1776824