Identification and Characterization of a Novel Chromosomal Aminoglycoside 2′-N-Acetyltransferase, AAC(2′)-If, From an Isolate of a Novel Providencia Species, Providencia wenzhouensis R33

Abstract

Multidrug-resistant bacteria from different sources have been steadily emerging, and an increasing number of resistance mechanisms are being uncovered. In this work, we characterized a novel resistance gene named <i>aac(2')-If</i> from an isolate of a novel <i>Providencia</i> species, <i>Providencia wenzhouensis</i> R33 (CCTCC AB 2021339). Susceptibility testing and enzyme kinetic parameter analysis were conducted to determine the function of the aminoglycoside 2'-<i>N</i>-acetyltransferase. Whole-genome sequencing and comparative genomic analysis were performed to elucidate the molecular characteristics of the genome and the genetic context of the resistance gene-related sequences. Among the functionally characterized resistance genes, AAC(2')-If shares the highest amino acid sequence identity of 70.79% with AAC(2')-Ia. AAC(2')-If confers resistance to several aminoglycoside antibiotics, showing the highest resistance activity against ribostamycin and neomycin. The recombinant strain harboring <i>aac(2')-If</i> (pUCP20-<i>aac(2')-If</i>/DH5α) showed 256- and 128-fold increases in the minimum inhibitory concentration (MIC) levels to ribostamycin and neomycin, respectively, compared with those of the control strains (DH5α and pUCP20/DH5α). The results of the kinetic analysis of AAC(2')-If were consistent with the MIC results of the cloned <i>aac(2')-If</i> with the highest catalytic efficiency for ribostamycin (<i>k _<i>cat</i> /K _<i>m</i> </i> ratio = [3.72 ± 0.52] × 10⁴ M^-1 ^⋅s^-1). Whole-genome sequencing demonstrated that the <i>aac(2')-If</i> gene was located on the chromosome with a relatively unique genetic environment. Identification of a novel aminoglycoside resistance gene in a strain of a novel <i>Providencia</i> species will help us find ways to elucidate the complexity of resistance mechanisms in the microbial population.