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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  aac(2′)-If  from an isolate of a novel  Providencia  species,  Providencia wenzhouensis  R33 (CCTCC AB 2021339). Susceptibility testing and enzyme kinetic parameter analysis were conducted to determine the function of the aminoglycoside 2′- N -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  aac(2′)-If  (pUCP20- aac(2′)-If /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  aac(2′)-If  with the highest catalytic efficiency for ribostamycin ( k  cat  /K  m    ratio = [3.72 ± 0.52] × 10 4  M –1  ⋅ s –1 ). Whole-genome sequencing demonstrated that the  aac(2′)-If  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  Providencia  species will help us find ways to elucidate the complexity of resistance mechanisms in the microbial population.

frontiers in microbiology

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