Identification and characteristics of a novel aminoglycoside phosphotransferase, APH(3′)-IId, from an MDR clinical isolate of Brucella intermedia

Abstract

Objectives To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3′)-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. Methods Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. Results APH(3′)-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3′)-IIb. Expression of aph(3′)-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3′)-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3′)-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3′)-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from ∼105 to 107 M−1 s−1. Genetic environment and comparative genomic analyses suggested that aph(3′)-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. Conclusions APH(3′)-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3′)-IId represents the fourth characterized example of an APH(3′)-II enzyme.

Nanopore Sequencing Using the Full-Length 16S rRNA Gene for Detection of Blood-Borne Bacteria in Dogs Reveals a Novel Species of Hemotropic Mycoplasma

“…In addition, a diverse range of putative pathogenic bacteria that show evidence in the literature of being opportunistic pathogens with disease-causing potential in humans were also detected by our method at read counts above the contaminant read threshold ( Table 4 ). Environmental bacteria that can opportunistically cause infections in humans, such as Brucella intermedia ( 57 – 59 ), Burkholderia gladioli ( 60 – 63 ), Haemophilus parainfluenzae ( 64 – 68 ), Paracoccus yeei, and Psychrobacter meningitidis ( 69 – 71 ), were detected from our Cambodian dog blood samples. Moreover, bacterial species that may be associated with the canine oral microbiome, and etiological agents responsible for canine bite wound infections, were also identified, including Pasteurella canis ( 72 – 74 ) and Neisseria animaloris ( 75 , 76 ).…”

ANT(9)-Ic, a Novel Chromosomally Encoded Aminoglycoside Nucleotidyltransferase from Brucella intermedia

“…In a recent study, we isolated a multidrug-resistant B. intermedia strain ZJ499 from the sputum of a cancer patient at a university hospital in Lishui, China ( 16 ). This strain possessed broad-spectrum resistance to most β-lactams, aminoglycosides, polymyxins, phenicols, and diaminopyrimidines ( 16 ).…”

“…In a recent study, we isolated a multidrug-resistant B. intermedia strain ZJ499 from the sputum of a cancer patient at a university hospital in Lishui, China ( 16 ). This strain possessed broad-spectrum resistance to most β-lactams, aminoglycosides, polymyxins, phenicols, and diaminopyrimidines ( 16 ). To investigate the resistance mechanisms of ZJ499, we have previously conducted whole-genome sequencing and identified several potentially novel antibiotic resistance genes in this isolate, including those predicted to provide resistance to aminoglycosides (for additional information, see reference 16 ).…”

“… Brucella intermedia (previously known as Ochrobactrum intermedium ) ( 13 ) is a Gram-negative bacillus that inhabits diverse terrestrial and aquatic environments and is considered to be an opportunistic human pathogen due to its ability to cause infections in both immunocompromised and immunocompetent hosts ( 14 , 15 ). In a previous study, we reported the results of a genome-based screening for antibiotic resistance genes in a clinical isolate of B. intermedia ( 16 ). Here, we describe the analysis of one of those putative resistance determinants, designated ant(9)-Ic .…”

Functional characterization of a novel aminoglycoside phosphotransferase, APH(9)-Ic, and its variant from Stenotrophomonas maltophilia

“…The kinetic assay used to monitor APH(9)-Ic activity was performed as reported previously ( Lu et al., 2021 ). Briefly, kinetic parameters for APH(9)-Ic activity were determined by a continuous spectrophotometric assay, which coupled the production of ADP generated upon AG phosphorylation to the oxidation of NADH using the enzymes pyruvate kinase (PK) and lactate dehydrogenase (LD).…”