Dataset Information

Identification and Characterization of a Novel Aminoglycoside 3''-Nucleotidyltransferase, ANT(3'')-IId, From Acinetobacter lwoffii.

ABSTRACT: A novel plasmid-encoded aminoglycoside 3''-nucleotidyltransferase ANT(3")-IId, was discovered in Acinetobacter lwoffi strain H7 isolated from a chick on an animal farm in Wenzhou, China. The whole-genome of A. lwoffii H7 consisted of one chromosome and five plasmids (pH7-250, pH7-108, pH7-68, pH7-48, and pH7-11). ant(3")-IId was identified as being encoded on pH7-250, sharing the highest amino acid identity of 50.64% with a function-known resistance gene, ant(3")-IIb (KB849358.1). Susceptibility testing and enzyme kinetic parameter analysis were conducted to determine the function of the aminoglycoside 3"-nucleotidyltransferase. The ant(3")-IId gene conferred resistance to spectinomycin and streptomycin [the minimum inhibitory concentration (MIC) levels of both increased 16-fold compared with the control strain]. Consistent with the MIC data, kinetic analysis revealed a narrow substrate profile including spectinomycin and streptomycin, with K _cat/K _m ratios of 4.99 and 4.45×10³M^-1 S^-1, respectively. Sequencing analysis revealed that the ant(3")-IId gene was associated with insertion sequences (IS) element [ΔISAba14-ΔISAba14-hp-orf-orf-orf1-ant(3")-IId], and ant(3")-IId were identified in plasmids from various Acinetobacter species. This study of the novel aminoglycoside 3"-nucleotidyltranferase ANT(3")-IId helps us further understand the functional and sequence characteristics of aminoglycoside 3"-nucleotidyltranferases, highlights the risk of resistance gene transfer among Acinetobacter species and suggests that attention should be given to the emergence of new aminoglycoside 3"-nucleotidyltranferase genes.

SUBMITTER: Liang J

PROVIDER: S-EPMC8438517 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Identification and Characterization of a Novel Aminoglycoside 3''-Nucleotidyltransferase, ANT(3'')-IId, From Acinetobacter lwoffii.

Liang Jialei J Zhou Kexin K Li Qiaoling Q Dong Xu X Zhang Peiyao P Liu Hongmao H Lin Hailong H Zhang Xueya X Lu Junwan J Lin Xi X Li Kewei K Xu Teng T Zhang Hailin H Bao Qiyu Q Zhu Mei M Hu Yunliang Y Ren Ping P

Frontiers in microbiology 20210831

A novel plasmid-encoded aminoglycoside 3''-nucleotidyltransferase ANT(3")-IId, was discovered in Acinetobacter lwoffi strain H7 isolated from a chick on an animal farm in Wenzhou, China. The whole-genome of A. lwoffii H7 consisted of one chromosome and five plasmids (pH7-250, pH7-108, pH7-68, pH7-48, and pH7-11). ant(3")-IId was identified as being encoded on pH7-250, sharing the highest amino acid identity of 50.64% with a function-known resistance gene, ant(3")-IIb ...[more]

PMID: 34531844

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Project description:Aminoglycoside-modifying enzymes are among the most important mechanisms of resistance to aminoglycoside antibiotics, typically conferring high-level resistance by enzymatic drug inactivation. Previously, we isolated a multidrug-resistant Brucella intermedia strain ZJ499 from a cancer patient, and whole-genome sequencing revealed several putative novel aminoglycoside-modifying enzyme genes in this strain. Here, we report the characterization of one of them that encodes an intrinsic, chromosomal aminoglycoside nucleotidyltransferase designated ANT(9)-Ic, which shares only 33.05% to 47.44% amino acid identity with the most closely related ANT(9)-I enzymes. When expressed in Escherichia coli, ANT(9)-Ic conferred resistance only to spectinomycin and not to any other aminoglycosides tested, indicating a substrate profile typical of ANT(9)-I enzymes. Consistent with this, deletion of ant(9)-Ic in ZJ499 resulted in a specific and significant decrease in MIC of spectinomycin. Furthermore, the purified ANT(9)-Ic protein showed stringent substrate specificity for spectinomycin with a Km value of 44.83 μM and a kcat/Km of 2.8 × 104 M-1 s-1, echoing the above observations of susceptibility testing. In addition, comparative genomic analysis revealed that the genetic context of ant(9)-Ic was conserved in Brucella, with no mobile genetic elements found within its 20-kb surrounding region. Overall, our results demonstrate that ANT(9)-Ic is a novel member of the ANT(9)-I lineage, contributing to the intrinsic spectinomycin resistance of ZJ499. IMPORTANCE The emergence, evolution, and worldwide spread of antibiotic resistance present a significant global public health crisis. For aminoglycoside antibiotics, enzymatic drug modification is the most common mechanism of resistance. We identify a novel chromosomal aminoglycoside nucleotidyltransferase from B. intermedia, called ANT(9)-Ic, which shares the highest identity (47.44%) with the previously known ANT(9)-Ia and plays an important role in spectinomycin resistance of the host strain. Analysis of the genetic environment and origin of ant(9)-Ic shows that the gene and its surrounding region are widely conserved in Brucella, and no mobile elements are detected, indicating that ANT(9)-Ic may be broadly important in the natural resistance to spectinomycin of Brucella species.

Project description:BackgroundThe mechanisms underlying the resistance of the genus Providencia to aminoglycosides are complex, which poses a challenge for the efficient treatment of infectious diseases caused by these pathogens. To help clinicians treat infections more effectively, a more comprehensive understanding of antibiotic resistance mechanisms is urgently needed.MethodsPlates were streaked to isolate bacteria from the intestinal contents of fish. The standard agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of the antimicrobial agents. Molecular cloning was carried out to study the function of the novel antibiotic inactivation gene ant(9)-Id. The kinetic parameters of ANT(9)-Id were measured by a SpectraMax multifunctional microplate reader. Whole-genome sequencing and bioinformatic analysis were conducted to elucidate the sequence structure and evolutionary relationships of similar genes.ResultsThe novel aminoglycoside O-nucleotidyltransferase gene ant(9)-Id was encoded on the chromosome of a species-unclassified isolate designated Providencia sp. TYF-12, which was isolated from the intestine of a marine fish. Among the 11 aminoglycosides tested, ant(9)-Id was resistant to only spectinomycin. The MIC of spectinomycin for the recombinant strain carrying ant(9)-Id (pUCP20-ant(9)-Id/DH5α) increased 64-fold compared with that of the control strain (pUCP20/DH5ɑ). ANT(9)-Id shares the highest amino acid (aa) identity of 46.70% with the known drug resistance enzyme ANT(9)-Ic. Consistent with the MIC results, ANT(9)-Id showed high affinity and catalytic efficiency for spectinomycin, with a K m of 8.94 ± 2.50 μM and a k cat/K m of 26.15 μM-1·s-1. This novel resistance gene and its close homologs are conserved in Providencia strains from various sources, including some of clinical significance. No mobile genetic elements (MGEs) surrounding the ant(9)-Id(-like) genes were identified.ConclusionThis work revealed and characterized a novel spectinomycin resistance gene, ant(9)-Id, along with its biological features. Identifying novel resistance genes in pathogens can assist in rational medication use and the identification of additional antimicrobial resistance mechanisms in microbial populations.

Dataset Information

Identification and Characterization of a Novel Aminoglycoside 3''-Nucleotidyltransferase, ANT(3'')-IId, From Acinetobacter lwoffii.

Publications

Identification and Characterization of a Novel Aminoglycoside 3''-Nucleotidyltransferase, ANT(3'')-IId, From <i>Acinetobacter lwoffii</i>.

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