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

Jialei Liang,Qiaoling Li,Hailin Zhang,Xu Dong,Peiyao Zhang,Mei Zhu,Hailong Lin,Yunliang Hu,Hongmao Liu,Kexin Zhou,Teng Xu,Qiyu Bao,Kewei Li,Xueya Zhang,Xi Lin,Junwan Lu,Ping Ren

doi:10.3389/fmicb.2021.728216

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 Kcat/Km 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.

Highlights

MATERIALS AND METHODSAcinetobacter lwoffii is a Gram-negative aerobic bacillus that inhabits the oropharynx, skin, and perineum (Rathinavelu et al, 2003)
The wild-type strain of Acinetobacter lwoffii H7 Escherichia coli DH5α was used as a host for cloning of the ant(3'')-IId gene Escherichia coli BL21 was used as a host for expression of the ant(3'')-IId gene Escherichia coli ATCC 25922 was used as a quality control for antimicrobial susceptibility testing DH5α carrying the recombinant plasmid pUCP20-ant(3'')-IId Escherichia coli C600 was used as the recipient in the conjugation experiment, RIFr Acinetobacter baumannii ATCC 19606 was used as the recipient in the conjugation experiment, AMPr
When analyzing the resistance mechanism of the isolate against the aminoglycoside antibiotic, we found an insert sequence (IS) element-related putative ant(3'')-II gene ( named ant(3'')-IId in this work) encoded on plasmid pH7-250 and cloned it to confirm whether it was functional

Summary

MATERIALS AND METHODS

Acinetobacter lwoffii (formerly Mima polymorpha, or Acinetobacter calcoaceticus var. lwoffii) is a Gram-negative aerobic bacillus that inhabits the oropharynx, skin, and perineum (Rathinavelu et al, 2003). We identified a novel aminoglycoside 3''-nucleotidyltransferase gene encoding an ANT(3'')-II-type enzyme, designated ANT(3'')-IId, encoded on a plasmid in A. lwoffii isolated from the environment. The wild-type strain of Acinetobacter lwoffii H7 Escherichia coli DH5α was used as a host for cloning of the ant(3'')-IId gene Escherichia coli BL21 was used as a host for expression of the ant(3'')-IId gene Escherichia coli ATCC 25922 was used as a quality control for antimicrobial susceptibility testing DH5α carrying the recombinant plasmid pUCP20-ant(3'')-IId Escherichia coli C600 was used as the recipient in the conjugation experiment, RIFr Acinetobacter baumannii ATCC 19606 was used as the recipient in the conjugation experiment, AMPr. Cloning vector for the PCR products of the ant(3'')-IId gene with its upstream promoter region, AMPr Expression vector for the PCR products of the ORF of the ant(3'')-IId gene, AMPr r, resistance; RIF, rifampin; and AMP, ampicillin. The nucleotide sequences of the chromosome, five plasmids (pH7-250, pH7-108, pH7-68, pH7-48, and pH7-11) of A. lwoffii H7 and the ant(3'')-IId gene reported in this study have been deposited in GenBank under accession numbers CP072549, CP072550, CP072551, CP072552, CP072553, CP072554, and MW984426, respectively

RESULTS AND DISCUSSION

DISCUSSION

CONCLUSION

DATA AVAILABILITY STATEMENT