Table 4_Identification and characterization of a novel chromosome-encoded aminoglycoside O-nucleotidyltransferase gene, ant(9)-Id, in Providencia sp. TYF-12 isolated from the marine fish intestine.docx

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 Km of 8.94 ± 2.50 μM and a kcat/Km 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.

研究背景：普罗威登斯菌属（Providencia）对氨基糖苷类（aminoglycosides）的耐药机制较为复杂，这为该类病原菌引发的感染性疾病的高效治疗带来了挑战。为助力临床医师更有效地开展感染治疗，当前亟需更为全面地阐明抗生素耐药机制。
研究方法：通过平板划线法从鱼类肠道内容物中分离细菌。采用标准琼脂稀释法测定抗菌药物的最低抑菌浓度（minimum inhibitory concentrations, MICs）。通过分子克隆技术探究新型抗生素灭活基因ant(9)-Id的功能。利用SpectraMax多功能微孔板检测仪测定ANT(9)-Id的动力学参数。通过全基因组测序与生物信息学分析，阐明同源基因的序列结构及进化关联。
研究结果：新型氨基糖苷类O-核苷酸转移酶基因ant(9)-Id位于一株未分类普罗威登斯菌（Providencia sp. TYF-12）的染色体上，该菌株分离自海水鱼肠道。在受试的11种氨基糖苷类药物中，ant(9)-Id仅对大观霉素（spectinomycin）表现出耐药性。携带ant(9)-Id的重组菌株（pUCP20-ant(9)-Id/DH5α）对大观霉素的最低抑菌浓度较对照菌株（pUCP20/DH5α）提升了64倍。ANT(9)-Id与已知耐药酶ANT(9)-Ic的氨基酸（amino acid, aa）同源性最高，达46.70%。与最低抑菌浓度实验结果一致，ANT(9)-Id对大观霉素展现出高亲和力与催化效率，其Km值为8.94±2.50 μM，kcat/Km值为26.15 μM⁻¹·s⁻¹。该新型耐药基因及其近缘同源基因在多种来源的普罗威登斯菌菌株中均保守存在，其中包含部分具有临床意义的菌株。未发现ant(9)-Id（类）基因周围存在可移动遗传元件（mobile genetic elements, MGEs）。
研究结论：本研究阐明并表征了新型大观霉素耐药基因ant(9)-Id及其生物学特性。在病原菌中鉴定新型耐药基因，有助于指导临床合理用药，并助力解析微生物群体中更多的抗菌耐药机制。