Zhang Shaoxing, Zhang Yuxin, Liu Ruijie, Yuan Shuying, Chen Yanwen, Li Wenjie, Lu Xinrong, Tong Yongliang, Hou Linlin, Chen Li, Sun Guiqin
School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People's Republic of China.
Clinical Laboratory Department, Jiaxing Maternity and Child Health Care Hospital, Jiaxing, Zhejiang, People's Republic of China.
Infect Drug Resist. 2023 Aug 22;16:5523-5534. doi: 10.2147/IDR.S423418. eCollection 2023.
(EM) is a multi-drug-resistant bacterium of global concern for its role in nosocomial infection and is generally resistant to aminoglycoside antibiotics. In the whole genome of an EM strain (FMS-007), an aminoglycoside-6-adenyl transferase gene () was predicted. This study aimed to characterize the biochemical function of ANT(6) and analyze the relationship between genotype and phenotype of in clinical EM isolates, so as to provide evidence for clinical precision drug use. This study could establish a method for the verification of known or unknown functionally resistant genes.
A total of 42 EM clinical isolates were collected from clinical departments during 2015-2023. The phenotype of aminoglycoside antibiotics was analyzed by broth microdilution (BMD) and Kirby-Bauer (K-B) methods. The whole-length from EM clinical isolates was analyzed by polymerase chain reaction (PCR) and sequencing. The biochemical function of predictive ANT(6) from the FMS-007 whole genome was identified by 3D plate experiment and mass spectrometry analysis. Candidate active sites were predicted by multi-species sequence alignment and molecular docking, and other important sites were identified in the comparison of genotypes and phenotypes of EM clinical isolates. Drug susceptibility test was used to verify the function of these sites.
The predictive ANT(6) protein could inactivate STR by modifying STR with ATP to form STR-AMP. Four active sites (Asp-38, Asp-42, Lys-95, and Lys-213) of ANT(6) were identified. Thirty-one EM clinical isolates (74%) carried the gene. Eight EM clinical isolates containing the gene had MIC values (<=32μg/mL) lower by at least 16-fold than FMS-007 (512μg/mL) for STR, and N59H and K204Q were the common mutations in the gene.
This assay verified the biochemical function of the predictive gene and could provide an alternative method to study resistant gene function in multi-drug-resistant bacteria. The inconsistency between genotype and phenotype of resistant genes indicated that the combination of resistance gene detection and functional analysis could better provide precision medicine for clinical use.
肠杆菌属（EM）是一种多药耐药菌，因其在医院感染中的作用而受到全球关注，并且通常对氨基糖苷类抗生素耐药。在一株EM菌株（FMS-007）的全基因组中，预测到一个氨基糖苷-6-腺苷转移酶基因（）。本研究旨在表征ANT(6)的生化功能，并分析临床EM分离株中该基因的基因型与表型之间的关系，从而为临床精准用药提供依据。本研究可以建立一种验证已知或未知功能抗性基因的方法。
2015年至2023年期间从临床科室收集了42株EM临床分离株。采用肉汤微量稀释法（BMD）和 Kirby-Bauer（K-B）法分析氨基糖苷类抗生素的表型。通过聚合酶链反应（PCR）和测序分析EM临床分离株的全长基因。通过3D平板实验和质谱分析鉴定FMS-007全基因组中预测的ANT(6)的生化功能。通过多物种序列比对和分子对接预测候选活性位点，并在EM临床分离株的基因基因型和表型比较中鉴定其他重要位点。采用药敏试验验证这些位点的功能。
预测的ANT(6)蛋白可通过用ATP修饰链霉素（STR）形成STR-AMP来使STR失活。鉴定出ANT(6)的四个活性位点（Asp-38、Asp-42、Lys-95和Lys-213）。31株EM临床分离株（74%）携带该基因。8株含有该基因的EM临床分离株对STR的最低抑菌浓度（MIC值）（<=32μg/mL）比FMS-007（512μg/mL）至少低16倍，N59H和K204Q是该基因中的常见突变。
本试验验证了预测基因的生化功能，并可为研究多药耐药菌中的抗性基因功能提供一种替代方法。抗性基因的基因型与表型不一致表明，抗性基因检测与功能分析相结合可以更好地为临床提供精准医学。
