摘要
      从临床铜绿假单胞菌分离株分离的pUM505质粒当转移到标准铜绿假单胞菌PAO1株时赋予对环丙沙星（CIP）的抗性。 CIP是用于治疗铜绿假单胞菌感染的喹诺酮家族的抗生素。在计算机分析中，进行鉴定CIP抗性基因，发现由pUM505中的orf131基因编码的65个氨基酸的产物与耻垢分枝杆菌氨基糖苷磷酸转移酶（磷酸化并使氨基糖苷类抗生素失活的酶）显示40％的氨基酸同一性。我们将orf131克隆到pUCP20穿梭载体中（重命名为用于环丙沙星抗性蛋白质粒编码的crpP）。得到的重组质粒pUC-crpP在大肠杆菌J53-3菌株中赋予对CIP的抗性，表明该基因的产物编码涉及CIP抗性的蛋白质。使用偶联的酶分析，我们确定CrPP对CIP的活性是ATP依赖性的，而对诺氟沙星检测到的活性很小，表明CIP可能发生磷酸化。使用重组His标记的CrpP蛋白和液相色谱 - 串联质谱，我们还显示CIP在其降解之前被磷酸化。因此，我们的研究结果表明，在pUM505质粒上编码的CrpP代表铜绿假单胞菌中CIP抗性的新机制，其涉及抗生素的磷酸化。

      The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa PAO1 strain. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP-resistance genes, revealed that the 65-amino acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed as crpP for ciprofloxacin-resistance protein plasmid-encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-crpP, conferred resistance to CIP in the Escherichia coli J53-3 strain, suggesting that the product of this gene encodes a protein involved in CIP resistance. Using a coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP-dependent, while little activity was detected towards norfloxacin, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography--tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP-resistance in P. aeruginosa, which involves phosphorylation of the antibiotic.

https://www.ncbi.nlm.nih.gov/pubmed/29581123