摘要

患有囊性纤维化（CF）的人的肺部长期感染铜绿假单胞菌，难以通过抗生素治疗根除。铜绿假单胞菌的两个主要抗生素耐药机制是降解β-内酰胺抗生素的AmpCβ-内酰胺酶和MexXYOprM，这是一种三蛋白外排泵，可从细菌细胞中排出氨基糖苷类抗生素。抗生素抗性基因表达水平可能是抗生素抗性的关键因素，但在感染过程中尚未确定。这项研究的目的是调查在CF患者以及从同一患者中分离并在实验室条件下生长的细菌感染过程中ampC和mexX基因的表达。在实验室培养中培养了来自36名CF患者的铜绿假单胞菌分离株，并通过逆转录定量PCR（RT-qPCR）测量了基因表达。在分离株之间，ampC的表达变化超过20,000倍，而mexX的表达变化超过2,000倍。由于存在亚抑制浓度的抗生素，两个基因的中值表达水平均增加。为了测量感染期间的铜绿假单胞菌基因表达，我们使用了从31位患者的新鲜痰液样本中提取的RNA进行了RT-qPCR。在患者之间，ampC的表达变化超过4,000倍，而mexX的表达变化超过100倍。尽管存在这些很大的差异，但痰中细菌中ampC的表达水平的中值与实验室培养细菌中的水平相似。痰液中mexX的表达高于实验室培养的细菌。总体而言，我们的数据表明，导致抗生素耐药性的基因可以在患者中高表达，但是分离株之间和分离株之间存在广泛的差异。

The lungs of individuals with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa that is difficult to eradicate by antibiotic treatment. Two key P. aeruginosa antibiotic resistance mechanisms are the AmpC β-lactamase that degrades β-lactam antibiotics and MexXYOprM, a three-protein efflux pump that expels aminoglycoside antibiotics from the bacterial cells. Levels of antibiotic resistance gene expression are likely to be a key factor in antibiotic resistance but have not been determined during infection. The aims of this research were to investigate the expression of the ampC and mexX genes during infection in patients with CF and in bacteria isolated from the same patients and grown under laboratory conditions. P. aeruginosa isolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription-quantitative PCR (RT-qPCR). The expression of ampC varied over 20,000-fold and that of mexX over 2,000-fold between isolates. The median expression levels of both genes were increased by the presence of subinhibitory concentrations of antibiotics. To measure P. aeruginosa gene expression during infection, we carried out RT-qPCR using RNA extracted from fresh sputum samples obtained from 31 patients. The expression of ampC varied over 4,000-fold, while mexX expression varied over 100-fold, between patients. Despite these wide variations, median levels of expression of ampC in bacteria in sputum were similar to those in laboratory-grown bacteria. The expression of mexX was higher in sputum than in laboratory-grown bacteria. Overall, our data demonstrate that genes that contribute to antibiotic resistance can be highly expressed in patients, but there is extensive isolate-to-isolate and patient-to-patient variation.

https://aac.asm.org/content/62/11/e01789-18.abstract