摘要

患有囊性纤维化（CF）的个体的肺部长期感染铜绿假单胞菌，其难以通过抗生素治疗根除。两种关键的铜绿假单胞菌抗生素抗性机制是降解β-内酰胺抗生素的AmpCβ-内酰胺酶和MexXYOprM，一种从蛋白质细胞中排出氨基糖苷类抗生素的三蛋白外排泵。抗生素抗性基因表达水平可能是抗生素抗性的关键因素，但在感染期间尚未确定。这项研究的目的是研究在患有CF的患者和从相同患者分离的细菌中感染期间ampC和mexX基因的表达。来自36个CF患者的铜绿假单胞菌分离株在实验室培养物中生长，并且通过逆转录定量PCR（RT-qPCR）测量基因表达。在分离株之间，ampC的表达变化超过20,000倍，而mexX的表达超过2000倍。亚抑制浓度的抗生素的存在平均增加了两种基因的表达水平。为了测量感染期间的铜绿假单胞菌基因表达，我们使用从31名患者获得的新鲜痰样品中提取的RNA进行RT-qPCR。在患者之间，ampC的表达变化超过4000倍，而mexX表达变化超过100倍。尽管存在这些广泛的变异，但痰中ampC的中值表达水平与实验室培养的细菌相似。痰中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，有助于抗生素抗性的基因可以在患者中高度表达，但存在广泛的分离 - 分离和患者 - 患者差异。

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 expression of the ampC and mexX genes during infection in patients with CF and in bacteria isolated from the same patients. P. aeruginosa isolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription quantitative PCR (RT-qPCR). Expression of ampC varied over 20,000-fold and that of mexX over 2000-fold between isolates. Expression levels of both genes were on average increased by the presence of sub-inhibitory 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. Expression of ampC varied over 4000-fold, while mexX expression varied over 100-fold, between patients. Despite these wide variations, median levels of expression of ampC in sputum were similar to those in laboratory-grown bacteria. 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://www.ncbi.nlm.nih.gov/pubmed/30201819