携带环境耐药基因blaCTX-M的质粒对临床ST131 ExPEC毒力的影响和机制

Bacterial resistance caused by antibiotic resistance gene contamination poses a very serious threat to human health. Meanwhile, antibiotic resistance gene transfer in human pathogens may result in major changes in bacterial virulence. However, the basic phenomenon of virulence changes and its molecular mechanisms remain unclear. We have collected environmental samples and 413 clinical isolates of extraintestinal pathogenic E.coli (ExPEC) in Tianjin. Compared to ESBL-negative isolates, adrA, csgA, csgD and csgE were significantly upregulated in ESBL-producing ST131 ExPEC isolates. Based on the results obtained, we put forward the following hypothesis about the mechanism of plasmids harbouring blaCTX-M genes on the virulence of ST131 ExPEC. Antibiotic resistance plasmids enter E.coli cells, which may lead to changes in the intracellular environment. The changes activate EnvZ/OmpR and CpxA/CpxR. EnvZ/OmpR and CpxA/CpxR regulate csgD gene expression, which results in changes in bacterial virulence. High-throughput sequencing, biofilm formation experiment, mouse bladder (kidney) colonization assays, etc. will be performed in this study to study polymorphism of blaCTX-M in environmental samples and clinical ExPEC isolates, and effect and mechanism of plasmids harbouring blaCTX-M genes present in environmental samples on the virulence of clinical ST131 ExPEC. We will take this study as an example to illustrate that changes in pathogen virulence caused by resistance gene contamination pose a serious threat to human health. This study will provide a theoretical basis for the prevention and treatment of ExPEC infection.

抗生素抗性基因污染引发的细菌耐药对人体健康构成巨大威胁，同时，耐药基因转移可能引起病原菌毒力的改变，但其基本现象和分子机制仍不清楚。我们已收集天津地区的环境样本和413株临床肠外致病性大肠埃希菌（ExPEC），并发现相比超广谱β-内酰胺酶（ESBLs）阴性菌株，adrA、csgA、csgD和csgE在ESBL阳性ST131 ExPEC中显著上调表达。据此，我们提出假设：耐药质粒进入E.coli可能引起细胞内环境的改变，激活EnvZ/OmpR和CpxA/CpxR，调控csgD表达，影响E.coli毒力。本项目将通过高通量测序、生物膜形成和小鼠膀胱（肾）定植等实验研究环境与临床ExPEC中blaCTX-M多态性特征和携带环境blaCTX-M的质粒对临床ST131 ExPEC毒力的影响和机制，以此为例说明耐药基因污染引起病原菌毒力变化对人体健康的威胁，也为临床上防治ExPEC提供理论基础。

肠外致病性大肠埃希菌（ExPEC）是临床上最为常见的分离株，常导致尿路感染、菌血症、新生儿脑膜炎等多种人体肠道外感染疾病。.411株临床ExPEC分离株被收集并进行分子分型和药敏实验。从这411株菌中选择了74株代表性菌株，进一步进行蜡螟实验和全基因组测序。CH40-30-ST131、CH37-27-ST405、CH40-41-ST131和CH13-5-ST12分型菌株对检测的抗生素有广泛耐药性；CH14-64-ST1193、CH40-30-ST131和CH35-27-ST69菌株有较高毒力。特定分子分型菌株携带特定耐药基因（ARGs）或VFs，如papB、papI、fimA、sat、kpsD、senB和aerobactin基因广泛存在CH14-64-ST1193菌株；blaCTX-M-15、aac(6′)-Ib-cr、papB、papI、sat、iucA、iucB、iucC、chuT、chuX和shuU广泛存在CH40-30-ST131菌株；tetB广泛存在CH35-27-ST69和CH13-5-ST12菌株。毒力评分在各分型菌株中存在显著差异。CH37-27-ST405和CH26-5-ST38菌株携带相对较多的ARGs和VFs，具有较高的耐药和毒力潜力。.在这411株临床ExPEC分离株中，菌株ECO3183对所检测的26种抗菌药物中的23种均不敏感。ECO3183属于系统发育A组、克隆复合群10（CC10）和ST167（ST167是ExPEC主要ST分型之一）。ECO3183含有一个基因组和两个质粒。我们在BacWGSTdb数据库中分析获得了与ECO3183系统发育关系较近的55株大肠埃希菌分离株（与ECO3183相比有24-100个不同的cgMLST等位基因）。这55株分离株也都属于ST167；其中69%（38/55）来自中国，47%（26/55）来源于人体，35%（19/55）来源于动物。基于上述分析，ST167型大肠杆菌很可能存在环境动物与人类之间的传播。.本研究系统地研究了ExPEC菌株耐药、毒力和分子分型，又报道了一株ST167型泛耐药ExPEC菌株，并分析了ST167型菌株在环境与临床的潜在传播。另外，也发现了大环内酯类抗生素能够诱导msrE-mphE操纵子表达。本研究为ExPEC的防治和设计抗大环内酯类耐药菌株的新药提供了基础。