Global gene expression profiling and antibiotic susceptibility after repeated exposure to the carbon monoxide-releasing molecule-2 (CORM-2) in multidrug-resistant ESBL-producing uropathogenic Escherichia coli. Escherichia coli

Treatment of urinary tract infections is today a challenge due to the increasing prevalence of multidrug-resistant ESBL-producing uropathogenic Escherichia coli (UPEC). There is an urgent need for new treatment strategies for multidrug-resistant UPEC and preferably with targets that have low potential for development of resistance. Carbon monoxide-releasing molecules (CORMs) are novel and potent antibacterial agents. The present study examines the transcriptomic targets of CORM-2 in a multidrug-resistant ESBL-producing UPEC isolate (ESBL7) in response to a single exposure to CORM-2 and after repeated exposure to CORM-2. The bacterial viability and minimal inhibitory concentration (MIC) were also examined after repeated exposure to CORM-2. Microarray analysis revealed that a wide range of processes were affected by CORM-2, including a general trend of down-regulation in energy metabolism and biosynthesis pathways and up-regulation of the SOS response and DNA repair. Several genes involved in virulence (ibpB), antibiotic resistance (marAB, mdtABC) and biofilm formation (bhsA, yfgF) were up-regulated, while some genes involved in virulence (kpsC, fepCEG, entABE), antibiotic resistance (evgA) and biofilm formation (artIP) were down-regulated. Repeated exposure to CORM-2 did not alter the gene expression patterns, the growth inhibitory response to CORM-2 or the MIC values for CORM-2, cefotaxime, ciprofloxacin and trimethoprim. Overall design: ESBL7 from the original isolate, or isolates pre-exposed 20 times to CORM-2 (250 µM) or vehicle (2.5% DMSO) for 4 hours at 37 °C, were used to inoculate MS-medium followed by exposure to CORM-2 (250 µM) or vehicle for 30 min at 37 °C. Microarray were performed on first exposure to sub-inhibitory levels of CORM-2 (250 µM), first exposure to vehicle (2.5% DMSO), pre-exposed 20 times to CORM-2 respectively pre-exposed 20 times to vehicle. Four independent experiments were included in each group.

当前，产超广谱β-内酰胺酶（ESBL）的多重耐药尿路致病性大肠埃希菌（UPEC）流行率持续攀升，尿路感染的临床治疗已面临严峻挑战。目前亟需开发针对多重耐药UPEC的新型治疗策略，且理想的治疗靶点应具备极低的耐药诱导潜力。一氧化碳释放分子（CORMs）是一类新型强效抗菌制剂。本研究以一株产ESBL的多重耐药UPEC菌株（ESBL7）为研究对象，分别考察其单次暴露于CORM-2以及反复暴露于CORM-2后的转录组靶点。同时，本研究还检测了反复暴露于CORM-2后的细菌存活率与最低抑菌浓度（MIC）。

微阵列（Microarray）分析结果显示，CORM-2可影响菌株大量生理过程，整体呈现能量代谢与生物合成通路下调、SOS应答及DNA修复通路上调的表达趋势。多个与毒力（virulence）、抗生素耐药（antibiotic resistance）及生物膜形成（biofilm formation）相关的基因出现上调，其中包括ibpB、marAB、mdtABC、bhsA、yfgF；而部分与毒力（kpsC、fepCEG、entABE）、抗生素耐药（evgA）及生物膜形成（artIP）相关的基因则发生下调。

反复暴露于CORM-2并未改变菌株的基因表达模式、对CORM-2的生长抑制应答，以及CORM-2、头孢噻肟（cefotaxime）、环丙沙星（ciprofloxacin）与甲氧苄啶（trimethoprim）的MIC值。

实验总体设计：将原始菌株ESBL7、经20次反复暴露于250 μM CORM-2的菌株，以及经20次反复暴露于2.5%二甲基亚砜（DMSO）溶剂对照的菌株，分别接种于MS培养基（MS-medium），随后于37 ℃条件下暴露于250 μM CORM-2或溶剂对照30分钟。本研究针对四组样本开展微阵列检测：首次暴露于亚抑菌浓度CORM-2（250 μM）的样本、首次暴露于2.5% DMSO溶剂对照的样本、经20次反复暴露于CORM-2的样本，以及经20次反复暴露于溶剂对照的样本。每组均设置4次独立重复实验。