Molecular Biology Department, Pasteur Institute of Iran, No. 69, Pasteur Ave, Tehran, 1316943551, Iran.
Microb Pathog. 2018 Sep;122:184-190. doi: 10.1016/j.micpath.2018.06.027. Epub 2018 Jun 20.
Urinary tract infection (UTI) is the second most frequent infection in human, and uropathogenic Escherichia coli is its most common cause. Although antibiotics are the standard treatment for UTI, they can cause harmful effects on gut microbiome and increase the rate of existing drug-resistant bacteria, which make the vaccine research reasonable. This study was conducted to construct a Killed but Metabolically Active (KBMA) E. coli strain, and to determine its characteristics as a possible vaccine candidate for UTI, which will be evaluated in further investigations.
The uvrB gene of uvrABC excision repair system of E.coli was deleted to construct a ΔuvrB mutant, lacking repairing system of intercross linkages between DNA strands. To construct KBMA strain, the ΔuvrB mutant was PUVA-treated, using different doses of 8-methoxypsoralen (8-MOP) followed by different doses of ultraviolet A (UVA) irradiation (365 nm), until the optimal doses of each were achieved. Then, different characteristics of the PUVA-treated E. coli (with the optimal doses) were assessed, using cell counting, colony formation assay, MTT and XTT assays, fluorescent staining, and flow cytometry.
PUVA treatment's optimal dose for E. coli isolates was 150 ng/ml 8-MOP plus 1000 mj/cm UVA. While the PUVA-treated isolates had a significant decrease in cell counting, the fluorescent dying of the un-grown parts of the culture plates revealed living bacteria with bizarre shapes. Meanwhile, MTT and XTT assays demonstrated the metabolic activity of these bacteria and flow cytometry confirmed their aliveness.
These PUVA-treated bacteria, with metabolic activity and proliferation inability, seem to be good enough to be tested in vitro and in vivo as a candidate for vaccine against UTI. Therefore it seems the first step toward development of a vaccine candidate is successfully done. The immunogenicity and protectivity of these treated bacteria is under evaluation.
尿路感染（UTI）是人类第二大常见感染，其最常见的病原体是尿路致病性大肠杆菌。尽管抗生素是治疗 UTI 的标准方法，但它们会对肠道微生物群产生有害影响，并增加现有耐药菌的发生率，这使得疫苗研究成为合理的选择。本研究旨在构建一种灭活但具有代谢活性（KBMA）的大肠杆菌菌株，并确定其作为 UTI 候选疫苗的特性，这将在进一步的研究中进行评估。
通过删除大肠杆菌 uvrABC 切除修复系统中的 uvrB 基因，构建了一个缺乏 DNA 链间交联修复系统的ΔuvrB 突变株。为了构建 KBMA 菌株，用不同剂量的 8-甲氧基补骨脂素（8-MOP）和不同剂量的紫外线 A（UVA）（365nm）对ΔuvrB 突变株进行 PUVA 处理，直到每种处理达到最佳剂量。然后，使用细胞计数、菌落形成试验、MTT 和 XTT 试验、荧光染色和流式细胞术评估 PUVA 处理后的大肠杆菌的不同特性。
大肠杆菌分离株的最佳 PUVA 处理剂量为 150ng/ml 8-MOP 和 1000mj/cm UVA。虽然 PUVA 处理后的分离株细胞计数明显减少，但培养板未生长部分的荧光染色显示出具有奇异形状的活菌。同时，MTT 和 XTT 试验表明这些细菌具有代谢活性，流式细胞术证实了它们的活力。
这些具有代谢活性但不能增殖的 PUVA 处理细菌似乎足以在体外和体内进行测试，作为治疗 UTI 的候选疫苗。因此，似乎已经成功地迈出了开发疫苗候选物的第一步。这些处理后的细菌的免疫原性和保护力正在评估中。
