Jafari Gholam Ali, Fotouhi Ardakani Reza, Akhavan Sepahi Mohsen, Nowroozi Jamileh, Soltanpour Mohammad Soleiman
Department of Biology, Tehran Science and Research Branch, Islamic Azad University, Facility of Basic Science, Tehran, Iran
Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran
Iran J Kidney Dis. 2021 May;15(3):190-198.
Oxalobacter formigenes, as a gram-negative anaerobic bacterium, metabolizes oxalate in the intestine by the enzymes oxalyl-CoA decarboxylase (OXC) and formyl-CoA transferase (FRC). Therefore, not only the presence of the bacterium but also microbial load may affect intestinal absorption and urinary exertion. We evaluated the relationship between Oxalobacter formigenes load and the formation of calcium oxalate urolithiasis using quantitative molecular methods.
By clinical manifestation and stone analysis, we selected the urine and stool specimens of 73 patients with calcium oxalate urolithiasis. First, the gene regions of the two genes FRC and OXC in Oxalobacter formigenes were selected utilizing bioinformatics and specific primers designed for these regions. Following DNA extraction from stool specimens by specific primers of each gene, PCR was carried out and positive samples were sequenced. Then, qPCR was applied to determine the effective load of Oxalobacter. Also, biochemical tests were performed to measure the excretion rate of oxalate in urine specimens.
In addition to oxalobacter identification by PCR, the load of bacteria was quantitatively assessed using qPCR by specific primers for both FRC and OXC gene regions. A significant negative relationship had found between the formation of calcium oxalate urolithiasis and the presence of Oxalobacter formigenes in patients with kidney stone disease. The mean excretion of oxalate and citrate in urolithiasis cases were 22.93 and 552.106 mg/24h, respectively.
The presence of Oxalobacter formigenes can highly inhibit the generation of calcium oxalate urolithiasis. Furthermore, molecular techniques are more effective than other methods such as culture for the isolation of this bacterium.
产甲酸草酸杆菌作为一种革兰氏阴性厌氧菌，通过草酸辅酶A脱羧酶（OXC）和甲酰辅酶A转移酶（FRC）在肠道内代谢草酸盐。因此，不仅该细菌的存在，而且微生物载量都可能影响肠道吸收和尿液排泄。我们使用定量分子方法评估了产甲酸草酸杆菌载量与草酸钙尿路结石形成之间的关系。
通过临床表现和结石分析，我们选取了73例草酸钙尿路结石患者的尿液和粪便标本。首先，利用生物信息学方法选择产甲酸草酸杆菌中FRC和OXC这两个基因的基因区域，并针对这些区域设计特异性引物。在用每个基因的特异性引物从粪便标本中提取DNA后，进行聚合酶链反应（PCR），并对阳性样本进行测序。然后，应用实时定量PCR（qPCR）来确定产甲酸草酸杆菌的有效载量。此外，还进行了生化测试以测量尿液标本中草酸盐的排泄率。
除了通过PCR鉴定草酸杆菌外，还使用针对FRC和OXC基因区域的特异性引物通过qPCR对细菌载量进行了定量评估。在肾结石疾病患者中，草酸钙尿路结石的形成与产甲酸草酸杆菌的存在之间发现了显著的负相关关系。尿路结石病例中草酸盐和柠檬酸盐的平均排泄量分别为22.93和552.106毫克/24小时。
产甲酸草酸杆菌的存在可高度抑制草酸钙尿路结石的产生。此外，分子技术在分离这种细菌方面比其他方法（如培养）更有效。
