Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Anaerobe. 2022 Jun;75:102546. doi: 10.1016/j.anaerobe.2022.102546. Epub 2022 Mar 12.
We aimed to identify the enterotoxigenic Bacteroides fragilis (ETBF) and bft subtypes among patients with diarrhea. In addition, we assessed whether DNA gyrase subunit B (gyrB) and neuraminidase (nanH) genes are useful determinants for identification of B. fragilis compared to 16S rRNA sequencing as a reference method.
The 530 fecal specimens were cultured on BBE agar. The colonies which supposed to be a member of B. fragilis group were subjected to 16S rRNA gene sequencing and PCR assays targeting the Bacteroides fragilis group (BFG), gyrB and nanH. The B. fragilis toxin (bft) gene and its subtype was detected by PCR. The specificity of PCR assays was calculated considering the 16S rRNA gene sequencing as the reference method.
A total of 111 Gram-negative anaerobic coccobacilli were isolated from 530 fecal specimens using BBE agar. Of the 111 isolates, 100 (90.09%) were assumed to be a member of Bacteroides fragilis group as they yielded an amplicon through PCR using the group-specific primers (Bfra-F/g-Bfra-R). However, only 28 isolates out of 100 were definitively identified as species of Bacteroides using16S rRNA gene sequencing; of which 15 isolates were B. fragilis and the remaining 13 isolates were identified as B. thetaiotaomicron (n = 6), Parabacteroides distasonis (n = 3), B. vulgatus (Phocaeicola vulgatus) (n = 1), B. ovatus (n = 1), B. congonensis (n = 1) and B. nordii (n = 1). Among the 15 isolates of B. fragilis, 4 were found to be ETBF. Compared to the reference method, the specificity and accuracy of the PCR targeting gyrB gene (64.7% and 65%) was higher than of nanH (36.4% and 46%, respectively.
This study demonstrated that more than one-fourth of B. fragilis isolates harbored bft gene and less than 1% of patients with diarrhea harbored ETBF. The slight agreement between the PCR assays -already used for identification of B. fragilis which targeting gyrB or nanH - and 16S rRNA gene sequencing as the reference method was noted.
我们旨在鉴定腹泻患者中的产肠毒素脆弱拟杆菌（ETBF）和 bft 亚型。此外，我们评估了与 16S rRNA 测序作为参考方法相比，DNA 拓扑异构酶亚单位 B（gyrB）和神经氨酸酶（nanH）基因是否可作为鉴定脆弱拟杆菌的有用决定因素。
将 530 份粪便标本接种于 BBE 琼脂上。将假定为脆弱拟杆菌群成员的菌落进行 16S rRNA 基因测序和针对脆弱拟杆菌群（BFG）、gyrB 和 nanH 的 PCR 检测。通过 PCR 检测脆弱拟杆菌毒素（bft）基因及其亚型。考虑到 16S rRNA 基因测序作为参考方法，计算了 PCR 检测的特异性。
使用 BBE 琼脂从 530 份粪便标本中分离出 111 株革兰氏阴性厌氧球菌。在 111 株分离株中，通过使用组特异性引物（Bfra-F/g-Bfra-R）进行 PCR，100 株（90.09%）被假定为脆弱拟杆菌群的成员。然而，只有 28 株通过 16S rRNA 基因测序明确鉴定为拟杆菌属物种；其中 15 株为脆弱拟杆菌，其余 13 株分别鉴定为拟杆菌属（n=6）、副拟杆菌属（n=3）、普通拟杆菌（Phocaeicola vulgatus）（n=1）、卵形拟杆菌（n=1）、congonensis 拟杆菌（n=1）和 nordii 拟杆菌（n=1）。在 15 株脆弱拟杆菌分离株中，发现 4 株为 ETBF。与参考方法相比，针对 gyrB 基因的 PCR 检测的特异性和准确性（分别为 64.7%和 65%）高于 nanH（分别为 36.4%和 46%）。
本研究表明，超过四分之一的脆弱拟杆菌分离株携带 bft 基因，腹泻患者中不到 1%携带 ETBF。已用于鉴定脆弱拟杆菌的 gyrB 或 nanH 靶向 PCR 检测与 16S rRNA 基因测序作为参考方法之间存在轻微一致性。
