Sárvári Károly Péter, Sóki József, Iván Miklós, Miszti Cecília, Latkóczy Krisztina, Melegh Szilvia Zsóka, Urbán Edit
Institute of Clinical Microbiology, University of Szeged, Szeged, Hungary.
Institute of Laboratory Medicine, Semmelweis University, Budapest, Hungary.
Anaerobe. 2017 Dec;48:98-102. doi: 10.1016/j.anaerobe.2017.07.005. Epub 2017 Jul 25.
Bacteroides fragilis as a commensal bacterium is a member of the human intestinal flora, but as an opportunistic pathogen it can cause serious infections as well. Some of them, harbouring an enterotoxin gene (bft), may cause diarrhoea mainly in young children. Recently it has been shown that a member of C11 proteases called fragipain (fpn) can activate the enterotoxin, while C10 protease (bfp) is suspected of playing an important role in the invasiveness of the B. fragilis isolates. The objective of this study was to investigate the prevalence and distribution of the bft isotypes in 200 Hungarian B. fragilis isolates collected recently; and in a subset of 72 strains, we wanted to determine the prevalence of bfp1-4 and fpn genes in bft-positive and bft-negative strains. Using the MALDI-TOF MS cfiA identification project file, 19 B. fragilis strains belonging to Division II were identified and the presence of the cfiA gene was confirmed by RT-PCR. Twenty six (13.0%) B. fragilis isolates turned out to be bft gene positive by RT-PCR; 20 isolates harboured bft-1 and six bft-2 isotypes, but no bft-3 isotype containing strains were found. A melting curve analysis and the PCR-RFLP were performed to differentiate between the bft-1 and bft-2 isotypes confirmed by sequencing. Thirty eight strains harboured bfp1, 58 isolates contained bfp2 gene, while 17 isolates proved positive for bfp3. Morever, no bfp4 positive isolate was found, and some of the B. fragilis strains tested harboured two or three bfp isotypes simultaneously. Among the 26 bft-positive strains, 24 contained the fpn gene, which confirms the role of fragipain in the activation of B. fragilis enterotoxin. In experiments, a significant negative correlation between fpn and cfiA was demonstrated (p < 0.000), a positive correlation was found between bfp2 and fpn genes (p = 0.0000803), and a negative correlation between bfp2 and cfiA genes (p = 0.011).
脆弱拟杆菌作为一种共生菌，是人类肠道菌群的成员，但作为一种机会致病菌，它也可引起严重感染。其中一些携带肠毒素基因（bft）的菌株，主要可导致幼儿腹泻。最近研究表明，一种名为脆弱蛋白酶（fpn）的C11蛋白酶成员可激活肠毒素，而C10蛋白酶（bfp）被怀疑在脆弱拟杆菌分离株的侵袭性中发挥重要作用。本研究的目的是调查最近收集的200株匈牙利脆弱拟杆菌分离株中bft亚型的流行情况和分布；在72株菌株的子集中，我们想确定bfp1 - 4和fpn基因在bft阳性和bft阴性菌株中的流行情况。使用MALDI - TOF MS cfiA鉴定项目文件，鉴定出19株属于第二组的脆弱拟杆菌菌株，并通过RT - PCR确认了cfiA基因的存在。通过RT - PCR检测，26株（13.0%）脆弱拟杆菌分离株的bft基因呈阳性；20株携带bft - 1，6株携带bft - 2亚型，但未发现含有bft - 3亚型的菌株。通过熔解曲线分析和PCR - RFLP对经测序确认的bft - 1和bft - 2亚型进行区分。38株携带bfp1，58株分离株含有bfp2基因，而17株分离株的bfp3呈阳性。此外，未发现bfp4阳性分离株，一些受试的脆弱拟杆菌菌株同时携带两种或三种bfp亚型。在26株bft阳性菌株中，24株含有fpn基因，这证实了脆弱蛋白酶在激活脆弱拟杆菌肠毒素中的作用。实验表明，fpn与cfiA之间存在显著负相关（p < 0.000），bfp2与fpn基因之间存在正相关（p = 0.0000803），bfp2与cfiA基因之间存在负相关（p = 0.011）。
