实验树鼩肠道螺杆菌属细菌特征分析

Analysis of Helicobacter spp.in the Gut of Laboratory Tree Shrew

近年来,树鼩(Tupaia belangeri)作为一种新型的实验动物被广泛应用于生物医学研究的各个领域。本研究组前期的研究结果显示,螺杆菌属(Helicobacter)是树鼩肠道微生物群落中相对丰度最高的一类细菌,但其具体的细菌种类和结构特征仍然不清楚。因此,本研究将开展实验树鼩肠道中螺杆菌属细菌的分布种类和特征分析,为后续实验研究工作提供资料。通过系统采集72只树鼩的粪便样本,提取核酸后采用巢氏PCR法应用属特异性引物扩增螺杆菌属特异性片段,再分别采用7个种特异性引物对属特异性阳性样本扩增螺杆菌种特异性片段,包括肝螺杆菌(H.hepaticus)、家鼠螺杆菌(H.muridarum)、胆汁螺杆菌(H.bilis)、啮齿类螺杆菌(H.rodentium)、弯曲螺杆菌(Flexispira rappini)、鼩螺杆菌(H.suncus)和盲肠螺杆菌(H.typhlonius)。属特异性引物扩增阳性但种引物扩增阴性的样本进行核酸序列测定和BLAST比对分析,确认其最终所属的螺杆菌种类。结果显示,72份树鼩粪便样本中,属特异性引物扩增阳性有18份,总体阳性率为25.0%。其中,盲肠螺杆菌阳性8株、胆汁螺杆菌阳性6株,其余8份阳性样本经过测序和BLAST比对分析后确认为同性恋螺杆菌(H.cinaedi)阳性5株、猫螺杆菌(H.felis)阳性2株和猕猴螺杆菌(H.macacae)阳性1株。有4份树鼩粪便样本出现同时携带盲肠螺杆菌和胆汁螺杆菌的情况。将螺杆菌属细菌携带结果与实验树鼩的性别和年龄组进行比较分析后发现,不同性别间以及不同年龄组间,属或种阳性样本情况均无差异(P>0.05)。本研究结果表明,实验树鼩具有较高的螺杆菌携带率,且不分性别和年龄,主要以盲肠螺杆菌、胆汁螺杆菌和同性恋螺杆菌为主。

Currently,the tree shrew has been widely used as an alternative laboratory animal in biomedical researches.Our previous study showed that Helicobacter spp.was the most distributed bacteria in gastrointestinal of tree shrew,however,the species of this genus were still unknown.To find out the characteristics of Helicobacter spp.species carried in laboratory tree shrew,we performed the current study to provide useful information for future investigation.Seventy-two tree shrew fecal samples were collected,and processed for DNA extraction,nested PCR to amplify specific gene fragments with the genus specific primers.The speciesspecific primers including those for Helicobacter hepaticus,H.muridarum,H.bilis,H.rodentium,Flexispira rappini,H.suncus and H.typhlonius were used to amplify species-specific fragments from genus-positive samples.The samples negative for species-specific primers but positive for genus primer were sent for sequencing.BLAST and alignment methods were used to confirm their Helicobacter species.The results showed that eighteen feces were positive for Helicobacter,and the positive rate was 25%,among which eight strains were H.typhlonius,six were H.bilis,five were H.cinaedi,two were H.felis and one was H.macacae(Fig.1).Four cases were mixed carrying both H.typhlonius and H.bilis.The carried Helicobacter genus and species had no statistical significance in tree shrew among different gender and age groups(P>0.05)(Table 2).The highly carried rate of Helicobacter spp.,mainly H.typhlonius,H.bilis and H.cinaedi,was found in laboratory tree shrew with different genders and ages.