口腔综合治疗台水路系统中假单胞菌属污染状况的检测

Determination of the contamination of Pseudomonas spp. in dental unit waterlines

目的:采用细菌培养技术与Miseq测序技术检测牙科综合治疗台水路系统(dental unit waterlines,DUWLs)中的假单胞菌属,以综合评估假单胞菌属在DUWLs中的污染情况。方法:选择南京医科大学附属口腔医院25台口腔综合治疗台作为研究对象。在医院开诊前收集三用枪水样400 mL,平均分为2份,一份用于细菌培养。另一份用于提取细菌基因组总DNA,经细菌通用引物PCR扩增后构建宏基因组文库,用于Miseq测序和生物信息学分析。结果:25个样本在不同浓度条件下经细菌培养法均未检出假单胞菌属。16个样本成功构建宏基因组文库,经Miseq测序均检测到假单胞菌属存在,检出率为100%,假单胞菌属在16个样本中的丰度为(3.02±2.12)%。结论:DUWLs中存在低水平的假单胞菌属污染,但假单胞菌属大部分可能处于死亡或"活的非可培养"状态,不具有较大的生物危害性。与传统细菌培养法相比,Miseq测序技术在特定病原菌的检测方面表现出了较高的敏感性,但无法区分样本中细菌的活性,若要将高通量测序技术用于活性病原菌的检测还需要进一步的研究。

Objective：To determine the contamination of Pseudomonasa spp. in dental unit waterlines（DUWLs） and water samples using bacterial culture technique as well as Miseq sequencing technology.Methods：Water samples were collected from 25 dental units（DU） at the Affiliated Stomatology Hospital of Nanjing Medical University.Before the opening of the clinic,a water sample of 400 mL was collected from each DU using triple change syringe.Each sample was divided into two parts.One part of the water samples was used for bacterial culture.The other one was used for extraction of the total bacterial DNA,which was subjected to PCR amplification using bacterial universal primers.The PCR products of each sample were constructed using Miseq sequencing technology to complete the sequencing analysis and biological information analysis.Results：No Pseudomonas spp. was detected by bacterial culture in 25 samples under the condition of different concentrations.The metagenomic libraries were successfully constructed by 16 samples.The detection rate of Pseudomonas spp. was 100%.The relative abundance of Pseudomonas spp. in 16 samples was accounted for（3.02±2.12）%.Conclusion：Low level of contamination of Pseudomonas spp.existed in DUWLs. Most clones of the Pseudomonas spp. in DUWLs were dead or in viable but nonculturable（VBNC） condition.Thus,Pseudomonas spp. presented a low biohazard risk in DUWLs.When compared to bacterial culture,Miseq sequencing technology is more accurate in detecting particular pathogens. However,Miseq sequencing technology has limitation on identification of viable bacterial from dead ones,which may lead to false positive results.It needs further researches using high-throughput sequencing technology for selectively detecting viable pathogens.