【作者】 陈曦；

【导师】 程磊；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2021， 硕士

【摘要】 幽门螺杆菌(Helicobacter pylori,Hp)是包括胃癌在内多种胃部疾病的危险因素,其全球感染率超过50%。随着抗生素的使用,其耐药率逐年上升,并且存在大量复发感染及再感染病例。因此幽门螺杆菌感染的防治已经成为全球亟待解决的重大问题。幽门螺杆菌可以通过粪-口、口-口等经口腔途径进行传播,然而口腔中仍不能大量分离培养幽门螺杆菌,且幽门螺杆菌在口腔的存在形式及状态仍不清楚。活而不可培养状态(viable but non culturable,VBNC)是细菌在不利生存环境下的一种休眠状态,它在保持细胞活性的同时丧失可培养性,并常常发生形态转变。已有研究表明幽门螺杆菌在一些不利生长的环境中及抗菌药物作用下,可以转化为VBNC状态以抵抗不利因素。幽门螺杆菌是否以VBNC状态存在于口腔环境(如唾液)中,促进其经口传播等科学问题仍不清楚。因此本实验将探究幽门螺杆菌在唾液中的存在形式及状态、进入VBNC状态的机制,以及唾液中的幽门螺杆菌从口腔到胃部的传播定植能力,为幽门螺杆菌经口传播及其感染防治提供新的策略。目的:研究幽门螺杆菌在唾液中的存在状态与机制,及其经口腔到胃部的定植传播能力,为其经口传播途径提供证据,为幽门螺杆菌感染的防治提供新的思路和方向。方法:通过人工唾液模拟口腔环境,对幽门螺杆菌进行体外培养,通过PMA-q PCR、Syto9/PI荧光染色等探究细菌菌量及活性变化;通过菌落实验探究唾液培养后幽门螺杆菌是否具备可培养能力;通过扫描电镜、透射电镜探究幽门螺杆菌在唾液环境中形态变化,以此确定幽门螺杆菌在唾液环境中的存在形式及状态。通过转录组测序、转录水平验证探究唾液中幽门螺杆菌基因表达情况及变化机制,并通过检测其对一线抗生素的耐受水平变化及代谢水平变化进行表型验证。最后通过体外实验及小鼠体内实验来探究唾液中幽门螺杆菌是否仍具备复苏及经口腔定植感染胃部的能力。结果:1、幽门螺杆菌在唾液环境中细胞形态由螺旋状或弧状变为短杆状或球状,以1×10~4CFU/m L长期(大于10天)存活,然而此状态下幽门螺杆菌丧失可培养性,在常规培养条件下无法复苏。说明幽门螺杆菌在唾液环境中处于VBNC状态。2、对VBNC状态进行转录组分析,发现唾液中的幽门螺杆菌通过双组份系统感应环境变化,从而显著下调代谢水平及上调细胞杆形决定蛋白基因mre B、mre C,以及细胞壁肽聚糖相关基因pbp1-A、pbp2、和ami A等进入VBNC状态;进入VBNC状态的幽门螺杆菌毒力显著下调,耐药相关通路显著上调,表现为对一线抗生素的显著耐受。3、唾液中处于VBNC状态的幽门螺杆菌可以通过逆转不利生存条件(如更换培养基、加入复苏促进因子等)进行复苏。唾液诱导的VBNC状态幽门螺杆菌通过接种小鼠口腔,能在胃部有长期大量定植,表明VBNC状态的幽门螺杆菌可经口腔传播到胃部定植。结论:综上所述,口腔环境中幽门螺杆菌通过双组份系统感应环境变化,从而上调细胞杆形决定蛋白基因mre B、mre C,以及细胞壁肽聚糖相关基因pbp1-A、pbp2、和ami A等使细胞形态向球形转变,在保持活性的同时丧失可培养性,以VBNC状态长期存活。其毒力因子表达及代谢水平明显下降,它可能通过此方式降低宿主免疫防御并适应口腔中不利的生存环境。同时它对不同类型抗生素耐药性明显上升,在药物作用后仍可保持存活状态,这可能导致幽门螺杆菌药物治疗后的复发感染。此状态下的幽门螺杆菌仍具备感染传播能力,可以通过口腔长期定植于胃部,进一步证明了VBNC状态是幽门螺杆菌经口途径定植于胃部的有效策略,通过阻断幽门螺杆菌经口传播途径可有效降低其感染。更多还原

【Abstract】 Helicobacter pylori(H.pylori)is a risk factor for a variety of gastric diseases including gastric cancer,and its global infection rate exceeds 50%.With the use of antibiotics,the drug resistance rate is increasing year by year,and there are a large number of recurrent infections and reinfection cases.Therefore,the prevention and treatment of H.pylori infection has become a major problem that needs to be solved urgently in the world.H.pylori can be transmitted through oral routes such as feces-oral,oral-oral,etc.However,the existence form and status of H.pylori in the oral cavity are still unclear.The viable but non culturable state(VBNC)is a dormant state of bacteria in a poor living environment.It loses cultivability while maintaining cell viability,and often undergoes morphological changes.Studies have shown that H.pylori can be transformed into VBNC state to resist unfavorable factors in some unfavorable growth environment and under the action of antibacterial drugs.Whether H.pylori exists in the oral environment(such as saliva)in the state of VBNC and promotes its oral transmission and other scientific issues remain unclear.Therefore,this experiment will explore the existence form and state of H.pylori in saliva,the mechanism of entering the VBNC state,and the ability of H.pylori in saliva to spread and colonize from the mouth to the stomach,providing new strategies for the oral transmission of H.pylori and its infection prevention and treatment.Objectives:To study the existence and mechanism of H.pylori in saliva,and its ability to colonize and spread through the oral cavity to the stomach,provide evidence for its oral transmission route,and provide new ideas and directions for the prevention and treatment of H.pylori infection.Methods:Through artificial saliva to simulate the oral environment,H.pylori was cultured in vitro,and the bacterial activity and the change of bacterial count were explored through PMA-q PCR and Syto9/PI fluorescent staining;colony experiment was used to explore whether H.pylori has cultivable ability after saliva culture;scanning electron microscopy and transmission electron microscopy explore the morphological changes of H.pylori in the saliva environment to determine the existence form and state of H.pylori in the saliva environment.Through transcriptome sequencing and transcription level verification,the expression of H.pylori genes in saliva and the mechanisms were explored,and the changes in the tolerance level of first-line antibiotics and metabolic level were tested for phenotypic verification.Finally,in vitro experiments and in vivo experiments in mice were conducted to explore whether H.pylori in saliva still has the ability to resuscitate and colonize the stomach to infect the stomach.Results:1.H.pylori cells changes from spiral or arc to rod or coccoids forms in saliva environment,and survive for a long time(more than 10 days)at 1×10~4CFU/m L.However,in this state,H.pylori loses its cultivability and cannot be recovered under conventional culture conditions.2.RNA-seq sequencing analysis of VBNC status revealed that H.pylori in saliva senses environmental changes through a two-component system,which significantly down-regulates the metabolic level and up-regulates the cell rod-determining protein genes mre B and mre C,and the cell wall peptidoglycan-related genes pbp1-A,pbp2,and ami A enter the state of VBNC;the virulence of H.pylori that enters the state of VBNC is significantly down-regulated,and resistance-related pathways are significantly up-regulated,showing significant resistance to first-line antibiotics.3.H.pylori in the VBNC state in saliva can be resuscitated by reversing unfavorable survival conditions(such as changing the culture medium,adding resuscitation promoting factors,etc.).Saliva-induced H.pylori in VBNC state can colonize the stomach in large quantities for a long time by inoculating the oral cavity of mice,indicating that H.pylori in VBNC state can be colonized by the oral cavity to the stomach.Conclusions:In summary,H.pylori in the oral environment senses environmental changes through a two-component system,thereby changing the cell morphology to a spherical shape by up-regulating the cell rod shape determining protein genes mre B and mre C,and the cell wall peptidoglycan-related genes pbp1-A,pbp2,and ami A.At the same time,it loses cultivability and survives in VBNC state for a long time.The expression of virulence factors and the level of metabolism decreased significantly,which may reduce the host’s immune defense and adapt to the unfavorable living environment in the oral cavity in this way.At the same time,its resistance to different types of antibiotics has increased significantly,and it can still remain alive after the drug is applied,which may lead to recurrent infections after H.pylori drug treatment.H.pylori in this state still has the ability to spread infection and can colonize the stomach for a long time through the oral cavity.This further proves that the VBNC state is an effective strategy for H.pylori colonization in the stomach via oral route.By blocking the oral transmission of H.pylori,the infection can be effectively reduced.更多还原