轻型链球菌促进铜绿假单胞菌致病性及机制研究

发布时间：2018-08-26 12:15

【摘要】：第一部分轻型链球菌对铜绿假单胞菌BF致病性的影响目的:机械通气广泛应用及生物膜(BF)在气管导管(ETT)的形成,新生儿呼吸机相关肺炎(VAP)仍然是临床上的棘手问题。我们前期发现铜绿假单胞菌(P.aeruginosa)和轻型链球菌(S.mitis),一种口咽部正常细菌,在新生儿ETT表面最常见;且S.mitis检出率在VAP组患儿中较高。鉴于BF内的不同种细菌可能促进或抑制对方,本研究目的在于探讨S.mitis能否影响P.aeruginosa的致病性。方法:将BF培养分为PAO1组、S.mitis组和PAO1+S.mitis组,并收集生物膜培养液(BCM)。结晶紫、平板计数以及光学显微镜观察细菌粘附能力。结晶紫、平板计数、CCK-8及激光共聚焦(CLSM)观察BF形成。BCM刺激人气道上皮细胞(BEAS-2B)后,检测细胞活性、IL-8表达以及TLR2、4的表达。结果:结果显示S.mitis几乎不能粘附于非生物介质表面,且不能形成BF,但它增强了PAO1的粘附能力和BF形成(P0.05)。混合BF内总活菌数和活P.aeruginosa数明显高于PAO1单独组(P0.05)。然而,与PAO1单独组BCM相比较,混合BCM的细胞毒性及其诱导的IL-8,TLR2、4表达明显降低(P0.05)。结论:S.mitis作为传统观点认为的口咽部正常细菌,一方面它能增强PAO1粘附力及BF形成,从而逃避宿主免疫及抗生素杀伤;另一方面S.mitis减轻了PAO1诱导的宿主免疫反应。可能是新生VAP患儿ETT BF表面S.mitis检出率高的原因。研究结果提示我们不能忽视传统观点认为的非致病菌在BF中的作用。对于新生儿VAP ETT表面BF致病机制提供了新的思路,该观点亦可能为其他BF相关感染提供新研究方向。第二部分轻型链球菌对浮游铜绿假单胞菌致病性的影响目的:第一部分已证实S.mitis能增强PAO1粘附力、BF形成能力,并减轻PAO1 BCM诱导的细胞毒性和免疫反应。BF成熟后亦可释放浮游细菌,对宿主产生影响。且研究表明某些口腔常驻菌群(如牙卟啉单胞菌、放线菌等)能促进某些呼吸道病原体对于宿主细胞及组织的粘附等,从而影响其致病性。那么S.mitis对于浮游状态的P.aeruginosa有无作用呢?这是本部分实验拟阐明的问题。方法:将浮游菌分组为PAO1组、S.mitis组和PAO1+S.mitis组。将细菌(MOI=100)加入人呼吸道上皮细胞(BEAS-2B)。侵袭实验和粘附实验观察细菌对BEAS-2B细胞的侵袭和粘附力。CCK-8法检测细胞存活率,ELISA法检测细胞IL-8表达。结果:与PAO1单独组相比较,S.mitis可促进PAO1对BEAS-2B的侵袭能力(P0.05),而对PAO1粘附BEAS-2B的能力无明显影响(P0.05)。PAO1+S.mitis组与PAO1单独组在细胞存活率及IL-8水平无显著差异(P0.05)。结论:S.mitis能增强PAO1对BEAS-2B细胞侵袭性,但对PAO1粘附BEAS-2B细胞无影响,提示两者机制不同。虽然在S.mitis存在下,浮游PAO1侵袭性增强,但细胞存活率及炎症无显著影响,这可能利于PAO1在宿主细胞内形成BF,逃逸宿主免疫。S.mitis对PAO1致病性的影响可能主要在其BF状态产生。第三部分轻型链球菌通过增强铜绿假单胞菌生物膜QS系统促进其生物膜形成目的:多菌种BF内部菌群间交流调控机制是BF形成的关键环节,其中密度感应(Quorum sensing,QS)系统是研究多菌种BF调控的关键。第一部分已证实S.mitis能增强PAO1粘附力、BF形成能力,但具体机制不明;而P.aeruginosa的QS系统与其粘附力、BF形成能力密切相关。那么,S.mitis对P.aeruginosa上述能力的促进作用是否与其作用于P.aeruginosa的QS系统相关呢?方法:BF分为PAO1组、P.aΔlas R组、P.aΔLas RΔrhl R组、S.mitis组、PAO1+S.mitis组、P.aΔlas R+S.mitis组、P.aΔLas RΔrhl R+S.mitis组。结晶紫、平板计数法以及光学显微镜观察细菌粘附能力。结晶紫、平板计数及激光共聚焦(CLSM)观察BF形成。q PCR法检测各组BF的QS基因表达(包括las I、las R、rhl I、rhl R)。结果:PAO1+S.mitis、P.aΔlas R+S.mitis、P.aΔLas RΔrhl R+S.mitis三个组的粘附力均分别明显强于单独PAO1、P.aΔlas R和P.aΔLas RΔrhl R组(P0.05);且平板计数法结果显示PAO1+S.mitis、P.aΔlas R+S.mitis、P.aΔLas RΔrhl R+S.mitis混合组中粘附的P.aeruginosa菌分别较单独PAO1、P.aΔlas R、P.aΔlas RΔrhl R组多(P0.05)。PAO1+S.mitis混合组的BF生物量明显多于PAO1单独组(P0.05);然而,P.aΔlas R+S.mitis组和P.aΔLas RΔrhl R+S.mitis组BF生物量与P.aΔlas R组、P.aΔLas RΔrhl R组BF生物量无明显差异(P0.05)。S.mitis能上调PAO1菌株的QS基因表达(包括las I、las R、rhl I、rhl R),明显高于单独PAO1组的表达(P0.05);然而P.aΔlas R+S.mitis组和P.aΔLas RΔrhl R+S.mitis组的QS基因表达(包括las I、las R、rhl I、rhl R)并无上调,与单独P.aΔlas R和P.aΔlas RΔrhl R组无差异(P0.05)。结论:S.mitis对于P.aeruginosa粘附力的促进不依赖于QS系统,有其他机制参与。S.mitis通过上调P.aeruginosa QS基因促进其BF形成。这种促进机制是通过S.mitis直接作用还是其分泌某些物质(如AI-2)对P.aeruginosa BF形成起作用待未来实验论证。
[Abstract]:Part I Effect of Streptococcus militaris on pathogenicity of Pseudomonas aeruginosa BF Objective: Mechanical ventilation is widely used and biofilm (BF) is formed in tracheal tube (ETT). Ventilator associated pneumonia (VAP) in newborns is still a difficult clinical problem. Normal bacteria in oropharynx were the most common on the surface of ETT in neonates, and the detection rate of S. mitis was higher in VAP group. Cell viability, IL-8 expression and TLR2,4 expression were detected after BCM stimulation of human airway epithelial cells (BEAS-2B). The total viable bacteria and P. aeruginosa in mixed BF were significantly higher than those in PAO1 alone (P 0.05). However, compared with BCM in PAO1 alone, the cytotoxicity of mixed BCM and the expression of IL-8, TLR2, 4 induced by mixed BCM were significantly lower (P 0.05). It is believed that normal oropharyngeal bacteria, on the one hand, can enhance the adhesion of PAO1 and BF formation, thereby escaping the host immunity and antibiotic killing; on the other hand, S. mitis reduces the host immune response induced by PAO1. This may be the reason for the high detection rate of S. mitis on the surface of ETT BF in neonatal VAP children. The role of non-pathogenic bacteria in BF is generally believed to provide new insights into the pathogenesis of BF on the surface of neonatal VAP ETT. This view may also provide new research directions for other BF-related infections. BF also releases planktonic bacteria after maturation and affects the host. Studies have shown that some oral colonies (such as Porphyromonas dentatus, actinomycetes, etc.) can promote the adhesion of certain respiratory pathogens to host cells and tissues, thereby affecting them. Pathogenicity. So does S. mitis have any effect on planktonic P. aeruginosa? This part of the experiment is to clarify the question. Methods: Planktonic bacteria were divided into PAO1 group, S. mitis group and PAO1 + S. mitis group. Bacteria (MOI = 100) were added to human respiratory epithelial cells (BEAS-2B). Invasion and adhesion experiments were used to observe the invasion of bacteria on BEAS-2B cells. Results: Compared with PAO1 alone group, S. mitis could promote the invasion ability of PAO1 to BEAS-2B (P 0.05), but had no significant effect on the adhesion ability of PAO1 to BEAS-2B (P 0.05). There was no significant difference in cell survival rate and IL-8 level between PAO1 + S. mitis group and PAO1 alone group (P 0.05). Conclusion: S. mitis can enhance the invasiveness of PAO1 on BEAS-2B cells, but has no effect on PAO1 adhesion to BEAS-2B cells, suggesting that the mechanisms are different. The effect of sex may be mainly produced in the BF state. Part 3 Streptococcus minimus promotes the formation of biofilm by enhancing the QS system of Pseudomonas aeruginosa biofilm. The mechanism of intercommunion regulation in multispecies BF is the key to the formation of BF, and the Quorum sensing (QS) system is the key to the study of BF regulation in multispecies. The first part has proved that S. mitis can enhance the adhesion of PAO1 and the formation of BF, but the specific mechanism is unclear; and P. aeruginosa QS system is closely related to its adhesion and BF formation ability. So, is S. mitis related to P. aeruginosa QS system? Group R, P.a_Las R_rhl R, S.mitis, PAO1+S.mitis, P.a_las R+S.mitis, P.a_Las R RHL R+S.mitis, P.a_Las R RHL R+S.mitis, crystal violet, plate counting and optical microscopy were used to observe bacterial adhesion, crystal violet, plate counting and laser confocal microscopy (CLSM) were used to observe the formation of BF. Results: The adhesion of PAO1+S.mitis, P.a91las R+S.mitis, P.a91las R+S.mitis, P.a91LasR91rhl R+S.mitis, P.a91R+S.mitis three groups were significantly stronger than PAO1 alone, P.a91lasR and P.a91lasR and P.a91LasR91rhl R group (P 0.05); and the plate count results showed that PAO1+S.mitis, P.a91las R+S.mitis, P.a91las R+S.las, P.a91las R+S.las, P.a91R+S.las, P.a91las, P.A91The adhesion of P. aeruginosa in RHL R + S. mitis mixed group was higher than that in RHL R + S. mitis mixed group. BF biomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomabiomaDifference (P 0.05). S. mitis could up-regulate the QS gene of PAO1 strain The expression of QS gene (including Las I, Las R, RHL I, RHL R, RHL I, RHL R) was significantly higher than that of PAO1 group (P 0.05), but there was no significant difference between P.a91las R+S.mitis group and P.a91las R+S.mitis group and P.a91las R91rhl R+S.mitis group (including Las I, Las R, RHL I, RHL R, RHL I, RHL R) and P.a91las91las91las91las R and P.a91Rmitmitmitmitmitaerl group (P 0.05). Conclusion: for P.a91las91las91las91rasrasrasrasrasrasrasadhesion force S. mitis promotes BF formation by up-regulating the P. aeruginosa QS gene. Whether this mechanism is directly mediated by S. mitis or whether certain substances (such as AI-2) secreted by S. mitis play a role in the formation of P. aeruginosa BF remains to be demonstrated by future experiments.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378.991

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