大肠杆菌密度感应调节子C（QseC）在生物材料植入感染中的作用研究
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摘要
[背景和目的]
随着生物材料被广泛应用于临床,生物材料植入感染(implant-associated infection)已经成为令人棘手的常见医院内感染,有报道占医院内感染的50%,往往给患者带来灾难性后果。大肠杆菌是临床生物材料植入感染优势菌种,在心脏瓣膜置换术、关节置换术、脑室腹腔分流术等术后生物材料植入感染中大肠杆菌检出率为3～10%。
大肠杆菌(Escherichia coli)是人体肠道中的条件致病菌,心脏大血管手术体外循环期间、神经外科或骨科等手术控制性降压期间、以及临床上常见的失血性休克,均可导致肠通透性升高及肠道细菌移位,造成菌血症,血液中的细菌为细菌在生物材料表面黏附提供菌源。细菌在生物材料表面一旦形成细菌生物膜,就能有效地抵御机体的防御反应和抗生素的治疗,是导致生物材料植入感染难以控制的根源。密度感应系统(quorum sensing system)在细菌生物膜形成过程中起重要作用。Sperandio等研究表明大肠杆菌密度感应调节子C (Quorum sensing E. coli regulator C, QseC)可以识别自诱导物3(AI-3)、肾上腺素(EPI)及去甲肾上腺素(NE)等信号分子,在浮游状态时细菌的密度感应系统中起重要调节作用。然而,在肠道大肠杆菌移位引发的生物材料植入感染中,大肠杆菌QseC的作用如何,目前相关报道甚少。
本研究采用Red同源重组技术构建大肠杆菌K-12菌株qseC阴性突变菌株；在前期建立的聚氯乙烯材料表面细菌生物膜模型基础上,采用激光扫描等技术,探讨大肠杆菌QseC对生物材料表面细菌生物膜形成能力的影响；通过失血性休克建立肠道细菌移位动物模型,探讨大肠杆菌QseC在肠道细菌移位引发生物材料植入感染中的作用；揭示防治大肠杆菌引发的生物材料植入感染的有效分子药物靶标,为防治生物材料植入感染提供实验依据。
[方法]
本研究分四部分：
1、应用Red同源重组技术构建大肠杆菌qseC阴性突变菌株：选用大肠杆菌K-12MC1000菌株为研究对象,应用PCR技术扩增两翼与目的基因上下游同源、含有氯霉素抗性基因片段,电击转化入大肠杆菌MC1000。在阿拉伯糖诱导下,含有质粒pKD46的菌株MC1000表达Red同源重组酶,利用含同源臂的氯霉素抗性片段置换目的基因qseC,并进一步利用FLP位点专一性重组将氯霉素抗性基因删除。最后用鉴定引物行PCR对大肠杆菌qseC阴性突变菌株进行鉴定。所得突变菌株标记为qseC
2、大肠杆菌QseC在细菌生物学表型变化中的作用：测定大肠杆菌MC1000和MC1000ΔqseC的生长曲线,及其在泳动能力检测培养基上的运动能力。探讨大肠杆菌QseC在细菌生物学表型变化中的作用。
3、大肠杆菌QseC在生物材料表面细菌生物膜形成中的作用：以两株大肠杆菌(MC1000菌株和MC1000ΔqseC菌株)为实验菌株,在培养基中添加EPI或NE,建立PVC材料表面细菌生物膜体外模型。实验中同时设置未经EPI或NE处理的野生菌株和阴性突变菌株作为对照。采用半定量检测方法测定大肠杆菌的细菌生物膜形成能力,应用激光共聚焦显微镜(CLSM)动态观察PVC材料表面大肠杆菌生物膜的形成过程、细菌群落数量及细菌生物膜厚度等,应用扫描电镜(SEM)观察PVC材料表面大肠杆菌细菌生物膜的表面结构。探讨大肠杆菌QseC在PVC材料表面细菌生物膜形成中的作用。
4、QseC在肠道细菌移位并在生物材料表面细菌生物膜形成中的作用：SPF级健康雄性SD大鼠随机分为6组：空白+假休克组(空白-Sham),空白+休克组(空白-HS),MC1000+假休克组(M-Sham), MC1000ΔqseC+假休克组(Δ-Sham),MC1000+休克组(M-HS),MC1000ΔqseC+休克组(Δ-HS),每组6只。将实验菌株定植于大鼠肠道内,经右下腹斜切口将PVC材料放入腹膜腔并建立失血性休克动物模型。在休克后24h处死大鼠,严格无菌条件下取门静脉血和肠系膜淋巴结、肝脾标本行细菌培养,采用平板菌落计数法计数细菌移位率及菌落数,并根据组织重量换算为每克组织的菌落总数(CFU/g);通过PCR反应对细菌进行鉴定,证实细菌来自于大鼠肠道内的实验菌株；取出PVC材料片作细菌培养和鉴定,同时用SEM对材料表面细菌生物膜的形成情况进行观察。
[结果]
1、大肠杆菌qseC阴性突变菌株的构建：PCR及DNA测序结果表明,qseC基因内部序列已被删除,且无氯霉素抗性基因,qseC基因已被成功敲除。
2、大肠杆菌QseC在细菌生物学表型变化中的作用：①MC1000和MC1000ΔqseC在LB培养基中的生长并无明显差异；②MC1000和MC1000ΔqseC在LBEPI(或LBNE)培养基中的生长并无明显差异；③相对于MC1000菌株,MC1000ΔqseC突变株的运动能力明显下降(P<0.05)；④MC1000菌株在LBEPI和LBNE培养基中的运动力较之在LB培养基中明显增强(P<0.05),而当存在EPI或NE时MC1000ΔqseC突变株的变化无统计学意义(P>0.05)。
3、大肠杆菌QseC在生物材料表面细菌生物膜形成中的作用：①PVC材料表面大肠杆菌生物膜的形成过程：PVC材料表面细菌群落数量及细菌生物膜厚度随时间的变化而变化,培育18-24h时细菌群落数量达到高峰,培育24h“时细菌生物膜厚度达到峰值；②QseC在PVC材料表面细菌生物膜形成中的作用：与MC1000组相比,MC1000ΔqseC组PVC材料表面大肠杆菌生物膜的形成能力明显降低(P<0.05),单位面积细菌群落数量减少(P<0.05),细菌生物膜厚度明显减小(P<0.05)；③EPI(或NE)对PVC材料表面大肠杆菌细菌生物膜形成的影响：添加EPI或NE后PVC材料表面MC1000细菌生物膜的形成能力明显增强(P<0.05),而添加EPI或NE后PVC材料表面MC1000ΔqseC细菌生物膜的形成能力并无明显改变(P>0.05)。
4、QseC在肠道细菌移位并在生物材料表面细菌生物膜形成中的作用：①在大鼠门静脉血、肠系膜淋巴结、肝脾组织内以及PVC材料片上,均分离出耐链霉素细菌,经PCR证实这些细菌就是来自于灌饲到大鼠肠道的实验菌株；②Δ-HS组大鼠细菌移位率和内脏组织细菌含量都比M-HS组明显降低(P<0.05);③Δ-HS组大鼠PVC材料上的细菌含量较之M-HS组有明显降低(P<0.05)。
[结论]
1、大肠杆菌QseC缺失后运动能力显著下降。儿茶酚胺对大肠杆菌运动能力有直接促进作用,而且该作用是通过QseC介导的。
2、生物材料表面大肠杆菌生物膜的形成是一个动态过程,生物材料表面细菌群落数量及细菌生物膜厚度的增加与时间的变化相关,这可能与生物材料植入感染的临床感染特征有关。
3、大肠杆菌QseC在生物材料表面细菌生物膜的形成中具有重要作用。儿茶酚胺对大肠杆菌在生物材料表面细菌生物被膜的形成有直接促进作用,而且该作用是通过QseC介导的。
4、大肠杆菌QseC缺失后,运动能力降低,同时QseC信号链中断,细菌对肠粘膜的侵袭力和穿透力减弱,导致细菌移位发生率下降。QseC-1肾上腺素信号通路在肠道大肠杆菌移位并在生物材料表面定植中起重要作用。
[Background and Objective]
With the increasing numbers of devices that are being implanted, implant-associated infection (IAI) has already become the commonly formidable infection in hospital. According to a report, IAI accounts for50%infections in hospital. Escherichia coli is one of the most common microorganisms associated with device-related infections. Infections of prosthetic heart valves, prosthetic joints, and ventriculoperitoneal shunts are often caused by Escherichia coli.
Cardiopulmonary bypass during cardiac surgery, controlled hypotension during neurosurgical or orthopedic procedure, and hemorrhagic shock result in the translocation of bacteria, leading to bacteremia. Bacterium in blood adhered to indwelling devices and then bacterial biofilm was formed. Bacterial biofilm has been shown to play an important role in implant-associated infections. In the biofilm state, microorganisms are relatively immune to antibodies and resistant to conventional antimicrobial agents. Regulation of the biofilm formation involves the qseC quorum-sensing system. The quorum sensing E.coli regulator C (QseC) is the histidine sensor kinase that detects autoinducer-3/epinephrine/norepinephrine in the planktonic state. However, the role of QseC in the implant-associated infection caused by Escherichia coli remains unclear.
We investigated biofilm formation on the surface of medical-grade polyvinyl chloride (PVC) by using the E.coli K-12strain as well as corresponding qseC mutant. E.coli qseC mutant was constructed using lambda Red mutagenesis. LB with epinephrine and without epinephrine was used, and then the experiment of bacterial biofilm formation on PVC material was taken. The thickness of bacterial community and bacterial community quantity in the unit area on PVC materials were measured by confocal laser scanning microscope (CLSM), and the surface structure of biofilm formation was observed by scanning electron microscope (SEM). Additionally, a rat model of nonlethal hemorrhagic shock was used. Whether the ability of E.coli to sense epinephrine/norepinephrine by QseC would have the effect on the bacterial translocation to indwelling devices in a rat model of hemorrhagic shock was studied. These results serve to give a deeper understanding of how Escherichia coli biofilm on indwelling devices works. Our results are helpful for understanding and preventing biofilm formation, as well as helpful for combating implant-associated infections.
[Methods]
1. Construction of the qseC gene knockout
The E.coli K-12MC1000qseC mutant was constructed by using the lambda Red mutagenesis strategy. The Cm resistance cassette from plasmid pKD3, including approximately50-bp extensions from the5'and3'ends of the predicted qseC gene was amplified. PCR products were electroporated into MC1000(pKD46) that had been induced to express the λRed recombinase. Cmr recombinants were selected. Mutations were confirmed by PCR. To remove the Cm cassette from the ΔqseC::Cm mutant, plasmid pCP20was introduced into the strain. The mutant, ΔqseC, was confirmed by PCR.
2. The role of QseC in growth curves and motility
The growth curves and motility of E. coli MC1000and MC1000ΔqseC were assayed.
3. The role of QseC in the biofilm formation on the surface of polyethylene chloride
We investigated the role of the QseC in the formation of biofilms on the surface of medical-grade poly vinyl chloride by using the E.coli K-12MC1000strain as well as a corresponding qseC mutant. LB with epinephrine and without epinephrine was used, and then the experiment of bacterial biofilm formation on PVC material was taken. The thickness of bacterial community and bacterial community quantity in the unit area on PVC materials were measured by confocal laser scanning microscope (CLSM), and the surface structure of biofilm formation was observed by scanning electron microscope (SEM).
4. The role of QseC in the bacterial translocation to indwelling devices
E. coli MC1000and MC1000ΔqseC were used to track BT after gavage in rats. A rat model of nonlethal hemorrhagic shock was used. The rats were divided into6groups:controls (SS), rats that received a sham shock and MC1000(M-SS), rats that received a sham shock and MC1000ΔqseC (A-SS), rats that received a hemorrhagic shock and were not fed with E. coli (HS), rats that received a hemorrhagic shock and MC1000(M-HS), and rats that received a hemorrhagic shock and MC1000ΔqseC (Δ-HS). At sacrifice (24h), portal venous blood, the mesenteric lymph nodes, the midsection of the spleen, and segment IV of the liver were aseptically removed for bacteriological examination, and segments of the ileum were harvested for histopathology. The polyethylene chloride from peritoneal cavity was observed by scanning electron microscope (SEM).
[Results]
1. Deletion of qseC using the lambda Red system
The qseC-deleted mutant of E. coli was confirmed by various PCR and DNA sequencing. Gene qseC was completely deleted.
2. The role of QseC in growth curves and motility
①There was no significant difference in growth ability between the qseC mutant strain and the wild-type strain MC1000in LB medium. Growth was not altered after qseC was deleted.②EPI and NE were used at concentrations of50μmol L-1. At these concentrations, no significant alterations in the growth of E.coli MC1000and MC1000ΔqseC were observed.③In the absence of EPI、NE, MC1000ΔqseC was less mobile than the isogenic wild-type strain.④MC1000motility increased upon the addition of EPI/NE. MC1000ΔqseC did not respond significantly to EPI/NE addition.
3. The role of QseC in the biofilm formation on the surface of polyethylene chloride
①E.coli biofilm formation on the surface of polyethylene chloride:Bacterium community quantity and thickness of bacterium biofilm on PVC have changed over time. After being cultured18-24h, bacterium community quantity reached peak. After being cultured24h, thickness of bacterium biofilm reached peak.②The effect of QseC on bacterium biofilm formation on PVC surface:Deletion of qseC decreased biofilm formation in LB. Deletion of qseC led to the reduction of bacterium community quantity and biofilm thickness.③Effect of EPI/NE on biofilm formation of the qseC-deleted mutant of Escherichia coli on biomaterial:Biofilm formation was stimulated significantly by EPI/NE in LB medium for E.coli MC1000. But that in E.coli MC1000ΔqseC, biofilm formation was not stimulated significantly by EPI/NE.
4. The role of QseC in the bacterial translocation to indwelling devices
①The streptomycin-resistant bacteria isolated from the portal venous blood, MLNs, spleen, liver and PVC in the M-HS group produced a band at1488bp, whereas the streptomycin-resistant extraintestinal bacteria in the Δ-HS group produced a band at237bp. So these bacteria were the E. coli MC1000or MC1000ΔqseC administered to the rats by gavage.②Rats treated with MC1000ΔqseC showed reduced levels of bacterial translocation following hemorrhagic shock compared with rats treated with MC1000.③The number of streptomycin-resistant bacteria found in the PVC in the A-HS group was considerably reduced and significantly lower than that of the M-HS group.
[Conclusions]
1. E.coli AqseC motility decreased. E.coli motility increased upon the addition of catecholamines. Catecholamines increased Escherichia coli motility through QseC.
2. The bacterium biofilm formation on the surface of implant is a dynamic process. Bacterium community quantity and thickness of bacterium biofilm has changed over time. It is possibly the important factor resulting in repetatus infection caused by the implant.
3. The qseC gene plays an important role in regulation of biofilm formation of Escherichia coli on the surface of implant. In E.coli AqseC, biofilm formation was not stimulated significantly by catecholamines, which indicates that catecholamines stimulate E.coli biofilm formation on the surface of implant through QseC.
4. Rats treated with E.coli AqseC, which has a weakened motility and does not respond to catecholamines, showed reduced levels of bacterial translocation following hemorrhagic shock. The blockade of the QseC receptor-mediated action may be useful to attenuate bacterial translocation to indwelling devices after hemorrhage.
随着生物材料被广泛应用于临床,生物材料植入感染(implant-associated infection)已经成为令人棘手的常见医院内感染,有报道占医院内感染的50%,往往给患者带来灾难性后果。大肠杆菌是临床生物材料植入感染优势菌种,在心脏瓣膜置换术、关节置换术、脑室腹腔分流术等术后生物材料植入感染中大肠杆菌检出率为3～10%。
大肠杆菌(Escherichia coli)是人体肠道中的条件致病菌,心脏大血管手术体外循环期间、神经外科或骨科等手术控制性降压期间、以及临床上常见的失血性休克,均可导致肠通透性升高及肠道细菌移位,造成菌血症,血液中的细菌为细菌在生物材料表面黏附提供菌源。细菌在生物材料表面一旦形成细菌生物膜,就能有效地抵御机体的防御反应和抗生素的治疗,是导致生物材料植入感染难以控制的根源。密度感应系统(quorum sensing system)在细菌生物膜形成过程中起重要作用。Sperandio等研究表明大肠杆菌密度感应调节子C (Quorum sensing E. coli regulator C, QseC)可以识别自诱导物3(AI-3)、肾上腺素(EPI)及去甲肾上腺素(NE)等信号分子,在浮游状态时细菌的密度感应系统中起重要调节作用。然而,在肠道大肠杆菌移位引发的生物材料植入感染中,大肠杆菌QseC的作用如何,目前相关报道甚少。
本研究采用Red同源重组技术构建大肠杆菌K-12菌株qseC阴性突变菌株；在前期建立的聚氯乙烯材料表面细菌生物膜模型基础上,采用激光扫描等技术,探讨大肠杆菌QseC对生物材料表面细菌生物膜形成能力的影响；通过失血性休克建立肠道细菌移位动物模型,探讨大肠杆菌QseC在肠道细菌移位引发生物材料植入感染中的作用；揭示防治大肠杆菌引发的生物材料植入感染的有效分子药物靶标,为防治生物材料植入感染提供实验依据。
[方法]
本研究分四部分：
1、应用Red同源重组技术构建大肠杆菌qseC阴性突变菌株：选用大肠杆菌K-12MC1000菌株为研究对象,应用PCR技术扩增两翼与目的基因上下游同源、含有氯霉素抗性基因片段,电击转化入大肠杆菌MC1000。在阿拉伯糖诱导下,含有质粒pKD46的菌株MC1000表达Red同源重组酶,利用含同源臂的氯霉素抗性片段置换目的基因qseC,并进一步利用FLP位点专一性重组将氯霉素抗性基因删除。最后用鉴定引物行PCR对大肠杆菌qseC阴性突变菌株进行鉴定。所得突变菌株标记为qseC
2、大肠杆菌QseC在细菌生物学表型变化中的作用：测定大肠杆菌MC1000和MC1000ΔqseC的生长曲线,及其在泳动能力检测培养基上的运动能力。探讨大肠杆菌QseC在细菌生物学表型变化中的作用。
3、大肠杆菌QseC在生物材料表面细菌生物膜形成中的作用：以两株大肠杆菌(MC1000菌株和MC1000ΔqseC菌株)为实验菌株,在培养基中添加EPI或NE,建立PVC材料表面细菌生物膜体外模型。实验中同时设置未经EPI或NE处理的野生菌株和阴性突变菌株作为对照。采用半定量检测方法测定大肠杆菌的细菌生物膜形成能力,应用激光共聚焦显微镜(CLSM)动态观察PVC材料表面大肠杆菌生物膜的形成过程、细菌群落数量及细菌生物膜厚度等,应用扫描电镜(SEM)观察PVC材料表面大肠杆菌细菌生物膜的表面结构。探讨大肠杆菌QseC在PVC材料表面细菌生物膜形成中的作用。
4、QseC在肠道细菌移位并在生物材料表面细菌生物膜形成中的作用：SPF级健康雄性SD大鼠随机分为6组：空白+假休克组(空白-Sham),空白+休克组(空白-HS),MC1000+假休克组(M-Sham), MC1000ΔqseC+假休克组(Δ-Sham),MC1000+休克组(M-HS),MC1000ΔqseC+休克组(Δ-HS),每组6只。将实验菌株定植于大鼠肠道内,经右下腹斜切口将PVC材料放入腹膜腔并建立失血性休克动物模型。在休克后24h处死大鼠,严格无菌条件下取门静脉血和肠系膜淋巴结、肝脾标本行细菌培养,采用平板菌落计数法计数细菌移位率及菌落数,并根据组织重量换算为每克组织的菌落总数(CFU/g);通过PCR反应对细菌进行鉴定,证实细菌来自于大鼠肠道内的实验菌株；取出PVC材料片作细菌培养和鉴定,同时用SEM对材料表面细菌生物膜的形成情况进行观察。
[结果]
1、大肠杆菌qseC阴性突变菌株的构建：PCR及DNA测序结果表明,qseC基因内部序列已被删除,且无氯霉素抗性基因,qseC基因已被成功敲除。
2、大肠杆菌QseC在细菌生物学表型变化中的作用：①MC1000和MC1000ΔqseC在LB培养基中的生长并无明显差异；②MC1000和MC1000ΔqseC在LBEPI(或LBNE)培养基中的生长并无明显差异；③相对于MC1000菌株,MC1000ΔqseC突变株的运动能力明显下降(P<0.05)；④MC1000菌株在LBEPI和LBNE培养基中的运动力较之在LB培养基中明显增强(P<0.05),而当存在EPI或NE时MC1000ΔqseC突变株的变化无统计学意义(P>0.05)。
3、大肠杆菌QseC在生物材料表面细菌生物膜形成中的作用：①PVC材料表面大肠杆菌生物膜的形成过程：PVC材料表面细菌群落数量及细菌生物膜厚度随时间的变化而变化,培育18-24h时细菌群落数量达到高峰,培育24h“时细菌生物膜厚度达到峰值；②QseC在PVC材料表面细菌生物膜形成中的作用：与MC1000组相比,MC1000ΔqseC组PVC材料表面大肠杆菌生物膜的形成能力明显降低(P<0.05),单位面积细菌群落数量减少(P<0.05),细菌生物膜厚度明显减小(P<0.05)；③EPI(或NE)对PVC材料表面大肠杆菌细菌生物膜形成的影响：添加EPI或NE后PVC材料表面MC1000细菌生物膜的形成能力明显增强(P<0.05),而添加EPI或NE后PVC材料表面MC1000ΔqseC细菌生物膜的形成能力并无明显改变(P>0.05)。
4、QseC在肠道细菌移位并在生物材料表面细菌生物膜形成中的作用：①在大鼠门静脉血、肠系膜淋巴结、肝脾组织内以及PVC材料片上,均分离出耐链霉素细菌,经PCR证实这些细菌就是来自于灌饲到大鼠肠道的实验菌株；②Δ-HS组大鼠细菌移位率和内脏组织细菌含量都比M-HS组明显降低(P<0.05);③Δ-HS组大鼠PVC材料上的细菌含量较之M-HS组有明显降低(P<0.05)。
[结论]
1、大肠杆菌QseC缺失后运动能力显著下降。儿茶酚胺对大肠杆菌运动能力有直接促进作用,而且该作用是通过QseC介导的。
2、生物材料表面大肠杆菌生物膜的形成是一个动态过程,生物材料表面细菌群落数量及细菌生物膜厚度的增加与时间的变化相关,这可能与生物材料植入感染的临床感染特征有关。
3、大肠杆菌QseC在生物材料表面细菌生物膜的形成中具有重要作用。儿茶酚胺对大肠杆菌在生物材料表面细菌生物被膜的形成有直接促进作用,而且该作用是通过QseC介导的。
4、大肠杆菌QseC缺失后,运动能力降低,同时QseC信号链中断,细菌对肠粘膜的侵袭力和穿透力减弱,导致细菌移位发生率下降。QseC-1肾上腺素信号通路在肠道大肠杆菌移位并在生物材料表面定植中起重要作用。
[Background and Objective]
With the increasing numbers of devices that are being implanted, implant-associated infection (IAI) has already become the commonly formidable infection in hospital. According to a report, IAI accounts for50%infections in hospital. Escherichia coli is one of the most common microorganisms associated with device-related infections. Infections of prosthetic heart valves, prosthetic joints, and ventriculoperitoneal shunts are often caused by Escherichia coli.
Cardiopulmonary bypass during cardiac surgery, controlled hypotension during neurosurgical or orthopedic procedure, and hemorrhagic shock result in the translocation of bacteria, leading to bacteremia. Bacterium in blood adhered to indwelling devices and then bacterial biofilm was formed. Bacterial biofilm has been shown to play an important role in implant-associated infections. In the biofilm state, microorganisms are relatively immune to antibodies and resistant to conventional antimicrobial agents. Regulation of the biofilm formation involves the qseC quorum-sensing system. The quorum sensing E.coli regulator C (QseC) is the histidine sensor kinase that detects autoinducer-3/epinephrine/norepinephrine in the planktonic state. However, the role of QseC in the implant-associated infection caused by Escherichia coli remains unclear.
We investigated biofilm formation on the surface of medical-grade polyvinyl chloride (PVC) by using the E.coli K-12strain as well as corresponding qseC mutant. E.coli qseC mutant was constructed using lambda Red mutagenesis. LB with epinephrine and without epinephrine was used, and then the experiment of bacterial biofilm formation on PVC material was taken. The thickness of bacterial community and bacterial community quantity in the unit area on PVC materials were measured by confocal laser scanning microscope (CLSM), and the surface structure of biofilm formation was observed by scanning electron microscope (SEM). Additionally, a rat model of nonlethal hemorrhagic shock was used. Whether the ability of E.coli to sense epinephrine/norepinephrine by QseC would have the effect on the bacterial translocation to indwelling devices in a rat model of hemorrhagic shock was studied. These results serve to give a deeper understanding of how Escherichia coli biofilm on indwelling devices works. Our results are helpful for understanding and preventing biofilm formation, as well as helpful for combating implant-associated infections.
[Methods]
1. Construction of the qseC gene knockout
The E.coli K-12MC1000qseC mutant was constructed by using the lambda Red mutagenesis strategy. The Cm resistance cassette from plasmid pKD3, including approximately50-bp extensions from the5'and3'ends of the predicted qseC gene was amplified. PCR products were electroporated into MC1000(pKD46) that had been induced to express the λRed recombinase. Cmr recombinants were selected. Mutations were confirmed by PCR. To remove the Cm cassette from the ΔqseC::Cm mutant, plasmid pCP20was introduced into the strain. The mutant, ΔqseC, was confirmed by PCR.
2. The role of QseC in growth curves and motility
The growth curves and motility of E. coli MC1000and MC1000ΔqseC were assayed.
3. The role of QseC in the biofilm formation on the surface of polyethylene chloride
We investigated the role of the QseC in the formation of biofilms on the surface of medical-grade poly vinyl chloride by using the E.coli K-12MC1000strain as well as a corresponding qseC mutant. LB with epinephrine and without epinephrine was used, and then the experiment of bacterial biofilm formation on PVC material was taken. The thickness of bacterial community and bacterial community quantity in the unit area on PVC materials were measured by confocal laser scanning microscope (CLSM), and the surface structure of biofilm formation was observed by scanning electron microscope (SEM).
4. The role of QseC in the bacterial translocation to indwelling devices
E. coli MC1000and MC1000ΔqseC were used to track BT after gavage in rats. A rat model of nonlethal hemorrhagic shock was used. The rats were divided into6groups:controls (SS), rats that received a sham shock and MC1000(M-SS), rats that received a sham shock and MC1000ΔqseC (A-SS), rats that received a hemorrhagic shock and were not fed with E. coli (HS), rats that received a hemorrhagic shock and MC1000(M-HS), and rats that received a hemorrhagic shock and MC1000ΔqseC (Δ-HS). At sacrifice (24h), portal venous blood, the mesenteric lymph nodes, the midsection of the spleen, and segment IV of the liver were aseptically removed for bacteriological examination, and segments of the ileum were harvested for histopathology. The polyethylene chloride from peritoneal cavity was observed by scanning electron microscope (SEM).
[Results]
1. Deletion of qseC using the lambda Red system
The qseC-deleted mutant of E. coli was confirmed by various PCR and DNA sequencing. Gene qseC was completely deleted.
2. The role of QseC in growth curves and motility
①There was no significant difference in growth ability between the qseC mutant strain and the wild-type strain MC1000in LB medium. Growth was not altered after qseC was deleted.②EPI and NE were used at concentrations of50μmol L-1. At these concentrations, no significant alterations in the growth of E.coli MC1000and MC1000ΔqseC were observed.③In the absence of EPI、NE, MC1000ΔqseC was less mobile than the isogenic wild-type strain.④MC1000motility increased upon the addition of EPI/NE. MC1000ΔqseC did not respond significantly to EPI/NE addition.
3. The role of QseC in the biofilm formation on the surface of polyethylene chloride
①E.coli biofilm formation on the surface of polyethylene chloride:Bacterium community quantity and thickness of bacterium biofilm on PVC have changed over time. After being cultured18-24h, bacterium community quantity reached peak. After being cultured24h, thickness of bacterium biofilm reached peak.②The effect of QseC on bacterium biofilm formation on PVC surface:Deletion of qseC decreased biofilm formation in LB. Deletion of qseC led to the reduction of bacterium community quantity and biofilm thickness.③Effect of EPI/NE on biofilm formation of the qseC-deleted mutant of Escherichia coli on biomaterial:Biofilm formation was stimulated significantly by EPI/NE in LB medium for E.coli MC1000. But that in E.coli MC1000ΔqseC, biofilm formation was not stimulated significantly by EPI/NE.
4. The role of QseC in the bacterial translocation to indwelling devices
①The streptomycin-resistant bacteria isolated from the portal venous blood, MLNs, spleen, liver and PVC in the M-HS group produced a band at1488bp, whereas the streptomycin-resistant extraintestinal bacteria in the Δ-HS group produced a band at237bp. So these bacteria were the E. coli MC1000or MC1000ΔqseC administered to the rats by gavage.②Rats treated with MC1000ΔqseC showed reduced levels of bacterial translocation following hemorrhagic shock compared with rats treated with MC1000.③The number of streptomycin-resistant bacteria found in the PVC in the A-HS group was considerably reduced and significantly lower than that of the M-HS group.
[Conclusions]
1. E.coli AqseC motility decreased. E.coli motility increased upon the addition of catecholamines. Catecholamines increased Escherichia coli motility through QseC.
2. The bacterium biofilm formation on the surface of implant is a dynamic process. Bacterium community quantity and thickness of bacterium biofilm has changed over time. It is possibly the important factor resulting in repetatus infection caused by the implant.
3. The qseC gene plays an important role in regulation of biofilm formation of Escherichia coli on the surface of implant. In E.coli AqseC, biofilm formation was not stimulated significantly by catecholamines, which indicates that catecholamines stimulate E.coli biofilm formation on the surface of implant through QseC.
4. Rats treated with E.coli AqseC, which has a weakened motility and does not respond to catecholamines, showed reduced levels of bacterial translocation following hemorrhagic shock. The blockade of the QseC receptor-mediated action may be useful to attenuate bacterial translocation to indwelling devices after hemorrhage.
引文
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