CpxR对鼠伤寒沙门菌连接蛋白基因pmrD的调控作用

前期发现黏菌素对鼠伤寒沙门菌acrB和cpxR双缺失后的cpxR回补株JS△acrB△cpxR::kan/pcpxR(JS△△/pR)的MIC值较标准株JS显著下降了16倍,且JS△△/pR中PhoPQ和PmrAB之间的连接蛋白基因pmrD的表达量显著降低。为探索cpxR对pmrD的调控作用,本研究构建了pmrD的单基因过表达菌株JS△acrB△cpxR::kan/ppmrD(JS△△/pD),并用overlapping PCR扩增得到cpxR和pmrD的共表达基因cpxR-pmrD,构建了cpxR、pmrD共表达菌株JS△acrB△cpxR::kan/pcpxR-pmrD(JS△△/pRD)。用微量肉汤稀释法测定了黏菌素对各菌株的最小抑菌浓度(MICs),同时测定了各菌株在LB肉汤中的生长曲线和黏菌素的杀菌曲线。结果表明,成功构建了鼠伤寒沙门菌pmrD的单基因过表达菌株JS△△/pD和cpxR、pmrD共表达菌株JS△△/pRD。MIC结果显示,黏菌素对JS△△/pR的MIC值较JS显著下降(16倍),JS△△/pD的MIC值没有变化,而JS△△/pRD的MIC值显著上升(16倍)。生长曲线显示,JS△△/pR的生长活性最低,JS△△/pD、JS△△/pRD的生长活性均高于JS△△/pR。黏菌素的杀菌曲线显示,JS△△/pRD在不同浓度黏菌素中的存活率显著高于JS△△/pD。这些结果表明:在JS△△背景下,pmrD对菌株黏菌素敏感性的影响依赖于cpxR,cpxR对pmrD的调控具有双向性,即对生理表达的pmrD具有负调控作用,而对过表达的pmrD具有正调控作用。本试验为全面系统阐明cpxR或cpxR与acrB相互作用调控沙门菌敏感性的分子机制奠定基础。     

STM1863分子特征分析及其基因缺失对鼠伤寒沙门菌生物学特性的影响

为揭示鼠伤寒沙门菌SL-1344菌株RcsCDB系统STM1863调控子分子特征,及对STM生物学特性的影响。本研究通过PCR扩增STM1863基因并进行克隆和测序,预测分析其编码蛋白的分子特征;利用λ-Red同源重组技术构建STM-ΔSTM1863基因缺失突变株,并对其生化特性、遗传稳定性、酸碱胁迫环境中的适应性及粘附侵袭小鼠巨噬细胞的能力进行研究。结果显示：STM1863基因全长159 bp,编码52个氨基酸,具有一个跨膜和d1p32a同系物结构域;与SL-1344亲本株相比,STM-ΔSTM1863缺失株生化特性与亲本株无明显差异,生长速度与亲本株生长速度一致,但STM-ΔSTM1863在p H4.0酸应激下对数期生长速度显著低于亲本株（P<0.05）;粘附和侵袭小鼠巨噬细胞能力与亲本株相比极显著降低（P<0.01）。提示STM1863因参与了STM酸应激和粘附侵袭宿主细胞的调控,本研究为深入揭示STM1863对STM粘附侵袭宿主细胞的调控机制提供了参考。     

CpxR对鼠伤寒沙门菌的黏菌素耐药相关基因pmrB和phoQ调控作用的研究

为了探索 cpxR 对鼠伤寒沙门菌的黏菌素耐药相关基因 pmrB 和 phoQ 的调控作用,用overlapping PCR扩增得到 cpxR 、 pmrB 和 cpxR 、 phoQ 共表达基因 cpxR - pmrB 、 cpxR - phoQ ,用2倍微量肉汤稀释法对构建的 pmrB 、 phoQ 单基因过表达菌株JSΔ acrB Δ cpxR :: kan /p pmrB (JSΔΔ/p B )、JSΔ acrB Δ cpxR :: kan /p phoQ (JSΔΔ/p Q )和 cpxR - pmrB 、 cpxR - phoQ 共表达菌株JSΔ acrB Δ cpxR :: kan /p cpxR - pmrB (JSΔΔ/p RB )、JSΔ acrB Δ cpxR :: kan /p cpxR - phoQ (JSΔΔ/p RQ )进行黏菌素最小抑菌浓度(MICs)的测定,同时以各菌株在LB中的OD 600 nm 值绘制生长曲线,以各菌株在不同浓度的黏菌素中的存活率绘制杀菌曲线。结果:成功构建的鼠伤寒沙门菌 pmrB 、 phoQ 的单基因过表达菌株JSΔΔ/p B 、JSΔΔ/p Q 和 cpxR - pmrB 、 cpxR - phoQ 共表达菌株JSΔΔ/p RB 、JSΔΔ/p RQ 的MIC结果显示,JSΔ acrB Δ cpxR :: kan /p cpxR (JSΔΔ/p R )的MIC值较JS下降93.75%,与本实验室前期研究结果一致。JSΔΔ/p B 、JSΔΔ/p Q 的MIC值较JS均上升3倍,而JSΔΔ/p RB 、JSΔΔ/p RQ 的MIC值较JS分别降低50%和75%。生长曲线结果显示JSΔΔ/p R 的生长活性最低,JSΔΔ/p B 、JSΔΔ/p Q 、JSΔΔ/p RB 和JSΔΔ/p RQ 的生长活性均低于JSΔ acrB Δ cpxR :: kan /pHisA(JSΔΔ/pHisA)。黏菌素的杀菌曲线结果显示JSΔΔ/p B 和JSΔΔ/p Q 在不同浓度的黏菌素中的存活率显著高于黏菌素较敏感的JSΔΔ/p RB 和JSΔΔ/p RQ 。上述结果表明:CpxR能够通过PmrB和PhoQ调控鼠伤寒沙门菌对黏菌素的敏感性。     

鼠伤寒沙门菌oppCDF基因缺失株的构建及生物学特性分析

利用λ-Red同源重组技术构建鼠伤寒沙门菌oppCDF基因缺失株,并检测其生长特性、运动性、生物被膜形成能力、胞内存活能力及LD₅₀毒力。结果显示,与野生型菌株相比,oppCDF基因缺失株的生长特性无明显变化;oppCDF基因缺失株可降低鼠伤寒沙门菌的运动性;oppC与oppF基因缺失后可提高鼠伤寒沙门菌生物被膜形成能力;同时,oppC基因缺失可降低在RAW267.4细胞内的存活能力和其在小鼠体内的毒力,oppF基因缺失可显著增强在RAW267.4内的存活能力和对小鼠毒力的影响,而oppD基因缺失后其在小鼠体内的毒力和RAW267.4内的存活率均无显著变化。研究表明,oppCDF基因与鼠伤寒沙门菌的运动性、生物被膜形成和毒力密切相关,为进一步揭示鼠伤寒沙门菌致病作用中的复杂调控和机制提供了理论基础。     

5株鼠伤寒沙门菌基因缺失株的生物学特性比较

为了探讨crp、cya、sipB、crp/cya、crp/sipB基因缺失株成为鼠伤寒沙门菌减毒候选活疫苗的可能性,对缺失株的生物学特性进行研究。结果显示,缺失株的血清型仍和亲本菌株相同,其中crp、cya、crp/cya、crp/sipB与亲本菌株相比失去了利用麦芽糖、乳糖、山梨醇等碳源的能力,也不能分解H2S、半乳糖和鼠李糖,但仍保留了利用葡萄糖的能力。通过口服方式把5株crp、cya、sipB、crp/cya、crp/sipB基因缺失株接种KM小鼠进行毒力测定和免疫保护性测定,结果它们的半数致死量比野生株的至少高700倍,双基因缺失株的毒力更弱,攻毒后crp、cya、sipB、crp/cya、crp/sipB基因缺失株的免疫保护率分别为90.0%,60.0%,50.0%,60.0%,60.0%。结果表明,crp基因缺失株可作为鼠伤寒沙门菌减毒活疫苗的候选株。     

鼠伤寒沙门菌ST1920株cpxR基因缺失株的构建及其对小鼠LD_(50)的影响

为了探索鼠伤寒沙门菌双组分信号系统CpxAR的应答调节蛋白基因cpxR对小鼠半数致死量(LD_(50))的影响,为下一步研究CpxAR系统对鼠伤寒沙门菌致病性的调控机制提供基础。本研究通过ERIC-PCR、MLST分型技术筛选流行克隆株JS_分,应用噬菌体转导技术构建基因缺失突变株JS_分△cpxR,并分别测定了小鼠腹腔注射JS_标、JS_标△cpxR、JS_分、JS_分△cpxR后的LD_(50)。结果显示:89株鼠伤寒沙门菌分为11个ERIC型、4个ST型,流行性克隆株为ST1920型(新的ST型)。LD_(50)结果显示,JS_标、JS_标△cpxR、JS_分、JS_分△cpxR对小鼠的LD_(50)分别为5.79×108、7.85×108、1.91×107、3.35×108 CFU。其中ST1920型代表株(JS_分)的LD_(50)较JS_标低约30.31倍,JS_标△cpxR突变株的LD_(50)比其亲本株JS_标升高了1.36倍,JS_分△cpxR突变株LD_(50)比其亲本株JS_分升高了17.54倍。结果表明cpxR基因缺失株毒力均明显下降,cpxR基因与鼠伤寒沙门菌毒力密切相关,对致病性有重要影响。     

减毒鼠伤寒沙门菌活疫苗载体研究进展

活载体疫苗技术的发展促使产生了更多的疫苗设计新思路.沙门菌是一种肠道细菌,目前已通过基因突变的方法构建了许多种减毒株,如GID101、GID105、X4064、ZJⅢ、Ty800、X4632、X4550、X4072和X3730等,这些减毒株被突变掉了两个或两个以上的基因,然而其基因型和血清型仍很稳定,所以人们常将其用作基因疫苗的表达载体或运送载体.现在已有许多种细菌、病毒和寄生虫的减毒沙门菌活载体疫苗被研制成功.文章对沙门菌的减毒方法,减毒沙门菌的免疫机理以及其用途和作为疫苗载体的优缺点做了简述.     

鼠伤寒沙门菌sapC基因缺失株的构建及生物学特性分析

旨在研究鼠伤寒沙门菌ABC转运膜蛋白SapC在沙门菌致病机制中的功能。本研究利用λ-Red重组技术构建了鼠伤寒沙门菌sapC基因缺失突变株SMΔsapC,对其进行生长特性、酸性应激试验、多黏菌素B敏感性试验、生物被膜检测、胞内存活和小鼠体内毒力试验。结果显示,基因缺失株SMΔsapC与亲本菌株和互补菌株相比,其生长速度无明显差异;在酸应激条件下,SMΔsapC存活率显著低于亲本菌株;sapC基因缺失降低了鼠伤寒沙门菌生物被膜的形成能力;同时,SMΔsapC基因缺失株在鼠源巨噬细胞内的增殖能力和小鼠体内的毒力显著低于亲本菌株。研究表明,sapC基因影响鼠伤寒沙门菌的抗酸能力、生物被膜形成能力,从而影响沙门菌在体内外的毒力。本研究为进一步阐释鼠伤寒沙门菌的致病机制奠定了基础。     

鹅源鼠伤寒沙门菌crp、hfq基因缺失株的构建及生物学特性分析

为构建鹅鼠伤寒沙门菌单基因缺失株并鉴定其生物学特性,本研究以广东地区鹅源鼠伤寒沙门菌流行株A29为研究对象,采用λ-Red同源重组技术,分别构建crp、hfq基因缺失株(A29Δcrp和A29Δhfq)及相应基因回补株,并分别采用相应引物经PCR鉴定。通过细菌培养,对各菌株生物学特性进行比较;通过PCR鉴定各菌株的遗传稳定性;采用K-B纸片扩散法检测各菌株的药物敏感性;将各菌株纯培养物经腹腔注射4周龄小鼠,测定各菌株对小鼠的毒力,并观察感染后小鼠的临床症状及各组织剖检病变与组织病理变化。PCR和测序结果表明,正确构建各基因缺失株和相应回补株。生物学特性试验结果显示,与亲本株相比,A29Δcrp菌落直径极显著变小(P<0.001),不产酸、不产生H₂S,菌体极显著变短(P<0.001);A29Δhfq菌落边缘不整齐,不产生H₂S,菌体极显著变长(P<0.001),表明crp、hfq基因影响细菌的菌落形态、生化特性和形态特征。菌株遗传稳定试验结果显示,菌株传至60代时,基因缺失株仍能够稳定缺失。药敏试验结果显示,与亲本株比较,A...     

鼠伤寒沙门菌烈性噬菌体的分离鉴定与生物学特性

旨在分离鼠伤寒沙门菌烈性噬菌体,为控制该病原菌感染或污染提供生物制剂.以1株从市售散装牛奶中分离到的多重耐药性鼠伤寒沙门菌为宿主菌,采用人工诱导方法,连续1周每日给3只试验鸡饲喂宿主菌悬液,7 d后,采用双层琼脂平板法从试验鸡粪便中分离培养噬菌体,并对其效价、核酸类型、宿主谱、热稳定性与酸碱耐受性以及对鼠伤寒沙门菌感染...     

鼠伤寒沙门菌sipB基因缺失对菌体糖酵解的影响

为研究鼠伤寒沙门菌sip B基因编码蛋白与菌体糖酵解的关系,本研究对sip B基因缺失前后菌株丙酮酸、乳酸以及ATP含量进行检测,结果显示,sip B基因缺失后菌体丙酮酸、乳酸含量降低,而ATP含量升高,表明菌体糖酵解减弱,获取能量的方式发生改变。该结果为分析sip B基因编码蛋白与菌体糖酵解的关系,以及为研究sip B基因与菌体死亡的关系奠定基础。     

白痢沙门菌CpxR的生物信息学分析和原核表达

对白痢沙门菌二元调控系统响应蛋白CpxR进行生物信息学分析,提示CpxR可能发挥了重要的生理功能。同时,构建了重组表达载体pET32a-CpxR,转化表达菌株BL21后,对其进行诱导表达,SDS-PAGE和Western blot结果表明CpxR获得了正确表达。纯化蛋白免疫小鼠制备了多抗血清,Western blot验证其有一定的免疫原性。为深入研究其生理功能和调控机制奠定了理论基础。     

Effects of subinhibitory concentrations of florfenicol on morphology, growth, and viability of Staphylococcus aureus

Staphylococcus aureus strain Newman was investigated for changes in its growth pattern, its morphology and its viability when grown in the presence of 3 microg/ml florfenicol (Ff). This concentration corresponds to the 0.75-fold strain-specific minimum inhibitory concentration (MIC). Under these conditions, S. aureus Newman showed a distinct retardation in its growth pattern and 20% dead cells were detected in a fluorescence microscopic viability assay. However, bactericidal activity - defined as a 3-log drop in the staphylococcal population - was not recorded at this Ff concentration. Further analysis of the cell wall revealed a significant increase in cell wall thickness of S. aureus Newman when grown in the presence of 3 microg/ml Ff. This might result in a compression of the protoplast with subsequent disruption of single staphylococcal cells. Accordingly, 20% of the staphylococcal cells analysed by electron microscopy proved to be disrupted. These observations suggest that Ff can cause a thickening of the cell wall accompanied by impaired viability of the staphylococcal cells.     

鼠伤寒沙门氏菌IsrB基因的克隆及其同源表达

为同源表达鼠伤寒沙门氏菌(S.typhimurium)的lsrB基因,本研究通过PCR扩增得到S.typhimurium SL1344株的lsrB基因,其全长1020 bp,编码340个氨基酸.通过序列分析,该基因与S.typhimurium LT2的neA基因同源性为100％,与克雷伯氏菌(Klebsiella) A...     

The effects of prior growth as a biofilm on the virulence of Salmonella typhimurium for mice

Relatively little is known about how growth as a biofilm affects the virulence of pathogenic bacteria. In this study, the virulence of Salmonella typhimurium grown as a biofilm, or as planktonic cells, was compared in mice. Increased numbers of colony forming units were recovered from the spleens of mice 5 days after i.p. injection with S. typhimurium grown as a biofilm, as compared with planktonic cells (P < 0.05). No significant difference in the CFU of S. typhimurium recovered from the liver was noted at the same time point, and no difference was noted in the CFU recovered from the spleen or liver at 5 days after i.v. or i.g. inoculation with 10(5) S. typhimurium. Nor were any differences noted at 7 days after i.p., i.v. or i.g. inoculation. Thus, any effect of growth as a biofilm has on the virulence of S. typhimurium seems to be limited to the first 5 days after i.p. inoculation.     

Studies on the pathogenesis of Salmonella typhimurium and Salmonella choleraesuis var kunzendorf infection in weanling pigs

Twenty-six 4-week-old pigs were randomly allotted to 4 groups: group 1--orally inoculated with Salmonella typhimurium; group 2--orally dosed with S choleraesuis; and groups 3 and 4, with surgically constructed intestinal loops--loops inoculated with either S typhimurium or S choleraesuis. One pig each from groups 1 and 2 was killed at 8, 12, 24, 48, 72, 96, and 120 hours after inoculation. One pig each from groups 3 and 4 was killed at 2, 4, 6, 8, 12, and 24 hours after intestinal loop inoculation. Inoculation of S typhimurium resulted in acute enterocolitis of variable severity, whereas inoculation of S choleraesuis resulted initially in septicemia followed by formation of large necrotic and ulcerative lesions in the colonic mucosa. The most consistent systemic lesion of S choleraesuis infection was interstitial pneumonia and multifocal hepatic necrosis. Salmonella typhimurium and S choleraesuis were ultrastructurally within enterocytes of ligated ileal loops. Intracellular bacteria were morphologically intact, occurred free in the cytoplasm and membrane bound, and caused no detectable cytotoxic effect to the cell. Both S typhimurium and S choleraesuis penetrated the intestinal mucosa and were isolated from mesenteric lymph nodes at 2 hours after inoculation.     

Comparing the minimum inhibitory and mutant prevention concentrations of selected antibiotics against animal isolates of Pasteurella multocida and Salmonella typhimurium

Historically, the use of antibiotics was not well regulated in veterinary medicine. The emergence of antibiotic resistance (ABR) in pathogenic bacteria in human and veterinary medicine has driven the need for greater antibiotic stewardship. The preservation of certain antibiotic classes for use exclusively in humans, especially in cases of multidrug resistance, has highlighted the need for veterinarians to reduce its use and redefine dosage regimens of antibiotics to ensure efficacy and guard against the development of ABR pathogens. The minimum inhibitory concentration (MIC), the lowest concentration of an antibiotic drug that will prevent the growth of a bacterium, is recognised as a method to assist in antibiotic dosage determination. Minimum inhibitory concentrations sometimes fail to deal with first-step mutants in bacterial populations; therefore dosing regimens based solely on MIC can lead to the development of ABR. The mutant prevention concentration (MPC) is the minimum inhibitory antibiotic concentration of the most resistant first-step mutant. Mutant prevention concentration determination as a complementary and sometimes preferable alternative to MIC determination for veterinarians when managing bacterial pathogens. The results of this study focused on livestock pathogens and antibiotics used to treat them, which had a MIC value of 0.25 µg/mL for enrofloxacin against all 27 isolates of Salmonella typhimurium. The MPC values were 0.50 µg/mL, with the exception of five isolates that had MPC values of 4.00 µg/mL. The MPC test yielded 65.52% (18 isolates) Salmonella isolates with florfenicol MICs in the sensitive range, while 11 isolates were in the resistant range. Seventeen isolates (58.62%) of Pasteurella multocida had MIC values in the susceptible range and 41.38% (12 isolates) had an intermediate MIC value. Mutant prevention concentration determinations as done in this study is effective for the antibiotic treatment of bacterial infections and minimising the development of resistance. The MPC method can be used to better control to prevent the development of antibiotic drug resistance used in animals.