噬菌体治疗小鼠肺部铜绿假单胞菌感染的研究

【目的】通过两种给药方式观察噬菌体鸡尾酒制剂对铜绿假单胞菌(Pseudomonas aeruginosa)肺部感染小鼠的疗效。【方法】将小鼠行气管切开术,经气管灌注2×10⁸ CFU/mL的铜绿假单胞菌悬液50 μL,0.5 h后分别给予噬菌体鸡尾酒滴鼻和腹腔注射,同时建立PBS滴鼻和腹腔注射对照组。待感染24 h后,观察小鼠的生理状态,并行细菌学、病理学与炎症因子水平等检查。【结果】噬菌体鸡尾酒制剂的治疗组小鼠,肺内的铜绿假单胞菌被清除;病理结果显示,治疗组小鼠肺部组织结构较完好、炎症程度较轻。【结论】该铜绿假单胞菌噬菌体鸡尾酒制剂在小鼠体内具有很强的抗菌作用,对肺部细菌感染具有一定的治疗效果。     

铜绿假单胞菌菌苗预防白色念珠菌感染的实验研究

目的利用铜绿假单胞菌菌苗探索阻断白色念珠菌感染的影响。方法家兔耳皮内注射白色念珠菌,进行活菌攻击,建立动物感染模型。观察用铜绿假单胞菌菌苗免疫的家兔状况;用试管凝集法测定家免的抗体效价;采用小白鼠体内吞噬法测定免疫组和空白对照组巨噬细胞的吞噬百分率和吞噬指数,以得出铜绿假单胞菌菌苗的免疫性。结果观察发现：与对照组比较,铜绿假单胞菌菌苗免疫组抗体效价,巨噬细胞的吞噬能力明显提高;注射区局部感染及全身症状明显减轻;肾脏感染灶减少,病理切片显示病情较轻。结论铜绿假单胞菌菌苗能够减轻注射区局部感染及全身症状,对肾脏的播散性白色念珠菌感染具有一定的阻断作用。     

宏基因组二代测序诊断肺隐球菌病2例

肺隐球菌病是最常见的肺部真菌感染之一,临床及影像学上缺乏特异性,容易误诊及漏诊。肺隐球菌病的诊断多依赖于病原学及组织病理学。近年来兴起的宏基因组二代测序技术,为感染性疾病的早期快速诊治提供了新的思路。本文报道2例既往无免疫抑制性基础疾病,无明显呼吸道症状及阳性体征,仅体检胸部CT发现肺野外带圆形实性结节灶,最终经支气管肺泡灌洗液宏基因组二代测序技术迅速诊断、及时治疗的肺隐球菌病患者的临床特征及诊治经过。     

反复铜绿假单胞菌感染诱导大鼠慢性阻塞性肺疾病模型探讨

目的探讨反复铜绿假单胞菌(PA)感染能否诱导大鼠产生慢性阻塞性肺疾病(COPD)的病理生理及病理学改变。方法将120只Wistar大鼠随机分为铜绿假单胞菌感染组(PA组)和对照组(NS组)。PA组通过气管穿刺,多次注入一定剂量铜绿假单胞菌菌液,建立大鼠慢性肺部感染模型,测定大鼠动脉血气,观察肺组织病理改变,测量气管壁厚度和血管壁厚度。结果自感染第4周开始,PA组大鼠体重较NS组显著减轻(P<0.05);自第12周开始,PA组大鼠血气PaO2显著低于NS组(0.01相似文献   

不同感染途径致大鼠肺炎模型制备的比较

目的探索铜绿假单胞菌致大鼠肺炎模型的制备方法,为后续研究奠定实验基础。方法将Sprague-Dawley(SD)大鼠48只随机分为4组:A对照组、B气管注射组、C气管插管感染组和D滴鼻感染组。适应饲喂3 d后,分别给予不同造模干预,分别于干预后5、10和15 d动态监测各亚组大鼠体重、体温、白细胞数、肺组织病理学变化等。结果各模型组大鼠行为学表现、体重、体温、白细胞、肺组织炎症病理学表现均与对照组差异有显著性;(2)各模型组均证实为铜绿假单胞菌感染,对照组则阴性。结论经滴鼻途径以铜绿假单胞菌感染大鼠,可得到合格大鼠肺炎模型;此感染途径可避免手术创口带来的炎症反应干扰,简便易行,可推广。     

铜绿假单胞菌肺部感染小鼠模型中L-17A的表达研究

摘要：目的 了解铜绿假单胞菌肺部感染小鼠模型中L-17A（白介素-17）的表达,指导铜绿假单胞菌肺部感染治疗。方法 择取60只SPF级昆明白雌性小鼠为研究对象,应用随机平行对照法将其分成两组,对照组自小鼠气管内注入相同量的生理盐水,实验组自小鼠气管内注入铜绿假单胞菌悬液0.03 mL,造模后处死两组小鼠,将其肺部组织制制作病理切片,行HE染色,并应用免疫组化法检测肺组织IL-17A表达量变化情况。结果 于3 h、9 h和24 h后,实验组肺组织中IL-17A灰度（182.2±4.7,184.0±9.0,182.8±9.9）明显优于对照组（170.2±4.3,161.8±6.5,147.4±7.3）,且9 h、24 h后血清中IL-17A浓度（132.6±5.5,149.8±5.7）pg/mL显著高于对照组（124.2±2.8,123.2±7.9）pg/mL,差异具有统计学意义（P＜0.05）。结论 IL-17A经由炎症细胞募集介入铜绿假单胞菌肺部感染炎症反应,并参与了病原菌清除过程,临床上应引起足够重视。     

噬菌体治疗对铜绿假单胞菌诱导的肺炎小鼠细菌负荷和炎症水平的影响

铜绿假单胞菌是医院获得性感染和肺部感染的主要致病菌之一,能够引发患者烧伤部位、呼吸道和泌尿道的慢性和急性感染,导致严重的发病率和死亡率。由于抗生素的滥用,铜绿假单胞菌已经对多种抗生素表现出耐药性,严重限制了可用于治疗感染患者的治疗方案。噬菌体依靠内寄生活的细菌进行生存和繁殖,其结构简单,分布广泛,被发现能够用于治疗人类细菌感染疾病,是治疗由铜绿假单胞菌引发的感染的可能方案之一。本研究构建了铜绿假单胞菌诱导的肺炎小鼠模型,对模型小鼠进行噬菌体和美罗培南的单独和联合给药处理,采用ELISA和Western blotting等方法检测了小鼠血清,支气管肺泡灌洗液和肺组织中的细菌负荷、常规生化指标和炎症因子水平。表明经铜绿假单胞菌感染后的小鼠,肺组织的细菌负荷显著增加,血清CRP和乳酸升高,TNF-α、IL-1β、IL-6和透明质酸水平也都显著上升。噬菌体和美罗培南的单独或联合处理能显著下调这些上升,其中噬菌体单独使用对乳酸的下调作用最为显著,而联合用药则对下调血清IL-1β水平具有协同优势。     

口腔细菌对铜绿假单胞菌黏附及侵入肺上皮细胞能力的影响

目的 通过比较铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入细胞的能力,探讨细菌间相互作用在呼吸道感染的最初阶段的作用机制.方法 应用培养法和抗生素保护法检测铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入肺上皮细胞的能力.结果 铜绿假单胞菌与口腔细菌共同作用于肺上皮细胞,牙龈卟啉单胞菌和伴放线放线杆菌降低了铜绿假单胞菌的黏附能力,却增强了其侵入能力;而铜绿假单胞菌能够影响口腔细菌对肺上皮细胞的黏附,同时增强口腔细菌侵入肺上皮细胞的能力.结论 口腔细菌,尤其是牙周可疑致病菌主要通过增强铜绿假单胞菌对肺上皮细胞的侵入而影响呼吸道感染过程.     

红霉素、氨溴索与环丙沙星雾化吸入对实验大鼠铜绿假单胞菌生物膜的影响

目的研究红霉素和氨溴索分别联合环丙沙星雾化吸入对铜绿假单胞菌成熟生物膜的干预效果。方法平板法培养成熟铜绿假单胞菌生物膜;微量肉汤稀释法测量红霉素和环丙沙星的最低抑菌浓度;制作气管插管铜绿假单胞菌生物膜感染模型;平板计数法计算红霉素、氨溴索分别联合环丙沙星对生物膜菌落数的影响;日本岛津紫外-可见光分光光度计UV1700测铜绿假单胞菌菌液的A值;石蜡切片HE染色定性观察肺组织的炎症情况;扫描电镜定性观察各处理组的生物膜结构变化。结果各处理组干预7 d后肺组织细菌菌落计数（×10^4CFU/m l）：干预组分为：生理盐水对照,氨溴索,红霉素,红霉素联合环丙沙星,氨溴索联合环丙沙星,各组分别为139.250±42.0162、101.625±40.4190、109.625±33.4747、57.750±37.8295和22.250±17.3184,前3组与后2组对比差异均有显著性（P〈0.05）,前3组之间对比差异没有显著性（P〉0.05）,后2组对比差异有显著性（P〈0.05）。导管生物膜细菌菌落计数（×10^4CFU/m l）：5组分别为170.000±48.3263、127.625±39.0163、133.500±33.6876、70.375±35.7768和38.125±19.1045,结论和肺组织菌落计数是一致的。导管生物膜电镜观察：第1组导管内表面均有较厚基质覆盖,2、3组减少不明显,而联合用药组导管内表面生物膜明显减少,其中第5组效果更好。结论氨溴索与红霉素分别联合环丙沙星雾化吸入在控制导管生物膜和呼吸系统相关感染均具有显著效果,其中氨溴索联合环丙沙星疗效更好。     

呼吸道合胞病毒感染兔呼吸系统组织病理学的改变和相关指数评分标准的建立

目的探讨RSV感染兔呼吸系统的组织病理学改变及其病理学指数评估方法。方法将RSV 100μL（108 TCID50/mL）鼻腔接种新西兰兔,分别于3和5 d时处死动物,取鼻黏膜、气管、肺门支气管、肺组织做石蜡包埋切片、HE染色,与正常对照组比较,显微镜下观察组织病理学改变。根据病变程度、参考相关文献,制定鼻黏膜、气管/支气管、肺组织的病理学指数评分标准并进行评分。结果与正常对照组相比,接种病毒动物可见鼻黏膜增厚甚至可见坏死脱落灶、单个核细胞浸润以及淋巴样组织增生;气管、支气管黏膜增厚、粗糙、排列紊乱,有坏死脱落,支气管内可见渗出物;肺组织以肺门部位病变显著,可见以围绕支气管或血管组织为主的炎细胞浸润灶,肺泡内、各级细支气管内存在炎性分泌物;接种RSV5 d组病理学指数评分与正常对照组相比有统计学意义。结论RSV鼻腔接种新西兰兔可致其鼻黏膜、气管、支气管及肺组织发生炎性损伤。我们制定的鼻黏膜、气管/支气管、肺组织的病理学指数评分标准适用于动物呼吸系统炎性损伤程度的评估。     

不同方法建立多重耐药铜绿假单胞菌肠道感染动物模型的比较和评价

目的探讨建立肠道多重耐药细菌感染动物模型的方法,为实验研究和治疗因耐药菌引起的感染提供良好的模型。方法 24只6周龄SPF级雄性BALB/c小鼠分成4组:正常对照组、MDR-PA组、MDR-PA+抗生素组和MDR-PA+禁食组,6只/组。分别给予生理盐水灌胃、PA悬液灌胃、自由饮用含头孢曲松钠水后PA悬液灌胃和禁食后PA悬液灌胃。实验结束后对结肠组织进行病理学观察和炎症评分;检测炎症因子TNF-α和INF-γ浓度。结果结肠组织病理表现出不同程度的炎症变化;炎症因子TNF-α和INF-γ浓度较对照组均明显增加,有显著统计学意义。结论三种方法均可成功建立肠道多重耐药铜绿假单孢菌感染的动物模型,可根据不同实验目的选择不同的建立模型方法。     

In vivo growth of Pseudomonas aeruginosa strains PAO1 and PA14 and the hypervirulent strain LESB58 in a rat model of chronic lung infection

Pseudomonas aeruginosa chronic lung infections are the major cause of morbidity and mortality in cystic fibrosis (CF) patients. The P. aeruginosa strains PAO1 and PA14 were compared with the Liverpool epidemic strain LESB58 to assess in vivo growth, infection kinetics, and bacterial persistence and localization within tissues in a rat model of chronic lung infection. The three P. aeruginosa strains demonstrated similar growth curves in vivo but differences in tissue distribution. The LESB58 strain persisted in the bronchial lumen, while the PAO1 and PA14 strains were found localized in the alveolar regions and grew as macrocolonies after day 7 postinfection. Bacterial strains were compared for swimming and twitching motility and for the production of biofilm. The P. aeruginosa LESB58 strain produced more biofilm than PAO1 and PA14. Competitive index (CI) analysis of PAO1, PA14, and LESB58 in vivo indicated CI values of 0.002, 0.0002, and 0.14 between PAO1-PA14, PAO1-LESB58, and LESB58-PA14, respectively. CI analysis comparing the in vivo growth of the PAO1 DeltaPA5441 mutant and four PA14 surface attachment-defective (sad) mutants gave CI values 10 to 1,000 times lower in competitions with their respective wild-type strains PAO1 and PA14. P. aeruginosa strains studied in the rat model of chronic lung infection demonstrated similar in vivo growth but differences in virulence as shown with a competitive in vivo assay. These differences were further confirmed with biofilm and motility in vitro assays, where strain LESB58 produced more biofilm but had less capacity for motility than PAO1 and PA14.     

Antigenicity and immunogenicity of the polyagglutinable antigen of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa strains isolated from cystic fibrosis patients agglutinate in antisera against anti-polyagglutinable antigen (PA). Anti-PA antibodies were formed in rabbits when immunization was carried out with bacteria possessing core-bound PA, independently of whether the strains were of S or R phenotype. For bacterial agglutination with anti-PA antibodies two prerequisites are essential: the bacterial cell must be of R phenotype and must possess the core-linked PA. In contrast, the PA in the isolated LPS's can be demonstrated in passive haemagglutination for both (S or R) phenotypes, provided the PA is core-linked. Two PA forms have been recognized, one found only in P. aeruginosa species, both in free and bound form. The other one is shared by all members of Pseudomonas genus but is present only in a free, unbound form.     

Antigenicity and immunogenicity of the polyagglutinable antigen of Pseudomonas aeruginosa

Pseudomonas aeruginosa strains isolated from cystic fibrosis patients agglutinate in antisera against anti-polyagglutinable antigen (PA). Anti-PA antibodies were formed in rabbits when immunization was carried out with bacteria possessing core-bound PA, independently of whether the strains were of S or R phenotype. For bacterial agglutination with anti-PA antibodies two prerequisites are essential: the bacterial cell must be of R phenotype and must possess the core-linked PA. In contrast, the PA in the isolated LPS's can be demonstrated in passive haemagglutination for both (S or R) phenotypes, provided the PA is core-linked. Two PA forms have been recognized, one found only in P. aeruginosa species, both in free and bound form. The other one is shared by all members of Pseudomonas genus but is present only in a free, unbound form.     

Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa

Nosocomial infections often cause lethal pneumogenic sepsis. Information on early bacteria-host interaction in the lung is limited. In the present study, mice were sacrificed 60 min and 4 h after Pseudomonas aeruginosa (PA) infection to investigate lung morphology by using electron microscopy and light microscopy. After 1 h, bacteria were found in the alveoli partly in contact with surfactant. Alveolar macrophages were seen with up to 10 intracellular bacteria close to protrusions of alveolar epithelial type I cells and the gas/blood barrier. A rare but surprising finding was bacteria and even replicating bacteria in alveolar epithelial type II cells (AEII). No bacteria were seen in capillaries. Neither engulfment of bacteria by neutrophils nor structural damage of the pulmonary barrier was visible. After 4 h, many neutrophils were found within the capillaries, but also in the alveolar space. Thus, we hypothesize that, in early stages of infection, the uptake of PA even by single AEII can influence the course of the disease.     

Kinase suppressor of Ras-1 protects against pulmonary Pseudomonas aeruginosa infections

Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe infections in immunocompromised individuals and individuals with cystic fibrosis or chronic obstructive pulmonary disease (COPD). Here we show that kinase suppressor of Ras-1 (Ksr1)-deficient mice are highly susceptible to pulmonary P. aeruginosa infection accompanied by uncontrolled pulmonary cytokine release, sepsis and death, whereas wild-type mice clear the infection. Ksr1 recruits and assembles inducible nitric oxide (NO) synthase (iNOS) and heat shock protein-90 (Hsp90) to enhance iNOS activity and to release NO upon infection. Ksr1 deficiency prevents lung alveolar macrophages and neutrophils from activating iNOS, producing NO and killing bacteria. Restoring NO production restores the bactericidal capability of Ksr1-deficient lung alveolar macrophages and neutrophils and rescues Ksr1-deficient mice from P. aeruginosa infection. Our findings suggest that Ksr1 functions as a previously unknown scaffold that enhances iNOS activity and is therefore crucial for the pulmonary response to P. aeruginosa infections.     

Omega-3 polyunsaturated fatty acids improve host response in chronic Pseudomonas aeruginosa lung infection in mice

Pseudomonas aeruginosa is a gram-negative bacilli frequently encountered in human pathology. This pathogen is involved in a large number of nosocomial infections and chronic diseases. Herein we investigated the effects of polyunsaturated fatty acids (PUFA) in chronic Pseudomonas aeruginosa lung infection. C57BL/6 mice were fed for 5 wk with specifically designed diets with high contents in either omega-3 (omega-3) or omega-6 PUFA and compared to a control diet. P. aeruginosa included in agarose beads was then instilled intratracheally, and the animals were studied for 7 days. On the 4th day, the mice fed with the omega-3 diet had a higher lean body mass gain and a lower omega-6:omega-3 ratio of fatty acids extracted from the lung tissue compared with the other groups (P < 0.05). The omega-3 group had the lowest mortality. Distal alveolar fluid clearance (DAFC) as well as the inflammatory response and the cellular recruitment were higher in the omega-3 group on the 4th day. The effect on DAFC was independent of alpha-epithelial Na(+) channels (alpha-ENaC), beta-ENaC, and alpha(1)-Na-K-ATPase mRNA expressions, which were not altered by the different diets. In conclusion, a diet enriched in omega-3 PUFA can change lung membrane composition and improve survival in chronic pneumonia. This effect on survival is probably multifactorial involving the increased DAFC capacity as well as the optimization of the initial inflammatory response. This work suggests that a better control of the omega-6/omega-3 PUFA balance may represent an interesting target in the prevention and/or control of P. aeruginosa infection in patients.     

Macrophage dysfunction and susceptibility to pulmonary Pseudomonas aeruginosa infection in surfactant protein C-deficient mice

To determine the role of surfactant protein C (SP-C) in host defense, SP-C-deficient (Sftpc-/-) mice were infected with the pulmonary pathogen Pseudomonas aeruginosa by intratracheal injection. Survival of young, postnatal day 14 Sftpc-/- mice was decreased in comparison to Sftpc+/+ mice. The sensitivity to Pseudomonas bacteria was specific to the 129S6 strain of Sftpc-/- mice, a strain that spontaneously develops interstitial lung disease-like lung pathology with age. Pulmonary bacterial load and leukocyte infiltration were increased in the lungs of Sftpc-/- mice 24 h after infection. Early influx of polymorphonuclear leukocytes in the lungs of uninfected newborn Sftpc-/- mice relative to Sftpc+/+ mice indicate that the lack of SP-C promotes proinflammatory responses in the lung. Mucin expression, as indicated by Alcian blue staining, was increased in the airways of Sftpc-/- mice following infection. Phagocytic activity of alveolar macrophages from Sftpc-/- mice was reduced. The uptake of fluorescent beads in vitro and the number of bacteria phagocytosed by alveolar macrophages in vivo was decreased in the Sftpc-/- mice. Alveolar macrophages from Sftpc-/- mice expressed markers of alternative activation that are associated with diminished pathogen response and advancing pulmonary fibrosis. These findings implicate SP-C as a modifier of alveolar homeostasis. SP-C plays an important role in innate host defense of the lung, enhancing macrophage-mediated Pseudomonas phagocytosis, clearance and limiting pulmonary inflammatory responses.