免疫抑制大鼠肺孢子菌合并呼吸道细菌感染的研究

目的 探讨免疫抑制兼肺孢子菌感染大鼠下呼吸道合并细菌感染的菌群种类及其影响因素.方法 用腹膜内注射地塞米松的方法建立免疫抑制大鼠并诱导肺孢子菌感染模型.免疫抑制的实验组动物再分为经呼吸道吸入乙醚麻醉组和不实施乙醚麻醉组,以了解乙醚对呼吸道感染的影响.此外,设不经任何处理的正常对照组.免疫抑制剂注射8周后处死大鼠,取肺组织制成肺印片,染色后进行肺孢子菌感染的病原学鉴定,同时取气管末端进行细菌培养和鉴定.结果 成功地建立了免疫抑制和肺孢子菌感染大鼠模型.乙醚麻醉组肺孢子菌感染率和菌体负荷均高于非乙醚麻醉组(P＜0.05).感染的菌种主要为双歧杆菌、大肠埃希菌、流感嗜血杆菌、丙酸丙酸杆菌、金黄色葡萄球菌、纤维单胞菌、放线菌及肺炎克雷伯杆菌.其中,麻醉组和非麻醉组下呼吸道细菌感染率有差异的是大肠埃希菌、流感嗜血杆菌、丙酸丙酸杆菌和金黄色葡萄球菌(P＜0.05).结论 用乙醚麻醉方法建立大鼠肺孢子菌模型能有效提高大鼠感染肺孢子菌感染率和感染度,下呼吸道感染细菌多为混合感染且受乙醚麻醉影响.     

高毒力肺炎克雷伯菌致病及耐药分子机制研究进展

近年来,肺炎克雷伯菌已成为医院内感染及社区获得性感染的常见致病菌,临床标本分离率仅次于大肠埃希菌.根据毒力特征差异,肺炎克雷伯菌可分为经典肺炎克雷伯菌和高毒力肺炎克雷伯菌2种类型.高毒力肺炎克雷伯菌是引起化脓性肝脓肿的主要病原菌,其感染可出现内源性转移,包括眼、肺和中枢神经系统;此外还与原发性肝外感染有关,包括菌血症、...     

肺炎克雷伯菌生物膜对小鼠巨噬细胞免疫功能的影响

目的研究肺炎克雷伯菌生物膜(BF)对小鼠腹腔巨噬细胞TLRs mRNA和细胞因子表达的影响,探索机体抗BF感染免疫的特点。方法将雄性昆明种小鼠40只随机分成2组,一组腹腔植入体外形成肺炎克雷伯菌BF的硅胶片,建立留置性医疗装置BF感染模型实验组,另一组植入与实验组同等量的浮游菌作为对照组。实时定量PCR分析2组巨噬细胞TLRs mRNA的表达水平,双抗体夹心ELISA法测定细胞因子的含量。结果实验BF组巨噬细胞TLR2、TLR4 mRNA表达量是对照浮游菌组的0.23和0.24倍;而TLR5、TLR9两组表达差异无显著性。实验BF组刺激前后IL-1、IL-2的差值明显低于对照浮游菌组,而IL-4则相反(P0.01)。结论与浮游菌相比,BF能下调小鼠腹腔巨噬细胞TLR2、TLR4的表达,机体的免疫应答朝着Th2型免疫反应发展,这可能是BF相对浮游菌更容易逃脱机体免疫防御系统、引起慢性感染的机制之一。     

产超广谱β-内酰胺酶肺炎克雷伯菌血流感染的耐药性、危险因素及临床结局分析

本研究旨在分析107例肺炎克雷伯菌血流感染患者的临床资料,探讨产超广谱β-内酰胺酶(extended-spectrumβ-lactamase,ESBL)肺炎克雷伯菌血流感染的耐药特点、危险因素及临床结局,为防治产ESBL肺炎克雷伯菌血流感染提供临床理论参考。选取2012年1月—2016年6月于深圳市南山区人民医院住院且血培养肺炎克雷伯菌阳性的107例患者,根据药敏结果分成产ESBL血流感染组(20例)和非产ESBL血流感染组(87例)。107例患者血流感染主要继发于肺部感染(38.32%)及泌尿系感染(14.02%),细菌对碳青霉烯类抗生素敏感性好。单因素及logistic回归分析结果显示,医院内感染和入院前有外伤史为产ESBL肺炎克雷伯菌血流感染的危险因素。总体肺炎克雷伯菌血流感染病死率为17.76%,产ESBL组与非产ESBL组之间病死率无显著性差异(25%vs.16.09%)。结果提示,产ESBL不是预测肺炎克雷伯菌血流感染患者死亡的独立危险因素。     

肺炎克雷伯菌生物膜对小鼠腹腔巨噬细胞TLRs受体表达的影响

目的研究肺炎克雷伯菌生物膜对小鼠腹腔巨噬细胞TEas受体表达的影响,探索机体抗生物膜（biofdm,BF）感染免疫的特点。方法将雄性昆明种小鼠40只随机分成2组,一组腹腔植入体外形成肺炎克雷伯菌生物膜的硅胶片,建立留置性医疗装置BF感染模型实验组,另一组植入与实验组同等量的浮游菌作为对照组。实时定量PCR分析2组巨噬细胞TLRsmRNA的表达水平,流式细胞仪检测分析蛋白的表达水平。结果实验生物膜组巨噬细胞TLR2、TLR4mRNA相对表达量是对照浮游菌组的0．23和0．24倍：实验组TLR2、TLR4蛋白表达率分别是（23．27±2．73）％和（15．83±2．04）％,明显低于对照组的（33．42±3．72）％、（21．75±1．25）％（P〈0．05）。结论与浮游菌相比,BF能下调小鼠腹腔巨噬细胞TLR2、TLR4表达,从而影响机体的免疫功能,这可能是BF相对浮游菌更容易逃脱机体免疫防御系统、引起慢性感染的机制之一。     

超广谱β-内酰胺酶肺炎克雷伯菌噬菌体F20的分离鉴定及其对小鼠败血症治疗效果

【背景】肺炎克雷伯菌是引起临床感染的重要条件致病菌之一,肺炎克雷伯菌中产超广谱β-内酰胺酶(Extended-spectrum beta-lactamases,ESBLs)的耐药菌株增多迫切需要找到一种新的治疗方法。【目的】自污水中分离超广谱β-内酰胺酶肺炎克雷伯菌噬菌体,并明确其生物学特性、观察其治疗小鼠产ESBLs肺炎克雷伯菌感染的疗效。【方法】电镜观察F20形态,调查其噬菌谱、生长曲线等生物学特性。建立小鼠败血症感染模型观察F20治疗小鼠肺炎克雷伯菌感染的疗效。【结果】F20在其宿主菌的菌苔上形成裂解性噬菌体所具有的完全透明的噬菌斑,电镜观察F20具典型的有尾噬菌体目长尾病毒科病毒的形态特征。一步生长曲线显示F20的潜伏期为18 min,裂解量为89 PFU/细胞。稳定性试验显示F20在pH 5.0-9.0及50°C环境均具良好稳定性。使用噬菌体F20对败血症小鼠治疗后,治疗组小鼠各外周血和各脏器(肺脏、肝脏、脾脏和肾脏)中的细菌数也显著小于对照组细菌数(P0.001),与对照组相比下降大约1–3数量级。F20治疗败血症小鼠存活率达到87.5%,无毒副作用,而对照组小鼠在1 d内全部死亡,可显著提高小鼠的存活率(P0.001)。【结论】新分离的裂解性噬菌体F20在小鼠体内能安全有效地治疗超广谱β-内酰胺酶肺炎克雷伯菌引起的败血症,可作为生物抗菌剂的有效成分。     

肺炎克雷伯菌生物被膜的体外模型建立

目的研究临床分离的肺炎克雷伯菌在体外形成生物被膜的情况,为进一步研究生物被膜肺炎克雷伯菌的耐药机制奠定基础。方法采用改良平板法建立肺炎克雷伯菌生物被膜模型,用喷金法和扫描电镜观察鉴定,并对生物被膜的形成进行定量分析。结果23株临床分离的肺炎克雷伯菌菌株生物被膜形成能力不同,以强阳性生物被膜形成能力者为最多数。结论绝大多数临床分离的肺炎克雷伯菌菌株具有较强的生物被膜形成能力。应用改良平板法能够较好的在体外建立其生物被膜模型。     

肺炎克雷伯菌血流感染的临床回顾性分析

目的探讨肺炎克雷伯菌血流感染的临床特点、治疗方案和预后。方法对诸暨市人民医院2014年3月-2015年9月诊断为肺炎克雷伯菌血流感染的住院患者临床资料进行回顾性分析。结果 66例肺炎克雷伯菌血流感染患者平均年龄64.6岁,检出前平均住院5.7d。13例(19.7%)合并有其他细菌感染,49例(74.2%)合并其他系统感染。肺炎克雷伯菌血流感染多见于糖尿病(33.3%)、实体肿瘤(30.3%)等免疫力低下疾病,最常见于肝胆外科(24.2%)、内分泌科(16.7%)等科室。患者预后好转47例(71.2%),APACHEⅡ评分为13.78±4.33;死亡19例(28.8%),APACHEⅡ评分为21.10±7.45,两组评分差异有统计学意义(P0.05)。合并其他感染状况与合并神经系统疾病状况病死率更高(P0.05)。结论肺炎克雷伯菌血流感染多见于糖尿病、实体肿瘤等免疫力低下的危重疾病患者,多发生在医院内环境的外科手术操作的科室,容易合并其他系统感染。APACHEⅡ评分可作为判断肺炎克雷伯菌血流感染预后的重要指标。控制其他感染情况可有助于临床防控肺炎克雷伯菌血流感染,改善其预后。     

老年慢性阻塞性肺疾病患者肺部感染病原菌与肠道定植菌的关系

目的探讨老年慢性阻塞性肺疾病(COPD)患者肺部感染病原菌与肠道定植菌的关系,为后续研究提供参考。方法选择2018年7月至2019年7月我院呼吸内科收治的60例COPD患者作为研究对象,根据是否合并肺部感染将患者分为感染组(n=38)和未感染组(n=22)。收集患者痰和粪便标本进行细菌培养,比较两组患者痰和粪便细菌检出情况,并对痰培养及粪便培养结果进行直线相关回归分析。结果感染组患者痰标本中铜绿假单胞菌、肺炎克雷伯菌、鲍曼不动杆菌及大肠埃希菌检出率高于未感染组(均P0.05)。感染组患者粪便标本中铜绿假单胞菌、肺炎克雷伯菌、鲍曼不动杆菌、阴沟肠杆菌、肠球菌、金黄色葡萄球菌检出率高于未感染组,肺炎链球菌检出率低于未感染组(均P0.05)。感染组患者粪便中各细菌检出率与痰培养细菌检出率呈正相关(r=0.745,P=0.009)。未感染组患者粪便中各细菌检出率与痰培养细菌检出率无显著相关性。结论老年COPD合并肺部感染患者病原菌与肠道定植菌具有一定相关性,临床治疗中应重视COPD患者肠道微生态平衡以降低肺部感染发生率。     

一株产1，3-丙二醇的克雷伯氏菌的噬菌体生物学特性

[目的]克雷伯氏菌发酵生产1,3-丙二醇过程中发生噬菌体感染会严重影响宿主菌的生长和目标产物的生成,因此分离克雷伯氏菌噬菌体并考察其生物学特性对预防和控制噬菌体感染具有重要意义.[方法]采用敏感指示菌法及Adams双层平板法从感染噬菌体的肺炎克雷伯氏杆菌发酵液中分离得到一株噬菌体;纯化后用磷钨酸负染法电镜观察;手工法提取噬菌体核酸,酶切后琼脂糖凝胶电泳分析;同时考察了其最佳感染复数、一步生长曲线及对温度、pH、紫外线、乙醚和氯仿等理化因素的敏感性等生理特性;最后考察了噬菌体对肺炎克雷伯氏杆菌生长和发酵的影响.[结果]分离出一株肺炎克雷伯氏杆菌溶源性噬菌体,其噬菌斑为无晕环透明圆斑,直径约1.5 mm;其头部为直径约60 nm-70 nm的球体,有一长约160 nm的丝状长尾;基因组核酸能被双链DNA内切酶EcoR Ⅰ及HindⅢ切开,大小约42 kb;对高温和紫外线敏感,耐碱性而受强酸抑制,对氯仿不敏感;最佳感染复数为1,潜伏期与裂解期均为50 min,裂解量为343个;感染噬菌体的肺炎克雷伯氏杆菌的细胞生长延滞约8h,代谢流偏向乳酸途径.[结论]该噬菌体属于无包膜长尾噬菌体,能改变克雷伯氏菌发酵生产1,3-丙二醇的代谢规律,为1,3-丙二醇发酵生产过程中噬菌体感染的预防和控制研究奠定了基础.     

Ubiquinone systems of the genus Cladosporium and morphologically similar taxa

Abstract We measured adenosine deaminase (ADA) activity in a guinea pig model of Legionella pneumophila infection. Female Hartley guinea pigs were inoculated intraperitoneally with one-quarter of the LD₅₀ dose of L. pneumophila Philadelphia-1 strain. Control groups were inoculated with clinical isolates of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae or Klebsiella pneumoniae . Each group consisted of 5 animals. ADA activity in plasma was assayed calorimetrically before and at various intervals after infection by measuring the amount of ammonia produced after adnosine was added to plasma samples. ADA activity before inoculation was 25.6±6.0 IU/1, it reached 174.4±60.0 IU/1 on day 3 after inoculation of L. pneumophila . ADA activity returned to normal levels on day 14. ADA activity did not increase significantly in guinea pigs infected with the other types of bacteria. These findings suggest that measurement of plasma ADA activity may be useful for the diagnosis of Legionella infection.     

Experimental chronic pulmonary infection in mice caused by Klebsiella pneumoniae

Examination of mouse strain differences in susceptibility to experimental respiratory tract infection with Klebsiella pneumoniae 27 revealed that a chronic pulmonary infection model could be established using CBA/J mice. After 6 X 10(5) colony-forming units of K. pneumoniae 27 were inoculated into the lung, the bacterial counts in the lungs changed with time showing four different phases: initial decrease, regrowth, steady-state, and final increase leading to death. Throughout the course of the infection, the challenge bacteria were isolated mainly from the respiratory organs. Pulmonary gross lesions appeared on day 2 after infection and persisted thereafter. Lobar consolidation was the primary lesion and occurred mainly in the anterior and middle lobes of the right lung, and the median lobe. Mice began to die from 4 weeks after aerosol exposure. This model may be useful for investigating the pathogenesis of chronic pulmonary infection by Klebsiella and its therapy.     

A comparative study of induction of pneumonia in mice with planktonic and biofilm cells of Klebsiella pneumoniae

In the present study, the course of acute pneumonia in normal BALB/c mice infected by intranasal inoculation of planktonic and preformed biofilm cells (3 days old) of Klebsiella pneumoniae B5055 was studied and compared. With both cell forms the peak of infection was observed on the third post infection day, as assessed on the basis of lung bacterial load and corresponding pathology. There was an intense neutrophil infiltration in bronchoalveolar lavage fluid. Tissue damage was assessed on the basis of increased amounts of nitrite, malondialdehyde and lactate dehydrogenase in lung homogenates. The phagocytic potential of alveolar macrophages was lower in biofilm cell-induced infection than in that induced by planktonic cells. Biofilm cell induced infection generated significantly greater production of tumor necrosis factor-α and interleukin-1β on the third and fifth days of infection, respectively. Production of interleukin-10 was, however, variable. There was no significant difference in the ability of planktonic and biofilm cell forms of K. pneumoniae to induce acute pneumonia in mice in terms of bacterial counts and histopathological changes. However, biofilm cell-induced infection showed delayed clearance as compared to infection induced with the planktonic form.     

Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response

Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community-acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)-1β and IL-8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram-negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response.     

生物被膜肺炎克雷伯菌产ESBLs的耐药性和基因型分析

目的检测临床分离的肺炎克雷伯菌在体外形成生物被膜后产超广谱β-内酰胺酶（Extended-Spectrum β-Laetamases,ESBLs）的情况,分析及研究其耐药性和耐药基因的分型情况。方法采用改良平板法在体外建立肺炎克雷伯菌生物被膜模型,用三维试验确认产ESBLs菌株,用K-B法进行药敏试验,用聚合酶链反应（Polymerase chain reaction,PCR）进行blaSHV、blaTEM和blaCTX—M基因扩增,产物分别克隆人pMD18-T载体后测定其核苷酸序列,分析其基因亚型。结果临床筛选出的60株ESBLs阴性肺炎克雷伯菌有46株在体外成功建立了生物被膜模型,并有9株产生了ESBLs表型。产酶后菌株的耐药性明显高于产酶前。PCR结果显示9株细菌均携带SHV基因,有4株同时携带TEM基因,没有检出携带CTX-M基因的菌株。9株细菌的SHV基因分别属于SHV-5、SHV-12和SHV-28亚型。4株携带TEM基因的细菌均为TEM-1亚型。结论生物被膜的形成能够诱导肺炎克雷伯菌产生ESBLs。本实验中检出的产ESBLs的基因型都是由SHV-1突变产生的。生物被膜的形成和产生ESBLs的协同作用是生物被膜肺炎克雷伯菌耐药性增强的主要原因之一。     

Synergism in biofilm formation between Salmonella enteritidis and a nitrogen-fixing strain of Klebsiella pneumoniae

A laboratory reactor, which simulates biofilm formation in water pipes, was used to study interactions in biofilm formation between a nitrogen-fixing strain of Klebsiella pneumoniae and Salmonella enteritidis. The level of attachment of Salm. enteritidis was higher in the binary biofilm than in the single species biofilm. In the initial colonization phase the binary biofilm contained a much higher proportion of metabolically active cells than in single species biofilms formed by either Salm. enteritidis or Kl. pneumoniae. When a pulse of Salm. enteritidis was passed over an already established biofilm of Kl. pneumoniae it rapidly became integrated into the biofilm, from where it was subsequently released into the water column, along with Kl. pneumoniae. Klebsiella pneumoniae fixed nitrogen in the presence of Salm. enteritidis in both types of biofilm.     

惰性材料表面细菌生物膜构建的研究

目的构建惰性材料塑料输液管内壁细菌生物膜体外模型,观察细菌生物膜的结构,探讨输液管内壁细菌生物膜形成影响因素。方法建立铜绿假单胞菌生物膜和铜绿假单胞菌、肺炎克雷伯菌混合生物膜,分别于培养1、3和7d用扫描电镜动态观察生物膜形成过程。结果混合菌生物膜的生长速度高于铜绿假单胞菌单独成膜。结论输液管是形成细菌生物膜的良好支持材料,混合细菌培养可以加速细菌形成生物膜。     

The use of ultrasonic imaging to evaluate the effect of protozoan grazing and movement on the topography of bacterial biofilms

AIMS: This study evaluated the effect of protozoan movement and grazing on the topography of a dual-bacterial biofilm using both conventional light microscopy and a new ultrasonic technique. METHODS AND RESULTS: Coupons of dialysis membrane were incubated in Chalkley's medium for 3 days at 23 degrees C in the presence of bacteria (Pseudomonas aeruginosa and Klebsiella aerogenes) alone, or in co-culture with the flagellate Bodo designis, the ciliate Tetrahymena pyriformis or the amoeba Acanthamoeba castellanii. Amoebic presence resulted in a confluent biofilm similar to the bacteria-only biofilm while the flagellate and ciliate created more diverse biofilm topographies comprising bacterial microcolonies and cavities. CONCLUSIONS: The four distinct biofilm topographies were successfully discerned with ultrasonic imaging and the method yielded information similar to that obtained with conventional light microscopy. SIGNIFICANCE AND IMPACT OF THE STUDY: Ultrasonic imaging provides a potential way forward in the development of a portable, nondestructive technique for profiling the topography of biofilms in situ, which might aid in the future management of biofouling.     

Biofilm formation by the Enterobacteriaceae: a comparison between Salmonella enteritidis, Escherichia coli and a nitrogen-fixing strain of Klebsiella pneumoniae

A simple laboratory reactor, which simulates biofilm formation in pipes, was used to compare biofilm formation by three members of the Enterobacteriaceae, namely, an environmental, nitrogen-fixing strain of Klebsiella pneumoniae , a pathogen, Salmonella enteritidis , and a faecal indicator, Escherichia coli. All three attached to CVCP pipe surfaces in the reactor and formed substantial biofilm populations of over a million bacteria cm^-2 within 24 h. These populations increased by approximately 10-fold over the next 48 h. Estimates of the numbers of metabolically active cells and the ratios of viable to direct counts showed that Kl. pneumoniae formed the densest and most metabolically active biofilms, followed by Salm. enteritidis and E. coli , respectively. Nitrogen fixation and polysaccharide production (EPS) by Kl. pneumoniae occurred only in mature biofilms and were of no selective advantage in the initiation of biofilms. Despite producing more EPS the rate of attachment of Salm. enteritidis was lower than for Kl. pneumoniae .