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。     

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。     

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。     

肺炎克雷伯菌研究进展

近年来由于各种抗菌药物的广泛使用,导致肺炎克雷伯菌多重耐药现象普遍存在,给临床治疗带来极大的困扰,造成医院获得性肺炎感染严重的现状。着重论述了肺炎克雷伯菌流行现状、耐药机制、致病因子及防治措施。     

Effects of Brazilian green propolis extract on planktonic cells and biofilms of multidrug-resistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa

Abstract

Propolis could represent an alternative therapeutic agent for targeting multidrug-resistant bacteria due to its antimicrobial potential. The effect of Brazilian green propolis (BGP) aqueous extract (AqExt) was evaluated on eight multidrug-resistant clinical strains of Klebsiella pneumoniae and Pseudomonas aeruginosa, as well as on one reference strain for each bacterial species. The minimum bactericidal concentration (MBC) was determined and optimal concentrations were further evaluated in comparison with 0.12% chlorhexidine. The natural extract was chemically characterized by HPLC-DAD analysis. The MBC values ranged between 3.12 and 27.5?mg ml^?1. Analysis of bacterial metabolic activity after treatment for 5?min with BGP-AqExt revealed a strong antimicrobial potential, similar to chlorhexidine. The extract comprised several active compounds including quercetin, gallic acid, caffeic and p-coumaric acid, drupani, galangin, and artepillin C. Altogether, the findings suggest that BGP-AqExt is fast and effective against multidrug-resistant strains of K. pneumoniae and P. aeruginosa in planktonic cultures and biofilms.     

Modulation of respiratory dendritic cells during Klebsiella pneumonia infection

Background

Klebsiella pneumoniae is a leading cause of severe hospital-acquired respiratory tract infections and death but little is known regarding the modulation of respiratory dendritic cell (DC) subsets. Plasmacytoid DC (pDC) are specialized type 1 interferon producing cells and considered to be classical mediators of antiviral immunity.

Method

By using multiparameter flow cytometry analysis we have analysed the modulation of respiratory DC subsets after intratracheal Klebsiella pneumonia infection.

Results

Data indicate that pDCs and MoDC were markedly elevated in the post acute pneumonia phase when compared to mock-infected controls. Analysis of draining mediastinal lymph nodes revealed a rapid increase of activated CD103⁺ DC, CD11b⁺ DC and MoDC within 48 h post infection. Lung pDC identification during bacterial pneumonia was confirmed by extended phenotyping for 120G8, mPDCA-1 and Siglec-H expression and by demonstration of high Interferon-alpha producing capacity after cell sorting. Cytokine expression analysis of ex vivo-sorted respiratory DC subpopulations from infected animals revealed elevated Interferon-alpha in pDC, elevated IFN-gamma, IL-4 and IL-13 in CD103⁺ DC and IL-19 and IL-12p35 in CD11b⁺ DC subsets in comparison to CD11c⁺ MHC-class II^low cells indicating distinct functional roles. Antigen-specific naive CD4⁺ T cell stimulatory capacity of purified respiratory DC subsets was analysed in a model system with purified ovalbumin T cell receptor transgenic naive CD4⁺ responder T cells and respiratory DC subsets, pulsed with ovalbumin and matured with Klebsiella pneumoniae lysate. CD103⁺ DC and CD11b⁺ DC subsets represented the most potent naive CD4⁺ T helper cell activators.

Conclusion

These results provide novel insight into the activation of respiratory DC subsets during Klebsiella pneumonia infection. The detection of increased respiratory pDC numbers in bacterial pneumonia may indicate possible novel pDC functions with respect to lung repair and regeneration.     

碳青霉烯酶在碳青霉烯类耐药肺炎克雷伯杆菌的分布及分子流行病学

目的了解碳青霉烯酶在碳青霉烯类抗生素耐药肺炎克雷伯杆菌的分布及其分子流行病学。方法收集我院2010年1月至2012年12月分离的碳青霉烯类耐药肺炎克雷伯杆菌120株,采用改良Hodge试验及EDTA协同试验进行碳青霉烯酶表型的确认,用PCR方法扩增碳青霉烯酶基因(KPC、IMP、NDM、VIM、OXA-48),经电泳检测扩增产物后纯化测序。通过MLST及ERIC-PCR进行分子流行病学分析。结果120株碳青霉烯类耐药肺炎克雷伯杆菌中,95株CRKP菌株改良Hodge试验阳性,64株菌株EDTA协同试验阳性。经PCR及测序确认,83株主要携带KPC-2型碳青霉烯酶,占69.2%;其次为IMP-4型金属酶,占28.3%(34/120),NDM-1型金属酶15株,占12.5%,未发现VIM和OXA-48碳青霉烯酶。ERICPCR及MLST分型显示出20个基因分型,其中ST395-A型和ST11-B型主要流行于产KPC-2型菌株中,占43.88%;ST263-B型和ST15-C型主要流行于产NDM-1菌株中,占13.27%;而ST11-B型主要分布于产IMP-4和KPC-2菌株中,占24.49%。结论我院碳青霉烯酶耐药肺炎克雷伯杆菌存在多种碳青霉烯酶,主要以KPC-2为主,产不同种类碳青霉烯酶菌株存在不同的流行基因型。     

肺炎克雷伯菌生物被膜豚鼠肺感染模型建立的实验研究

目的采用吸入法建立肺炎克雷伯菌生物被膜（BF）肺感染模型,探讨肺内肺炎克雷伯菌BF的发生发展变化及其BF菌周围炎性细胞的变化。方法40只豚鼠随机分为两组：A组和B组动物分别通过喷雾器接种灭菌生理盐水及肺炎克雷伯菌菌悬液,取材日进行活菌计数、光学及电子显微镜检查。结果肺炎克雷伯菌BF在肺内以特异的肉芽肿结节的形式存在,有体内炎性细胞的参与。扫描电镜可见结节内有多糖蛋白复合物包绕的细菌,菌体之间相互以粘液丝相连。结论吸入法BF肺感染模型方法简单、重复性好,可用于BF菌相关肺感染的研究。     

肺炎克雷伯菌耐药性与I类整合子关系的研究

目的检测I类整合子在肺炎克雷伯菌临床分离株中的分布,分析整合子对细菌耐药性的影响。方法采用K—B纸片扩散法对127株肺炎克雷伯菌临床分离株进行药敏试验;并用WHONET5．6软件分析菌株药敏情况;采用聚合酶链反应（PCR）分析127株肺炎克雷伯菌株的I类整合子。并对I类整合子阳性株与阴性株的耐药性进行对比分析。结果127株菌中有53株（41．70％）含有I类整合子,I类整合子阳性菌株对氨基糖苷类、喹诺酮类及大多数B一内酰胺类的耐药率高于整合子阴性的菌株。结论I类整合子在肺炎克雷伯菌临床分离株存在较广,含有I类整合子的肺炎克雷伯菌更易获得耐药性。     

A Dam methylation mutant of Klebsiella pneumoniae is partially attenuated

In Klebsiella pneumoniae, a chromosomal insertion mutation was constructed in the dam gene, which encodes DNA adenine methylase (Dam), resulting in a mutant unable to methylate specific nucleotides. In some bacteria, the Dam methylase has been shown to play an important role in virulence gene regulation as well as in methyl-directed mismatch repair and the regulation of replication initiation. Disruption of the normal Dam function by either eliminating or greatly increasing expression in several organisms has been shown to cause attenuation of virulence in murine models of infection. In K. pneumoniae, a mutation-eliminating Dam function is shown here to result in only partial attenuation following intranasal and intraperitoneal infection of Balb/C mice.     

高毒力肺炎克雷伯菌的分子致病机制

肺炎克雷伯菌是一种常见致病菌,根据毒力和致病特点不同,可分为毒力相对较弱的经典肺炎克雷伯菌和高毒力肺炎克雷伯菌。目前,关于肺炎克雷伯菌分子致病机制研究较多且较清楚的主要有荚膜、脂多糖、黏附素和铁载体。这四大类毒力因子在经典肺炎克雷伯菌中也存在,但在高毒力肺炎克雷伯菌中存在的频率更高,并引发不同的免疫应答,从而使高毒力肺炎克雷伯菌具有特征性表型。本文就此进行详细介绍和讨论。     

非产超广谱β-内酰胺酶肺炎克雷伯杆菌的耐药性分析

目的了解临床分离肺炎克雷伯杆菌中非产超广谱β-内酰胺酶（ESBLs）菌株对18种常见抗菌药物的耐药性。方法CLSI表型确证试验-纸片增强法检测非产ESBLs肺炎克雷伯菌,K-B法测定非产ESBLs肺炎克雷伯杆菌对18种常见抗菌药物的敏感性。结果非产ESBLs肺炎克雷伯杆菌株对头孢唑啉、头孢呋辛的耐药率〉50．0％,对其余抗生素的耐药率均低于25．0％,对亚胺培南、美罗培南非常敏感,耐药率分别为2．3％和2．0％;痰标本的分离株对头孢唑啉、头孢呋辛的耐药率明显高于血、尿液标本分离株,差异有统计学意义（P〈0．05）。结论非产ESBLs肺炎克雷伯杆菌对第一、二代头孢菌素耐药显著,对第三代头孢菌素、亚胺培南、美罗培南等抗生素比较敏感。     

耐碳青霉烯类肺炎克雷伯菌的耐药机制研究

目的了解碳青霉烯类耐药肺炎克雷伯菌及耐药机制。方法对2012-2013年临床分离的耐碳青霉烯类肺炎克雷伯菌共计12株进行分析,药敏采用MIC方法检测,用WHONET 5.6软件进行分析,KPC表型检测采用改良Hodge试验,基因检测采用PCR方法。结果 12株碳青霉烯类耐药肺炎克雷伯菌改良Hodge试验阴性,基因测序为KPC-2型。结论 KPC-2基因是引起本院肺炎克雷伯菌耐药的主要原因。     

Biotyping,virulotyping and biofilm formation ability of ESBL-Klebsiella pneumoniae isolates from nosocomial infections

The aim of this study was to investigate the frequency, molecular characterization, virulence genes, resistance genes and antimicrobial profile of nosocomial extended spectrum beta lactamase producing Klebsiella species. A total of 22 (12.2%) K. pneumoniae strains were isolated from 180 clinical samples collected from hospitalized patients in Egypt. K. pneumoniae biotypes were B1 (72.8%), B3 (13.6%) and B4 (13.6%). The isolates were classified for the capsular serotypes, 86.4% (20/22) were of K1 serotype, while only two isolates (13.64%) were of K2 serotype. Hypermucoviscous K. pneumoniae isolates accounted for 68.2%. Biofilm formation ability of K. pneumoniae was determined by microtitre plate method. The majority of the isolates (40.9%) were moderate biofilm producers, while 27.3% were strong biofilm producers. All K. pneumoniae strains were positive for fimH and traT genes, while magA was identified in only 63.6% of the isolates. The antibiotic susceptibility profile of the isolates (n = 22) was determined by the disc diffusion technique using 23 different antibiotics. Streptomycin and imipenem are the most effective antibiotics against 22 tested K. pneumoniae isolates with sensitivity rates of 63.64% and 54.54% respectively. All tested K. pneumoniae isolates showed high resistance to amoxicillin∕clavulanate (100%), cefuroxime (100%) and ceftazidime (95.45%). Extended spectrum beta lactamases (ESBL) production and the presence of ESBL-related genes were tested in the isolates. All the isolates tested positive for blaVIM, NDM1 and blaTEM, while only 81.8 %tested positive for the blaSHV gene. Increasing antimicrobial resistance in K. pneumoniae causing nosocomial infections limits the use of antimicrobial agents for treatment. Furthermore, the spread of biofilm, multiple drug resistant and ESBL-producing K. pneumoniae isolates is a public threat for hospitalized patients.     

A Klebsiella pneumoniae plasmid which codes for isocitrate dehydrogenase

Abstract A naturally occurring Klebsiella pneumoniae plasmid of 114 MDa was able to complement isocitrate dehydrogenase deficiencies in K. pneumoniae and Salmonella typhimurium . The plasmid encoded isocitrate dehydrogenase, which differed from that of S. typhimurium in the kinetic parameters for NADP and isocitrate, in its isoelectric point, and in its response to repression by cAMP. This is the first report of a naturally occurring plasmid encoding a Krebs cycle enzyme.     

多重耐药肺炎克雷伯菌肺部感染的抗感染治疗新策略

目的 尝试使用新的抗感染治疗策略治疗多重耐药肺炎克雷伯菌肺部感染。方法 选择10例转入ICU的院内获得性肺部感染患者,入选患者使用抗生素治疗效果不佳,多次痰培养结果提示多重耐药的肺炎克雷伯菌生长。患者先予以头孢美唑抗感染治疗,在48~72 h后痰培养结果均提示对亚胺培南等敏感的铜绿假单胞菌生长,然后根据患者肺部感染是否好转选择继续使用头孢美唑或调整为对铜绿假单胞菌有效的抗生素。结果 10例患者治疗7 d后C反应蛋白（CRP）、降钙素原（PCT）、临床肺部感染评分（CPIS）均显著下降（P＜0.05）,所有患者均顺利脱机拔管转出ICU。结论 根据肺部微生态特点,利用菌群之间存在的拮抗作用,可以使用抗生素来调节微生态失衡。对于合适的患者,这种新的抗感染策略可以有效治疗多重耐药肺炎克雷伯菌肺部感染,可作为临床抗感染策略之一。     

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5.7 d。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（P<0.05）。合并其他感染状况与合并神经系统疾病状况病死率更高（P<0.05）。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。     

耐亚胺培南肺炎克雷伯菌腹腔感染患者的临床特征及预后

目的 研究耐亚胺培南肺炎克雷伯菌所致腹腔感染患者的临床特征、感染危险因素、治疗方案及临床预后。方法 回顾性调查2011年1月至2014年10月浙江大学医学院附属第一医院住院患者中由耐亚胺培南肺炎克雷伯菌所致腹腔感染患者的基础疾病、临床表现、实验室指标、微生物学检测结果等临床特征;了解腹腔感染发生前的侵入性操作、手术、抗菌药物使用、免疫抑制剂使用等高危因素;分析腹腔感染的治疗措施及抗菌药物的使用情况,患者对治疗的临床反应及30 d病死率。目标变量使用单因素、多因素分析,χ2检验及风险评估进行分析。结果 本研究入选43例腹腔感染患者,其病原菌均为亚胺培南耐药的肺炎克雷伯菌。病例年龄17~92岁,平均（54.8±15.9）岁;男性31例（72.1%）;APACHEⅡ评分22~33分,平均（27.7±2.2）分。全部患者在其样本培养阳性前2周内有侵入性操作,其中100.0%的患者留置腹腔引流管、95.3%的患者留置导尿管、93.0%的患者留置中心静脉管、76.7%的患者气管插管或切开;有36例患者（83.7%）感染前90 d内有手术史（均为经腹腔手术）;32例（74.4%）患者曾有30 d内ICU入住病史;38例（88.4%）患者在其标本培养阳性前2周内曾接受抗生素治疗。发热（74.4%）为腹腔感染耐亚胺培南肺炎克雷伯菌的主要临床表现,其次为腹痛（44.2%）。分别有13例（30.2%）和6例（14.0%）患者合并血流感染和/或痰液中检出耐亚胺培南肺炎克雷伯菌,17例（37.2%）患者出现感染性休克。在多因素分析中,感染性休克是30 d病死唯一的危险因素（OR：0.693,95%CI：1.591~30.245,P=0.010）。累计30 d病死率37.2%,存活时间2~28 d,平均（12.3±9.4）d;合并血培养阳性者的病死率为46.2%。使用替加环素治疗后30 d病死率较未用替加环素治疗低（19.0% vs 54.5%,P=0.027）。结论 腹腔感染耐亚胺培南肺炎克雷伯菌患者的病死率较高,感染性休克是30 d内病死的危险因素。使用替加环素进行抗菌治疗能降低30 d病死率、改善临床预后。