肺炎链球菌的转化缺陷导致细菌毒力减弱

的探讨肺炎链球菌的自然转化与其条件致病的关系。方法采用插入失活的方法制备肺炎链球菌的转化缺陷菌株，通过黏附实验和小鼠毒力实验观察它们毒力的变化，应用RT-PCR测定黏附因子PsaA在野生和缺陷菌株中的表达。结果实验获得了3株肺炎链球菌(1、2和22)的转化缺陷菌株(1d、2d和22d)。毒力研究发现转化缺陷菌株对ECV 304细胞的黏附能力显著弱于相应的野生菌株，1和2株肺炎链球菌腹腔感染BALB／c小鼠的能力显著强于相应的转化缺陷菌株；研究还发现PsaA基因在2和22株肺炎链球菌中的表达量显著高于其缺陷菌株。结论肺炎链球菌具有自然转化的能力有助于其毒力的表现，且可能通过黏附因子PsaA等相关毒力因子表达增加而增强其毒力。     

肺炎链球菌感受态的形成影响毒力因子mRNA的表达

目的：通过体外实验诱导的转化过程，探索肺炎链球菌（Streptococcus pneumoniae,S.pn)毒力因子的表达是否与环境诱导细菌感受态形成与转化有关。方法：采用插入失活的方式断裂S.pn感受态形成的关键基因comE，获得感受态缺陷菌株。以实时定量RT－PCR测定毒力因子在感受态缺陷菌与野生菌的表达是否存在差异。所测毒力因子有自溶酶、溶血素、胆碱结合蛋白、S-IgA、神经氨酸酶、透明质酸酶。结果：S.pn在540nm处吸光度（A）值＝0.044－0.127之间产生感受态。自溶酶、神经氨酸酶、透明质酸酶3种毒力因子的表达，野生菌组表达高于缺陷菌组，两均数间比较的t值分别为2．96（P＜0．05）、2．8（P＜0．05）、4．56（P＜0．05）。结论：细菌感受态能启动自溶酶、神经氨酸酶、透明质酸酶3种毒力因子的表达，提示S.pn的感受态能影响其毒力因子的表达。     

SsPep contributes to the virulence of Streptococcus suis

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen responsible for a spectrum of disease in pigs and that can be transmitted to humans with fatal consequences. Despite the socioeconomic importance of this infection, the pathogenesis of SS2 is poorly understood. The protein SsPep (05SSU0153) has been characterized as an extracellular protein. A deletion mutant of the gene encoding SsPep showed significantly decreased virulence in the pig infection model. Three groups challenged with different doses 5 × 10⁵ CFU, 1 × 10⁶ CFU, and 5 × 10⁶ CFU of the wild type strain, as the results all the pigs died, while those given the SsPep deletion mutant all survived challenge with 5 × 10⁵ CFU and 1 × 10⁶ CFU doses; four pig in the high dose group challenged with 5 × 10⁶ CFU and two pigs died at last. These findings suggest that SsPep plays a critical role in the pathogenesis of SS2.     

A C-terminal truncated mutation of licC attenuates the virulence of Streptococcus pneumoniae

LicC has been identified as a virulence factor of Streptococcus pneumoniae. However, its role in virulence is still not fully understood because deletion of licC is lethal for the bacterium. In this study, a mutant with 78-bp truncation at the C-terminus of licC was obtained from a signature-tagged mutagenesis (STM) library. The mutant was viable with a large reduction in enzymatic activity as CTP:phosphocholine cytidylyltransferase detected in vitro using a firefly luciferase assay. The mutation attenuated the adhesion and invasion of S. pneumoniae ST556 (serotype 19F) to epithelial cells by 72% and 80%, respectively, and increased the phagocytosis by macrophages for 16.5%, compared to the parental strain. When the mutation was introduced into the encapsulated D39 strain (serotype 2), it led to attenuated virulence in mouse models either by intranasal colonization or by intraperitoneal infection. In addition, the phosphocholine (PCho) on cell surface was decreased, and the choline binding proteins (CBPs) were impaired, which may explain the attenuated virulence of the mutant. These observations indicate that C-terminus of licC is accounted for the main activity of LicC in PCho metabolism and is essential for the virulence of S. pneumoniae, which provides a novel target for drug design against pneumococcal infection.     

肺炎链球菌ply基因缺陷菌株的构建及毒力变化的初步研究

目的 构建肺炎链球菌(Streptococcus pneumoniae,Sp)溶血素基因(pneumolysin,ply)缺陷菌株,并对其毒力作初步研究,为进一步探索宿主对溶血素的防御应答奠定基础.方法 采用长臂同源多聚酶链反应(LFH-PCR)技术将ply基因替换为红霉素耐药基因(erm)后同源重组于肺炎链球菌,在含红霉素的血平板上筛选出ply缺陷菌株.用PCR鉴定缺陷菌株,观察体外缺陷菌株生长情况,并在小鼠体内感染模型研究其毒力侵袭变化.结果 PCR结果显示ply基因完全被erm基因所替代,构建ply缺陷菌成功;单个菌落培养基生长情况表明ply基因缺陷并未对细菌的体外生长造成影响;但在小鼠鼻腔感染模型中,缺陷菌株入血时间(6 h)明显晚于野生菌株(2 h),且各时间点的菌量均显著低于野生菌株,两者比较差异有统计学意义(P＜0.01);小鼠腹膜感染模型显示野生菌株半数致死时间为3 d,而缺陷菌株半数致死时间为18d,两者比较差异有统计学意义(P＜0.01).结论 采用LFH-PCR技术作基因突变完全替代ply基因,方法简便快捷;ply的缺陷不影响细菌在体外的生长,但可显著降低细菌在宿主体内的毒力和侵袭.     

Differential activation of the immune system by virulent Streptococcus pneumoniae strains determines recovery or death of the host

Streptococcus pneumoniae infection may result in asymptomatic carriage, mucosal or invasive disease. We hypothesize that self-limiting or fatal disease outcome follows infection with S. pneumoniae differential activation of the host immune response. BALB/c and C57BL/6 mice were inoculated intranasally with S. pneumoniae serotype 3 strain WU2 and serotype 14 strain DW14 and mortality, bacterial load, pathological changes in the lungs and cytokines mRNA levels in the spleen were analysed. No differences between the C57BL/6 and the BALB/c inbred mice were observed except for the severity of their lung pathology and IL-4 expression. Infection of the two mouse strains with S. pneumoniae WU2 resulted in sepsis and death that occurred within 4 days post-inoculation. This death was preceded, in both mouse strains, in an increase over time of the lung bacterial load and bacteraemia. The lung pathology was characterized by diffuse pneumonia with marked congestion of the lungs. Analysis of mRNA expression of cytokines in the spleen revealed no alterations in tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, interleukin (IL)-12 and interferon (IFN)-gamma and induction of IL-10 and IL-4. The two strains of mice survived infection with S. pneumoniae DW14. This was accompanied by a reduction over time of lung bacterial load and bacteraemia. The lung pathology was characterized by focal lymphocyte infiltration and preserved architecture of the organ. Analysis of mRNA expression of cytokines in the spleen revealed a significant decrease in the levels of TNF-alpha, TGF-beta, IL-12 and IFN-gamma mRNA expression, which usually precedes cytokine protein expression. Interestingly, a significant increase in the levels of IL-4 mRNA expression was found in BALB/c mice only. This study suggests that differential activation or evasion of cytokine expression by S. pneumoniae virulent strains determines disease outcome regardless of the host's immunogenetic background.     

肺炎链球菌体内启动子诱捕文库的构建与初步分析

构建肺炎链球菌体内启动子诱捕文库(promoter-trap library),用于筛选肺炎链球菌体内诱导的基因。以穿梭质粒pEVP3为骨架,将无启动子的galU基因作为体内报告基因定向克隆到pEVP3上,与无启动子的体外报告基因lacZ基因融合,构建用于筛选体内诱导基因的载体pEVP3-galU;再把肺炎链球菌基因组DNA的随机酶切片段(200~500bp)克隆到此载体galU基因上游的Bgl II位点,构建了启动子诱捕文库,并转化肺炎链球菌galU缺陷菌株,获得相应的菌株库。文库大约覆盖基因组全长的5倍,插入率达到90%以上,保持了较高的复杂性。对得到的约450 000个肺炎链球菌转化子进行体内、外初步分析,那些从小鼠体内分离出的细菌,并在X-gal平板上为白色的菌落表明galU报告基因上游含仅在体内表达的启动子,提示所构建的启动子诱捕文库可用于筛选肺炎链球菌的体内诱导基因。     

临床分离肺炎链球菌的毒力基因及血清型分析

目的 了解肺炎链球菌的5种常见毒力基因(psaA、pspA、nanA、ply、lytA)在临床分离株中的分布情况.方法 以2006-2008年从临床分离133株肺炎链球菌为研究对象,采用PCR法对5种常见的毒力基因进行检测.结果 lytA、psaA、nanA、pspA、ply这5种基因在133株菌中的检测阳性率分别为94.7%、85.0%、84.2%、60.2%、82.7%;5种毒力基因在87株常见血清型菌中的阳性率分别为100%、87.4%、89.7%、67.8%、86.2%.结论 5种毒力基因在本地区肺炎链球菌中的阳性检测率均较高,在常见血清型菌株中的阳性检测率较其他血清型高.这5种基因是肺炎链球菌致病的主要毒力基因,可能作为新刑肺炎蛋白疫苗的候选基因.     

DC-SIGN specifically recognizes Streptococcus pneumoniae serotypes 3 and 14

The Gram-positive bacterium Streptococcus pneumoniae is the leading causative pathogen in community-acquired pneumonia. The ever-increasing frequency of antibiotic-resistant S. pneumoniae strains severely hampers effective treatments. Thus, a better understanding of the mechanisms involved in the pathogenesis of pneumococcal disease is needed; in particular, of the initial interactions that take place between the host and the bacterium. Recognition of pathogens by dendritic cells is one of the most crucial steps in the induction of an immune response. For efficient pathogen recognition, dendritic cells express various kinds of receptors, including the DC-specific C-type lectin DC-SIGN. Pathogens such as Mycobacterium tuberculosis and HIV target DC-SIGN to escape immunity. Here the in vitro binding of DC-SIGN with S. pneumoniae was investigated. DC-SIGN specifically interacts with S. pneumoniae serotype 3 and 14 in contrast to other serotypes such as 19F. While the data described here suggest that DC-SIGN interacts with S. pneumoniae serotype 14 through a ligand expressed by the capsular polysaccharide, the binding to S. pneumoniae serotype 3 appears to depend on an as yet unidentified ligand. Despite the binding capacity of the capsular polysaccharide of S. pneumoniae 14 to DC-SIGN, no immunomodulatory effects on the dendritic cells were observed. The immunological consequences of the serotype-specific capacity to interact with DC-SIGN should be further explored and might result in new insights in the development of new and more potent vaccines.     

青岛地区部分医院2005-2008年肺炎链球菌的耐药性分析

目的 监测青岛地区肺炎链球菌的耐药性,为临床合理应用抗菌药物提供依据.方法 采集青岛地区部分医院2005年1月到2008年12月门诊与住院感染患者呼吸道、血液、脑脊液等标本,培养、分离和鉴定肺炎链球菌.根据NCCLS的推荐,采用琼脂微茸稀释法测定分离出的231株肺炎链球菌对11种常用抗菌药物的耐药性,分析耐药趋势及年龄差异.结果 231株肺炎链球菌对青霉素不敏感率为23.38%[耐青霉素肺炎链球菌(PRSP):9.52%;低耐青霉素肺炎链球菌(PISP):13.85%].对头孢噻肟耐药率最低为9.96%(23/231),其次阿莫西林为12.55%(29/231).对红霉素耐药率最高为90.48%(209/231).14岁以下患者PRSP检出率为27.91%(12/43),明显高于成人的PRSP检出率5.38%(10/186).结论 本地区PRSP检出率较2004年前明显增加,并有逐年增加的趋势,肺炎链球菌的耐药性也有逐年上升的趋势.本地区对感染低耐青霉素肺炎链球菌的患者头孢噻肟、阿莫西林可为首选药物.     

79株肺炎链球菌的耐药性测定

目的调查北京地区肺炎链球菌对青霉素等抗生素的耐药率。方法用Ｅｔｅｓｔ及琼脂稀释法测定临床分离的７９株肺炎链球菌１５种抗生素的最低抑制浓度（ＭＩＣ）。结果Ｅｔｅｓｔ测得１０株（１２．７％）低耐青霉素（ＭＩＣ０．１２５～１μｇ／ｍｌ），１株（１．３％）高耐青霉素（ＭＩＣ４μｇ／ｍｌ）；仅１株处于头孢曲松、头孢噻肟中介范围（ＭＩＣ１μｇ／ｍｌ）．琼脂稀释法测得阿莫西林、阿莫西林／棒酸、头孢呋肟、环丙沙星、氯霉素、四环素、红霉素的耐药率分别为１．３％、１．３％、２．５％、２．５％、１６．５％、４９．４％、４０．５％，所有菌株对头孢曲松、万古霉素敏感。结论北京地区１４％的肺炎链球菌耐青霉素，耐三代头孢菌素及多重耐药株罕见。     

Mitogenic effect contributes to increased virulence of Streptococcus suis sequence type 7 to cause streptococcal toxic shock-like syndrome

Streptococcus suis serotype 2 sequence type 7 strains emerged in 1996 and caused a streptococcal toxic shock-like syndrome in 1998 and 2005 in China. Evidence indicated that the virulence of S. suis sequence type 7 had increased, but the mechanism was unknown. The sequence type 7 strain SC84, isolated from a patient with streptococcal toxic shock-like syndrome during the Sichuan outbreak, and the sequence type 1 strain 31533, a typical highly pathogenic strain isolated from a diseased pig, were used in comparative studies. In this study we show the mechanisms underlying cytokine production differed between the two types of strains. The S. suis sequence type 7 strain SC84 possesses a stronger capacity to stimulate T cells, naive T cells and peripheral blood mononuclear cell proliferation than does S. suis sequence type 1 strain 31533. The T cell response to both strains was dependent upon the presence of antigen-presenting cells. Histo-incompatible antigen-presenting cells were sufficient to provide the accessory signals to naive T cell stimulated by the two strains, indicating that both sequence type 7 and 1 strains possess mitogens; however, the mitogenic effect was different. Therefore, we propose that the difference in the mitogenic effect of sequence type 7 strain SC84 compared with the sequence type 1 strain 31533 of S. suis may be associated with the clinical, epidemiological and microbiological difference, where the ST 7 strains have a larger mitogenic effect.     

Regulation of Streptococcus pneumoniae distribution by Toll-like receptor 2 in vivo

The phagocyte pattern recognition receptor Toll-like receptor 2 (TLR2) and the multi-receptor adaptor MyD88 contribute to the reduction of bacterial load in infections with intra- and extra-cellular Gram-positive bacteria. Their mechanism of antibacterial action is mostly unresolved but evident in vivo by an increased pathogen burden in infected TLR2-/- and MyD88-/- compared to C57BL/6 wild type (wt) mice. We had previously observed higher bacterial numbers in brains of TLR2-/- than of wt mice with meningitis. Here we study bacteria-phagocyte interaction by comparing S. pneumoniae distribution and localization in wt and TLR2-/- brain by confocal microscopy using a green fluorescent protein-transformed encapsulated S. pneumoniae (C5017). Colony-forming units were similarly distributed in TLR2-/- and wt mice and exclusively localized in meninges and ventricles. Bacteria were more abundant in ventricles, in and around TLR2-/- than wt GLT1v+ plexus choroideus epithelial cells. S. pneumoniae were also found in and around Gr-1+ granulocytes, but never in F4/80+ macrophages, Iba1+ microglia, GFAP+ astrocytes, Meca-31+ endothelial cells or Neun+ neurons of either mouse strain. The results indicate that TLR2 does not change bacterial distribution, but may contribute to antibacterial defense by modulating S. pneumoniae adherence and uptake in plexus epithelia.     

E-cadherin is a receptor for the common protein pneumococcal surface adhesin A (PsaA) of Streptococcus pneumoniae

Streptococcus pneumoniae (Pnc) binds to nasopharyngeal (NP) epithelial cells in the first steps of nasopharyngeal carriage and colonization through bacterial adhesins. The pneumococcal surface adhesin A (PsaA) has previously been reported to play a significant role in pneumococcal adherence and colonization. Identification of a receptor for PsaA on human epithelium will aid in understanding the pathogenesis of this bacterium. Using recombinant PsaA covalently bound to fluorescent spheres (fluospheres), we show PsaA binds to NP cells through interaction with the human cellular receptor, E-cadherin. SDS-PAGE silver stain analysis demonstrates binding of PsaA to E-cadherin. Recombinant human E-cadherin binds to and blocks PsaA-coated fluospheres and whole transparent bacteria from adhering to NP cells, but does not block a Pnc PsaA(-) mutant. Recombinant E-selectin and human alpha(5)beta(1) integrin did not bind to or block PsaA-coated fluosphere adherence to NP cells. Likewise, if NP cells were preincubated with anti-E-cadherin antibody, there was a significant decrease (46%, P=0.05) in PsaA-coated fluosphere adherence to the cells. Additionally, when using E-cadherin transfected cells, we observed PsaA-coated fluospheres bind more efficiently to cells which express E-cadherin. This work identifies E-cadherin as a receptor on human epithelial cells for the pneumococcal surface adhesin, PsaA.     

Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.     

The antibacterial effects of ciprofloxacin and trovafloxacin against Streptococcus pneumoniae in an in vitro dynamic model

Objective: To determine whether the newer fluoroquinolone antibiotics such as trovafloxacin posses enhanced activity against Gram-positive organisms, including Streptococcus pneumoniae , because the clinical activity of older quinolones against pneumococci has been questioned.
Methods: In this study, the bactericidal activities of ciprofloxacin and trovafloxacin against six strains of penicillin-resistant and -sensitive strains of Streptococcus pneumoniae were compared using an in vitro model that simulates human pharmacokinetics. Ciprofloxacin was administered at 750 mg every 12 h, higher than the usual daily dose of 500 mg twice a day. Trovafloxacin was administered at 300 mg every 24 h for the six strains and at 200 mg every 24 h for three of the strains.
Results: The single 300-mg dose of trovafloxacin killed five of the six strains in 4 h, with no bacterial regrowth. Ciprofloxacin reduced the initial inoculum by 3–5 logs by 24 h. Although the 300-mg dose of trovafloxacin more rapidly eradicated susceptible strains, the activity of trovafloxacin at 200 mg every 24 h was similar to that of ciprofloxacin at 750 mg every 12 h against the three strains tested.
Conclusion: Trovafloxacin (and ciprofloxacin at high doses) eradicates susceptible strains of pneumococci in an in vitrc dynamic model.     

Streptococcus uberis internalizes and persists in bovine mammary epithelial cells

Streptococcus uberis is one of the most important emerging bovine mastitis pathogens and chronic persistent intramammary infections (IMI) are often described. To define the ability of S. uberis to persist intracellularly, studies on time-dependent internalization and survival of S. uberis strains in bovine mammary epithelial cells were conducted. Two S. uberis strains (UT366 and UT888) and a Staphylococcus aureus strain used as positive control, all isolated from cows with clinical mastitis were cocultured with bovine mammary epithelial cells (MAC-T) and persistent survival in host epithelial cells for extended periods (120 h) studied. Of S. uberis strains tested, UT366 showed highest internalization values at 60 min of incubation whereas at 8 h of incubation the corresponding values for UT888 were the highest. Of both strains of S. uberis tested, UT366 seems to internalize bovine mammary cells more efficiently initially, however, during the first 8 h, UT888 seems to survive intracellularly better than UT366. Results showed that both S. uberis strains could survive intracellularly up to 120 h without apparent loss of host cells viability. S. aureus internalized more efficiently than all strains tested and host cell death was observed after 72 h of incubation. These results indicate that S. uberis can survive within mammary epithelial cells for extended time without apparent loss of host cells viability. Intracellular persistence of S. uberis may be associated with the spread of the infection to deeper tissues and development of persistent IMI.     

肺炎链球菌青霉素结合蛋白基因pbp2x新的变异与青霉素及头孢噻肟耐药

目的 调查研究肺炎链球菌临床分离株的pbp2x基因和氨基酸序列的变异特点,探讨本地区的肺炎链球菌对青霉素及头孢噻肟的耐药机制.方法 2006年1月-2007年2月收集肺炎链球菌临床分离株34株,进行青霉素及头孢噻肟药敏试验,对青霉素不敏感的肺炎链球菌(PNSP)的青霉素结合蛋白pbp2x基因进行PCR扩增和测序,并进行BLAST分析.结果 有12株PNSP(青霉素及头孢噻肟MIC≥0.5 mg/L)发生了2个重要位点的氨基酸的替换:第一个保守基序STMK内Thr338→Ala及第三个保守基序KSG之前的Leu546→Val氨基酸替换.另外,菌株15发生了第二个保守基序SSN之前的His394→Leu氨基酸替换,而且本研究首次发现了紧邻第一个保守基序STMK后,Met342→Ile位点的氨基酸替换.有17个菌株的pbp2x基因的核苷酸及氨基酸序列出现了新的变异,已向GenBank提交,获得序列号:EU044831、EU089706-EU089709、EU106881-EU106884、EU124672.结论 本地区大多数PNSP的pbp2x核苷酸及氨基酸变异序列高度相似,提示肺炎链球菌对青霉素及头孢噻肟的耐药与pbp2x基因变异相关.     

Antimicrobial susceptibility of invasive isolates of Streptococcus pneumoniae in Ireland

Between January 1999 and June 2002, 646 invasive isolates of Streptococcus pneumoniae were collected in Ireland. MICs of penicillin, ciprofloxacin, cefotaxime, moxifloxacin and linezolid were determined by Etest methodology. Eighty-seven (13.5%) isolates showed intermediate resistance to penicillin, while seven (1.1%) showed high-level resistance. Eighty-seven (13.5%) isolates were resistant to erythromycin, but all isolates were susceptible to cefotaxime, moxifloxacin and linezolid. The prevalence of pneumococcal isolates non-susceptible to penicillin in Ireland is worryingly high, but currently there are alternative agents available to treat invasive infection.