水苏糖对艰难梭菌在小鼠肠道内定植与菌群的影响

目的利用艰难梭菌感染(CDI)小鼠模型,评价水苏糖是否具有抑制小鼠肠道内艰难梭菌定植的作用,并分析其对肠道菌群结构的影响。方法将C57BL/6雌鼠随机分为3组,其中不做任何处理的设定为空白对照组,水苏糖干预组和磷酸盐缓冲液(PBS)模型组在建立CDI小鼠模型后分别连续10 d每日给予水苏糖和PBS灌胃处理。利用荧光定量聚合酶链式反应(PCR)检测感染后第10 d各组小鼠粪便中艰难梭菌的含量,并利用16S rRNA基因测序分析各组小鼠肠道菌群结构的变化。结果水苏糖干预导致CDI小鼠粪便中艰难梭菌含量显著降低。水苏糖干预组小鼠粪便的菌群丰富度显著高于PBS模型组,但没有恢复到空白对照组的水平。在门水平上,水苏糖干预可导致CDI小鼠粪便的拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)的相对丰富度显著增高,变形菌门(Proteobacteria)的相对丰富度显著降低。在种水平上,水苏糖干预导致CDI小鼠粪便中格氏副拟杆菌(Parabacteroides goldsteinii)、汉森氏布氏菌(Blautia hansenii)、多形拟杆菌(Bacteroides thetaiotaomicron)的相对丰度显著增高;Parasutterella excrementihominis、狄氏副拟杆菌(Parabacteroides distasonis)的相对丰度显著降低。结论水苏糖干预可有效降低CDI小鼠肠道内艰难梭菌定植量,增加CDI小鼠肠道微生物丰富度,特异性地改变多形拟杆菌和格氏副拟杆菌等菌种的相对丰度。     

GeneXpert实时荧光定量PCR快速检测艰难梭菌的临床评估

目的对GeneXpert实时荧光定量聚合酶链反应(PCR)在快速检测临床粪便标本中艰难梭菌的应用进行评估。方法采用双拭子蘸取临床未成形粪便标本,一支拭子用于GeneXpert实时荧光定量PCR检测艰难梭菌毒素基因tcdB,另一支用于常规厌氧菌培养检测;对GeneXpert实时荧光定量PCR检测结果与常规厌氧菌培养结果的一致性进行统计学分析,并计算GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值等参数。结果临床收集到141例未成形粪便标本,GeneXpert实时荧光定量PCR检出艰难梭菌毒素基因tcdB阳性42例,其中常规厌氧菌培养阳性34例,两者一致性较好(Kappa=0.775 0,P0.01),GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值分别为87.2%、92.2%、81.0%和94.9%。结论 GeneXpert实时荧光定量PCR直接检测粪便标本中的艰难梭菌具有检测快速、操作简便等优点,有重要的临床应用价值。     

实时荧光定量PCR直接检测粪便标本艰难梭菌tcdB基因

目的建立艰难梭菌实时荧光定量PCR检测方法,直接检测粪便标本艰难梭菌tcd B基因,并探讨其应用价值。方法根据艰难梭菌基因组设计tcd B基因特异性引物及探针,用ATCC 43255标准株评价其敏感性,选取艰难梭菌生物学特征相近的临床菌株验证其特异性;收集2015年10-12月临床腹泻患者稀便标本115份,提取基因组总DNA后进行tcd B基因检测,并以产毒培养法为参考方法,探讨该方法的临床诊断价值。结果荧光定量PCR法最低检测限为1×10-3ng,并且仅特异性地扩增产毒艰难梭菌;临床样本评价荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值分别为75.0%、99.1%、85.7%和98.1%。结论实时荧光定量PCR直接检测稀便标本中的艰难梭菌tcd B基因可用于艰难梭菌相关腹泻的快速诊断。     

快速检测粪便中艰难梭菌的方法研究

目的建立可用于粪便中艰难梭菌快速检测的环介导恒温扩增(LAMP)方法。方法针对艰难梭菌毒素A基因,设计引物。采用LAMP对艰难梭菌和其他腹泻干扰菌及不同浓度艰难梭菌的扩增检测,对其特异性和灵敏度进行评价。同时采用LAMP和荧光定量PCR对100例疑似艰难梭菌感染的临床腹泻患者粪便进行检测,对两种方法进行比较。结果 LAMP对艰难梭菌及6种腹泻干扰菌的检测,只针对艰难梭菌有扩增,显示了较好的特异性。LAMP最低检测限为10CFU/mL,灵敏度较高。对100例临床粪便标本LAMP检测结果与荧光定量PCR比较差异无统计学意义(P>0.05,χ2=0.1429)。结论本研究建立的粪便中艰难梭菌的LAMP快速检测方法特异性好、灵敏度较高、操作简便,适用于艰难梭菌的临床快速检测及现场检测。     

2种艰难梭菌感染检测方法的比较与分析

目的比较酶联免疫荧光分析(ELFA)与实时荧光定量PCR(PCR)2种艰难梭菌感染(Clostridium difficile infection,CDI)检测方法的检验性能,从中选择灵敏度高、特异性好且操作简便的CDI检验方法开展临床检验。方法对来自重症监护病房(ICU)、消化内科及感染科等临床科室的126份粪便标本进行艰难梭菌毒素或基因检测。以产毒培养试验(toxigenic culture,TGC)结果作为参考标准,评价ELFA与PCR方法的性能,同时分析我院及各科室的CDI流行情况。结果 TGC检测126份腹泻患者的粪便标本,阳性率为19.0%(24/126),其中ICU、消化内科感染率较高。ELFA和PCR方法的检测敏感性分别为66.7%和91.7%,差异有统计学意义(χ2=4.55,P0.05);特异性、阳性预测值、阴性预测值与准确度差异无统计学意义。结论我院CDI形势较为严峻,以ICU和消化内科最为突出。PCR与ELFA均能够提供更为准确、可靠的检测结果,但前者敏感性较好。     

实时荧光定量PCR法检测产气荚膜梭菌

目的:运用实时荧光定量PCR法检测产气荚膜梭菌,为早期快速诊断气性坏疽提供新方法.方法:以产气荚膜梭菌16s rDNA基因序列为模板,在其保守区域设计特异性引物与探针,将PCR扩增所得产物片段克隆,作为定量检测的标准品,绘制标准曲线.并对荧光定量PCR体系与反应条件进行优化,验证方法的特异性、敏感性、重复性及可行性.结果:实时荧光定量PCR法对产气荚膜梭菌的检测具有高度特异性,与创伤弧菌等24种相关细菌等均无交叉反应;检测灵敏度以纯菌计数达9×102cfu/mL,相当于9 cfu/反应;反应体系有较高稳定性;整个操作过程仅需3 h;300例创伤可疑分泌物样本的荧光定量PCR检测结果与细菌培养结果一致.结论:实时荧光定量PCR法特异、灵敏、快速,适用于产气荚膜梭菌的临床检测及突发事件的批量检测.     

GeneXpert实时荧光定量PCR在诊断艰难梭菌感染中的临床应用

目的评价Gene Xpert实时荧光定量PCR法在诊断艰难梭菌感染中的应用价值。方法收集住院腹泻患者非重复粪便标本296份,同时进行Gene Xpert试验和产毒素培养(toxigenic culture,TC)。TC包括厌氧培养、菌株基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)鉴定、PCR扩增艰难梭菌毒素基因。以TC结果为参考,评价Gene Xpert试验的敏感性、特异性、阳性预测值、阴性预测值,并评价Gene Xpert试验和TC 2种方法结果的一致性。结果 Gene Xpert试验的敏感性、特异性、阳性预测值和阴性预测值分别为98.5%、92.6%、91.7%和98.7%。Gene Xpert试验与TC一致性好(Kappa=0.905)。Gene Xpert试验报告2株RT027型高毒力菌株,经核糖体分型确证为RT027型艰难梭菌。结论 Gene Xpert实时荧光定量PCR可快速、准确检测粪便标本中的艰难梭菌毒素基因,且可报告高毒力RT027型菌株,具有重要的临床应用价值。     

实时荧光定量PCR检测人粪便中空肠弯曲杆菌方法的建立与评价

目的：建立一种快速检测人粪便样本中空肠弯曲杆菌的实时荧光定量 PCR方法。方法根据空肠弯曲杆菌保守序列设计特异引物,建立空肠弯曲杆菌的实时荧光定量 PCR检测法。对该方法的线性范围、抗干扰能力进行考察。同时采用培养法和实时荧光定量 PCR法对150例粪便样本进行检测,以培养法检测结果作为参照标准,对实时荧光定量 PCR方法的灵敏度、特异度、准确度和重复性进行考察。采用 Kappa检验对两种检测方法的结果进行统计学分析。结果建立的实时荧光定量PCR方法标准曲线线性关系良好Y＝-3.51 Log（X）＋37.09,相关系数为0.996,理论检测下限为102 CFU/ml。抗干扰能力强,仅空肠弯曲杆菌出现特异性扩增曲线。与培养法相比,其灵敏度、特异度、准确度分别为92.4％,95.8％和94％;检测结果重复性良好（CV％＜5％）。统计学分析显示两种方法检测结果具有一致性,且一致性强度为极强（Kappa＝0.88,P＜0.05）。结论实时荧光定量PCR法能准确快速检测粪便样品中的空肠弯曲杆菌。     

检测艰难梭菌耐莫西沙星gyrA基因点突变的双重荧光PCR方法的建立和评价

目的 建立一种检测艰难梭菌耐莫西沙星gyrA基因点突变的双重荧光PCR方法.方法 设计针对艰难梭菌gyrA基因的特异性引物,并针对莫西沙星耐药株和敏感株的gyrA基因突变位点设计不同的TaqMan-MGB探针,优化可同时检测ATT、ACT突变点的双重荧光定量PCR方法,验证该方法的灵敏性、特异性和重复性,并进行应用评价...     

实时荧光定量PCR对艰难梭菌感染诊断价值的meta分析

目的系统评价实时荧光定量PCR(real-time PCR)对艰难梭菌感染的诊断价值。方法制定原始文献的纳入、排除标准及检索策略,检索有关real-time PCR诊断粪便中艰难梭菌感染且同时采用细胞培养毒素中和试验(CCCNA)或厌氧产毒培养(TC)作为诊断金标准的文献,采用诊断准确性研究的质量评价工具QUADAS进行质量评价,并用Meta DiSc 1.4软件进行meta分析。结果纳入研究文献共42篇,异质性检验提示无阈值效应,但存在其他原因导致的非阈值性异质性。real-time PCR诊断粪便艰难梭菌感染的合并敏感性、特异性、阳性似然比、阴性似然比及诊断优势比(95%CI)分别为0.925(0.913,0.936)、0.965(0.962,0.969)、28.385(23.394,34.441)、0.093(0.076,0.115)和378.15(282.17,506.77)。ROC曲线下面积(AUCSROC)和Q*指数分别为0.987 8和0.953 2,漏斗图显示无发表偏倚,meta回归结果显示各个影响因素中以发表年份和样本含量为主要异质性来源,对发表年份和样本含量进行亚组分析,发现2010年之后的诊断效能大于2010年之前,样本量大于200的诊断效能大于样本量小于200者。结论通过对real-time PCR对艰难梭菌感染的诊断价值进行meta分析,发现realtime PCR具有较高的敏感性、特异性、阳性似然比及阴性似然比。因其快速准确,可广泛应用于临床。     

Effects of piperacillin/tazobactam on Clostridium difficile growth and toxin production in a human gut model

OBJECTIVES: Clostridium difficile infection (CDI) is a major cause of morbidity in the nosocomial environment. Antimicrobial agents such as the third-generation cephalosporins, lincosamides and aminopenicillins are well known for their propensity to induce CDI, but the definitive reasons why remain to be elucidated. Despite their broad spectrum of activity against both aerobic and anaerobic bacteria, the ureidopenicillins remain a class of antimicrobials infrequently associated with the development of CDI. METHODS: We used a triple-stage chemostat model that simulates the human gut to study the effects of the ureidopenicillin/beta-lactamase inhibitor combination piperacillin/tazobactam on gut bacterial populations and C. difficile. RESULTS: Piperacillin/tazobactam rapidly reduced all enumerated gut bacterial populations (including bacteroides, bifidobacteria and lactobacilli) below the limits of detection by the end of the piperacillin/tazobactam instillation period. Despite such widespread disruption of gut bacterial populations, C. difficile populations remained principally as spores, with no sustained proliferation or high-level cytotoxin production observed. CONCLUSIONS: Factors other than reduced colonization resistance must be responsible for determining whether CDI develops following antimicrobial administration. We believe the gut model is a promising approach for the study of C. difficile pathogenesis reflecting in vivo events likely to occur in CDI.     

Amixicile, a novel inhibitor of pyruvate: ferredoxin oxidoreductase, shows efficacy against Clostridium difficile in a mouse infection model

Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ～250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-μg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.     

空肠弯曲菌 TaqMan-MGB双重探针实时荧光定量 PCR快速检测

目的　开发一种特异、灵敏的TaqMan MGB 双重探针实时荧光定量PCR 方法,用于空肠弯曲菌的快速定量检 测。方法　针对空肠弯曲菌鞭毛蛋白A 和马尿酸酶基因设计特异性引物和探针,建立一种新型TaqMan-MGB 双重探针 实时荧光定量PCR 检测空肠弯曲菌方法,对该方法的定量检测线性范围、特异度、灵敏度、重复性、稳定性进行评价, 应用该方法对临床标本中的空肠弯曲菌进行检测,同时用细菌培养、普通PCR、基因克隆和测序鉴定。结果　建立的 空肠弯曲菌TaqMan MGB 双重探针实时荧光定量PCR 检测方法专属性强,能准确检出空肠弯曲菌,而与其他细菌无交 叉反应,特异度为100%。该技术灵敏度高,能精确定量检测空肠弯曲菌DNA 线性范围达10 个数量级,最低检测限为 4 个菌落形成单位。重复性和稳定性良好,组内和组间相对标准偏差均小于1%。应用该方法成功从78 例临床标本中定 量检出28 例空肠弯曲菌阳性标本,用普通PCR 和基因克隆测序分析确认,细菌培养方法仅获得6 株存活的空肠弯曲菌。 结论　TaqMan MGB 双重探针实时荧光定量PCR 具有快速简便、可靠稳定、特异灵敏的优点,可用于临床标本中空肠 弯曲菌定量检测,值得推广应用。     

Fidaxomicin: first-in-class macrocyclic antibiotic

The incidence of Clostridium difficile has doubled over the past 15 years, and rising mortality rates associated with this infection have followed in its wake. C. difficile infection (CDI) has supplanted methicillin-resistant Staphylococcus aureus as the major cause of nosocomial infection. An insufficient response rate to currently available CDI therapies has prompted the search for new and alternative treatment modalities for this disease. The investigational pipeline includes evaluation of new antimicrobial agents that exhibit good activity against C. difficile without altering normal gut flora, C. difficile toxin-absorbing compounds, and preformed antibodies and vaccines against C. difficile toxin. In two robust clinical trials comparing fidaxomicin to vancomycin in the treatment of CDI, treatment with fidaxomicin demonstrated a superior global cure (cure without recurrence) rate compared with the current gold standard, vancomycin. Fidaxomicin, the first of a new class of macrocyclic antimicrobial agents, represents an advance in the management of CDI.     

Effects of Tigecycline and Vancomycin Administration on Established Clostridium difficile Infection

The glycylcycline antibiotic tigecycline was approved in 2005 for the treatment of complicated skin and soft tissue infections and complicated intra-abdominal infections. Tigecycline is broadly active against both Gram-negative and Gram-positive microorganisms, including Clostridium difficile. Tigecycline has a low MIC against C. difficile in vitro and thus may represent an alternate treatment for C. difficile infection (CDI). To assess the use of tigecycline for treatment of established CDI, 5- to 8-week-old C57BL/6 mice were colonized with C. difficile strain 630. After C. difficile colonization was established, mice (n = 10 per group) were treated with either a 5-day course of tigecycline (6.25 mg/kg every 12 h subcutaneously) or a 5-day course of vancomycin (0.4 mg/ml in drinking water) and compared to infected, untreated control mice. Mice were evaluated for clinical signs of CDI throughout treatment and at 1 week posttreatment to assess potential for disease development. Immediately following a treatment course, C. difficile was not detectable in the feces of vancomycin-treated mice but remained detectable in feces from tigecycline-treated and untreated control mice. Toxin activity and histopathological inflammation and edema were observed in the ceca and colons of untreated mice; tigecycline- and vancomycin-treated mice did not show such changes directly after treatment. One week after the conclusion of either antibiotic treatment, C. difficile load, toxin activity, and histopathology scores increased in the cecum and colon, indicating that C. difficile-associated disease occurred. In vitro growth studies confirmed that subinhibitory concentrations of tigecycline were able to suppress toxin activity and spore formation of C. difficile, whereas vancomycin did not. Taken together, these data show how tigecycline is able to alter C. difficile pathogenesis in a mouse model of CDI.     

Clostridium difficile infection in cancer patients and hematopoietic stem cell transplant recipients

Clostridium difficile has become the most common bacterial cause of nosocomial diarrhea. High rates of C. difficile infection (CDI) coupled with increasing morbidity and mortality attributed to CDI have sparked a renewed interest in this disease. Emergence of hypervirulent strains, rising rates of severe and recurrent infection and associated infection control challenges, and diagnostic and therapeutic dilemmas are major issues in the non-oncology population. Scant data on CDI exist in the cancer/transplant population. The purpose of this article is to describe the epidemiology, pathogenesis and management of CDI in patients receiving cancer chemotherapeutic agents, and in hematopoietic stem cell transplant recipients.     

Effects of cefotaxime and desacetylcefotaxime upon Clostridium difficile proliferation and toxin production in a triple-stage chemostat model of the human gut

Clostridium difficile is recognized as an important nosocomial pathogen. C. difficile infection (CDI) is thought to arise as a result of depletion of the normal gut flora by antimicrobial agents. Cefotaxime (CTX) is well-known for its propensity to cause CDI, but the reasons behind its particular predisposition to the disease remain unclear. Previous investigations have so far relied upon the hamster model of CDI or human volunteers. We have used a triple-stage chemostat model of the human gut to investigate the behaviour of C. difficile and components of the normal gut flora, in response to exposure to CTX alone, and in combination with its active metabolite desacetylcefotaxime (dCTX). C. difficile remained in a steady state during non-antibiotic exposed periods, with no detectable cytotoxin. During both antibiotic exposure regimens, proliferation of C. difficile and elevated cytotoxin levels were observed. Cessation of antibiotic instillation produced a reduction in cytotoxin levels and viable counts. Decreases in bacterial counts were observed in response to both antibiotic exposure regimens, notably for bifidobacteria and bacteroides. Numbers of bacteroides were profoundly affected by exposure to the CTX/dCTX combination, and this may indicate a possible role for bacteroides in colonization resistance. We believe that the gut model is a promising method for studying C. difficile pathogenesis in conditions analogous to the in vivo situation.     

建立检测产气肠杆菌的TaqMan荧光定量PCR和普通PCR方法

目的 建立敏感、特异的普通聚合酶链反应(PCR)方法和TaqMan 荧光定量PCR方法对产气肠杆菌进行快速检测。方法 以产气肠杆菌组氨酸脱氢酶基因(hdc)为靶基因设计引物以及TaqMan FAM探针,建立对产气肠杆菌进行检测的普通PCR方法和TaqMan 荧光定量PCR方法,并评价该方法的特异性、灵敏性和稳定性。结果 普通PCR和TaqMan 荧光定量PCR方法均能对产气肠杆菌进行特异检测;普通PCR方法对质粒标准品和粪便模拟标本的检测下限分别为 100 copies/l和1.0105 cfu/g,TaqMan 荧光定量PCR方法对质粒标准品和粪便模拟标本的检测下限分别为33 copies/l和1.0104 cfu/g;TaqMan 荧光定量PCR方法对质粒标准品和粪便模拟标本检测的扩增曲线良好;在稳定性评价试验中,普通PCR方法的重复性良好,TaqMan 荧光定量PCR方法对质粒标准品检测Ct值的组内差异为0.15%~0.98%,组间差异为0.55%~1.63%。结论 本研究建立的检测产气肠杆菌的普通PCR方法和TaqMan 荧光PCR方法特异性好、灵敏度高,能够用于产气肠杆菌的快速检测。     

Multiplex PCR for detection of Enterobacteriaceae in blood

BACKGROUND: Rapid and sensitive methods are needed to detect the small numbers of bacteria that may sometimes contaminate units of blood during collection. A multiplex 5'-nuclease TaqMan PCR assay (PE Applied Biosystems) was used to detect several bacterial species that may contaminate blood. STUDY DESIGN AND METHODS: Oligonucleotide primers were made for regions of the 16S rRNA gene conserved in four different bacterial species: Yersinia enterocolitica and Serratia, Klebsiella, and Enterobacter species. Two probes were designed: SL-1 detected Serratia, Klebsiella, and Enterobacter species, and YE-3 detected Y. enterocolitica. RESULTS: When TaqMan PCR was performed with chromosomal DNA isolated from pure cultures of Serratia liquefaciens, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter agglomerans, the limit of detection with probe SL-1 was 1 to 2 CFUs. For S. marcescens, the sensitivity was 8 CFUs. The limit of detection for Y. enterocolitica with probe YE-3 was 2 CFUs. When total chromosomal DNA was extracted from whole-blood samples spiked with different numbers of Y. enterocolitica, S. liquefaciens, E. cloacae, or K. pneumoniae bacteria, the TaqMan PCR detected 12 to 16 organisms in 1 mL of blood. CONCLUSION: The 5'-nuclease TaqMan PCR assay takes only 3 hours to perform and has the potential to detect very small numbers of bacteria.