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1.
目的:建立艰难梭菌分离培养与鉴定的表型诊断技术,并对分离获得的艰难梭菌进行分子学特征研究。方法采集杭州某医院ICU住院患者肛拭标本,接种厌氧血琼脂平皿,置厌氧培养箱,可疑菌株采用厌氧菌鉴定试剂条鉴定;用VIDAS荧光酶联免疫技术检测A/B毒素;用PCR技术检测艰难梭菌毒素基因和核糖体分型。结果28例 ICU 患者检出艰难梭菌9株,检出率为32.14%。9株艰难梭菌中, A/B 毒素表型阳性7株(77.78%),毒素基因检测均阳性。9株艰难梭菌可分为3种核糖型,其中R3型为优势株,占77.78%。结论杭州市ICU患者检出艰难梭菌的比例较高,均携带A/B毒素基因,且存在优势型别。  相似文献   

2.
目的对不同老年人群粪便中艰难梭菌进行分离培养、鉴定以及分子型别特征研究,分析其规律特征,为积极有效地预防与控制老年人群艰难梭菌感染提供数据支持。方法收集2010年1月~2016年12月不同老年人群的粪便样本,厌氧培养艰难梭菌,并使用多位点序列分型(MLST)、毒素基因A、B和二元毒素检测、核糖体分型的方法,对老年人群分离的艰难梭菌进行研究分析。结果 116名老年人的粪便样品中分离出艰难梭菌;MLST分型得到32个型别,其中ST3、ST2、ST35、ST11为主要型别,新发现型别ST332及ST473;老年人群中艰难梭菌以产A毒素、B毒素并二元毒素阴性的菌株为主;经核糖体分型鉴定其中1株为RT027型(ST1型),发现6株RT078型(ST11型),RT001型最为常见。结论老年住院患者是艰难梭菌感染的高危人群,老年人群分离的艰难梭菌多为产毒株,ST37型非主要型别,与以往报道略有不同,并发现引起暴发流行的高毒株,应注重加强老年人群的艰难梭菌检测和预防措施。  相似文献   

3.
目的研究中国艰难梭菌A-B+型分离株BJ08和美国艰难梭菌A-B+型暴发流行株US1的毒力编码区域PaLoc各基因转录及B毒素的表达,为预防和控制中国可能暴发的艰难梭菌感染提供理论支持。方法每隔3 h提取BJ08与US1艰难梭菌和培养上清,用荧光定量聚合酶链反应(PCR)法测定各时间段两菌株毒力编码区域PaLoc各基因(tcdA、tcdB、tcdC、tcdR、tcdE)的表达;用酶联免疫吸附试验(ELISA)法检测BJ08和US1各时间段的细胞和培养液上清中B毒素的含量。结果US1的生长速率稍快于BJ08,衰退速率显著快于BJ08(P<0.05);它们都不表达A毒素,但是都检测到tcdA基因的转录,而且tcdA转录没有明显差异。BJ08的tcdB、tcdC和tcdE基因的转录要比US1早3 h。B毒素在两种菌株的胞内和胞外合成或分泌没有明显差异。结论中国艰难梭菌A-B+型高分离株BJ08与美国艰难梭菌A-B+型暴发流行株US1相比,有相似的毒力表达或更强的基因调控能力,要警惕中国艰难梭菌BJ08暴发流行的可能。  相似文献   

4.
目的 初步了解农村社区艰难梭菌流行状况及分子生物学特征.方法 对河南省济源市某乡村医疗机构送检的7株艰难梭菌进行生化鉴定和分子生物学方法鉴定,并进行MLST分型分析,同时与北京某三甲医院的84株艰难梭菌进行对比,用eBURST软件聚类分析,寻找其中可能的关联.结果 源自河南济源市某乡村医院的7株菌均属艰难棱菌,PCR毒素基因鉴定结果显示,其中两株为A-B+菌株,其MLST分型结果是ST37;5株为A+B+菌株,其中4株对应ST35,1株对应ST3;两重来源的菌株中均是毒素基因A+B+菌株占优势;乡村医疗机构的7株艰难棱菌中未发现新的与David MLST数据库中不同的ST型别,未发现不同于北京城市医院的ST型别.结论 David方案适合种群结构和全球流行病学研究,且其数据库全球共享,便于数据交换;该次研究中未发现新的ST型别,所有菌株ST呈散在分布.  相似文献   

5.
目的建立多重PCR方法检测毒力型艰难梭菌。方法设计艰难梭菌种特异tpi引物和毒力基因tcdA、tcdB特异性引物,建立多重PCR方法。利用已知菌株,验证方法的特异性和最低检出限。与厌氧培养法、ELISA法比较其检测临床菌株和其毒素分泌的准确性。结果多重PCR方法检测艰难梭菌的最低检出浓度为0.7 pg/μ1,特异性为100%。53株厌氧培养法鉴定的艰难梭菌临床分离株,多重PCR方法检测tpi基因均为阳性,其中tcdA+/tcdB+为39株,tcdA-/tcdB-为14株。ELISA法检测毒素A/B显示23株为阳性、30株为阴性。23株ELISA法毒素A/B阳性的艰难梭菌多重PCR方法检测结果均为tcdA+/tcdB+。结论多重PCR方法可用于检测毒力型艰难梭菌具有较高的临床应用价值。  相似文献   

6.
目的 通过对住院腹泻患者粪便中艰难梭菌的分离培养和毒力基因检测及分型,了解郴州市住院腹泻患者艰难梭菌感染状况。方法 收集2020年10-12月湘南学院附属医院、郴州市第一人民医院和郴州市第三人民医院住院腹泻患者粪便标本306例,厌氧培养法分离艰难梭菌菌株,采用荧光聚合酶链反应(RT-PCR)检测A、B毒素基因tcdA、tcdB及二元毒素基因cdtA、cdtB,并对分离的艰难梭菌菌株进行多位点序列分型(MLST)。结果 郴州市住院腹泻患者产毒艰难梭菌核酸阳性率为8.17%(25/306),>60岁患者感染风险更高(χ2=5.499,P=0.019); 306份标本中分离出17株艰难梭菌,粪便标本荧光PCR检测有25份阳性,二者比较有统计学差异(P=0.008),毒力基因检测均为tcdA2=5.499,P=0.019); 306份标本中分离出17株艰难梭菌,粪便标本荧光PCR检测有25份阳性,二者比较有统计学差异(P=0.008),毒力基因检测均为tcdA+tcdB+tcdB+,未检出二元毒素;选取7株艰难梭菌进行MLST分型,分出5个ST型,ST54型(3株)、ST129型(1株)、ST98型(1株)、ST53(1株)和ST631(1株)。结论 郴州市住院腹泻患者艰难梭菌感染率较低,感染毒素类型为tcdA+,未检出二元毒素;选取7株艰难梭菌进行MLST分型,分出5个ST型,ST54型(3株)、ST129型(1株)、ST98型(1株)、ST53(1株)和ST631(1株)。结论 郴州市住院腹泻患者艰难梭菌感染率较低,感染毒素类型为tcdA+tcdB+tcdB+,呈多种ST流行。  相似文献   

7.
目的通过研究临床艰难梭菌菌株的临床特征和耐药性,为临床防治艰难梭菌提供理论依据。方法收集2015年全年分离自临床的艰难梭菌,采用PCR技术检测其毒素基因,采用琼脂稀释法检测其耐药性,对患者的临床资料进行分析。结果共收集临床送检粪便标本144份,分离培养出艰难梭菌33株,艰难梭菌阳性检出率为22.92%;科室分布以神经外科、神经内科和消化科为主;分离所得到的艰难梭菌体外实验均对万古霉素、甲硝唑和替加环素敏感,对红霉素和亚胺培南耐药检出株数最多,其次为克林霉素;33株艰难梭菌毒素基因检测结果 A+B+菌株24株,A-B+菌株1株;未检测到二元毒素基因cdtA和cdtB;产毒菌(25株)和非产毒菌(8株),两组菌株均对万古霉素、甲硝唑和替加环素敏感;非产毒艰难梭菌和产毒菌株对抗菌药物红霉素、亚胺培南、克林霉素、利福平和左氧氟沙星的耐药率比较,虽差异无统计学意义,但均高于产毒菌株;患者感染艰难梭菌后,均出现不同程度的腹泻,使患者病情加重,住院时间延长,并出现2例死亡病例。结论非产毒菌株对多种抗菌药物耐药率均高于产毒菌株,并出现对万古霉素敏感性降低的菌株,提示本地区艰难梭菌耐药性可能增加,甚至可能出现耐万古霉素的艰难梭菌。  相似文献   

8.
目的探讨同时检测艰难梭菌毒素A、B基因和二元毒素基因的多重聚合酶链反应(PCR)方法。方法收集2014年1-9月医院120例住院腹泻患者的粪便标本,用艰难梭菌选择性培养基CCFA分离培养艰难梭菌;采用法国生物梅里埃公司厌氧菌鉴定试剂盒鉴定;采用酶免夹心与终点荧光检测相结合技术(ELFA),检测艰难梭菌毒素A、B基因,并分析其毒素特征;将艰难梭菌纯培养出的菌落用多重PCR方法测定艰难梭菌的毒素A、B基因和二元毒素基因。结果 120例腹泻患者送检粪便标本中,分离培养出19株艰难梭菌,艰难梭菌分离率达15.8%;应用免疫学方法检测毒素检出率为52.63%;用多重PCR方法艰难梭菌A、B毒素基因检出率为94.7%;15例患者送检粪便标本检测艰难梭菌A、B毒素阳性中,10株分离培养并鉴定出艰难梭菌;105例患者送检粪便标本检测艰难梭菌A、B毒素阴性中,有9例分离培养并鉴定出艰难梭菌。结论利用多重PCR检测艰难梭菌毒素A、B基因和二元毒素基因,较传统的免疫学方法敏感性高,能够准确地区分艰难梭菌毒素基因的类型,多重PCR是一种快速、特异的一步筛选艰难梭菌毒素基因的方法。  相似文献   

9.
目的对2018-2020年昆明地区腹泻患者中艰难梭菌感染特征进行分析, 为后续监测和防治提供数据支持。方法收集2018-2020年云南省4家哨点医院腹泻患者粪便标本共388份, 使用实时荧光定量PCR方法进行艰难梭菌粪便毒素基因检测, 对结果阳性的粪便标本进行菌株的分离, 用基质辅助激光解析电离飞行时间质谱鉴定菌株。提取分离菌株的基因组DNA进行多位点序列分型(MLST)。分析毒素阳性和菌株分离阳性与患者的临床特征以及艰难梭菌阳性与其他病原共感染的情况。结果 388份粪便标本中, 艰难梭菌内参tpi基因阳性标本47份, 总阳性率为12.11%。其中, 非产毒艰难梭菌4份(8.51%), 产毒艰难梭菌43份(91.49%)。47份阳性标本分离得到18株艰难梭菌, 阳性标本的分离率为38.30%。其中tcdA、tcdB、tcdC、tcdR和tcdE基因均为阳性的菌株14株。18株艰难梭菌的二元毒素均为阴性。所有分离菌株的MLST结果共形成10种序列型(ST), 其中ST37型5株(27.78%);ST129、ST3、ST54和ST2型各2株;ST35、ST532、ST48、ST27和ST3...  相似文献   

10.
目的探讨学龄前患儿艰难梭菌毒素基因分型及患儿临床特点。方法选取2017年10月—2020年10月在嘉兴市第二医院儿科住院的148例腹泻患儿为研究对象,所有患儿均留取48 h内粪便,将粪便标本置于-80℃冰箱中,进行艰难梭菌培养,实时荧光定量PCR完成DNA提取及毒素基因检测,酶联免疫吸附法测毒素A/B。结果148例腹泻患者的粪便标本培养出24株艰难梭菌,分离率16.21%。发现粪便标本培养阳性及培养阴性患儿近2个月抗生素使用情况、血红蛋白含量、白蛋白含量及粪便性状差异有统计学意义(P<0.05)。用PCR法对24株艰难梭菌毒素基因检测,tcdA和tcdB均为阳性菌株9株(37.50%),均为阴性3株(12.50%)。tcdA或tcdB阳性有12株(50.00%),其中tcdA^(-)tcdB^(+)菌株9株,tcdA^(+)tcdB^(-)菌株3株。酶联免疫吸附法检测,测得毒素阳性21例,阳性率为87.50%,其中毒素A^(+)B^(-)9例,毒素A^(+)B^(+)6例,毒素A^(-)B^(+)6例。建立logistic回归模型分析,抗生素过去2个月、白蛋白与粪便性状是艰难梭菌感染的危险因素(P<0.05)。结论艰难梭菌培养以产毒株tcdA^(-)tcdB^(+)、A^(+)B^(-)为主,艰难梭菌感染与抗生素过去2个月、白蛋白与粪便性状体有关,为艰难梭菌监测、预防具有指导价值。  相似文献   

11.
Fifty isolates of the most common UK strain of Clostridium difficile [polymerase chain reaction (PCR) ribotype 001] were analysed by three PCR-based typing methods in order to determine genomic diversity within this strain that may form the basis of a subtyping method. The three methods used were repetitive extragenic palindromic elements (REP), conserved repetitive DNA elements (BOX), and enterobacterial repetitive PCR intergenic consensus sequences (ERIC). The performance of each typing method was assessed by comparing powers of discrimination, typeability and reproducibility. All methods had satisfactory levels of typeability and reproducibility as determined by blind-coded repeats, but REP-PCR typing proved to be the most discriminatory method, yielding seven distinct amplicon profiles consisting of up to eight major bands. BOX-PCR generated between two and five major amplicons with four distinct BOX profiles. ERIC-PCR primers, however, could not discriminate between isolates. These results suggest that PCR ribotype 001 is not clonal in nature at present, and that REP-PCR subtyping methods offer promise to further our understanding of the epidemiology of C. difficile PCR ribotype 001 disease in UK hospitals.  相似文献   

12.
A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to 50 isolates of the UK epidemic strain of Clostridium difficile, polymerase chain reaction (PCR) ribotype 001, to develop a PFGE-based subtyping scheme. This protocol overcame the inherent DNA degradation problems associated with typing this strain of C. difficile by this method, and whole genomic digestion with SmaI restriction enzyme yielded seven distinct and reproducible PFGE banding patterns. Modified PFGE is an appropriate method for subtyping C. difficile PCR ribotype 001 that could be used to improve epidemiological investigations.  相似文献   

13.
We recently attempted to clarify an increased incidence of Clostridium difficile-associated diarrhoea (CDAD) in our hospital by arbitrarily primed polymerase chain reaction (AP-PCR) typing of isolates from 147 consecutive patients collected during a 12 month period (Wullt et al. J Hosp Infect 1999;43:265-273). In the present study we compared the results based on previous AP-PCR data with those based on recent PCR ribotyping of the same isolates and re-analysis of a subset of isolates by AP-PCR typing. The pattern of PCR ribotypes was similar among inpatients and outpatients. A cluster of three closely related PCR ribotypes, related to those of the serogroup H and A8 type strains, dominated and comprised 31% of inpatient and 28% of outpatient C. difficile isolates. The apparent nosocomial transmission rate among inpatients with CDAD was only 9% by AP-PCR typing compared with 18 or 36% by PCR ribotyping depending on the definition used (proportion of patients sharing C. difficile type and ward within two or 12 months). Corresponding rates for all CDAD patients were 5% by AP-PCR and 11 or 21% by PCR ribotyping. Thus, most CDAD patients apparently became ill due to their endogenous strain of C. difficile. Because of the low concordance between the two typing methods the proportion of patients fulfilling the criteria for nosocomial transmission by both methods was only 1%. Re-examination of isolates from patients with recurrences revealed a reproducibility problem with AP-PCR typing. We conclude, that of these two PCR-based options for typing of C. difficile PCR ribotyping offers a superior experimental robustness compared with AP-PCR typing.  相似文献   

14.
Of 40 ready-to-eat salads, 3 (7.5%) were positive for Clostridium difficile by PCR. Two isolates were PCR ribotype 017 (toxin A–, B+), and 1 was PCR ribotype 001. Isolates were susceptible to vancomycin and metronidazole but variably resistant to other antimicrobial drugs. Ready-to-eat salads may be potential sources for virulent C. difficile.  相似文献   

15.
Outbreaks due to Clostridium difficile polymerase chain reaction (PCR) ribotype 027, toxinotype III, were detected in 7 hospitals in the Netherlands from April 2005 to February 2006. One hospital experienced at the same time a second outbreak due to a toxin A-negative C. difficile PCR ribotype 017 toxinotype VIII strain. The outbreaks are difficult to control.  相似文献   

16.
We compared 30-day case-fatality rates for patients infected with Clostridium difficile possessing genes for toxins A and B without binary toxin (n = 212) with rates for patients infected with C. difficile possessing genes for A, B, and binary toxin. The latter group comprised patients infected with strains of PCR ribotype 027 (CD027, n = 193) or non-027 (CD non-027, n = 72). Patients with binary toxin had higher case-fatality rates than patients without binary toxin, in univariate analysis (relative risk [RR] 1.8, 95% confidence interval [CI] 1.2-2.7) and multivariate analysis after adjustment for age, sex, and geographic region (RR 1.6, 95% CI 1.0-2.4). Similar case-fatality rates (27.8%, 28.0%) were observed for patients infected with CD027 or CD non-027. Binary toxin either is a marker for more virulent C. difficile strains or contributes directly to strain virulence. Efforts to control C. difficile infection should target all virulent strains irrespective of PCR ribotype.  相似文献   

17.
BACKGROUND: Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Between August 2003 and January 2004, we experienced an increase in the incidence of C. difficile-associated disease. We describe the investigation into and management of the outbreak in this article. METHODS: A total of 73 consecutive patients with nosocomial C. difficile-associated diarrhea were identified. C. difficile isolates were characterized using toxin-specific enzyme immunoassays, a tissue-culture fibroblast cytotoxicity assay, polymerase chain reaction (PCR), and antimicrobial susceptibility tests. Rates of recurrence and of C. difficile colitis were recorded. Changes in antibiotic use and infection control policies were documented. RESULTS: The incidence of C. difficile-associated diarrhea peaked at 21 cases per 1,000 patient admissions. Of the C. difficile isolates recovered, 85 (95%) were identical toxin A-negative and toxin B-positive strains, corresponding to toxinotype VIII and PCR ribotype 017. All clonal isolates were resistant to multiple antibiotics, including ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin (minimum inhibitory concentrations [MICs] of greater than 32 micro g/mL) and erythromycin, clarithromycin, and clindamycin (MICs of greater than 256 micro g/mL). Recurrent C. difficile-associated disease occurred in 26 (36%) of the patients. At least 10 (14%) of the patients developed C. difficile colitis. Additional infection control measures introduced included the use of ward memos, a hand-hygiene awareness campaign, increased environmental cleaning, attention to prescribing practices for antibiotics, increased awareness of diarrheal illness, and early isolation of affected patients. Total use of fluoroquinolones did not change throughout the study period. Despite persistence of this toxin-variant strain, the incidence of C. difficile-associated disease in our institution decreased to fewer than 5 cases per 1,000 admissions. CONCLUSIONS: We report on the emergence of a fluoroquinolone- and clindamycin-resistant, toxin A-negative, and toxin B-positive strain of C. difficile associated with an outbreak of C. difficile-associated disease in our institution during a 6-month period. We found that careful attention to improvement of infection control interventions was the most important means of controlling this nosocomial pathogen.  相似文献   

18.
We characterized 670 Clostridium difficile isolates collected from patients in 84 hospitals in Germany in 2008. PCR ribotyping showed high prevalence of ribotype 001 and restricted dissemination of ribotype 027 strains. Fluoroquinolone resistance and associated gyrase mutations were frequent in various ribotypes, but no resistance to metronidazole or vancomycin was noted.  相似文献   

19.
目的 对阪崎肠杆菌食品分离株进行核糖体分型,并分析其分型效果.方法采用杜邦Riboprinter~(TM)指纹图谱分析仪对2株阪崎肠杆菌标准菌株和28株分离株进行核糖体分型,运用BioNumerics数据库软件建立相应的核糖体指纹图谱数据库,进行指纹图谱分析.结果 核糖体分型将2株阪崎肠杆菌标准菌株和28株分离株分成26种核糖体带型,其中22种带型分别对应1株阪崎肠杆菌试验菌株,其他4种带型分别对应2株试验菌株,不同带型之间的最低相似度为31.58%.所有菌株的酶切条带数为10个左右,酶切条带分子量范围位于1~50 kb之间.同时,使用BioNumerics软件对结果进行分群和多维尺度分析,可见核糖体分型主要分为4个群.结论 自动化核糖体分型可以方便快捷的实现阪崎肠杆菌的分型.  相似文献   

20.
We investigated Clostridium difficile in calves and the similarity between bovine and human C. difficile PCR ribotypes by conducting a case-control study of calves from 102 dairy farms in Canada. Fecal samples from 144 calves with diarrhea and 134 control calves were cultured for C. difficile and tested with an ELISA for C. difficile toxins A and B. C. difficile was isolated from 31 of 278 calves: 11 (7.6%) of 144 with diarrhea and 20 (14.9%) of 134 controls (p = 0.009). Toxins were detected in calf feces from 58 (56.8%) of 102 farms, 57 (39.6%) of 144 calves with diarrhea, and 28 (20.9%) of 134 controls (p = 0.0002). PCR ribotyping of 31 isolates showed 8 distinct patterns; 7 have been identified in humans, 2 of which have been associated with outbreaks of severe disease (PCR types 017 and 027). C. difficile may be associated with calf diarrhea, and cattle may be reservoirs of C. difficile for humans.  相似文献   

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