41株艰难梭菌临床分离株分子特征及感染危险因素分析

目的:了解我国山东省济南地区艰难梭菌临床分离株的多位点序列分型情况及感染危险因素。方法:收集2018年1月至2019年6月山东第一医科大学附属省立医院成人腹泻患者分离的41株艰难梭菌,对其进行PCR毒素基因检测、多位点序列分型及感染危险因素分析。结果:所分离的41株艰难梭菌中,tcdA⁺ tcdB⁺tcdC⁺tcdR⁺ tcdE⁺cdtA^- cdtB^-为最常见的毒素表达谱型;共检出13种ST型别,最常见的型别依次为ST3(11株,占26.8%)、ST2(6株,占14.6%),并发现5株ST1型高毒力RT027艰难梭菌。艰难梭菌感染患者流行病学分析显示,高龄、慢性肺部疾病等基础疾病及服用抗生素等为感染的高危因素。结论:增强对艰难梭菌感染的认知,提升艰难梭菌感染检测能力,建立艰难梭菌感染监测体系,将有助于应对艰难梭菌感染带来的严峻挑战。     

广东省东莞市脑膜炎奈瑟菌多位点序列分型研究

目的 对东莞市2004—2017年分离的41株脑膜炎奈瑟菌（Neisseria meningitidis,Nm）进行基因分型研究,了解菌株间的遗传进化关系。方法 采用多位点序列分型（MLST）技术、进化分析软件BioNumerics 7.5及聚类分析软件eBURST V3对菌株进行核酸序列分析。结果 41株脑膜炎奈瑟菌分为A、B、C、W135四个群,其中1株A群、9株B群、23株C群、8株W135群。共有11个不同STs,分别属于ST-4821、ST-11、ST-5、ST-41/44克隆群及4个无序列分类（UA）,另有1株B群及1株C群菌株无法得到ST型。其中ST-4821 Complex及ST-11 Complex为主要克隆群,分别占61.54%、23.08%。不同血清群其STs/ST-Complex 有明显不同：C群菌株以ST-4821 Complex为主,有 ST-4821、ST-4831、ST-4833、ST-5798型,其中ST-4821型有3种基因序列;W135群全为ST-11 Complex,有ST-11和ST-5412型,ST-11集中在2015年分离出的菌株,均同源;A群仅有1株ST-5型;B群较分散。eBURST V3 聚类分析发现,东莞市ST-11 Complex基因型相对稳定,ST-4821Complex 基因型已具遗传多样性。结论 东莞市Nm 分子型具有基因多样性,以C群（ST-4821/ST-4821 Complex）和W135群（ST-11/ST-11 Complex）两大克隆群占绝对优势,B 群呈分散状态出现在不同的ST克隆群中,可能不同血清群之间已产生荚膜转换,提示B群出现基因重组进化特征。     

院内感染艰难梭菌的毒素检测及核糖体分型研究

目的 了解艰难梭菌临床分离株的毒力情况,并运用核糖体分型技术进行流行病学研究.方法 采集住院腹泻患者的未成形粪便标本,无水乙醇处理后接种CDMN选择培养基行艰难梭菌分离培养,并通过革兰染色、耐氧试验和凝集试验行菌落鉴定;提取菌株DNA,选择特异性引物用PCR法扩增毒素基因tcdA和tcdB;核糖体分型针对细菌基因组16S～23S rDNA间区序列进行扩增,并根据电泳带型的多态性实现分型.结果 共计分离得44株艰难梭菌,3种毒素型分别为A+B+型、A-B+型和A-B-型,分别占 57％ 、34％和9％;18种核糖体型中以R8 型和R4 型为主,分别占 20％和18％.结论 本研究临床分离的艰难梭菌菌株中,毒素型以A+B+型为主,核糖体分型存在相对优势的型别;无证据提示存在院内艰难梭菌感染的暴发流行.     

广州地区鲍曼不动杆菌的多位点序列分型及流行克隆

目的 本研究对广州地区5家教学医院的鲍曼不动杆菌进行分子流行病学分析.方法 5家教学医院共采集138株鲍曼不动杆菌,利用多位点序列分型(multilocus sequence typing,MLST)及eBURST算法评价菌株之间的遗传关系.结果 MLST将138株鲍曼不动杆菌分为8个已有序列类型(STs),分别为ST...     

上海地区某三甲医院艰难梭菌感染临床分析及微生物学特征研究

背景：近年来,艰难梭菌作为医院感染的重要病原菌逐渐受到国内外研究者的重视,然而国内相关报道较少。本研究拟对上海地区某三甲医院艰难梭菌感染情况进行临床分析和微生物学特征研究。 方法：本研究收集2010年12月至2013年3月艰难梭菌感染的住院病人,对临床数据进行回顾性分析;同时分离病原体艰难梭菌,采用微生物学和分子生物学方法对其进行药物敏感试验、毒素检测、芽孢及生物膜形成、动力试验和多位点序列分型等研究。 结果：本研究共入选94例艰难梭菌感染患者,其中12例为严重感染。对临床信息进行分析表明,所有患者均接收过质子泵抑制剂和（或）抗生素治疗,且分布在不同的科室,其中消化科较多,占29.79%（28/94）。通过比较感染严重病例组和感染轻微病例组,感染的病原体之间在微生物学特征上未发现有统计学意义的差异。94株艰难梭菌中,31株为A毒素阴性B毒素阳性（A-B ）且为序列型ST37,为流行克隆株。与其他克隆来源的菌株相比,流行克隆株产较少的毒素和芽孢,生物膜形成和动力也相似,但都对头孢菌素类、喹诺酮类、大环内酯类-林可酰胺类链霉素类和四环素类具有较高的耐药性。 结论：在艰难梭菌感染严重病例中,没有发现特异的临床症状和微生物学特征。高耐药性可能是艰难梭菌感染流行和传播的主要潜在原因之一。     

ICU病房血流感染白色念珠菌的多位点序列分型研究

目的调查四川大学华西医院ICU病房白色念珠菌血流感染现状,探讨其分子流行病学特征。方法收集2009～2011年四川大学华西医院全院送检的血培养分离所得白色念珠菌。对ICU病房血流感染白色念珠菌采用多位点序列分型(multilocus sequencing typing,MLST)分析菌株间的亲缘关系。结果 2009～2011年华西医院送检的血培养标本共分离念珠菌135株,以白色念珠菌为主(51株,37.8%)。进行MLST分型的17株白色念珠菌来自ICU病房的15名患者,标本来源包括外周血15份,中央静脉导管2份(有两名患者分别取外周血和中央静脉导管各1份且均培养出白色念珠菌)。17株菌经MLST分型得到15个序列型(sequence type,ST),其中3株菌具有相同的ST型,另外14株菌分别具有14个不同ST型。经聚类分析,5株菌(29.4%)归为Group 46克隆组,2株菌(11.8%)归为Group 47克隆组,其余10株菌为单体型。分离自同一患者外周血和中央静脉导管的2株菌(Calb-36和Calb-40)经MLST分型证实是同一克隆株。结论白色念珠菌为华西医院念珠菌血症的首要致病菌。ICU病房血流感染白色念珠菌的主要流行株属于Group46和Group47克隆组。我院ICU病房目前尚不存在白色念珠菌血流感染的暴发流行,但有导管相关性白色念珠菌血症的感染发生。     

重复序列PCR与多位点分型技术在白假丝酵母菌基因分型中的比较

目的应用多位点序列分型技术（MLST）和重复序列聚合酶链反应（REP—PCR）对临床分离出的白假丝酵母菌进行分型,并对两种方法作出应用评价。方弦收集上海地区医院中真菌性阴道炎分离出的40株白假丝酵母菌,选择合适引物进行扩增,通过电泳比较分析获得REP—PCR分型。选择7对管家基因进行PCR反应并测序,将测序结果与标准序列进行比对,获得由7对管家基因组成的等位基因谱,最终得到相应的序列分型。结果REP-PCR中Ca22-Ca22引物对得到电泳条带最优,40株白假丝酵母菌共分成7种REP—PCR型;MLST分型得出29种,且均为新型。结论MLST分辨率较REP—PCR高,但由于REP—PCR分型方法更为快捷、经济,更适用于实验室大量菌株分型和临床分型。MLST则更客观,更适合用于进化学的研究及全球性流行病学的调查研究。     

潜水人群铜绿假单胞菌基因多位点序列遗传分型

目的 研究海军潜水人群铜绿假单胞菌分离株的遗传基因型,明确海军潜水人群铜绿假单胞菌携带株的流行特征.方法 用多位点序列分型(multilocus sequence typing,MLST)技术、以铜绿假单胞菌7个管家基因acsA、aroE、guaA、mutL、nuoD、ppsA和trpE内部特定核酸片段作检测靶,对随机抽样海军潜水员携带的64株铜绿假单胞菌进行目的基因扩增和测序,应用Pseudomonas aeruginosa MLST数据库对测序结果进行分析,以获得检测靶基因的多位点序列信息;应用Bionumerics 4.0的LIAN,SplitsTree和eBURST分析程序对获得的MLST进行进一步的生物信息学分析,以获得流行株的遗传特征.结果 64株铜绿假单胞菌中53株可分为19个ST型,11株未能分型;其中ST274和ST260是优势克隆株,分别占18.75％(12/64)、15.62％ (10/64).结论 海军潜水人群携带的铜绿假单胞菌具有遗传多样性和优势基因型,MLST分型对于研究海军潜水人群携带的铜绿假单胞菌遗传差异与流行特征具有重要意义.     

鲍曼不动杆菌多位点序列分型与抗生素耐药表型的关系

目的：对鲍曼不动杆菌菌株进行分子流行病学分型及耐药性分析,为临床防控提供指导。方法收集非重复鲍曼不动杆菌42株,采用多位点序列分型（ MLST）进行分子流行病学分型,eBURST分析菌株亲缘性,纸片扩散法进行药敏试验。结果42株鲍曼不动杆菌被分为10个ST型,其中ST195及ST208最常见,占总数的69.0％,eBURST分析提示主要流行克隆为CC92。根据纸片药敏结果,菌株对多黏菌素B的敏感性最高为100％,其余抗生素的敏感率均小于30％,42株菌株可分为多重耐药（ MDR）菌株11株,广泛耐药（ XDR）菌株31株。优势克隆CC92与非CC92的MDR、XDR菌株的比例差异无统计学意义（P＝0.676）。结论鲍曼不动杆菌CC92广泛流行,CC92与非CC92菌株的耐药性无差别,CC92的成功流行可能更依赖于对医院环境的高度适应性。     

Diarrhoea associated with Clostridium difficile in a hospital population

The incidence of disease associated with Clostridium difficile was investigated in a general hospital population over a period of six months. In 26 (14.5%) of 179 patients studied, C. difficile was either isolated or faecal cytotoxin was detected. The incidence of other enteropathogenic bacteria, except Aeromonas hydrophila, was low. Faecal cytotoxin was not detected in nine patients (35%), and non-cytotoxigenic strains of C. difficile were isolated from these patients. In seven patients, a selective broth was required to isolate C. difficile, suggesting their presence in low numbers. Although some C. difficile diarrhoeal disease which was not associated with antibiotic therapy was recorded, prior exposure to antibiotic agents still appears to be the major predisposing factor in this population. Faecal cytotoxin detection, sigmoidoscopy, and rectal biopsy were not reliable investigations for disease associated with C. difficile; hence, we advocate increased emphasis on isolation of the causative organism. Routine culturing for C. difficile in a hospital population appears to be warranted.     

Optimization of Bartonella henselae multilocus sequence typing scheme using single-nucleotide polymorphism analysis of SOLiD sequence data

Background  Multi-locus sequence typing (MLST) is widely used to explore the population structure of numerous bacterial pathogens. However, for genotypically-restricted pathogens, the sensitivity of MLST is limited by a paucity of variation within selected loci. For Bartonella henselae (B. henselae), although the MLST scheme currently used has been proven useful in defining the overall population structure of the species, its reliability for the accurate delineation of closely-related sequence types, between which allelic variation is usually limited to, at most, one or two nucleotide polymorphisms. Exploitation of high-throughput sequencing data allows a more informed selection of MLST loci and thus, potentially, a means of enhancing the sensitivity of the schemes they comprise.

Methods  We carried out SOLiD resequencing on 12 representative B. henselae isolates and explored these data using single nucleotide polymorphism (SNP) analysis. We determined the number and distribution of SNPs in the genes targeted by the established MLST scheme and modified the position of loci within these genes to capture as much genetic variation as possible.

Results  Using genome-wide SNP data, we found the distribution of SNPs within each open reading frame (ORF) of MLST loci, which were not represented by the established B. henselae MLST scheme. We then modified the position of loci in the MLST scheme to better reflect the polymorphism in the ORF as a whole. The use of amended loci in this scheme allowed previously indistinguishable ST1 strains to be differentiated. However, the diversity of B. henselae was still rare in China.

Conclusions  Our study demonstrates the use of SNP analysis to facilitate the selection of MLST loci to augment the currently-described scheme for B. henselae. And the diversity among B. henselae strains in China is markedly less than that observed in B. henselae populations elsewhere in the world.     

Serological and molecular capsular typing, antibiotic susceptibility and multilocus sequence typing of Streptococcus pneumoniae isolates from invasive and non-invasive infections

Background Streptococcus pneumoniae (S. pneumoniae) is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, and has become a major public health concern. We report the pneumococcal serotype and sequence type (ST) distribution, and antimicrobial resistance of 39 S. pneumoniae strains from seven hospitals in China.

Methods Blood/cerebrospinal fluid (CSF) and sputum isolates from patients were analyzed to determine S. pneumoniae serotypes by polymerase chain reaction (PCR) and the Neufeld Quellung reaction, the multilocus sequence types (MLST) by PCR and sequencing, and susceptibility to antimicrobial agents by the VITEK Gram Positive Susceptibility Card.

Results A total of 39 isolates were collected including 21 blood/CSF and 18 sputum isolates. Conventional serotyping by the Quellung reaction required 749 reactions. In contrast, PCR based typing needed only 106 PCR reactions. The most frequent serotypes from the blood/CSF isolates were 14 (38.1%), 19A (14.3%), 23F (9.5%), and 18C (9.5%). In the sputum isolates the most frequent serotypes were 19F (33.3%), 23F (16.7%), 19A (11.1%), and 3 (11.1%). The incidence of penicillin resistance in the blood/CSF and sputum isolates was 66.7% and 55.6%, respectively. Statistical analysis showed that patients ≤5 years old had a higher resistance to penicillin when they compared with the patients ≥65 years old (P=0.011). Serotypes 14, 19A and 19F were significantly associated with penicillin resistance (P <0.001). ST320, ST271, and ST876 isolates showed high resistant rates to several antibiotics including penicillin (P=0.006). All of the isolates of serotype 19A were resistant to both penicillin and erythromycin, and they were all multi-drug resistant (MDR) isolates.

Conclusions The specificity and sensitivity of multiplex-PCR are good, and this method represents a substantial savings of time and money, and can be widely used in the laboratory and clinical practice. Data from this research showed an extremely high prevalence of penicillin resistance and an increasing prevalence of multi-drug resistant (MDR) rate in S. pneumoniae. A distinctive emergence of serotype 19A was observed which was also associated with the increasing prevalence of antimicrobial resistance. Therefore, nationwide surveillance of pneumococcal resistance and serotypes is strongly warranted.

     

Clinical and microbiological characterization of Clostridium difficile infection in a tertiary care hospital in Shanghai, China

Background Over the last decade,Clostridium difficile infection （CDI） has emerged as a significant nosocomial infection,yet little has been reported from China.This study aimed to characterize the clinical and microbiological features of CDI from a hospital in Shanghai.Methods Patients with CDI seen between December 2010 and March 2013 were included in this study,of which clinical data were retrospectively collected.The microbiological features of corresponding isolates were analyzed including genotype by multi-locus sequence typing （MLST）,antimicrobial susceptibility,toxin production,sporulation capacity,biofilm formation,and motility.Results Ninety-four cases of CDI were included during this study period,12 of whom were severe cases.By reviewing the clinical data,all patients were treated empirically with proton pump inhibitor or antibiotics or both,and they were distributed widely across various wards,most frequently to the digestive ward （28/94,29.79％）.Comparing the severe with mild cases,no significant differences were found in the basic epidemiological data or the microbiological features.Among the 94 isolates,31 were toxin A-negative toxin B-positive all genotyped as ST37.They generated fewer toxins and spores,as well as similar amounts of biofilm and motility percentages,but exhibited highest drug resistance to cephalosporins,quinolones,macrolide-lincosamide and streptogramin （MLSB）,and tetracycline.Conclusions No specific clinical genotype or microbiological features were found in severe cases; antimicrobial resistance could be the primary reason for epidemic strains leading to the dissemination and persistence of CDI.     

我院ICU多重耐药鲍曼不动杆菌聚集性病例与环境污染调查分析

［摘要］ 〖HTH〗目的〖HTSS〗〖KG*2〗调查我院重症监护病房（intensive care unit,ICU）多重耐药鲍曼不动杆菌（multidrug-resistant Acinetobacter baumannii,MDR-AB）医院获得性肺炎聚集性病例与医院环境污染的相关性,为预防和控制ICU患者MDR-AB医院感染提供环境流行病学依据。 〖HTH〗方法〖HTSS〗〖KG*2〗采用现场流行病学调查方法,对短时间内我院ICU出现的3例MDR-AB医院获得性肺炎患者、周围环境以及医务人员标本进行干预前后大面积环境卫生学采样监测,用VITEK-2 Compact全自动细菌鉴定及药敏分析系统进行菌株鉴定与药敏分析。采取分区分色管理方法实施强化环境清洁消毒等综合干预措施,追踪干预效果。 〖HTH〗结果〖HTSS〗〖KG*2〗对干预前80份环境标本进行微生物监测,其中护理站电脑键盘、医护人员手、床单、手消压盖、抢救车剪刀、回风口、患者胸部皮肤、隔离衣袖口、床挡板扶手等14份标本分离出鲍曼不动杆菌,其耐药谱与痰培养耐药菌株相一致。干预后连续2个月环境采样146份标本,均未检出MDR-AB。连续实施干预措施6个月后,标本合格率由77.50%上升至96.57%,医院感染率由干预前2.15%（214/9 965）下降至1.56%（185/11 866）,差异均有统计学意义（P＜0.05）。 〖HTH〗结论〖HTSS〗〖KG*2〗MDR-AB医院感染的发生与患者周围环境污染密切相关,分区分色强化环境清洁消毒等综合干预措施可以有效降低医院感染暴发风险。     

Pathogenesis and treatment of Clostridium difficile infection

This paper reviews the pathogenesis and management of Clostridium difficile diarrhoea, in particular the management of recurrent episodes.     

重症监护室鲍曼不动杆菌感染及药敏试验研究

目的 了解重症监护室(ICU)患者鲍曼不动杆菌感染及耐药情况,指导临床医师选择有效抗生素进行抗感染治疗,控制院内感染的发生.方法 2008年1月至2010年1月期间自重症监护室患者分离鲍曼不动杆菌,细菌鉴定用VITEK-GNI+,药敏试验采用K-B法.结果 分离出172株鲍曼不动杆菌,主要来自呼吸道感染患者痰液,占87...