感染过程中钩端螺旋体外膜蛋白抗原表达水平变化及其调控机制

目的：了解感染人巨噬细胞过程中问号钩端螺旋体（简称钩体）黄疸出血群赖型赖株主要外膜蛋白（OMP）抗原表达水平变化及其OmpR相关基因调控机制。 方法：采用生物信息学技术预测问号钩体赖株OmpR及其组氨酸激酶（HK）基因和结构功能域。采用实时荧光定量RT-PCR检测钩体感染人THP-1巨噬细胞前后钩体主要OMP编码基因mRNA水平的变化。采用HK胞外区多肽抗血清封闭试验及氯氰碘柳胺阻断试验,检测OmpR及其HK对感染过程中问号钩体OMP编码基因mRNA水平变化的影响。 结果：生物信息学分析结果显示,LB015和LB333可能是问号钩体赖株OmpR编码基因,LB014可能是其HK编码基因。问号钩体赖株感染THP-1细胞后,lipL21、lipL32、lipL41基因mRNA水平迅速并持续下降（P<0.01）,groEL、mce、loa22、ligB基因mRNA水平瞬时迅速升高（P<0.01）。氯氰碘柳胺或HK抗血清处理可使感染THP-1细胞而显著下降的问号钩体lipL21和lipL48基因mRNA水平明显回升（P<0.01）,氯氰碘柳胺处理可使感染THP-1细胞而显著升高的groEL、mce、loa22、ligB基因mRNA水平明显下降（P<0.01）。 结论：问号钩体赖株感染人巨噬细胞后主要OMP抗原表达水平发生明显变化。问号钩体赖株染色体中存在一组编码OmpR及其HK的基因,其主要功能可能是下调感染过程中部分OMP抗原表达水平。     

贵州省2010－2014年间钩体宿主动物带菌监测结果与分析

目的：了解贵州省鼠类宿主动物钩端螺旋体（简称钩体）带菌情况、菌型分布,为钩体病的防控提供技术手段和科学依据。方法：对2010～2014年对贵州省黔东南州钩体疫源地采用夜夹捕鼠法进行鼠间动物监测捕获鼠类,分离钩体后用PCR方法进行鉴定。结果：2010－2014年鼠间动物监测有效布夹数6750夹次,共捕鼠646只（鼠密度9．57％）,以黑线姬鼠最多240只,占总数的37．15％。共分离钩体菌株56株（带菌率8．67％）经PCR检测为黄疸出血群。结论：黑线姬鼠为贵州省钩体病常见的带菌鼠种;黄疸出血群钩体是贵州省鼠间的主要传染源。     

广东省茂名市副溶血弧菌涉水产品分离株病原学特征

目的 了解茂名市涉水产品中副溶血弧菌分离株的病原学特征。方法 对茂名市食品安全风险监测中分离到的副溶血弧菌进行血清分型、抗生素敏感性实验、毒力基因检测、脉冲场凝胶电泳( PFGE) 分子分型和多位点序列分型（MLST）。结果 2017—2018年共分离到涉水产品中副溶血弧菌分离株44株,O型共分7个血清型,无明显优势血清型。44株分离株除对氨苄西林和头孢唑林表现出高度的耐药性外,对其它抗生素均敏感,未发现有分离株携带tdh和trh基因。44株分离株分成44种不同的PFGE带型,MLST分型发现有30个新的ST型,呈现一定的遗传多样性。结论 广东省茂名市涉水产品中副溶血弧菌分离株血清型别多样,对大部分的抗生素敏感,均未发现携带tdh和trh 基因,且呈现一定的遗传多态性,需要继续加大监测的力度和范围。     

Establishment and Comparison of Pulsed-field Gel Electrophoresis, Multiple-locus Variable Number Tandem Repeat Analysis and Automated Ribotyping Methods for Subtyping of Citrobacter Strains

ObjectiveTo establish and compare the pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) and automated ribotyping for subtyping of Citrobacter strains.MethodsPFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system.ResultsWe optimized the plug preparation procedure, focused on the cell suspension concentration (turbidity of 2.5 to 3.5), SDS addition (no SDS needed) and lysis time (1 h), and selected the appropriate restriction enzyme (XbaI) and the electrophoretic parameters (1.0 s-20.0 s for 19 h) of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources (with the same species from the same place at the same time identified as the same source) and divided strains from different sources (from different years, places and hosts) into different subtypes.ConclusionPFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.     

Molecular Characteristics of Neisseria meningitidis Isolated during an Outbreak in a Jail: Associatition with the Spread and Distributition of ST-4821 Complex Serogroup C Cltione in China

Objective To characterize the meningococcal strains isolated from cases and close contacts with meningococcal disease associated with an outbreak in a jail in May 2010 by investigating the national distribution of hyperinvasive ST-4821 serogroup C clone associated with this outbreak. Methods The cases were described based on the clinical symptoms and laboratory results. Pharyngeal swabs were cultured for N. meningitidis from men in the jail. Meningococcal isolates were identified by serogrouping, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), respectively. Four hundred and sixteen serogroup C N. meningitidis strains were collected from 27 provinces between 2003 and 2010 for a nationwide survey and analyzed by PFGE and MLST. Results Three persons in a jail system were infected with invasive N. meningitidis serogroup C. All isolates tested had matching PFGE patterns and belonged to the multilocus sequence type (ST) 4821 clonal complex. All 47 N. meningitidis strains were identified from the pharyngeal swabs of 166 peoples in the jail, and 26 of them belonged to ST-4821 serogroup C clone, and 90.14% (375/416) serogroup C strains identified in the nationwide survey belonged to the ST-4821 complex. The ST-4821 serogroup C clone was spread nationwide, distributed in 24 provinces, especially in eastern provinces between 2003 and 2010. Conclusion Endemic transmission and carriage rate of ST-4821 serogroup C clone are high in this jail system. The ST-4821 serogroup C clone is spreading in China and nationwide distributed despite the existence of some effective vaccines.     

Seroprevalence of bovine leptospiral antibodies by microscopic agglutination test in Southeast of Iran

ObjectiveTo evaluate serological findings of bovine leptospirosis which is a zoonotic disease with worldwide distribution caused by Leptospira interrogans.MethodsOne hundred and sixty seven sera were collected from 9 commercial dairy herds in jiroft suburbs, from July to October 2011. Microscopic agglutination test (MAT) was used to evaluates serological findings of bovine leptospirosis in Jiroft suburb dairy farms, Kerman province, Iran.ResultsAntibodies were found by MAT at least against one serovar of Leptospira interrogans in 29 samples (17.36%) among 167 sera at a dilution 1:100 or higher, and Leptospira pomona was the most prevalent serovar. Positive titers against more than one serovar were detected in 6 sera of the positive samples.ConclusionsThis study is the first report of leptospirosis in Southeast Iran and showed that Leptospira pomona was the most and Leptospira icterohaemorrhagiae the least prevalent serovars in Southeast Iran.     

问号钩端螺旋体诱导J774A.1细胞凋亡及caspase-3、-6活化对凋亡的影响

目的：了解问号钩端螺旋体（钧体）诱导小鼠单核巨噬样细胞（J774A．1）凋亡的作用，以及caspase-3、-6活化对凋亡的影响。方法：建立问号钩体黄疸出血群赖型56601株J774A．1细胞感染模型。采用FITC—AnnexinV／PI荧光标记流式细胞术检测细胞凋亡或坏死情况。分别采用荧光比色法和Western Blot，检测感染细胞caspase-3、-6活性及其底物（PARA和LaminA／C）剪切情况。结果：多种不同浓度的问号钩体赖株感染均可使J774A．1细胞发生凋亡，其中以问号钩体：细胞=100：1的比例感染时凋亡率最高（48．81％&#177;5．950o）。感染细胞caspas-3和-6最大活性分别为（1453．41&#177;36．07）FU和（618．65&#177;39．82）FU，是未感染细胞的16．38和9．98倍。感染细胞的PARP和LaminA／C均被剪切。Caspase-3、-6抑制剂对问号钩体赖株诱导的J774A．1细胞凋亡有明显的阻断作用。结论：问号钩体可诱导J774A．1细胞凋亡，easpase-3和-6与该细胞凋亡密切相关。     

问号钩端螺旋体胶原酶体内和体外的活性研究

目的通过检测致病性钩端螺旋体（钩体）胶原酶在体内、外的活性,探讨问号钩体黄疸出血群赖型赖株（赖株）假定的胶原酶（LA0872）在钩体病出血中的可能作用。方法在大肠杆菌中克隆、表达重组赖株la0872,并行相关鉴定及蛋白酶学活性检测。比较不同毒力菌株赖株、赖株减毒株和双曲钩体三宝垄群montevalerio型Monte Valerio株钩体胶原酶的基因序列特异性及转录和酶活水平的差异。测定豚鼠和沙鼠赖株感染模型的血清胶原酶活性水平变化。结果成功构建的重组质粒经酶切和测序鉴定显示位点连接正确,插入序列与GenBank公布的la0872序列完全一致,并证实其具有胶原酶活性;不同毒力菌株中的钩体胶原酶基因序列一致,转录和酶活水平均无显著性差异;豚鼠和沙鼠感染赖株后,宿主体内血清胶原酶活性水平与未感染赖株动物比较差异无统计学意义（P〉0.05）。结论首次表达和鉴定了有活性的钩体胶原酶,但其在钩体病出血中的作用尚待证实和探讨。     

北京地区宋内志贺菌分子流行病学特征分析

目的 了解北京地区宋内志贺菌毒力基因分布及分子流行病学特征. 方法 采用实时荧光PCR方法检测毒力基因、脉冲场凝胶电泳(PFGE)分型,多位点序列分型(MLST)及多位点可变数目串联重复序列分析(MLVA)等方法对北京地区2001～2009年50株志贺菌菌株进行分子流行病学特征分析. 结果 50株菌中,ipaH、set1、sen三种毒力基因的携带率分别为98％,18％和78％.选取19株流行病学和遗传背景无关的菌株,进行MLST、MLVA与PFGE分型方法的比较,MLST为1个序列型ST152 complex:adk(11)、fumC (63)、gyrB(7)、icd(1)、mdh(14)、purA (7)、recA(7); MLVA被分成15个型别,D值为0.9708;PFGE分成12个型别,D值为0.9532.MLVA初筛得到的8个可变数目串联重复(VNTR)位点用于扩大菌株分析,发现50株志贺菌被分为21个MLVA的型别,TS002和TS001为主要型别. 结论 北京地区近年来流行的宋内志贺菌存在毒力基因的丢失,MLVA分子型别复杂,存在多克隆来源,提示需要进行连续系统的分子流行病学监测.     

问号钩端螺旋体胶原酶体内和体外的活性研究

目的 通过检测致病性钩端螺旋体(钩体)胶原酶在体内、外的活性,探讨问号钩体黄疸出血群赖型赖株(赖株)假定的胶原酶(LA0872)在钩体病出血中的可能作用.方法 在大肠杆菌中克隆、表达重组赖株la0872,并行相关鉴定及蛋白酶学活性检测.比较不同毒力菌株赖株、赖株减毒株和双曲钩体三宝垄群montevalerio型Monte Valerio株钩体胶原酶的基因序列特异性及转录和酶活水平的差异.测定豚鼠和沙鼠赖株感染模型的血清胶原酶活性水平变化.结果 成功构建的重组质粒经酶切和测序鉴定显示位点连接正确,插入序列与GenBank公布的la0872序列完全一致,并证实其具有胶原酶活性;不同毒力菌株中的钩体胶原酶基因序列一致,转录和酶活水平均无显著性差异;豚鼠和沙鼠感染赖株后,宿主体内血清胶原酶活性水平与未感染赖株动物比较差异无统计学意义(P>0.05).结论 首次表达和鉴定了有活性的钩体胶原酶,但其在钩体病出血中的作用尚待证实和探讨.     

我国西沙群岛鼠形动物和吸血昆虫的鉴定并首次记述按蚊和蠓

目的 研究我国西沙群岛有害的鼠形动物和吸血昆虫的种类和分布情况,为当地传染病防控提供资料。 方法 2013年11月至12月和2014年2月至3月两次赴我国海南省西沙群岛的永兴岛和石岛,用鼠笼和诱蚊灯采集鼠形动物和吸血昆虫,结合形态和分子特征进行种类鉴定。 结果 捕获鼠形动物3种共计160只,分别为：褐家鼠、黄胸鼠和鼩鼱,优势种为褐家鼠（61.25%）。捕获的315只蚊成虫隶属4属5种,优势种是骚扰阿蚊（52.38%）,按蚊经分子鉴定为浅色按蚊B。捕获蠓成虫121只,隶属3亚科4属8种,帛琉库蠓为优势种（61.98%）。 结论 共发现西沙群岛的永兴岛和石岛的鼠形动物3种,蚊5种,蠓8种,其中按蚊和蠓均为西沙群岛首次记述,斑美铗蠓和泥泽铗蠓是我国的新记录种。     

Mycoplasma pneumoniae Macrolide Resistance and MLVA Typing in Children in Beijing,China, in 2016: Is It Relevant*

ObjectiveThe aim of this study is to investigate the macrolide resistance rate and molecular type with multiple-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae of Beijing in 2016 in pediatric patients.MethodsReal-time quantitative polymerase chain reaction (PCR) was used to identify M. pneumoniae, and MLVA was performed. The domain V of the 23S rRNA was sequenced to detect macrolide-resistant point mutations. We also investigated the activities of antibiotics against M. pneumoniae isolates in vitro.ResultsThe PCR detection rate of M. pneumoniae in children in Beijing was 40%, and the macrolide resistance rate was 66%. The A2063G mutation in the 23S rRNA V region is the dominant mutation (137/146, 93.84%), whereas the A2064G mutation is rare (9/146, 6.16%). Seventy-three samples were typed successfully by MLVA typing, including 86.3% (63/73) were MLVA type 4-5-7-2, and 13.7% (10/73) were MLVA type 3-5-6-2. No other types were found. No strains were resistant to levofloxacin or tetracycline.ConclusionIn 2016, a specific decrease in the macrolide resistance rate occurred in Beijing. The detection rate and macrolide resistance rate of outpatients are lower than those of inpatients. The A2063G mutants M. pneumoniae have high levels of resistance to erythromycin and azithromycin. The primary MLVA type is 4-5-7-2, followed by 3-5-6-2. No other MLVA types were detected. No strains resistant to tetracycline or levofloxacin were found in vitro.     

问号状钩端螺旋体外膜单克隆抗体凝集反应特性的研究

Three McAb were produced against an outer envelope preparation from Leptospira, interrogans, serovar Lai by fusion of SP2/0 myeloma cells with immune BALB/c mice spleen cells. The fusion rate was 96% and the antibody positive rate was 50%. One of the hybridomas, E4B11C9, reacted with 13 of the 13 serovars of the Icterohaemorrhagiae serogroup in microscopic agglutination test (MAT) but did not react with the 18 representative serovars of L. interrogans and L. biflexa serovar patoc and Leptonema illini. For all non-reactive serovars the MAT titres were greater than 1:25. The McAb, E4B7G5, reacted similarly with all serovars except smithi and tonkini. E4B7D4 reacted also similarly with all serovars except serovars birkini, ndambari, bogvere, smithi and tonkini. Therefore, 3 McAb showed serogroup specificity and partial serogroup specificity by agglutination. The agglutination titres were high and hybridomas were stable, so it might be useful in providing a simple, rapid method for the classification and identification of clinical isolates such as pathogenic L. interrogans in place of the complicated and time-consuming conventional methods.     

应用分子杂交对钩端螺旋体DNA同源性的研究

Homology of leptospires from different genus, different serogroups were studied with molecular hybridization. Leptospiral DNAs were extracted and purified with phenolchloroform-isoamylalcohol method. Alpha 32P-dCTP was used to label DNA from L. interrogans serogroup icterohaemorrhagiae serovar lai strain 017 as a DNA probe, and hybridized with DNAs of 2 genus, 5 serogroups of leptospires represented by 6 strains on NC filter. Four serogroups of pathogenic leptospires which caused endemic disease in Sichuan Province were also detected by the probe. The results showed that L. interrogans serogroup icterohaemorrhagiae, serogroup autumnalis and serogroup hebdomadis had a high degree of homology while there was a low degree of homology between L. interrogans and L. biflexa and Leptonema illini. Four major serogroups of pathogenic leptospires in Sichuan Province, with their high degree of homology, could be detected by a radiolabelled probe from serogroup icterohaemorrhagiae. Hybridization may be used as a tool for diagnosis of leptospirosis in human beings and animals.     

Genotypic Characterization of Methicillin-resistant Staphylococcus aureus Isolated from Pigs and Retail Foods in China

Objective To investigate the genotypic diversity of Methicillin-resistant Staphylococcus aureus (MRSA) isolated from pigs and retail foods from different geographical areas in China and further to study the routes and rates of transmission of this pathogen from animals to food. Methods Seventy-one MRSA isolates were obtained from pigs and retail foods and then characterized by multi-locus sequencing typing (MLST), spa typing, multiple-locus variable number of tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. Results All isolated MRSA exhibited multi-drug resistance (MDR). Greater diversity was found in food-associated MRSA (7 STs, 8 spa types, and 10 MLVA patterns) compared to pig-associated MRSA (3 STs, 1 spa type, and 6 MLVA patterns). PFGE patterns were more diverse for pig-associated MRSA than those of food-associated isolates (40 vs. 11 pulse types). Among the pig-associated isolates, CC9-ST9-t899-MC2236 was the most prevalent clone (96.4%), and CC9-ST9-t437-MC621 (20.0%) was the predominant clone among the food-associated isolates. The CC9-ST9 isolates showed significantly higher antimicrobial resistance than other clones. Interestingly, CC398-ST398-t034 clone was identified from both pig- and food-associated isolates. Of note, some community- and hospital-associated MRSA strains (t030, t172, t1244, and t4549) were also identified as food-associated isolates. Conclusion CC9-ST9-t899-MC2236-MDR was the most predominant clone in pigs, but significant genetic diversity was observed in food-associated MRSA. Our results demonstrate the great need for improved surveillance of MRSA in livestock and food and effective prevention strategies to limit MDR-MRSA infections in China.     

江苏省张家港市2017年人源沙门菌血清型特征及分子分型

目的 分析2017年张家港检出的92株人源沙门菌的血清学特征,用脉冲场凝胶电泳（PFGE）技术对优势血清群进行分子分型研究,为该地区沙门菌菌型库积累数据。方法 收集和提取2017年张家港地区人源沙门菌患者血清学特征,对优势群B群沙门菌进行PFGE分子分型检测,利用PulseNet China网络获得相关性指纹图谱并进行聚类分析。结果 92株沙门菌分离株分布7个血清群和16个血清型,以B群为主（33.70％）,30株B群沙门菌PFGE指纹图谱可分为26个基因型（相似值100% 为同一PFGE 型别）,19个克隆系（相似度在0.85~1.00的为同一PFGE的克隆系）。其中9株德尔卑沙门菌可分为5个克隆系7个基因型,7个基因型相似度60.92％~100％;7株阿贡那沙门菌PFGE指纹图谱5个克隆系6个基因型,6个基因型相似度0~100%;6株鼠伤寒沙门菌可分为3个克隆系5个基因型,5个基因型相似度48.51％~100％。结论 张家港市人源沙门菌血清型多样化分布,B群沙门菌为优势血清群。沙门菌PFGE带型较多,基因多态性较高,同一血清型可获得不同PFGE指纹图谱。其中德尔卑沙门菌亲缘关系近,鼠伤寒部分菌株存在较近的亲缘关系,阿贡那沙门菌各克隆系存在明显差异,亲缘关系较远。     

布鲁氏菌分型研究进展

布鲁氏菌是严重危害人畜健康的病原菌之一,其分型技术主要有生物分型、脂肪酸分型和基因分型等.前者是传统的分型法,得到广泛应用,后二者是近年来研究的重点.其中,基因分型法作为布鲁氏菌溯源的主要方法,目前以多位点可变数量串联重复序列分型(MLVA)受到国内外的普遍关注,其他基因分型技术如Rep-PCR分型、扩增片段长度多态性分型(AFLP)、随机扩增多态性分型、染色体DNA的酶切脉冲场凝胶电泳法(PFGE)、核酸探针及多位点序列分型(MLST)也得到应用,本文综述了布鲁氏菌分型的研究进展.     

应用分子杂交对钩端螺旋体DNA同源性的研究

作者以~(32)P标记的黄疸出血群赖型017株钩体DNA为探针,分别与2个属5个血清群的6株钩体DNA进行分子杂交。结果表明,黄疸出血群赖型017株与同群、型的56601株同源性较高,与秋季群(56606株)、七日热群(56610株)次之,与双曲钩体patoc型Patoc Ⅰ株、细螺旋体属illini细螺旋体同源性很低;~(32)P标记的钩体DNA探针能检测四川地区流行的致病性钩体。     

问号钩体（狭义）种特异探针重组质粒pCX7插入片段核苷酸序 …

为了建立敏感特异的鉴别致病性钩体菌侏的方法，用ＡＢＩ自动测序仪对问号钩体（Ｌ．ｉｎｔｅｒｒｏｇａｎｓｓｅｎｓｕｓｔｒｉｃｔｏ）种异探针ＤＮＡ重组质粒ＰＣＸ７，１．７ｋｂＰｓｔＩ部分插入片段进行了核苷酸序列测定。结果：该部分插入片段ｂｐ总数为１－５０１ｂｐ，其中可读框１个，序列为８７－３９３，共３０６ｂｐ。     

海南省首例B群流行性脑脊髓膜炎病例病原学分析

目的对海南省海口市2015年2月发现的1例新生儿B群流行性脑脊髓膜炎(流脑)病例进行病原学分析。方法采集新生儿血液接种于血培养瓶,对培养出的菌株采用生化鉴定、血清学分型和普通聚合酶链反应(polymerasechain reaction,PCR)技术鉴定、基因分型;分离株进行多位点序列分型(multilocus sequence typing,MLST)及14种抗生素敏感性试验。结果从新生儿血液中培养出的菌株经生化鉴定、血清学分型与PCR技术鉴定、基因分型结果一致,均是B群脑膜炎奈瑟菌。该菌株MLST结果为序列型ST-7962,属无序列群分类。该菌株对青霉素、氨苄西林、头孢噻肟、头孢曲松、美罗培南、氯霉素、阿奇霉素、米诺环素、利福平敏感,对左氧氟沙星中介,对复方新诺明、磺胺、环丙沙星、萘啶酸耐药。结论海南省发现新生儿感染B群脑膜炎奈瑟菌病例,应加强病原菌监测与研究,以便采取针对性的防控措施,合理使用抗生素。