嗜肺军团菌血清I型菌株随机扩增多态性DNA指纹图谱研究

目的采用随机扩增多态性DNA(RAPD)方法研究我国58株嗜肺军团菌血清1型(Lp1)菌株的基因型特征。方法58株嗜肺军团菌血清1型菌株来自北京和深圳市的集中空调冷却塔水和温泉水,随机引物采用5’-CGGCGGCG-GCGG-3’序列,分析Lp1型军团菌RAPD基因型和温度、pH值的关系。结果58株Lp1型菌株共分为17个RAPD基因型。与集中空调冷却塔水Lp1型菌株相比,温泉水分离的菌株基因型具有较高的多态性。深圳市29株集中空调冷却塔水分离的Lp1菌株,共呈现出3个RAPD基因型,北京市24株来自温泉水的Lp1菌株分为12个RAPD型,5株来自集中空调冷却塔水的Lp1型菌株分为两个RAPD型。温泉水Lp1菌株的基因型差异和水温度和pH值无明显的关联。结论RAPD分型方法可用于我国Lp1菌株的基因分型,不同地区和来源的Lp1型军团菌菌株具有特征性的RAPD基因型,温泉水中分离的Lp1菌株较集中空调冷却塔水分离菌株基因型呈现较高的基因多态性。     

温泉水中嗜肺军团菌污染调查及分子分型研究

目的 探讨温泉水环境中嗜肺军团菌的污染状况和分布规律,了解其主要血清型别及基因型别。方法 采取随机抽样方法采集温泉场所蓄水池、温泉池、客房淋浴水等,浓缩后用细菌培养法分离得到可疑菌株,用血清学与荧光定量 PCR技术进行嗜肺军团菌的鉴定。对分离的菌株用脉冲场凝胶电泳(PFGE)和多位点序列分型方法(SBT)进行基因分型。结果 65份温泉水中35份检出嗜肺军团菌,检出率为53.85%。嗜肺军团菌计数范围在20 CFU/100 mL-16 000 CFU/100 mL之间。血清分型以LP1、LP3为主。分离的嗜肺军团菌菌株均携带dot基因。对40株嗜肺军团菌菌株进行了PFGE聚类分析,得到10种不同的带型。40株菌株13种ST型可以被分为3个克隆群和1个单态群。结论 本地区温泉水嗜肺军团菌污染程度较高,分子分型结果表明这些菌株具有遗传多样性。     

嗜肺军团菌血清1型基因检测及分子分型研究

目的了解南昌市宾馆业中央空调冷却塔水中分离的嗜肺军团菌菌株的基因特征。方法 6株分离嗜肺军团菌菌株分别经血清学凝集试验、胶乳凝集试验确定为Lp1型嗜肺军团菌后,利用PCR技术对军团菌属5SrRNA基因和嗜肺军团菌mip毒力基因的特异序列进行扩增,应用多位点测序(SBT)分析,获得相应的SBT型别。使用脉冲场凝胶电泳(PFGE)分型技术,对菌株进行全染色体DNA酶切电泳分型,并用BioNumerics数据库软件进行分型分析。结果所有的菌株均带有属特异性5SrRNA基因及mip毒力基因,SBT分型6株菌均为ST1型,PFGE聚类分析结果显示菌株带型之间有差异,6株菌可分为4种带型。结论南昌市公共场所冷却塔水中分离的Lp1型嗜肺军团菌菌株基因呈多样性。     

公共场所冷却塔水嗜肺军团菌的基因序列分型研究

目的 为了解温州市中央空调冷却塔水中嗜肺军团菌(Legionella pneumophila,LP)基因特征,采用序列分型方法(sequenced-based typing,SBT)对公共场所中央空调冷却塔水中的嗜肺军团菌进行分子分型研究。方法 选择嗜肺军团菌的7 个管家基因 flaA、asd、mip、pilE、mompS、proA 和 neuA 作为目的基因, 对温州市公共场所中央空调冷却塔水中分离的31株嗜肺军团菌进行PCR扩增并测序。测序结果上传欧盟军团菌感染工作组(EWGLI)数据库进行比对、分型。运用DNAsp 5.0 、Bionumerics5.1及SplitsTree 等软件对分型结果进行分析。结果 31株嗜肺军团菌中,11株LP1型被分为2种ST型;17株非LP1型被分为8种ST型,其中7种ST型是新发现的序列型,并新发现1个等位基因;另3株非LP1型因缺少neuA基因未被数据库确认 。7个管家基因的核苷酸多态性 (Pi)范围为0.00995(mip)～0.02311(flaA)。 flaA具有最大的核苷酸多态性 ( 0.02311 )。经聚类分析得到本地分离的嗜肺军团菌株的遗传进化关系。结论 温州市公共场所冷却塔水中的嗜肺军团菌与国内外其他地区报道的嗜肺军团菌具有一定的亲缘关系,同时也具有地区特异性和遗传多样性。     

四川省部分地区温泉水嗜肺军团菌污染状况及基因分型

目的了解四川省部分地区温泉水中嗜肺军团菌污染状况,分析嗜肺军团菌SBT基因型别。方法随机采集省内部分温泉水进行嗜肺军团菌分离、鉴定和计数,对分离到的菌株进行血清分型、毒力基因(lvh、rtxA)检测、SBT基因分型。结果共采集水样27份,检出13株嗜肺军团菌,检出率为48.15%;分离到的嗜肺军团菌血清型包括Lp1、Lp3、Lp6、Lp9(或14)、Lp11和Lp13,以Lp1为主(占46.15%,6/13)。13株菌中,lvh、rtxA基因全部为阳性;13株嗜肺军团菌分为8种SBT型别,其中发现4种SBT新型。结论四川部分地区温泉水嗜肺军团菌污染比较严重,菌株均具有毒力,致病力强;温泉水中嗜肺军团菌SBT基因分型较分散,发现4个新的SBT型别。     

南昌市宾馆空调冷却塔水军团菌污染状况调查

目的了解南昌市宾馆空调冷却塔水军团菌污染状况。方法按分层随机抽样的方法,抽取南昌市13家宾馆中央空调26个冷却塔48份水样品进行细菌培养鉴定和聚合酶链式反应(PCR)检测。结果所调查宾馆中7家空调冷却塔有军团菌污染,污染率53.8%,48份水样分离到14株嗜肺军团菌,阳性检出率29.3%,其中12株为嗜肺军团菌Lp1型,2株为嗜肺军团菌Lp2型。结论南昌市宾馆中央空调冷却塔存在军团菌的污染,需加强对军团菌的监测,防止军团菌病发生。     

5株嗜肺军团菌的初步鉴定

本文检查了５株从国内分离的军团菌菌株。从培养特性、革兰氏染色镜检、生化反应、凝集试验、直接荧光抗体试验初步鉴定出３株为嗜肺军团菌血清１型，另２株为嗜肺军团菌血清５型。最后，通过扩增军团菌８７ｏｂｐＤＮＡ和１６ＳｒＲＮＡ基因的方法进一步证实这５株菌均为嗜肺军团菌种。     

浙江省首次从室外中央空调冷却塔水中分离到嗜肺军团菌

采集浙江省9家医院、6家超市中央空调冷却塔的15份水样中有8份检出嗜肺军团菌,阳性率高达5333%。玻片凝集试验结果证实,4份医院水样、1份超市水样存在两种不同血清型的嗜肺军团菌,13株嗜肺军团菌中Lp1型8株、Lp6型4株、Lp7型1株。上述菌株PCR扩增结果均为阳性。     

美国《Emerging Infectious Diseases》2019年第11期有关人兽共患病论文摘译

P20132016年美国密歇根州弗林特市自来水和临床菌株中嗜肺军团菌的全基因组序列的比较//Emily Garner,Connor,L.Brown,David Otto Schwake等2014―2015年美国密歇根州弗林特市用水安全发生危机期间,在杰纳西郡暴发了2起嗜肺军团菌病.在第二次暴发期间,收集了10份来自临床的嗜肺军团菌分离株,及第二年收集的103份源于自来水的嗜肺军团菌分离株,提交给杰纳西郡实验室进行全基因组的序列比对.记录了来自临床和自来水分离出的嗜肺军团菌株的遗传多样性.分离株来自1个分支(3个临床分离株,3个来自弗林特医院自来水的分离株,1个来自弗林特住宅的自来水分离株,及参考的巴黎菌株)具有高度的相似性(2-1062个单核苷酸多态性),所有嗜肺军团菌均为序列1型,血清组1型.血清组6型分离株属于2518型序列,在弗林特医院的水样中广泛分布,但与临床分离株无相似之处.弗林特自来水中的嗜肺军团菌株在暴发后具有多样性,与某些致病菌株相似.     

广东省布鲁氏菌菌株脂肪酸成分分析

目的探讨脂肪酸成分分析方法对广东布鲁氏菌菌株进行分型的可能性,获得广东布鲁氏菌脂肪酸成分的本底资料。方法选择历年从广东不同地区分离到的29株布鲁氏菌进行了菌体脂肪酸成分分析,利用MIDI公司Sherlock系统的软件进行聚类分析。结果广东布鲁氏菌的主要脂肪酸成分为19：0cycloω8c酸、16：0酸、18：0酸,其中牛种菌的19：0cycloω8c酸含量最高,羊种次之,猪种最低,其中羊种和猪种布鲁氏菌19：0cycloω08c酸、18：0酸含量差异有统计学意义,1965年和近3年的羊种布鲁氏菌株19：0cycloω8c酸、18：0酸含量差异有统计学意义。结论菌株的脂肪酸成分稳定,但各种间脂肪酸含量存在差异,一定的欧氏距离时,可以在种的水平上区分布鲁氏菌的3个种。     

上海口岸管道水中军团菌污染状况及其细胞内生长能力研究

目的 了解口岸公共场所管道水军团菌污染状况和细菌在细胞内的生长能力。方法 采集上海市口岸管道水132份,采用GVPC(军团菌选择性培养基)培养计数法对水样中军团菌进行定性和定量分析。用小鼠巨噬细胞J774细胞对分离菌株进行菌株致病性研究。结果 平板培养法检测管道水中军团菌阳性率为18.94%, 25份培养阳性标本中军团菌定量数值为50～1 320菌落形成单位(CFU)/L。分离的25株军团菌中17株是嗜肺军团菌;其中血清1型2株、8型13株、7型1株、5型1株;其余8株为米克戴德军团菌。细胞内生长实验结果显示, 4个血清型的嗜肺军团菌均具有很高的细胞内生长能力,而米克戴德军团菌细胞内生长能力较低。结论 管道水中军团菌污染率和含菌量较高,且菌株具有很高的细胞内生长能力,提示其具有一定致病性,有引起军团菌病的危险,应采取相应消毒和预防措施。     

Presence of colonization factor antigens on fresh isolates of fecal Escherichia coli: a prospective study

In Dhaka, Bangladesh, fresh isolates of Escherichia coli from 197 patients with diarrhea were investigated for production of enterotoxin and possession of colonization factor antigen (CFA) I or II. Enterotoxigenic E. coli (ETEC) was isolated from 34% of the patients, and of the 67 enterotoxin-positive strains, 75% carried CFAs. Among 68 healthy control persons no strains positive for both enterotoxin and CFA were found. The CFAs in general were restricted to certain serotypes of E. coli. In a subgroup of patients, part of an ongoing surveillance study, mixed infection was seen in 23% of those from whom recognized pathogens were identified. There was a tendency to more severe dehydration when the two virulence factors, enterotoxin and CFA, were simultaneously present.     

mip基因聚合酶链反应结合地高辛标记探针杂交鉴定嗜肺军团菌

目的对嗜肺军团菌国外参考菌株及国内分离菌株的巨噬细胞感染增强子（ｍｉｐ）基因分析，建立一种快速、敏感、特异的嗜肺军团菌基因检测鉴定方法。方法利用嗜肺军团菌种特异性ＤＮＡ片段ｍｉｐ基因通过聚合酶链反应（ＰＣＲ）扩增，对ＰＣＲ阳性产物进行地高辛标记的ｍｉｐ基因探针杂交，观察其反应结果。并对２６株环境和患者中分离的疑似嗜肺军团菌菌株进行分析。结果所有实验用嗜肺军团菌国外参考菌株及国内分离菌株的ｍｉｐ基因的ＰＣＲ扩增结果都为阳性，探针杂交结果也为阳性，而非嗜肺军团菌及与非军团菌对照菌株的ＰＣＲ扩增结果及探针杂交结果都为阴性，本实验其灵敏度和特异度均为１００％。另外２６株疑似嗜肺军团菌菌株有６株被鉴定为嗜肺军团菌。结论所建立嗜肺军团菌的快速初步鉴定方法具有很高的特异性和敏感性，并具有较高的应用价值     

Enterotoxins,colonization factors,serotypes and antimicrobial resistance of enterotoxigenic Escherichia coli (ETEC) strains isolated from hospitalized children with diarrhea in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is recognized as the main cause of bacterial diarrhoea among children in Asia, Africa and Latin America but less investigated in Bolivia. Objective: To determine the relation between enterotoxins, CFs and serotypes as well as the antimicrobial resistance patterns in a set of ETEC isolates collected from hospitalized children with acute diarrhea. In the present study we characterized 43 ETEC strains isolated from 2002 to 2006 from hospitalized children (0–5 years) with acute diarrhea in Bolivia. The strains were analyzed for heat-labile (LT) and heat-stable (ST) enterotoxins and colonization factor (CF) profiles, as well as for serogroups and antimicrobial resistance using phenotypic (ELISA, dot blot, slide agglutination and disc diffusion) and genotypic (Multiplex PCR) methods. Among the ETEC isolates tested, 30 were positive for LT, 3 for STh and 10 for LT/STh. Sixty-five percent (28/43) of the strains expressed one or more CF. The most common CFs were CS17 (n = 8) and CFA/I (n = 8). The phenotypical and genotypical results for toxins and CFs were congruent except for CS21 that was amplified in 10 of the strains by multiplex PCR, but CS21 pili was only detected phenotypically in four of these strains. The ETEC strains had diverse O and H antigens and the most common types were O8:H9 LT CS17 (n = 6; 14%) and O78:HNM LT-ST CFA/I (n = 4; 9%). The analysis of antibiotic resistance showed that 67% (n = 29/43) of the strains were resistant to one or several of the antimicrobial agents tested. Presence of CFs was associated with antibiotic resistance. Conclusion: The most common toxin profile was LT 70%, LT/STh 23% and STh 7%. High antimicrobial resistance to ampicillin among serogroups O6, O8 and O78 were the most common.     

2013年贵州省急性弛缓性麻痹病例中非脊髓灰质炎肠道病毒的鉴定分析

目的 研究2013年贵州省急性弛缓性麻痹(acute flaccid paralysis,AFP)病例中非脊髓灰质炎肠道病毒(non-polio enterovirus,NPEV)的基因型别,并对分离的柯萨奇病毒A4型(coxsackievirus,CVA4)进行基因特征分析。方法 2013年贵州省共报告15岁以下AFP病例235例,采用RD细胞和L20B细胞对235例AFP病例粪便标本进行病毒分离,对分离到的NPEV进行VP1基因序列测定分型。通过MEGA7.0软件采用邻位相接法(neighbor joining method)构建遗传进化树进行基因特征分析,可靠性通过1 000 bootstrap值评估。结果 235例AFP病例中共检测到24株NPEV(10.2%),有2株无法定型,另外22株共包括12个血清型,其中人类肠道病毒(human enterovirus,HEV)A组包括4个血清型6株,其中3株为CVA4,均属于C2基因亚型;HEV-B组包括7个血清型15株,1株为HEV-C组,未分离到HEV-D组病毒。结论 2013年贵州省AFP病例病原谱包括多种型别的NPEV,其中CVA4基因特征分析表明2013年贵州地区流行的CVA4毒株为C2基因亚型。     

High Diversity of Human Non-Polio Enterovirus Serotypes Identified in Contaminated Water in Nigeria

Human enteroviruses (EVs) are highly prevalent in sewage and have been associated with human diseases with complications leading to severe neurological syndromes. We have used a recently developed molecular method to investigate the presence of EVs in eight samples collected in 2017–2018 from water streams contaminated by drainage channels in three different locations in Nigeria. A total of 93 human EV strains belonging to 45 different serotypes were identified, far exceeding the number of strains and serotypes found in similar samples in previous studies. Next generation sequencing analysis retrieved whole-capsid genomic nucleotide sequences of EV strains belonging to all four A, B, C, and D species. Our results further demonstrate the value of environmental surveillance for the detection of EV transmission of both serotypes commonly associated with clinical syndromes, such as EV-A71, and those that appear to circulate silently but could eventually cause outbreaks and disease. Several uncommon serotypes, rarely reported elsewhere, were detected such as EV-A119, EV-B87, EV-C116, and EV-D111. Ten EV serotypes were detected in Nigeria for the first time and two of them, CV-A12 and EV-B86, firstly described in Africa. This method can be expanded to generate whole-genome EV sequences as we show here for one EV-D111 strain. Our data revealed phylogenetic relationships of Nigerian sewage strains with EV strains reported elsewhere, mostly from African origin, and provided new insights into the whole-genome structure of emerging serotype EV-D111 and recombination events among EV-D serotypes.     

Ganglioside GM1 mimicry in Campylobacter strains from sporadic infections in the United States

To determine whether GM1-like epitopes in Campylobacter species are specific to O serotypes associated with Guillain-Barré syndrome (GBS) or whether they are frequent among random Campylobacter isolates causing enteritis, 275 random enteritis-associated isolates of Campylobacter jejuni were analyzed. To determine whether GM1-like epitopes in Campylobacter species are specific to O serotypes associated with Guillan-Barre syndrome (GBS) or whether they are frequent among random Campylobacter isolates causing enteritis, 275 enteritis-associated isolates, randomly collected in the United States, were analyzed using a cholera-toxin binding assay [corrected]. Overall, 26.2% of the isolates were positive for the GM1-like epitope. Of the 36 different O serotypes in the sample, 21 (58.3%) contained no strains positive for GM1, whereas in 6 serotypes (16.7%), >50% of isolates were positive for GM1. GBS-associated serotypes were more likely to contain strains positive for GM1 than were non-GBS-associated serotypes (37.8% vs. 15.1%, P=.0116). The results suggest that humans are frequently exposed to strains exhibiting GM1-like mimicry and, while certain serotypes may be more likely to possess GM1-like epitopes, the presence of GM1-like epitopes on Campylobacter strains does not itself trigger GBS.     

中国小肠结肠炎耶尔森菌分布及分子流行病学特征

目的 通过中国1980-2019年不同地区小肠结肠炎耶尔森菌的病原学与PFGE分子型别特征分析,为小肠结肠炎耶尔森菌病的防控提供依据。方法 对中国不同区域,不同来源标本分离到的6 847株小肠结肠炎耶尔森菌,进行血清型别、生化表型、毒力基因分布研究,对致病性菌株进行脉冲场凝胶电泳(PFGE)分析。结果 小肠结肠炎耶尔森菌中致病性菌株2 047株、非致病菌4 800株。致病性菌株仅存在两种血清型,O∶3占比88.96%(1 821/2 047)、O∶9占比11.04%(226/2 047)。18个省市自治区的致病株以O∶3血清型为主,宁夏回族自治区以O∶9血清型为主。非致病菌血清型众多,普遍比致病株具有更多的阳性生化反应。1 821株O∶3致病株分为93个PFGE型别,分布于19个省市自治区,其中K6GN11C30021、K6GN11C30012为优势带型(分别占比46.35%、22.5%),75.38%的人源菌株与分离自当地的动物、食品菌株带型一致;226株O∶9致病株共分为14个PFGE型别,分布于8个省市自治区,77.78%人源菌株与分离自当地的动物、食品菌株带型一致。结论 我国小肠结肠炎耶尔森菌分布广泛、宿主众多,致病株仅有两个血清型,非致病株血清型众多、可能比致病株具有更强的生化代谢能力与环境适应力。致病性菌株PFGE优势带型相对集中,地域分布广泛,多数人源菌株与分离自当地的动物、食品菌株带型一致,应警惕通过家畜家禽、食品及外环境等多种来源感染人的风险。     

Detection and serotyping of human adenoviruses from patients with influenza-like illness in mongolia

Human adenoviruses (HAdVs) are responsible for approximately 5%-10% of acute respiratory infections. The serotypes of commonly detected respiratory HAdV in Asian countries are diverse. However, there are no well-documented reports of circulating HAdV serotypes in Mongolia. Between January 2010 and May 2011, 1,950 influenza-negative samples from patients with influenza-like illness, including eye swabs from patients with eye symptoms, were screened for HAdV, and 40 samples (2.1%) were positive for HAdVs. Among these 40 samples, 31 samples were positive for the hexon gene used in phylogenetic analysis, as determined by PCR. We identified 7 different serotypes. We constructed the phylogenetic trees of HAdV-B7 and HAdV-B3, the 2 most commonly detected serotypes in this study. All detected HAdV-B7 and -B3 Mongolian strains had identical sequences. HAdV-D8, known to be associated with epidemic keratoconjunctivitis (EKC), was detected from nasopharyngeal and eye swabs. There was no difference between the amino acid sequences of the hexon and fiber genes that may affect tissue tropism in Mongolian strains and those in EKC-causing strains.