多位点序列分型和脉冲场凝胶电泳在肠炎沙门菌分子分型的比较

目的 比较多位点序列分型技术和脉冲场凝胶电泳（PFGE）技术在肠炎沙门菌菌株间分型的分辨率。方法 分别建立肠炎沙门菌的6个管家基因thrA、pure、sucA、aroC、hemD、dnaN和一个特异性的DNA标记Sdf Ⅰ的多位点序列分型技术，及以寡切点的XbaⅠ、SpeⅠ作为限制性内切酶的PFGE方法，并应用上述方法对食品中的分离株进行分型，比较两种方法的分辨率。结果 PFGE可以将50株肠炎沙门菌分为11个型。并且通过双酶系统的双重PFGE分型，还可以将PFGE型别再精细划分为亚型；MLST则揭示了在同一血清型内部，各菌株之间的管家基因碱基序列高度保守，而在沙门菌不同血清型的核苷酸序列之间，则分别存在不同数量的碱基差异。即MLST方法只能用于血清型之间，不能在血清型内部进行分型研究。结论 与MLST比较，PFGE方法在肠炎沙门菌的分型方面显示了比较高的分辨率。     

多位点基因序列分型在沙门菌鉴定中的应用

目的探讨多位点基因序列分型方法在沙门菌分型鉴定研究中的应用,初步掌握广州地区沙门菌多位点基因序列分型数据。方法选取沙门菌7对保守管家基因aroC、dnaN、hemD、hisD、purE、sucA、thrA作为研究靶标,对其进行扩增、测序及序列分析。结果 41株沙门菌分离株有8个血清群和14个ST型,食品来源沙门菌菌株的血清群有7个,ST型为10个,患者来源菌株血清群为5个,ST型为10个,实验中还发现2个新的ST型。结论沙门菌血清学分群结果与MLST分型结果的对应关系密切,很多ST型菌株的血清群固定,相同血清群菌株也有较固定的ST型,因而,可运用多位点基因序列分型方法对沙门菌进行分型鉴定。     

2011-2012年杭州市肠道沙门菌临床分离株的分子型别分析

目的研究2011-2012年杭州市肠道沙门菌临床分离株的型别,了解本地菌株分子流行病学特征。方法对66株肠道沙门菌临床分离株进行血清分型和多位点序列分型(MLST)。对其中主要血清型:鼠伤寒、甲型副伤寒、萨雷甲尼和肠炎沙门菌菌株进行脉冲场凝胶电泳(PFGE)分型。结果分布于21个血清型的66株沙门菌分成26个ST型别。发现一株纽波特沙门菌为新型ST1690。菌株血清型与MLST型别数据库中所对应的血清型符合率为100.00%。9株甲型副伤寒沙门菌的PFGE带型完全一致(P7型),与先前杭州流行菌株有差异(P1-P6型)。6株肠炎沙门菌分成4个PFGE型,型间最小相似性为92.70%。13株鼠伤寒沙门菌分为11个PFGE型,型间最小相似性为71.70%。7株萨雷甲尼沙门菌分成4个PFGE型别,型间最小相似性为91.00%。结论近年杭州腹泻病人中流行的肠道沙门菌菌株主要血清型为鼠伤寒、甲型副伤寒、萨雷甲尼和肠炎等。甲型副伤寒沙门菌菌株在杭州出现了新PFGE型别。MLST数据可以对沙门菌血清学鉴定提供一定的帮助。     

沙门菌鞭毛素C蛋白基因型与血清型关系的研究

目的探讨沙门菌鞭毛素C蛋白基因型与血清表型的关系. 方法从几种沙门菌临床分离株提取基因组,以此为模板通过聚合酶链反应(PCR)扩增相应的鞭毛素C基因片段,对扩增的片段进行测序和琼脂糖凝胶电泳分析. 结果从鞭毛血清型为H-1-d的细菌基因组中可以扩增出特异性PCR产物,这些细菌包括慕尼黑沙门菌(Salmonella muenchen), 伤寒沙门菌(S. typhi)和鼠伤寒沙门菌(S. typhimurium),但是从其他血清型的菌株[丙型副伤寒沙门菌,(S. paratyphoid C)和肠炎沙门菌(S. enteritidis)]基因组中则不能扩增出相应的片段. 结论本实验提示,同菌株鞭毛抗原血清型存在特异性一样,沙门菌鞭毛素C中间可变区核苷酸序列也存在特异性,这一结论可能为检测鞭毛血清型为H-1-d沙门菌提供一种快速、灵敏、准确的新方法.     

侵袭性非伤寒沙门菌血清型和耐药性分析

目的分析7株侵袭性非伤寒沙门菌(iNTS)的血清型和耐药性。方法针对收集的7株iNTS菌株,开展血清学鉴定、药物敏感性实验以及全基因组测序,并对血清型、MLST型和耐药基因进行鉴定、注释和分析。结果7株iNTS菌株中,包含1株鼠伤寒血清型和2株Ⅰ4,[5],12:i:-(ST34型)、2株肠炎血清型、1株科瓦利斯血清型和1株未知血清型Ⅰ4,[5],12:d:-(ST279型),其中6株为单相菌,沙门菌二相鞭毛基因缺失或假基因化可能有助于增强沙门菌侵袭性。未发现替加环素、氨曲南、阿米卡星及头孢类、碳青霉烯类抗生素耐药株,存在1株八重耐药鼠伤寒沙门菌。耐药基因与耐药表型基本相符。结论本研究中的iNTS菌株存在八重耐药株,整体耐药水平尚低但不可忽视。     

贵州省动物性食品中沙门菌耐药及分子特征相关性研究

目的 了解贵州省动物性食品中沙门菌的耐药和分子型别特征及二者相关性,为我省食源性沙门菌感染及防控提供科学依据。方法 对贵州省9个市(州)2017—2021年市售动物性食品中分离的146株沙门菌进行血清学分型,采用微量肉汤稀释法测定菌株对15种抗生素的耐药性,脉冲场凝胶电泳(pulsed-field gel electrophoresis, PFGE)和多位点序列分型(multi-locus sequence typing, MLST)对菌株进行分子分型。结果 146株沙门菌分出27种血清型,以鼠伤寒(31/146,21.2%)、德尔卑(26/146,17.8%)、肠炎沙门菌(16/146,11.0%)为优势血清型。146株菌耐药率为79.5%(116/146),多重耐药率达60.3%(88/146),共有72种耐药谱。对氨苄西林、四环素耐药率分别达61%、64.4%。129株菌经PFGE共分出52个聚类76种带型,相似度在53.5%～100%,146株菌经MLST共检出29种ST型,以ST40(26/146,17.8%)、ST34(17/146,11.6%)、ST11(16/146、1...     

杭州市2002-2008年甲型副伤寒沙门菌分子分型研究

目的研究2002-2008年杭州市甲型副伤寒疫情分离株的亲缘性,并探讨脉冲场凝胶电泳（PFGE）、多位点序列分型（MLST）在甲型副伤寒沙门菌分子分型中的应用。方法对404株甲型副伤寒沙门菌运用脉冲场凝胶电泳（PFGE）进行分子分型,并用K-B法测定菌株抗生素敏感性。挑选9株甲型副伤寒沙门菌进行多位点序列分型（MLST）以及稳定性试验。结果 404株甲型副伤寒沙门菌可分为6个PFGE型和4个耐药型,P1型和P2型属于同一个克隆系,该克隆系菌株占试验菌株数的99%,分离自临安市的310株菌均为P1型,分离自杭州市西湖区的主要菌型是P2型和P1型。9株甲型副伤寒沙门菌根据SucA位点的差异可分为2个MLST型。结论杭州市2002-2008年的甲型副伤寒疫情由同一克隆系的菌株主导。PFGE、MLST分型方法各有侧重,可互为补充。     

中国2000-2008年间甲型副伤寒沙门菌脉冲场凝胶电泳和多位点序列分析

目的 分析我国2000-2008年间甲型副伤寒沙门菌流行株的分子分型及病原进化上的特征.方法 应用以Spe Ⅰ为限制性内切酶的脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)方法 和基于9个管家基因位点(aroC、thrA、hisD、purE、sucA、dnaN、hemD、adk和purA)的多位点序列分析(multilocus sequence typing,MLST)分型方法 ,对我国2000-2008年间分离自10个地区的118株甲型副伤寒沙门菌流行株进行分析.结果 应用PFGE方法 将118株甲型副伤寒沙门菌分为32个型,其中包含5株以上的优势PFGE带型有5种.而应用MLST方法 ,所有菌株只分出了2个序列分型(sequence type,ST),各菌株的管家基因序列高度保守,呈现高度克隆化.结论 中国2000-2008年间甲型副伤寒沙门菌菌株应用MLST这种分型方法 很难区分,MLST不适于甲型副伤寒沙门菌的暴发调查和流行病学监测.目前中国的甲型副伤寒是由高度克隆化的菌株引起全国范围的扩散 流行.随着年份的变迁,也积累了散在的变异.     

2015—2016年淮安市售禽畜肉中沙门菌污染及其病原学特征北大核心CSCD

目的了解市售禽畜肉中沙门菌的污染情况,并研究分离菌株的病原学特征。方法 2015—2016年以随机采样方式从淮安市8个县区的超市和农贸市场采集禽畜肉样品,按照GB 4789.4—2010《食品安全国家标准食品微生物学检验沙门氏菌检验》方法检测沙门菌。对分离的沙门菌株进行血清分型,耐药特性分析,毒力基因携带情况分析以及脉冲场凝胶电泳(PFGE)分型研究。结果 368份畜禽肉中共有37份检出沙门菌,总检出率为10.05%,其中,鸡肉、鸭肉、猪肉和牛羊肉中沙门菌的检出率分别为14.14%(14/99)、11.11%(10/90)、10.34%(12/116)和1.59%(1/63)。37株沙门菌共有8种血清型,优势血清型为肠炎沙门菌和鼠伤寒沙门菌。药敏试验说明本地禽畜源沙门菌对氨苄西林和萘啶酸的耐药率最高(72.97%),对头孢西丁的耐药率最低(8.11%),不同样本来源及不同血清型沙门菌的耐药表型存在差异。肠炎沙门菌中inv H和sop E的携带率最高,鼠伤寒沙门菌中inv H、sop E、rhu M的携带率最高。PFGE分型研究发现,所有肠炎沙门菌的带型相似度均在81.56%以上,相同样本来源的肠炎菌株之间的亲缘关系更近;鼠伤寒沙门菌的带型各不相同,分离株的基因型别呈现多样性。结论淮安市售禽畜肉中沙门菌的污染率较高,优势血清型为肠炎沙门菌和鼠伤寒沙门菌。沙门菌分离株的耐药状况严重,毒力基因携带率高。     

广东省2007年度非伤寒沙门菌监测及病原学特征分析

目的 了解广东省腹泻病患者中非伤寒沙门菌感染情况和菌株的血清型别、分布、耐药性变化.方法 对纳入研究的腹泻病患者进行非伤寒沙门菌检测,对分离到的菌株进行血清分型、药物敏感性试验和脉冲场凝胶电泳(PFGE)分型.结果 2007年度共检测1128份腹泻粪便标本,分离到71株沙门菌,阳性检出率为6.29%;共分得29种血清型,肠炎和鼠伤寒沙门菌居多;大多数沙门菌对常用的头孢类和喹诺酮类抗生素敏感,鼠伤寒沙门菌的耐药率普遍较肠炎和斯坦利沙门菌高;除肠炎沙门菌外,其余的血清型没有同一PFGE型别的菌株;用Xba Ⅰ酶切17株肠炎沙门菌可分为PFGE-XbaⅠ 1～8型,其中PFGE-XbaⅠ 4型为优势型别.用Sfi Ⅰ和Not Ⅰ酶对12株肠炎沙门菌进行再分型,综合用Xba Ⅰ/Sfi Ⅰ/Not Ⅰ三种酶的结果 发现仍有三组菌的PFGE图谱是完全一致的.结论 2007年度广东省非伤寒沙门菌的感染多数为散发病例,头孢类和喹诺酮类抗生素是治疗非伤寒沙门菌感染的首选药物.     

2008-2009年北京市沙门菌流行特征和分子分型

目的 分析2008-2009年北京市沙门菌流行特征及脉冲场凝胶电泳(PFGE)分子分型. 方法 对2008-2009年通过WHO全球沙门菌监测系统及北京市肠道门诊监测系统分离到的137株沙门菌进行生化鉴定、血清分型和相关流行病学分析;利用PFGE进行分子分型. 结果 北京市2008-2009年沙门菌的流行具有明显季节性,6-9月份高发,共分离菌株84株,占64.1%(84/131);患者年龄多为18～40岁,占46.1%(58/128);男性80例,女性51例,男女比例为1.57:1.137株沙门菌分属于20种血清型,其中肠炎沙门菌和鼠伤寒沙门菌为优势菌型,分别占46.7%(64/137)和17.5%(24/137).共分71种PFGE带型,其中肠炎沙门菌和鼠伤寒沙门菌都有16种PFGE型别.肠炎沙门菌的4种PFGE型别(JEGX01.CN0001、JEGX01.CN0003、JFGX01.CN0002、JEGX01.CN0019)和鼠伤寒沙门菌的JPXX01.CN0001为优势分子型别. 结论 2008-2009年北京市沙门菌的流行具有性别、年龄和季节性分布特征;PFGE分子型别较多,且存在差异明显的多个克隆系.

Abstract：

Objective To study the epidemiological characteristics and molecular phenotypes of Salmonella by pulsed-field gel electrophoresis (PFGE) in Beijing from 2008 to 2009. Methods A total of one hundred thirty-seven isolates recovered from the WHO Global Samonella Surveillance system and entero clinic surveillance system were identified by biochemical tests and serotyping.The related epidemiological informations were also analyzed.The isolates were further typed by PFGE. Results The prevalence of Salmonella from 2008 to 2009 showed obvious seasonal character.High incidence occurred from June to September,and 64.1% (84/131) isolates were recovered in this period.Patients of 18-40 year-old were 46.1% (58/128) and 80 patients were male and 40 patients were female with the ratio of 1.57:1.These 137 Salmonella isolates belonged to 20 serotypes,including Enteritidis (46.7%,64/137) and Typhimurium (17.5%,24/137) as the dominant serotype.In total,71 PFGE profiles were identified.Four PFGE patterns of S.Enteritidis isolates (JEGX01.CN0001,JEGX01.CN0003,JEGXO1.CN0002,JEGX01.CN0019) and S.Typhimurium pattern of JPXX01.CN0001 were dominant patterns. Conclusion The prevalence of Salmonella from 2008 to 2009 showed distribution characteristics of sex,age and seasons.The numerous PFGE patterns of Salmonella showed diversity of these isolates and different clones existed in Beijing.     

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis.

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.     

Web-based surveillance and global Salmonella distribution, 2000-2002

Salmonellae are a common cause of foodborne disease worldwide. The World Health Organization (WHO) supports international foodborne disease surveillance through WHO Global Salm-Surv and other activities. WHO Global Salm-Surv members annually report the 15 most frequently isolated Salmonella serotypes to a Web-based country databank. We describe the global distribution of reported Salmonella serotypes from human and nonhuman sources from 2000 to 2002. Among human isolates, S. Enteritidis was the most common serotype, accounting for 65% of all isolates. Among nonhuman isolates, although no serotype predominated, Salmonella enterica serovar Typhimurium was reported most frequently. Several serotypes were reported from only 1 region of the world. The WHO Global Salm-Surv country databank is a valuable public health resource; it is a publicly accessible, Web-based tool that can be used by health professionals to explore hypotheses related to the sources and distribution of salmonellae worldwide.     

Recent trends in the epidemiology of non-typhoidal Salmonella in Israel, 1999-2009

The aim of the present study was to assess the recent trends in the epidemiology of non-typhoid Salmonella in Israel using a sentinel laboratory-based surveillance network. Between 1999 and 2009, 8758 Salmonella stool isolates were reported by five sentinel laboratories. There was a significant decrease in the incidence rate of Salmonella isolates from 70·5/100,000 in 1999 to 21·6/100,000 in 2005 followed by a slight increase to 30·3/100,000 in 2009. Of all Salmonella, 64·3% were isolated from children in the 0-4 years age group. Up to 2008, S. Enteritidis was the most prevalent serotype and in 2009 S. Infantis emerged as the most common Salmonella serotype. The decrease in the incidence of S. Enteritidis and S. Typhimurium and increase in S. Infantis among humans were associated with a similar trend among breeding flocks, which followed significant preventive interventions conducted against S. Enteritidis and S. Typhimurium infections in poultry. Tight surveillance and education of food handlers and consumers should be enhanced to reduce the foodborne transmission of Salmonella in Israel.     

Laboratory-based surveillance of non-typhoidal Salmonella infections in Guangdong Province, China

Salmonella is one of the most common foodborne pathogens in humans. Laboratory-based surveillance for non-typhoidal Salmonella infection was conducted in Guangdong Province, China to improve understanding about the disease burden and detection of dispersed outbreaks. Salmonella isolated from patients with diarrhea were sent from 16 sentinel hospitals to local public health laboratories for confirmation, serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). PFGE patterns were analyzed to identify clusters representing potential outbreaks. Between September 2009 and October 2010, 352 (4%) Salmonella isolates were obtained from 9167 stool specimens. Salmonella enterica serotype Typhimurium (45%) and Salmonella enterica serotype Enteritidis (13%) were the most common serotypes, and multidrug resistance was high, especially in Salmonella Typhimurium isolates. PFGE patterns of obtained Salmonella isolates were found to be diverse, but a unique PFGE pattern comprising 53 Salmonella Typhimurium isolates were found to occur almost exclusively in infants. Epidemiologic studies are ongoing to determine whether a common exposure is the source of the Salmonella Typhimurium strain frequently isolated from infants.     

A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources

PurposeTo evaluate the abilities of these subtyping methods, we distinguished Salmonella Enteritidis (S. Enteritidis) isolated from food products and human clinical samples between 2009 and 2010 in Seoul using five subtyping methods.MethodsWe determined the subtypes of 20 S. Enteritidis isolates from food and human sources using phage typing, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multilocus sequence typing (MLST).ResultsA total of 20 tested isolates were differentiated into six antimicrobial susceptibility patterns, three different phage types, four different PFGE profiles, seven rep-PCR patterns, and one MLST type. Food isolates were considerably more susceptible to antibiotics than human isolates. We were best able to discriminate among S. Enteritidis isolates using rep-PCR, and obtained the highest Simpson's diversity index of 0.82, whereas other methods produced indices that were less than 0.71. PFGE pattern appeared to be more related to antimicrobial resistance and phage types of S. Enteritidis isolates than rep-PCR. MLST revealed identical alleles in all isolates at all seven loci examined, indicating no resolution.ConclusionThe results of this study suggest that rep-PCR provided the best discriminatory power for phenotypically similar S. Enteritidis isolates of food and human origins, whereas the discriminatory ability of MLST may be problematic because of the high sequence conservation of the targeted genes.