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。     

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。     

甲型副伤寒沙门菌株的脉冲电场凝胶电泳分型研究

目的探讨台州地区甲型副伤寒沙门菌株的流行病学特征及分型情况。方法用脉冲电场凝胶电泳(PFGE)方法进行分型。结果根据细菌染色体DNA的XbaⅠ酶切图谱,可将台州地区患者中分离的65株甲型副伤寒沙门菌分成9个PFGE型别(X1～X9)。来自温岭菌株有6个PFGE型,三门菌株有4个PFGE型,玉环菌株仅有2个PFGE型,其中X2为优势菌型。结论分型研究表明,甲型副伤寒沙门菌核型上虽在台州地区存在着县区性差异,但均以X2为主要流行菌型,从而提示了同一个克隆群内的菌株广泛传播可能是造成台州地区甲型副伤寒流行的主要原因。     

多位点序列分型技术在沙门菌鉴定中的应用研究

目的:探讨多位点序列分型技术(Multilocus sequence typing,MLST)在沙门菌鉴定中的应用。方法:对1株分离自腹泻病人的沙门菌疑似菌株用肠杆菌科鉴定试条API 20E进行生化鉴定并进行血清学鉴定,应用MLST分型方法对该菌株分子分型。结果:API 20E鉴定为沙门菌属,Vi因子血清不凝集,O因子血清A-F O多价不凝集。该菌的MLST型别为ST64型,提示为鸭沙门菌。结论:MLST分子分型技术有助于沙门菌的鉴别。     

甲型副伤寒沙门菌株的脉冲电场凝胶电泳分型研究

目的探讨台州地区甲型副伤寒沙门菌株的流行病学特征及分型情况。方法用脉冲电场凝胶电泳(PFGE)方法进行分型。结果根据细菌染色体DNA的XbaⅠ酶切图谱，可将台州地区患者中分离的65株甲型副伤寒沙门菌分成9个PFGE型别(X1～X9)。来自温岭菌株有6个PFGE型，三门菌株有4个PFGE型，玉环菌株仅有2个PFGE型，其中X2为优势菌型。结论分型研究表明，甲型副伤寒沙门菌核型上虽在台州地区存在着县区性差异，但均以X2为主要流行菌型，从而提示了同一个克隆群内的菌株广泛传播可能是造成台州地区甲型副伤寒流行的主要原因。     

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。     

2008年-2010年上海市部分伤寒副伤寒沙门菌耐药分析及分子分型

目的:了解上海市2008年-2010年伤寒、副伤寒沙门菌的耐药及分子分型特点,初步掌握伤寒、副伤寒沙门菌的分子流行特点,为今后的防制工作提供科学依据。方法:采用WHO推荐的改良K-B纸片法,对18株伤寒、副伤寒沙门菌进行13种抗生素敏感性试验;运用脉冲场凝胶电泳分型(PFGE)方法对18株伤寒、副伤寒沙门菌进行分子分型及聚类分析。结果:18株伤寒副伤寒沙门菌对利福平100%耐药,对奈啶酸的耐药率为44.4%,其中伤寒沙门菌对奈啶酸的耐药率为37.5%,5株甲型副伤寒沙门菌对奈啶酸100%耐药,乙型副伤寒对奈啶酸无耐药株。所有菌株对其他11种抗生素均敏感;18株伤寒、副伤寒沙门菌共产生16种PGFE带型,有3株甲型副伤寒沙门菌表现为同一PFGE型别。结论:2008年-2010年伤寒、副伤寒沙门菌对抗生素的敏感性较高;甲型副伤寒沙门菌之间有较高的同源性,伤寒沙门菌的PFGE带型比较分散。     

伤寒与甲型副伤寒沙门菌基因分型与耐药性研究

目的:通过追溯菌株的流行优势型,了解菌株的耐药性,分析流行因素,为查明宁波地区伤寒、副伤寒疫情回升原因提供科学依据。方法:水产品检测采用GB、PCR和mini VIDAS联合检测方法;病人、从业人员检测采用全国临床操作规程或GB方法;菌株鉴定采用VITEK、ATP方法;药物敏感试验采用K-B纸片扩散法;基因分型采用PFGE方法。结果:从11类1500份海产品中检出6株沙门菌,检出率为0.40%,其中甲型副伤寒沙门菌2株,检出率为0.13%,肠炎等沙门菌3株,检出率为0.20%,伤寒沙门菌1株,检出率为0.07%;住院病人及暴发疫情标本中分离到沙门菌593株,其中伤寒沙门菌25株,占4.22%,乙型副伤寒沙门菌1株,占0.17%,甲型副伤寒沙门菌567株,占95.62%;食品、公共场所从业人员检出甲型副伤寒沙门菌2株;病人胆汁中分离到甲型副伤寒沙门菌1株。182株甲型副伤寒沙门菌和3株伤寒沙门菌药敏试验显示,大多数菌株对常用多种抗生素均敏感,其中1株分离于水产品伤寒沙门菌出现多重耐药。14株伤寒沙门菌可分为3个PFGE型,364株甲型副伤寒沙门菌可分为12个PFGE型。结论:宁波地区伤寒、副伤寒疫情回升与食源性有关,同源性试验提示菌株来是同一克隆系的可能性较大,优势流行型为PFGE 2型,且发现宁波地区副伤寒疫情的流行强度与此型细菌相关。检出的伤寒、副伤寒沙门菌对氧哌嗪青霉素、妥布霉素及头孢哌酮等第3代头孢菌素药物显示良好的敏感性,可作为当前预防和治疗的首选药物。伤寒出现多重耐药株,应引起我们的关注。PFGE分型有助于病原的追溯,分析菌株的变迁,为流行病学进一步分析病因提供支持,在确定传播途径等方面具有重要意义。     

中国四省甲型副伤寒分离株脉冲场凝胶电泳分析及分型数据库建立

目的对中国4个甲型副伤寒流行省份的分离株进行标准化脉冲场凝胶电泳（PFGE）分型分析。方法对来自4个省的甲型副伤寒沙门菌利用XbaⅠ酶切染色体DNA进行PFGE分析，利用BioNumerics建立数据库和进行相似性聚类。结果分离自1998-2002年的89株甲型副伤寒分离株共有11种PFGE带型，这些菌株中具有优势带型。有3种带型相似性在96．3％，这3种带型的菌株占到分析菌株的865％，并在不同省份和年度中出现。结论建立了中国甲型副伤寒沙门菌的用于网络监测分析的PFGE技术和数据库，近年中国部分省份的甲型副伤寒沙门菌分离株PFGE型别集中，存在同型菌株的广泛流行。     

浙江省伤寒副伤寒沙门菌耐药及分子分型

目的了解浙江省伤寒副伤寒沙门菌耐药及分子分型特点,为伤寒副伤寒防制工作提供依据。方法采用WHO推荐的改良K-B纸片法,对209株甲型副伤寒沙门菌和5株伤寒沙门菌进行14种抗生素敏感性试验;运用脉冲场凝胶电泳分型(PFGE)方法对119株甲型副伤寒沙门菌进行分子分型及流行病学特征分析。结果209株甲型副伤寒沙门菌对红霉素、萘啶酸和利福平100%耐药,对阿米卡星100%敏感,对环丙沙星的敏感率仅为58%;5株伤寒沙门菌对红霉素和利福平100%耐药,对头孢噻吩、头孢噻肟、庆大霉素、环丙沙星、氨苄西林、阿米卡星和阿莫西林/克拉维酸100%敏感;不同地区间的甲型副伤寒沙门菌对大部分药物敏感率一致,只有多西环素存在较大差异;119株甲型副伤寒沙门菌共产生20种PFGE带型,并主要集中在2种同源性较高的型别。结论2008年浙江省伤寒副伤寒以甲型副伤寒菌株为优势株,不同地区甲型副伤寒沙门菌对大部分抗生素敏感率一致;高发地区(台州)的菌株PFGE带型比较分散,可能与菌株变异有关。     

Molecular characteristics of Salmonella enterica Paratyphi A in Yunnan Province,southwest China

Previously, the prevalence of Salmonella enterica Paratyphi A in Yunnan was high; and recently Yunnan was the predominant endemic province in China. To identify the molecular epidemiology, antibiotic resistance profile and genotypic diversity of the S. Paratyphi A isolates from 1995 to 2013 in Yunnan, we performed the study. Antibiotic susceptibility tests, pulse-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to identify the characteristics of the bacterial isolates. The results showed from 1995 to 2013, 366 S. Paratyphi A were isolated: 295 isolates (80.6%) from Yuxi and 68 isolates (18.58%) from Honghe. All of the strains were resistant to nalidixic acid, and some were resistant to ampicillin and trimethoprim/sulfamethoxazole in different years. All the isolates were sensitive to cefotaxime and ciprofloxacin. Identical PFGE with two enzyme digestion patterns were found for 339 isolates. Some environmental isolates in different years were homologous with the strains isolated from food and patients. MLST showed 349 strains were ST85, only 17 isolates were ST129. S. Paratyphi A isolates from Yunnan showed a high similarity, and we found the pathogen isolated from patients, the environment and food had the close epidemiological relationship, forming a transmission circulation. These findings have important implications for paratyphoid-control strategies.     

2008 - 2014年北京市山夫登堡沙门菌耐药性及分子流行病学特征分析

目的 分析北京市2008 - 2014年山夫登堡沙门菌临床分离株的分子流行病学特征。方法 对从腹泻病例分离到的91株山夫登堡沙门菌进行药敏试验、脉冲场凝胶电泳（PFGE）分子分型及多位点序列分型（MLST）。结果 2011 -2012年山夫登堡沙门菌在北京地区高水平流行,占所有分离株的87.9%,2008 - 2010年,2013 - 2104年各年份高度散发,病例主要集中在20～59岁成人组,5 - 6月为发病高峰;所有菌株对萘啶酸有较高耐药性（76.9%）,对磺胺异噁唑（53.9%）和链霉素（49.5%）中度耐药,对其他抗菌药物均较敏感;PFGE分型将91株腹泻株分成23种型别,形成三大克隆群,引起2011 - 2012年暴发流行的64株菌形成绝对优势克隆株;42株菌的MLST分型结果显示包括暴发流行在内的绝大多数菌株属于常见型ST14,5株散发菌株为新型ST1923。结论 北京市山夫登堡沙门菌具有明显的克隆株优势带型,引起2011 - 2012年的本地流行,对抗生素敏感性较高。     

肠炎沙门菌多位点序列分型技术的研究

目的研究肠炎沙门菌菌株间的分子特征,建立了分子流行病学研究方法——多位点基因序列分型技术(MLST),并对食品中的肠炎沙门菌分离株进行分型研究。方法选择肠炎沙门菌的6个管家基因thrA、purE、sucA、aroC、hemD、dnaN及一个特异性的DNA标记SdfΙ作为目标基因。对上述基因分别进行PCR扩增及产物测序,用sequencer2.0软件对测序结果进行分析、比对核苷酸的差异。同时,将肠炎沙门菌株与GenBank中鼠伤寒沙门菌LT2相应的基因序列进行比较。结果在肠炎沙门菌标准菌株50041和18株食品分离株之间,6个基因PCR产物范围内的近60000个核苷酸序列完全相同,未观察到同一血清型内的基因突变。而与LT2相比,基因产物发生了1到6个点突变。在对SdfΙ的核苷酸序列比较研究中发现,肠炎沙门菌标准菌株与食品中分离株SdfΙ的核苷酸序列与GenBank中该序列的碱基对比,发生了C182T的点突变。即MLST技术揭示了在同一血清型内各菌株管家基因碱基序列的高度保守。结论MLST方法适用于不同血清型沙门菌株间的分型研究,而不适于同一血清型的菌株分型。     

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.     

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.     

Molecular epidemiological investigation of a diffuse outbreak caused by Salmonella enterica serotype Montevideo isolates in Osaka Prefecture, Japan

In Osaka Prefecture, Japan, three foodborne outbreaks were caused by Salmonella enterica serotype Montevideo in rapid succession between September 2007 and May 2008. Further, Salmonella Montevideo was also isolated from several sporadic diarrhea patients and asymptomatic carriers examined during approximately the identical period. To investigate the relatedness of the isolates, we performed antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) analysis, and multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) for 29 Salmonella Montevideo isolates obtained in this region between 1991 and 2008. Although antimicrobial susceptibility tests had low discriminatory power, PFGE patterns revealed 17 unique types with <90% similarity in combined analyses involving XbaI and BlnI. Moreover, we detected three VNTR loci that were useful to genotype Salmonella Montevideo isolates, with our method ultimately classifying the isolates into 11 MLVA types based on differences in repeat unit number in each examined locus. Six isolates obtained from patients of two separate foodborne disease outbreaks, one sporadic patient, and three different carriers between 2007 and 2008 had nearly identical PFGE patterns and were classified into the identical MLVA type; further, the isolates with this PFGE and MLVA pattern appeared only at that time between 1991 and 2008. These data strongly suggest that genetically identical Salmonella Montevideo strains may have caused the 2007 and 2008 outbreaks in Osaka Prefecture. Our results demonstrate that PFGE using XbaI and BlnI is useful for discriminating between Salmonella Montevideo isolates, even within a limited area, and reconfirm that continuous epidemiological surveillance for bacterial intestinal infections such as salmonellosis may be useful to not only monitor changes in the genetic diversity of isolates, but to also detect diffuse outbreaks.     

青岛地区医院内感染耐甲氧西林金黄色葡萄球菌分子分型研究

目的 探讨青岛地区医院内感染的耐甲氧西林金黄色葡萄球菌(MRSA)分子流行病学特征及脉冲场凝胶电泳(PFGE)型别与菌株表型、一般临床资料间的关系.方法 收集2003-2007年间青岛地区主要医院内感染MRSA 360株,Sma Ⅰ酶切菌株染色体DNA后,进行PFGE电泳,用Bionumericus 2.0软件对电泳图谱进行比较和聚类分析,绘制进化树.同时对患者的性别、年龄、菌株来源等进行多变量统计分析.应用纸片扩散法测定分离菌株的药物敏感谱,并与PFGE型别进行比较分析.PCR扩增不同PFGE型别MRSA代表株25株分离株的7个管家基因进行序列测定和多位点测序分型分析(MLST).结果 所有菌株经PFGE电泳后共分为5型(M0～M4型),其中M1型为优势菌型,M2型次之,M4型相对少见,M0为独特型,明显不同于其他已知PFGE型别.统计学分析发现5种PFGE型别在患者性别、年龄分布上的差异无统计学意义,但在菌株分离部位、来源有统计学的差异:M2型多分离自伤口感染,而M3型菌株多来自ICU病房,5种PFGE型在不同医院间及医院内的分布存在差异.M1与M2两型构成各医院分离菌株的主要型别.抗生素敏感性测定中未发现万古霉素耐药菌株,亦未发现某种PFGE型别与某种特定抗生紊抗性之间的直接相关性.MIST分型发现优势型M1与M3共属于国内常见ST239型,M2型则归类于ST5,M4型属于ST240,独特型中的2种PFGE谱型则分属于ST45及ST398.结论 ST239菌株为青岛地区医院内感染MRSA优势菌株;医院内MRSA的PFGE分型与菌株来源明显相关,与患者年龄、性别无关,MRSA感染普遍存在于各年龄人群中.