O139群霍乱弧菌的核糖体基因分型及其与药物抗性相关性研究

目的了解杭州市及浙江省部分地区O139群霍乱弧菌分离株的核糖体基因分型特征及其与抗生素抗性关系。方法对1994~2004年杭州市分离的105株及2000年浙江省部分地区分离的5株O139群菌株,PCR法检测ctxA和tcpA基因,纸片扩散法测定抗生素抗性,应用核糖体基因分型技术进行分子分型;并与1997~2001年分离的20株O1群霍乱弧菌流行株代表菌株的分子分型结果进行比较。结果杭州市105株O139群菌株中,95株ctxA阳性菌株分为8个型(Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ和Ⅷ型),其中Ⅱ型菌株为2000年后的优势克隆群(占73.7%,70/95),并于2004年引起一起较大规模的爆发;10株ctxA阴性菌株被分为3个型(α、β和γ型)。2000年浙江绍兴市4株ctxA阳性O139群分离株属Ⅱ型,浙江湖州市1株ctxA阴性分离株属γ型。20株O1群流行代表菌株被分为5个型(A、B、C、D和E型),其中A型和B型分别与O139群中的Ⅵ型和Ⅶ型的带谱相同。ctxA阳性和ctxA阴性O139群菌株间的耐药谱显著不同。67株Ⅱ型O139群菌株中共有28种耐药谱,最少的仅对2种抗生素同时耐药,最多对11种抗生素同时耐药;分别有对11种和10种抗生素耐药的菌株在2002~2004年间成为优势菌(81.5%,22/27)。结论1994~2004年间多种核糖体基因型O139群霍乱弧菌在杭州同时或替换流行,多重耐药的核糖体基因型Ⅱ型O139群菌株于2002~2004年间成了杭州优势流行菌株。     

贵州省2000年甲型副伤寒沙门菌分离株16SrRNA基因型分析

近年来贵州省出现甲型副伤寒的流行。为探讨贵州省不同地区甲型副伤寒流行菌株的分子流行病学特征以及菌株间的相关关系 ,对贵州省 6个地 (市 ) 2 0 0 0年部分甲型副伤寒沙门菌分离株进行染色体DNA基因限制性酶切 16srRNA探针杂交图谱分析。PstⅠ酶切后进行的杂交显示 6 4株均为一个基因型 ,然后进一步采用BglⅠ、SalⅠ两个内切酶对其中部分菌株进行酶切杂交分析 ,核糖体杂交谱上仍没有差异 ,分析认为同一个克隆群的菌株广泛传播是造成贵州省甲型副伤寒流行的主要原因.     

Fingerprinting of Salmonella enterica subsp. enterica serovar Enteritidis by ribotyping

Objective: To carry out an epidemiologic evaluation of Salmonella enterica subsp. enterica serovar Enteritidis outbreaks in households and small communities by means of rRNA gene restriction pattern analysis (ribotyping).
Method: One hundred Enteritidis isolates dating from 1989 to 1994 which could be allocated epidemiologically to different sources or to small community outbreaks were investigated with ribotyping, a fingerprinting method in which bacterial DNA is hybridized with the biotin-labeled plasmid pKK 3535 containing a ribosomal RNA operon of Escherichia coli to determine the ribosomal RNA gene restriction patterns.
Results: Four different ribotyping patterns were found with the restriction endonuclease Sma I and nine with Sph I. Ribotypes of isolates which could be allocated epidemiologically to a common source usually corresponded. Almost 60% of the Enteritidis infections had the ribotyping pattern Sph I-A. In contrast, this pattern was not found in any of the five Enteritidis strains isolated in 1989. The suspicion that Enteritidis phage type 4 infections are caused by consumption of insufficiently heated eggs is supported by the fact that the ribotyping pattern Sph 1-A was found in isolates from eggs and from human specimens.
Conclusions: As patterns Sph I-A and Sma I-J appeared in 58% and 75% of the isolates, respectively, ribotyping cannot be used for the differentiation between various outbreaks with these two patterns. In cases where the Enteritidis strains showed less frequent patterns, ribotyping seems to be a practical tool for the identification of infection chains. In addition newly appearing ribotyping patterns can give information about the epidemiologic development of Enteritidis infection.     

2008年海南省霍乱暴发分离株的分析

目的 分析海南省2008年霍乱暴发菌株的分子分型成簇性,为疫情分析提供分离株的病原学特征,协助疫情判断.方法 从本次霍乱疫情患者中分离69株,外环境分离7株[其中患者家厕所1株,水样1株,患者家附近鱼(池)塘3株,患者上卫生所就诊时呕吐地面的涂抹拭子1株和海南大学食堂菜刀涂抹拭子1株]共76株,对分离株进行常规病原学检测,利用脉冲场凝胶电泳(PFGE)技术获取受试菌株的DNA分子指纹图谱并对其结果进行聚类分析,将PFGE图像应用BioNumerics(Version4.0)数据库软件(Applied Maths BVBA,Belium)进行处理,识别图像条带,自动生成得出相似系数.结果 2008年海南省暴发霍乱疫情中分离的76株霍乱菌株中,6月份分离到的5株分离菌株图谱相同,相似系数达100%;10-11月份自患者和环境水体中分离到的70株菌图谱完全一致,相似系数达100%,但与6月份分离株的图谱不同;1株在暴发中分离自食堂菜刀的菌株,其图谱与其他菌株差异较大,相似系数为79.7%.结论 海南2008年存在不同的霍乱暴发,10-11月出现在不同市县的疫情可能属于一起较大范围的流行,环境水体污染是传播的不可忽视的环节.     

14株伯克霍尔德菌的鉴定分析

目的 对从菌种保藏中心获得以及样品分离的14株伯克霍尔德菌进行鉴定分析，为该菌的鉴定和溯源分析提供参考。方法 分别用生理生化(API20NE和Biolog碳源分析系统)、基因型(自动化核糖体分型、16S rDNA和recA序列分析)的方法对11株标准菌株和3株未知菌进行了鉴定，并对结果进行了分析比较。结果 生理生化的方法能够鉴定到属的水平，Biolog能够区分BCC与非BCC类群微生物；16S rDNA序列分析能够鉴定到属水平，对于非BCC的部分菌可以鉴定到种水平，并且能够将BCC正确分类；recA序列分析能够将包含BCC的部分菌正确鉴定到种水平；Riboprinter技术能够鉴定到属水平，但是该技术能够对菌进行溯源分析。结论 在进行该菌的鉴定时，可以参考中国药典2015年版四部9204微生物鉴定技术指导原则的要求，根据自身的需求、技术能力以及能够承担的成本，选择适宜的鉴定方法和技术，依据多相鉴定原则进行鉴定分析，第一步判断是否属于伯克霍尔德菌属，第二判断是否属于BCC，第三尽可能准确鉴定到种水平，有必要的情况下进行溯源分析以确认污染来源。     

Epidemiological relationships between Aeromonas strains isolated from symptomatic children and household environments as determined by ribotyping

Ribotyping was used to study the epidemiology of Aeromonasassociated gastro-enteritis in young children. Ribotyping patterns of 29 Aeromonasstrains (16 Aeromonas caviae, 8 Aeromonas hydrophila, 3 Aeromonas eucrenophila, 1 Aeromonas veronii, and 1 Aeromonas encheleia) isolated from primary stool cultures of sick children were compared using the GelCompare software with patterns of 104 strains (39 Aeromonas eucrenophila, 29 Aeromonas caviae, 11 Aeromonas encheleia, 10 Aeromonas hydrophila, 6 Aeromonas bestiarum, 3 Aeromonas veronii, 3 Aeromonas popoffiiand 3 Aeromonas media) isolated from their household environment in order to investigate the route of transmission of these bacteria. Fifteen strains (47%) isolated from stool cultures of patients showed the same riboprofile as strains found in contacts or environment. In particular, three strains isolated from patients shared the same riboprofile with strains found in their domestic environment. The wide diffusion of potentially pathogenic Aeromonasstrains in our household samples, and the high rate of asymptomatic carriers among family members, suggested that predisposing factors of the host could make children prone to an Aeromonas-related intestinal disease.     

Comparison of serotyping, ribotyping and pulsed-field gel electrophoresis for distinguishing group A Streptococcus strains isolated in Albania

Conventional serotyping for T antigens, rRNA gene restriction fragment length polymorphism analysis (ribotyping) and pulsed-field gel electrophoresis were compared for distinguishing among group A streptococci isolated in Albania between 1980–1982 and in 1995. A total of twelve serotypes were identified among seventy GAS strains. Ribotyping revealed eight and eleven distinct patterns after digestion with HindIII and PvuII, respectively. Twenty-three strains of serotype T12 were subdivided in 10 ribotypes and 11 strains of T2 serotype were differentiated in 5 ribotypes. By comparison, PFGE generated 37 patterns after SmaI digestion. The index of discrimination, using the Hunter–Gaston formula, was applied to assess the value of these methods for interpretation of the epidemiological data. For serotyping the value of index was 0.85. The ribotyping system revealed an ID of 0.83 when the combination HindIII and PvuII was used. This index reached 0.97 for PFGE. The methods used were useful to subtype the isolates of GAS.     

阪崎肠杆菌自动化核糖体分型的研究

目的 对阪崎肠杆菌食品分离株进行核糖体分型,并分析其分型效果.方法采用杜邦Riboprinter~(TM)指纹图谱分析仪对2株阪崎肠杆菌标准菌株和28株分离株进行核糖体分型,运用BioNumerics数据库软件建立相应的核糖体指纹图谱数据库,进行指纹图谱分析.结果 核糖体分型将2株阪崎肠杆菌标准菌株和28株分离株分成26种核糖体带型,其中22种带型分别对应1株阪崎肠杆菌试验菌株,其他4种带型分别对应2株试验菌株,不同带型之间的最低相似度为31.58%.所有菌株的酶切条带数为10个左右,酶切条带分子量范围位于1～50 kb之间.同时,使用BioNumerics软件对结果进行分群和多维尺度分析,可见核糖体分型主要分为4个群.结论 自动化核糖体分型可以方便快捷的实现阪崎肠杆菌的分型.     

Molecular typing of thermotolerant species of Campylobacter with ribosomal RNA gene patterns

Ribosomal RNA gene restriction patterns (ribopatterns) of 72 strains representing Campylobacter jejuni (subspecies jejuni and doylei), C. coli, C. upsaliensis and C. lari including the urealytic (UPTC) biovar were determined using four common restriction endonucleases (HaeIII, HindIII, PstI and PvuII). The relative effectiveness of these enzymes for general molecular typing of the thermotolerant campylobacters was assessed. Ribotypes (HaeIII) were defined on the basis of computer-assisted numerical analysis. For C. jejuni, C. coli and C. lari, the HaeIII ribopatterns provided a high level of typability and discrimination, with patterns that were reproducible and easy to code for numerical analysis. There was evidence of diversity within three of the HaeIII types, and PstI ribopatterns proved the most reliable for detecting such differences. C. upsaliensis also could be ribotyped with HaeIII, but HindIII, PvuII and PstI were less satisfactory for this species. For such campylobacters, the HindIII ribopatterns generally were complex and difficult to compare, and the PvuII profiles provided the least discrimination. We conclude that the choice of restriction endonuclease is of critical importance when applying ribotyping to different species of Campylobacter. HaeIII ribopatterns were the most effective means of typing strains of different thermotolerant species of campylobacters and, when combined with PstI ribopatterns, offered a highly discriminatory basis for molecular typing.     

贵州省伤寒沙门菌分离株16s核糖体基因型的多态性及其流行病学意义

目的：探讨贵州省不同时间、不同地区伤寒流行菌株的遗传多态性及内在联系。方法：利用southern杂交技术，对贵州省9个地区26个县、市1959－1999年伤寒沙门菌分离株进行染色体DNA基因限制性酶切16s rDNA探针杂交图谱分析及药物敏感性试验。结果：分析发现选择的209株分属26个RT型，以RT1和RT2为优势型；在局部发生伤寒流行时，均由独特的RT型引起。耐药菌株以RT7和RT1型为主。结论：贵州省不同地区、不同时间的分离菌株在核糖体杂交图谱上有明显的多态性，分析认为具有多重耐药性以及存在众多克隆群菌株是引起贵州伤寒发病率居高不下的主要原因。