Pulsed Field Gel Electrophoresis Analysis of Borrelia burgdorferi Sensu Lato Isolated in Japan and Taxonomic Implications with Lyme Disease Spirochetes

Genomic DNAs of Borrelia burgdorferi sensu lato isolates obtained in Japan sharing different rRNA gene ribotypes were digested with rare-cutter restriction endonucleases and the fragments obtained were separated by pulsed field gel electrophoresis (PFGE). The sizes of large restriction cleavage bands with MluI endonuclease were quite similar among isolates in each ribotype group. On the other hand, the PFGE profiles obtained with the other enzymes (NruI, Sal I or SplI) were rather divergent, and Japanese isolates were distinguishable from the United States and European isolates. The Japanese isolates classified as ribotypes group II (Borrelia garinii) and III (B. afzelii) showed different PFGE patterns from that of European isolates. The isolates grouped into ribotype IV revealed distinctively different PFGE profiles. These results indicate that the Japanese isolates may be genetically divergent and distinct from the United States and European isolates.     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

Bacterial genome mapping by two-dimensional pulsed-field gel electrophoresis (2D-PFGE)

Macrorestriction mapping is often the first step toward a thorough physical and genetic characterization of a bacterial genome. The problem of deducing the order of partially or completely digested macrorestriction fragments to yield a physical genome map may readily be solved by applying twodimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. These powerful methods are quick and technically easy to perform; specifically, they are independent of DNA probes and should therefore be applicable to any bacterial species irrespective of its prior genetic characterization. In this article, detailed step-by-step protocols are given to set up, run, and evaluate 2D pulsed-field gels. Two basic methods are described: partial/complete 2D gels of one restriction enzyme and complete/complete 2D gels of two different restriction enzymes. Other topics include preparation of bacterial genomic DNA, screening for suitable rare-cutting restriction enzymes and determination of optimal running conditions. Accompanied by many notes, these protocols are meant to offer the novice a sound and rapid access to these important methods.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Characterization of Borrelia Species Isolated from Ixodid Ticks,Ixodes ovatus

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.     

An Arrayed Bacteriophage P1 Genomic Library of Pneumocystis carinii

We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system. The library consists of ∽4800 independent clones with an average insert size of ∽55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device. Screening of the library for unique P. carinii sequences detected an average of 4–5 positive clones for each, consistent with a several-fold coverage of the ∽10-mbp P. carinii genome. Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts. Thus, this arrayed, large insert library off. carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen.     

Current methods for molecular typing of Campylobacter species

Campylobacter remains one of the most common bacterial causes of gastroenteritis worldwide. Tracking sources of this organism is challenging due to the large numbers of human cases, and the prevalence of this organism throughout the environment due to growth in a wide range of animal species. Many molecular subtyping methods have been developed to characterize Campylobacter species, but only a few are commonly used in molecular epidemiology studies. This review examines the applicability of these methods, as well as the role that emerging whole genome sequencing technologies will play in tracking sources of Campylobacter spp. infection.     

A new clade of Dickeya spp. plays a major role in potato blackleg outbreaks in North Finland

Until recently Dickeya was regarded as a pathogen not established in Finland. As a result the blackleg symptom observed on potato was often associated with Pectobacterium atrosepticum. The occurrence of Dickeya spp. on potato in Finland was first reported in 2004. Since then the prevalence of Dickeya has been monitored through surveys and routine test of seed lots produced in the country. The results of monitoring of Dickeya spp. in seed lots produced in Finland between the years 2004 and 2008 indicated a steady increase in the incidence of Dickeya spp. The highest incidence was observed in samples from the 2006 growing season where about 37% were positive for Dickeya spp. The summer in 2006 was one of the warmest summers recorded in 100 years in Finland. The majority of infected lots were imported varieties. Since recently heavy blackleg outbreaks have occurred in production fields in the High Grade (HG) zone. A detailed study of these incidents of blackleg outbreaks in North Finland during the years 2008 and 2010 indicated that Dickeya spp. was the major component in the observed blackleg complex. It was detected and isolated from almost all symptomatic plants investigated. Repetitive sequences PCR (REP‐PCR) and Pulsed Field Gel Electrophoresis (PFGE) analysis of strains isolated in Finland showed identical pattern with those isolated recently in other European countries with a proposed name ‘Dickeya solani’. Moreover, the dnaX gene sequence of the representative strains isolated in Finland indicated 100% similarity to the dnaX sequences of ‘D. solani’. The study presents the first report of a detailed analysis of bacteria involved in potato blackleg complex from natural field outbreaks in North Finland HG zone and characterisation of the ‘D. solani’ strains playing the major role in the disease complex.     

Insecticidal and Acaricidal Activity of Certain O,O-Dialkyl O-3-Pyridazinyl Phosphorothioates

Forty-three 3-pyridazinyl phosphorothioates were evaluated for insecticidal and acaricidal activities against two-spotted spider mite, turnip aphid, smaller brown planthopper, mosquito larvae and American cockroach. Approximate acute oral toxicity of these compounds in mice was also examined. In general, the toxicity in mice was in parallel with the pesticidal activity, but a few compounds clearly showed a high degree of selectivity between pests and mice. Especially O,O-dimethyl O-(6-cyclohexyloxy-3-pyridazinyl)phosphorothioate showed substantially reduced mammalian toxicity but maintained high insecticidal and acaricidal activity.     

沙门氏菌分型研究进展

沙门氏菌是重要的致病菌之一,该菌属型别繁多。沙门氏菌的分型方法可分为以表型特征为依据的表型分型方法和以基因特征为依据的分子分型方法。沙门氏菌的表型分型主要有血清分型和噬菌体分型。分子分型主要有分子血清分型、脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)、多位点序列分析(Multi-locus sequence typing,MLST)、多位点可变重复序列分析(Multi-locus variable numbers tandem repeat analysis,MLVA)以及成簇的规律间隔的短回文重复序列分析(Clustered regularly interspaced short palindromic repeats,CRISPR)等。与表型分型相比,分子分型技术具有快速、准确、重复性好等特点,现已得到广泛应用。其中,分子血清分型是鉴定沙门氏菌血清型的新方法,PFGE被认为是病原微生物分子分型的"金标准",MLST和MLVA以其分辨率高、可重复性好和可比性强等优势满足了全球流行病学发展的要求,能实现序列数据交换和共享,成为新一代的分子分型新工具,而近年发现的CRISPR分型对同源性较高的同种血清型具有较高的分型能力。但每种分型方法也有各自的优缺点和使用条件及适用范围,因此可以根据菌株特性、分型目的和实验室拥有的条件选择最合适的分型方法。