Localization of the sapA gene on a physical map of Campylobacter fetus chromosomal DNA

     We constructed a physical map of Campylobacter fetus TK(+) chromosomal DNA digested by either SmaI, SalI, or NotI using pulsed-field gel electrophoresis and Southern hybridization data. The genome size of C. fetus TK(+) is 2016 kb, larger than that reported by the others. To locate the sapA gene, which encodes the surface array protein (SAP), on the physical map, we performed Southern hybridizations with probes based on the conserved region of the sapA gene. The results showed that more than seven copies of the conserved region were present on C. fetus chromosomal DNA and that the sapA gene was located on a limited number of fragments forming a cluster of genes. By comparing fingerprint patterns of strain TK(+) and strain TK(–), which lost the ability to produce SAP during culture on agar medium, an approximately 10 kb deletion was observed in the fragments of strain TK(–). The results of Southern hybridization with two probes, one from the upstream region and the other from the variable region of sapA, suggest that the loss of SAP expression might not be the result of the loss of the sapA gene itself, but only a loss of its control systems. Received: 25 May 1994 / Accepted: 1 September 1994     

A deletion in the sapA homologue cluster is responsible for the loss of the S-layer in Campylobacter fetus strain TK

The surface array protein (SAP) of Campylobacter fetus strain TK is encoded by seven homologous sapA genes clustered on the chromosomal DNA. The spontaneously arising variant strain TK(SAP^–) produces no SAP and carries an approximately 10-kb chromosomal deletion. To elucidate the mechanism underlying the loss of SAP synthesis, we analyzed the region containing the sapA homologues and the deletion. We constructed a physical map of the sapA cluster region by aligning the clones that contain sapA homologues. These analyses demonstrated that all sapA homologues were located within a limited region of about 50 kb of chromosomal DNA of strain TK. The TK(SAP^–) deletion was located within this cluster and was 13.3 kb in size. The deletion occurred between two sapA homologues and resulted in the formation of a chimeric sapA homologue in the variant strain. Sequence analysis of the upstream regions and the conserved regions of all sapA homologues revealed a high degree of similarity. However, only one sapA homologue contained a putative promoter sequence. This promoter sequence was located in the deleted region. Thus, the deletion of the promoter appears to be responsible for the loss of SAP expression in TK(SAP^–). Received: 17 May 1996 / Accepted: 6 December 1996     

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.     

辽宁沿海地区溶藻弧菌的耐药性及分子分型

目的 了解辽宁沿海地区溶藻弧菌污染情况,分析其耐药性及分子分型特点。方法 在辽宁沿海地区采集牡蛎,分离溶藻弧菌,选取15种抗生素对其进行耐药分析,运用脉冲场凝胶电泳对其进行聚类分析。结果 分离出52株阳性菌株,夏季检出率高于春秋两季,对头孢唑林、氨苄西林、红霉素的耐药率分别为53.85%、26.96%、9.62%,同时对2种抗生素耐药菌株占23.08%;经聚类分析52株阳性菌中有3种优势群。结论 辽宁沿海海域溶藻弧菌对一代β-内酰胺类抗生素头孢唑林、氨苄西林耐药率高,PFGE结果表明存在遗传多样性,并有优势菌存在,为今后进一步研究提供数据支持。     

32株金黄色葡萄球菌分子分型及肠毒素分析

目的 对辽宁省内2016-2018年分离出的食源性金黄色葡萄球菌采用脉冲场电泳(PFGE)和肠毒素分型进行分析,为今后公共卫生等领域提供技术保障.方法 将32株金黄色葡萄球菌用限制性内切酶SmaI酶切以进行PFGE分析,并用BioNumerics(7.6版本)软件对分离株的指纹图谱进行聚类分析;用PCR方法对菌株进行肠...     

Differentiation of bacterial strains by thermal gradient gel electrophoresis using non-GC-clamped PCR primers for the 16S-23S rDNA intergenic spacer region

The method for DNA fingerprinting of the 16S-23S rDNA intergenic spacer region was modified to increase resolution of bacterial strains by thermal gradient gel electrophoresis (TGGE) analysis. By utilizing the high melting temperature region of the tRNA gene located in the middle of the 16S-23S rDNA intergenic spacer region as an internal clamp for TGGE, multiple melting domain problems were solved. PCR primers lacking a stretch of GC-rich sequences (GC-clamp) amplified the intergenic spacer region more efficiently than GC-clamped primers. Therefore, PCR artifacts were avoided by using low, 17-cycle, PCR. The method was successfully applied to diverse bacterial species for strain differentiation by TGGE without requiring a special PCR primer set.     

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus

AIM: DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. METHODS AND RESULTS: RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. CONCLUSIONS: Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.     

Persistence of a Salmonella enterica serotype typhimurium clone in Danish pig production units and farmhouse environment studied by pulsed field gel electrophoresis (PFGE)

The clonal relationship among Salmonella enterica serotype Typhimurium isolates from selected pig production units in Denmark was investigated by the pulsed field gel electrophoresis (PFGE) typing method to determine environmental survival and spread of Salmonella in different herds. Thirty-four Typhimurium isolated during 1996-1998 from porcine faeces and environmental samples from three pig farms designated 1, 3 and 5 were characterised by PFGE using two restriction enzymes. Farm 5 supplied piglets to farm 1 and the herds were located close to each other. Results of PFGE analysis showed both intra- and inter-relationships, i.e. identical PFGE patterns among the faecal and environmental isolates from farm 1 and farm 5. All the isolates from farm 3 irrespective of the source showed identical PFGE patterns, but were different from samples from farms 1 and 5. This study indicates spread between farms and survival of a farm-specific clone. Furthermore, identical PFGE patterns of isolates from piglet supplier and finisher herds indicate that the farrow-to-grower herd of farm 5 was sub-clinically infected prior to delivery to farm 1 and thereby caused the transmission of Salmonella.     

Size variation of rDNA clusters in the yeasts Saccharomyces cerevisiea and Schizosaccharomyces pombe

Summary The higher-order organization of rRNA genes was investigated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We used pulsed-field gel electrophoresis (PFGE) in combination with frequent cutter endonucleases having no recognition sites within rDNA repeating units to characterize tandem arrays of ribosomal genes in these two species. Large variations in rDNA cluster length were detected in various S. cerevisiae and S. pombe strains commonly used as PFGE molecular weight markers. This wide range of variability implies that the sizes currently assessed for chromosomes bearing rRNA genes in these organisms are unreliable since they may vary within strains by several hundreds of kilobase pairs, depending on the size of the tandem arrays of rRNA genes. Consequently, there is now a lack of reliable PFGE size standards between 1.6 Mb and 4.5 Mb, even when established yeast strains with calibrated chromosomes are used.     

Epidemiological evaluation of Neisseria meningitidis serogroup B by pulsed-field gel electrophoresis

Abstract Genomic DNA from 25 strains of serogroup B Neisseria meningitidis was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Spe I. N. meningitidis genomic DNA displayed considerable diversity. The diversity we observed among these strains was stable and included isolates from an outbreak that were phenotypically identical. This confirms the value of macrorestriction profiling and PFGE in providing epidemiologically stable strain markers for typing meningococci.     

121株甲型副伤寒病原菌的噬菌体型和脉冲场凝胶电泳型

目的认识甲型副伤寒疫病区甲型副伤寒沙门菌的噬菌体型和脉冲场凝胶电泳（PFGE）型,确定噬菌体型和PFGE型之间的关系以及菌型的分布和流行率。方法采用沙门菌组合噬菌体和SpeI、XbaI消化染色体DNA的PFGE对来自玉溪市7县（区）的121株甲型副伤寒菌进行分型。结果121株菌存在4个完全噬菌体型或1个噬菌体型;用SpeI或XbaI消化产物分别得出以SpeI01、SpeI02或XbaI01占优势的5种或4种PFGE型,SpeI01型和SpeI02型分别占37．2％和57．9％,XbaI01型占95．1％。结论121株菌的噬菌体型与PFGE型之间无一致性联系,PFGE型的SpeI01和SpeI02或XbaI01是玉溪地区的主要流行型,采用SpeI和XbaI的PFGE是鉴别甲型副伤寒菌流行克隆的一项有用技术。     

Lysogenic phages of Closrtidium perfringens: mapping of the chromosomal attachment sites

The sites of insertion for two lysogenic bacteriophages have been mapped on the chromosome of Clostridium perfringens strain CPN50 using two techniques based on pulsed field gel electrophoresis. Phage phi 29 was mapped to the 1 Mb region of the 3.6 Mb genome, near nanH which encodes a potential virulence factor, while phi 59 was found to have inserted at 2.9 Mb.     

Magnification of the rDNA cluster in Kluyveromyces lactis

Summary By employing pulsed field gel electrophoresis we find that slow growing strains of Kluyveromyces lactis have only 43%–55% of the wild-type level of ribosomal DNA (rDNA) repeats. When subjected to prolonged vegetative growth these strains can increase both the number of rDNA repeats and their growth rate.     

血流感染碳青霉烯类耐药肺炎克雷伯菌的耐药分析及同源性研究

本研究旨在探讨血流感染碳青霉烯类耐药肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumonia, CRKP)的耐药特点、分子分型特征和菌株同源性,为CRKP感染的临床诊治和预防控制提供理论参考。收集2015年4月至2018年3月期间就诊于上海市某三甲医院患者血标本分离的肺炎克雷伯菌(KP)非重复菌株,采用VITEK 2 Compact全自动细菌鉴定仪鉴定细菌并进行药物敏感性试验,共获得51株CRKP。对CRKP菌株使用纸片扩散法或琼脂稀释法进行15种抗菌药物的敏感性试验;采用聚合酶链式反应(polymerase chain reaction,PCR)检测碳青霉烯酶基因型;通过脉冲场凝胶电泳和多位点序列分型(MLST)技术分析菌株同源性。结果显示,51株CRKP对检测的15种临床常用抗菌药物呈广泛耐药,对其中的哌拉西林、头孢唑啉、头孢吡肟、头孢噻肟、头孢呋辛、头孢他啶、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦和环丙沙星耐药率为100%;对阿米卡星、庆大霉素和复方新诺明的耐药率较高,分别为76.5%、90.2%和62.8%;对替加环素耐药率较低,为3.9%。51株CRKP均检测出blaKPC基因,经测序鉴定为blaKPC-2基因,提示产KPC-2是CRKP对碳青霉烯类耐药的主要机制。MLST检测到4种ST型别,以ST11型为主,共 43株(84.3%),另有ST15型6株(11.8%),ST4845型1株及1株新分型。51株CRKP的PFGE图谱相似性系数在62.9%～100%,可分为19个簇(A-S簇),每簇分别包含1～12个菌株。其中A簇(13.7%)和G簇(23.5%)包含的菌株相对较多,且MLST分型均为ST11型,为优势簇。G簇包含7个型别,G4型为主要克隆菌株。 ST11型为CRKP的主要型别,PFGE分析表明该院存在菌株的克隆传播,应规范抗菌药物使用,同时加强细菌耐药性监测和医院内感染的防控工作。     

Immotile phenotype of an Escherichia coli mutant lacking the histone-like protein HU

The histone-like protein HU in Escherichia coli are encoded by the hupA and hupB genes. A hupA-hupB double deletion mutant has now been shown to express an immotile phenotype. The motility of hupA or hupB single mutants was similar to that of wild-type cells. SDS-polyacrylamide gel electrophoresis revealed that the amount of flagellin in the hupA-hupB double deletion mutant was markedly reduced compared with the wild-type strain, suggesting that the immotile phenotype of the double deletion mutant is caused by a loss of flagella.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

Molecular organization of Chlorella vulgaris chromosome I: presence of telomeric repeats that are conserved in higher plants

The unicellular green alga Chlorella vulgaris (strain C-169) has a small genome (38.8 Mb) consisting of 16 chromosomes, which can be easily separated by CHEF gel electrophoresis. We have isolated and characterized the smallest chromosome (chromosome 1, 980 kb) to elucidate the fundamental molecular organization of a plant-type chromosome. Restriction mapping and sequence analyses revealed that the telomeres of this chromosome consist of 5-TTTAGGG repeats running from the centromere towards the termini; this sequence is identical to those reported for several higher plants. This sequence is reiterated approximately 70 times at both termini, although individual clones exhibited microheterogeneity in both sequence and copy number of the repeats. Subtelomeric sequences proximal to the termini were totally different from each other: on the left arm, unique sequence elements (14–20 bp) which were specific to chromosome I, form a repeat array of 1.7 kb, whereas a 1.0 kb sequence on the right arm contained a poly(A)-associated element immediately next to the telomeric repeats. This element is repeated several times on chromosome I and many times on all the other chromosomes of this organism.     

Identification of some chromosomes of the brewing yeast Saccharomyces uvarum

Abstract Chromosomal DNA molecules of Saccharomyces uvarum and Saccharomyces cerevisiae were separated using Orthogonal Field Alteration Gel Electrophoresis (OFAGE). Hybridization with specific probes of S. cerevisiae chromosomes allowed the identification of seven chromosomes of S. uvarum . The majority of the studied chromosomal DNA molecules show the same OFAGE mobility as the corresponding molecules of S. cerevisiae , with some minor differences.
Hybridizations with two distinct bands of S. uvarum were observed with each URA1 (marker of chromosome XI) and ARG80 (marker of chromosome XIII) probes, demonstrating the presence of at least two copies of these genes in the brewing yeast.     

《中国药典》中标准菌种质控新方法的研究

目的 研究《中华人民共和国药典》(简称《中国药典》)纳入的标准菌种质控新方法,并评价不同批号标准菌种的质量稳定性。方法 对脉冲场凝胶电泳(PFGE)技术进行比较研究,同时整合16SrRNA基因序列分析、多位点序列分型和基质辅助激光解吸电离飞行时间质谱等质控新方法,进行标准菌种质控新方法的建立,并对标准菌种的质量进行评价。结果 形成了适用于《中国药典》中标准菌种的方法,并通过整合的质控新方法对不同批号的标准菌种进行评价,结果显示,菌种质量稳定,遗传信息无改变。同时,建立了标准菌种16SrRNA基因标准序列、PFGE标准指纹图谱和标准基因型。结论 标准菌种质控新方法的研究,为更加全面、深入地评价标准菌种的质量提供了依据;建立的标准菌种质量控制体系及标准菌种质控鉴定信息,为标准菌种持续的质量控制奠定了重要的参比信息基础。