Serotyping and RAPD profiles of Salmonella enterica isolates from Mauritius

AIMS: The genus Salmonella is a common agent of gastroenteritis in Mauritius, generating more cases of the disease during summer than during winter. The aims of this study were to assess the genetic diversity of isolates of Salmonella enterica by RAPD fingerprinting, and to establish the relationship between human and chicken isolates. METHODS: Twenty-six isolates were obtained from hospital laboratories and commercial poultry producers locally. RESULTS: The RAPD profiles, biochemical and serological analyses showed that two of the chicken isolates were mistakenly identified as Salmonella. The genetic diversity of the remaining 24 isolates (five chicken and 19 human), confirmed as Salmonella, was analysed using four arbitrary primers, OPA-10, OPR-03, OPI-06 and OPJ-09, chosen from an initial set of 10 decamers. Seventy RAPD markers were generated in four individual DNA profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Cluster analysis (UPGMA) performed using the NTSYS-pc V 1.8 computer software, confirmed that some strains of Salmonella isolated from chicken were genetically similar to those isolated from humans. Furthermore, a 1 kbp band amplified using primer OPA-10 was specific for the Salmonella genus as it was not amplified in any of the control bacteria.     

Molecular fingerprinting of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica derby isolated from tropical seafood in South India

Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby strains isolated from different seafood were genotyped by PCR-ribotyping and ERIC-PCR assays. This study has ascertained the genetic relatedness among serovars prevalent in tropical seafood. PCR-ribotyping exhibited genetic variation in both Salmonella serovars, and ribotype profile (II) was most predominant, which was observed in 10/18 of Salmonella enterica subsp. enterica Typhimurium and 7/17 Salmonella enterica subsp. enterica Derby isolates. Cluster analysis of ERIC-PCR for Salmonella enterica subsp. enterica Typhimurium strains exhibited nine different banding patterns and four strains showed >95% genetic homology within the cluster pairs. ERIC-PCR produced more genetic variations in Salmonella enterica subsp. enterica Typhimurium; nevertheless, both methods were found to be comparable for Salmonella enterica subsp. enterica Derby isolates. Discrimination index of PCR-ribotyping for Salmonella enterica subsp. enterica Typhimurium isolates was obtained at 0.674 and index value 0.714 was observed for Salmonella enterica subsp. enterica Derby strains. Molecular fingerprinting investigation highlighted the hypothesis of diverse routes of Salmonella contamination in seafood as multiple clones of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby were detected in same or different seafood throughout the study period.     

Difference and consensus of whole cell Salmonella enterica subsp. enterica serovars matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectra

AIM: Application of MALDI-TOF MS for characterization of strains of Salmonella enterica subsp. enterica. METHODS AND RESULTS: Whole cells were analysed by MALDI-TOF MS. Spectra with a maximum of 500 mass peaks between (m/z) 0 and 25000 were examined for consensus peaks manually and by a computer software algorithm. Consensus peaks were observed by both methods for spectra of Salmonella enterica serovars Derby, Hadar, Virchow, Anatum, Typhimurium and Enteritidis. CONCLUSIONS: Differences in numbers of consensus peaks in spectra obtained by manual and computer comparison indicated that development of the software involving statistical analysis of peak accuracy is necessary. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of an analysis system for peak profiles in whole cell MALDI-TOF MS spectra to enable intra and interlaboratory comparison.     

The colanic acid gene cluster of Salmonella enterica has a complex history

The colanic acid gene cluster of Salmonella enterica LT2 was sequenced and compared with that of Escherichia coli K-12. The two clusters are similar with divergence slightly higher than average for genes of the two species. The cluster was divided into four blocks by GC content and seems likely to have transferred from a higher GC content species to the ancestor of E. coli and S. enterica. All 19 genes of K-12 and 13 genes of LT2 appear to have undergone random genetic drift with amelioration of the GC content. However, in the case of S. enterica, we believe that the six genes of the GDP-fucose pathway group were replaced relatively recently by genes closely related to those of the original donor species. Two repetitive elements were observed: a bacterial interspersed mosaic element in the intergenic region between wzx and wcaK in K-12 only and a RSA (repetitive sequence element) sequence between wcaJ and wzx in LT2 only.     

Restriction enzyme analysis of randomly amplified polymorphic DNA amplicons of Salmonella enterica ser. Enteritidis PT4 and Typhimurium DT104

Salmonella enterica serotype Enteritidis PT4 and Typhimurium DT104 isolates were characterized using a random amplification of polymorphic DNA (RAPD) protocol found previously to be highly discriminatory for isolates of Salmonella . Profiles generated with a single primer 1254, and independently 1283, successfully characterized an outbreak strain of Enteritidis PT4 but could not differentiate epidemiologically unrelated strains of Enteritidis PT4 from the outbreak strains. Primer 1254 differentiated one strain, and 1283 two strains of Typhimurium DT104 previously undifferentiated on the basis of biochemical and physical properties. Subsequent analysis using a combination of RAPD and restriction enzyme analysis could not provide additional differentiation of Enteritidis PT4 and Typhimurium DT104 isolates but did, however, exhibit the potential to be a useful combination of molecular techniques.     

Complete nucleotide sequence of a virulence plasmid of Salmonella enterica serovar Dublin and its phylogenetic relationship to the virulence plasmids of serovars Choleraesuis, Enteritidis and Typhimurium

The complete nucleotide sequence of pOU1113 (pSDVu), one of the two types of virulence plasmids of Salmonella enterica serovar Dublin, was determined. It contained 80 156 bp with 53.8 mol% G+C content. Approximately 70 genes could be discerned. Compared with pSTV, the virulence plasmid of serovar Typhimurium, pOU1113 was shorter owing to a missing region amounting to c. 10 kb; furthermore, except for a unique 10 849-bp region, the nucleotide as well as deduced amino acid sequences of pOU1113 were nearly identical to the corresponding regions of three S. enterica virulence plasmids, namely pSCV (virulence plasmid of Choleraesuis), pSTV and pSEV (virulence plasmids of Enteritidis), confirming their close phylogenetic relationship. Comparative analysis indicated that these virulence plasmids appeared to have descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. From a biological and evolutionary point of view, if the decreasing sizes of pOU1113 and pSCV truly reflect a process in which the virulence plasmid has been shedding unnecessary genes during evolution, our data suggest that some genes in the missing region, such as the pef and tra operons, could have a minimal role in maintaining the survival of the bacteria in their environmental niche.     

Genetic rearrangements in the tyrB-uvrA region of the enterobacterial chromosome: a potential cause for different class B acid phosphatase regulation in Salmonella enterica and Escherichia coli

Unlike in Escherichia coli, in Salmonella enterica production of class B acid phosphatase (AphA) was detectable also in cells growing in the presence of glucose. Characterization of the aphA locus from a S. enterica ser. typhi strain showed that the aphA determinant is very similar to the E. coli homolog, and that its chromosomal location between the highly conserved tyrB and uvrA genes is retained. However, the aphA flanking regions were found to be markedly different in the two species, either between tyrB and aphA or between aphA and uvrA. The differences in the aphA 5'-flanking region, which in S. enterica is considerably shorter than in E. coli (183 vs. 1121 bp) and includes potential promoter sequences not present in E. coli, could be responsible for the different regulation of class B acid phosphatase observed in the two species.     

无核沙田柚的RAPD研究——无核机理研究(Ⅱ)

选用经筛选的２０个短序列引物（１０ｎｔ）和随机抽取的２个随机长序列引物,对激光诱导产生的无核沙田柚（处理组）及未经激处理的有核沙田柚（对照组）进行了ＲＡＰＤ（随机扩增多态性ＤＮＡ）分析。结果有６个引物扩增出了共２０条多态性片段,从而表明在沙田柚的处理组与对照组之间,存在着ＤＮＡ分子水平上差异,也就量说,已经引起了沙田柚后代基因组的显著变异,且这种诱导所引起的性状改变是可以遗传的。     

Competition among isolates of Salmonella enterica ssp. enterica serovar Typhimurium: role of prophage/phage in archived cultures

Previously, we reported extensive diversity among survivors of Salmonella enterica ssp. enterica serovar Typhimurium that were stored for four decades in sealed agar stabs. Thus raising the question: was there selection for greater fitness among eventual survivors? To address this, we cocultured archived LT2 survivors with nonarchived (parental) LT2 strains in competition experiments. Selected archived strains outgrew a nonarchived LT2 sequenced strain. Although we initially assumed this was the result of mutations empowering greater nutritional utilization, we found phage selection was also involved. Phage fels- 1 and fels- 2 in supernatants were identified by primer/PCR as a putative selective force following single plaque isolations on a prophage-free strain and testing on appropriate hosts. In confirmatory experiments, instead of coculture in Luria–Bertani requiring antibiotic marker insertions, competing strains without markers were inoculated at opposite edges of motility plates. Not only did the archived LT2 population overgrow the nonarchived LT2 population, but also clear zones appeared at edges of encounters from which phage fels- 1 and fels- 2 (but not gifsy- 1 nor gifsy- 2) were recovered. However, in competitions of an archived strain with S . Typhimurium ATCC 14028, phage emerged that had a DNA base sequence segment of prophage ST64B but the sequence differed from the reported homologous segment in ST64B.     

Osmoprotection of Salmonella enterica serovar Typhimurium by Ngamma-acetyldiaminobutyrate, the precursor of the compatible solute ectoine

N(gamma)-acetyl-2,4-diaminobutyrate (NADA), the precursor of the compatible solute ectoine, was shown to function as an osmoprotectant for the non-halophilic bacterium Salmonella enterica serovar Typhimurium. The addition of NADA-containing extracts of an ectoine synthase mutant of the broad salt-growing halophile Chromohalobacter salexigens DSM 3043(T) could alleviate the inhibitory effects of high salinity in S. enterica, which lacks the ectoine biosynthetic pathway. NADA, purified from extracts of the mutant, protected S. enterica against salinity stress. This osmoprotective effect was slightly lower than that of ectoine, but more potent than that of hydroxyectoine. Accumulation of purified NADA by S. enterica was demonstrated by (13)C-NMR spectroscopy and HPLC analysis. In addition, it was shown that NADA was taken up by S. enterica via the ProP and ProU transport systems, which are known to transport glycine betaine and proline. This finding provides evidence that these permeases can recognize a diaminoacid that carries an unsubstituted alpha-amino group. This is the first time that NADA has been connected with osmoprotective functions in non-halophilic bacteria.     

应用RAPD分子标记技术探讨3种石斛属植物的种间关系

采用RAPD分子标记技术,分析了金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的种间关系。10个引物产生的113条DNA扩增片段中,106条（93.81%）具有多态性,利用113个RAPD标记,计算遗传距离,利用非加权组平均法建立聚类图。结果表明,RAPD标记技术较好地从分子水平揭示金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的遗传背景、亲缘关系,并为后期在DNA水平上对药用石斛的开发利用提供资料。     

稗草致病菌——尖角突脐孢菌菌株RAPD指纹图谱的分析

以我国主要稻区的稗草植株上分离的17株尖角突脐孢菌菌株为试验材料,采用改良的SDS法提取其基因组DNA,并运用优化的RAPD分析体系对其进行了分子标记遗传差异研究。从25个随机引物中筛选出20个扩增效果好的引物,对全部试验材料进行了RAPD扩增,共得到239条有效带,其中多态性带229条（占95.8%）。依据扩增结果建立了17株尖角突脐孢菌基因型的DNA指纹图谱并对其进行了有效区分。根据RAPD分析结果计算了菌株间的遗传距离,分析了它们的遗传差异并进行了聚类分析,结果表明,RAPD分子标记技术是能够用于杂草致病菌资源的鉴定的,并可以进一步应用于特定性状的基因标记研究。     

福建省枳椇种质资源遗传多样性RAPD分析

建立和优化枳椇Hovenia acerba的RAPD反应体系,并对福建省12个种源地35份枳椇材料进行遗传多样性及亲缘关系研究。利用筛选出的10个RAPD引物共检测基因组DNA中193个位点,其中多态性位点161个(83.42%)。供试材料观测等位基因数(Na)1.8342、有效等位基因数(Ne)1.4155,Nei’s基因多样性(H)0.2493,Shannon多样性指数(I)0.3822。以遗传相似系数0.754为阈值,可将35份枳椇分成五大组。RAPD标记可有效揭示福建省枳椇种质资源的遗传多样性,所测枳椇种质资源遗传多样性丰富,极具开发利用价值。     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

弯孢类炭疽菌菌株的RAPD分析与分类研究

通过RAPD分析对38个不同寄主来源的弯孢类炭疽菌菌株的系统发育及分类进行研究。结果表明:许多引物的RAPD扩增带型在种内是相似或一致的,而种间差异较大。UPGMA聚类分析的结果表明:38个弯孢类炭疽菌菌株被聚为6个类群,群内菌株之间遗传相似性较高,而群间遗传相似性较低;群与群之间分界明显,表明种的分界相当明显。因此,RAPD分析所反映的种间亲缘关系,将有助于疑难种分类地位的确定和近似种的区分。RAPD分析的结果还揭示了一些近似种的分类关系,如按传统方法分别鉴定为Colletotrichumtruncatum、C.circinans和辣椒炭疽菌C.capsici的许多菌株聚类在同一群内,有很高的遗传相似性,实验结果支持它们为同一个种。实验中还发现5条辣椒炭疽菌的特有片断,分别由引物OPE-14、OPH-15(2条)、OPM-12和OPM-20扩增产生。这些特异带的发现对该菌的快速检测和鉴定具有重要意义。     

Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.     

珍稀濒危植物天目木兰（Magnolia amoena）遗传多样性的RAPD分析

运用随机扩增多态DNA(RAPD)技术,对天目木兰(Magnolia amoena)居群的遗传多样性进行了研究．从40个10-mer随机引物中筛选出14个能得到清晰、稳定扩增带的引物进行扩增,14个引物共检测了94个位点,其中多态性位点为23,占24．4％,计算了12个居群之间的遗传相似度和遗传距离,并运用UPGMA法进行了聚类分析,结果显示相同严地个体间(居群内)的遗传距离较小,遗传多样性水平很低;不同产地个体间(居群间)遗传距离较大,遗传多样性水平较前者高,即天目木兰个体间遗传多样性水平与它的地理分布有关,天目木兰总体较低的遗传多样性是导致它濒危的原因之一。     

一种新的分子标记方法－随机微卫星扩增多态DNA (RMAPD)

随机微卫星扩增多态DNA（RMAPD）是利用随机引物和微卫星的上游或下游引物一起作为该扩增的引物,在Taq DNA聚合酶、MgCl2、dNTPs和模板DNA等共同作用下进行PCR扩增的一种新型分子标记方法。其核心是RMAPD引物的有效性问题。通过对西农萨能奶山羊群体RMAPD电泳检测、数据统计分析及验证实验等证明RMAPD的引物是有效的。通过与微卫星和RAPD标记比较,发现RMAPD标记在扩增引物、扩增程序和重复性等方面区别于微卫星和RAPD标记;它是RAPD标记的一种广义的延伸,但又不完全等同于RAPD标记。因此,确定RMAPD是一种新的分子标记方法。该方法也具有DNA标记的特点,在群体遗传结构和亲缘关系分析以及标记辅助选择等遗传育种领域具有广阔的应用前景。     

26个唐菖蒲品种RAPD分析

唐菖蒲是世界著名的切花之一,由于是纯粹的外来品种,目前国际上对其分类尚无统一的方法,主要是依据其生物习性等形态特征进行分类,受环境影响很大。同工酶是基因的产物,利用同工酶可以进行唐菖蒲品种鉴定和分类,但它在植物的不同器官组织发育的不同时期存在差异,而分子标记技术具有不受环境、器官、组织发育不同时期影响、遗传性稳定、分析简单等特点而被广泛应用在花卉品种分类上。利用RAPD技术对26个主要唐菖蒲切花品种进行分类和亲缘关系的研究,从520个随机引物中筛选出33个用于PCR反应,共在206个位点上扩增出条带,平均每个引物扩增位点6.24个,多态性位点185个,占总带数的89.8%,可以进行品种的鉴定。从分子水平揭示出唐菖蒲切花品种种质资源遗传基础狭窄,夏花大花型唐菖蒲品种间存在一定的亲缘关系。