Detection of Shigella spp. in food with a nested PCR method – sensitivity and performance compared with a conventional culture method

A nested PCR method was developed and its performance evaluated by detection of Shigella flexneri in food. The nested PCR amplifies sequences within an invasion-associated locus (ial) on the invasion plasmid specific for Shigella and enteroinvasive Eschrichia coli (EIEC). The nested PCR detected Sh. flexneri in lettuce inoculated with 2, 20 and 200 cfu g-1 after 1, 7 and 18 d of storage, respectively. In comparison, a culture method (NMKL no. 151) detected 10(5) cfu g-1 after 1 but not after 7 d of storage. The presence of inhibitors in blue cheese and shrimps reduced the sensitivity of the PCR assay. To overcome this inhibition, a sample preparation step based on buoyant density centrifugation was developed. This treatment resulted in a successful recovery of Sh. flexneri in lettuce, milk, shrimp and blue cheese inoculated with 10 cfu g-1. The proposed method, which includes a combination of enrichment, buoyant density centrifugation and a nested PCR, can be completed in less than two working days.     

PCR for Detection of Shigella spp. in Mayonnaise

The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.     

驱除痢疾杆菌侵袭大质粒的新方法

用质粒不相容性原理驱除痢疾杆菌福氏 2a 2 4 5 7T和宋内S7的侵袭大质粒 ,先从福氏2a侵袭大质粒分别扩增ori和inc基因 ,将它们克隆至 pMD18 T载体 ,得重组质粒pMDori和 pMDinc ,然后转化 2 4 5 7T和S7,不管是pMDori还是 pMDinc都能竞争驱除痢疾杆菌福氏 2a 2 4 5 7T和宋内S7的侵袭大质粒。     

The Shigella virulence gene regulatory cascade: a paradigm of bacterial gene control mechanisms

Shigella flexneri is the causative agent of bacillary dysentery and is a facultative intracellular pathogen. Its virulence regulon is subject to tight control by several mechanisms involving the products of over 20 genes and an array of environmental signals. The regulon is carried on a plasmid that is prone to instability and to integration into the chromosome, with associated silencing of the virulence genes. Closely related regulons are found in other species of Shigella and in enteroinvasive Escherichia coli . A wealth of detailed information is now available on the Shigella virulence gene control circuits, and it is becoming clear that these share many features with regulatory systems found in other bacterial pathogens. All of this makes the S. flexneri virulence gene control system a very attractive topic for those interested in the nature of gene regulatory networks in bacteria.     

Partial sequence of acid phosphatase-1(1) gene (Aps-1(1)) linked to nematode resistance gene (Mi) of tomato.

A partial amino acid sequence of acid phosphatase-1(1) (apase-1(1)), one of acid phosphatase isozymes of tomato, was identified. This information enabled us to synthesize degenerated primer pools of oligonucleotides for polymerase chain reactions (PCR) using cDNA for poly(A)+ RNA of tomato leaves as a template. As a result, a 135-bp, then a 467-bp PCR product were obtained. Nucleotide sequencing of these two PCR products gave a total of 522-bp sequence that was identified as a part of the Asp-1(1) gene judging from the amino acid sequence deduced from it. Using the 135-bp PCR product as a probe, we detected the restriction fragment length polymorphism (RFLP) in two different lines of tomato by genomic Southern blot analysis. We also did pulsed-field gel electrophoresis (PFGE) and Southern blot analysis to search for suitable fragments to clone into a YAC vector. As a result, a single band with a size that could be cloned into a YAC vector was detected when the genomic DNA was digested with some kinds of restriction enzymes.     

Partial Sequence of Acid Phosphatase-11 Gene (Aps-11) Linked to Nematode Resistance Gene (Mi) of Tomato

A partial amino acid sequence of acid phosphatase-1¹ (apase-1¹), one of acid phosphatase isozymes of tomato, was identified. This information enabled us to synthesize degenerated primer pools of oligonucleotides for polymerase chain reactions (PCR) using cDNA for poly(A)⁺ RNA of tomato leaves as a template. As a result, a 135-bp, then a 467-bp PCR product were obtained. Nucleotide sequencing of these two PCR products gave a total of 522-bp sequence that was identified as a part of the Asp-1¹ gene judging from the amino acid sequence deduced from it. Using the 135-bp PCR product as a probe, we detected the restriction fragment length polymorphism (RFLP) in two different lines of tomato by genomic Southern blot analysis. We also did pulsed-field gel electrophoresis (PFGE) and Southern blot analysis to search for suitable fragments to clone into a YAC vector. As a result, a single band with a size that could be cloned into a YAC vector was detected when the genomic DNA was digested with some kinds of restriction enzymes.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

Identification and characterization of phoN-Sf, a gene on the large plasmid of Shigella flexneri 2a encoding a nonspecific phosphatase.

A gene encoding a nonspecific phosphatase, named PhoN-Sf, was identified on the large virulence plasmid (pMYSH6000) of Shigella flexneri 2a YSH6000. The phosphatase activity in YSH6000 was observed under high-phosphate conditions. However, it was found that low-phosphate conditions induced a slightly higher level of activity. The nucleotide sequence of the phoN-Sf region cloned from pMYSH6000 possessing the phoN-Sf gene encoded 249 amino acids with a typical signal sequence at the N terminus. The deduced amino acid sequence of the PhoN-Sf protein revealed significant homology to sequences of nonspecific acid phosphatases of other bacteria, such as Providencia stuartii (PhoN, 83.2%), Morganella morganii (PhoC, 80.6%), Salmonella typhimurium (PhoN, 47.8%), and Zymomonas mobilis (PhoC, 34.8%). The PhoN-Sf protein was purified, and its biochemical properties were characterized. The apparent molecular mass of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was calculated to be 27 kDa. The 20 amino acids at the N terminus corresponded to the 20 amino acid residues following the putative signal sequence of PhoN-Sf protein deduced from the nucleotide sequence. The PhoN-Sf activity had a pH optimum of 6.6, and the optimum temperature was 37 degrees C. The enzymatic activity was inhibited by diisopropyl fluorophosphate, N-bromosuccinimide, or dithiothreitol but not by EDTA. The subcellular localization of the PhoN-Sf protein in YSH6000 revealed that the protein was found predominantly in the periplasm. Examination of Shigella and enteroinvasive Escherichia coli strains for PhoN-Sf production by immunoblotting with the PhoN-specific antibody and for the presence of phoN-Sf DNA by using a phoN-Sf probe indicated that approximately one-half of the strains possessed the phoN-Sf gene on the large plasmid and expressed the PhoN-Sf protein. The Tn5 insertion mutants of YSH6000 possessing phoN-Sf::Tn5 still retained wild-type levels of invasiveness, as well as the subsequent spreading capacity in MK2 epithelial cell monolayers, thus suggesting that the PhoN-Sf activity is not involved in expression of the virulence phenotypes of Shigella strains under in vitro conditions.     

志贺菌的质粒图谱分析

目的：分析志贺菌的质粒图谱及其与细菌药物敏感性的相关性。方法：从菌痢患者粪便标本中分离6株福氏志贺菌和4株宋内志贺菌,分别对其质粒图谱及药物敏感性进行分析。结果：不同菌株的质粒图谱具有明显的差异,但福氏志贺菌的5株以及宋内志贺菌的3株具有分子量23Kb的质粒带。各菌株的质粒图谱与其对头孢三嗪,头孢唑啉,环丙沙星,诺氟沙星,氯霉素的耐药特性无明显相关性。结论：获自患者的福氏志贺菌和宋内志贺菌具有不同的质粒图谱以及抗菌药物敏感性,提示在我市引起菌痢的志贺菌具有不同的来源。     

Identification of the Hind III polymorphic site in the PAI-1 gene: analysis of the PAI-1 Hind III polymorphism by PCR

The identification of the Hind III polymorphic site in the 3' end of the plasminogen activator inhibitor 1 (PAI-1) gene and a simple method to identify the Hind III polymorphism rapidly in the PAI-1 gene using PCR is described. The Hind III restriction site was identified by restriction site mapping and sequence analysis from a cosmid DNA clone. Genomic DNA was isolated from individual human umbilical cords and a 754-bp fragment of the human PAI-1 gene was amplified by PCR. Aliquots of the PCR products were digested with Hind III and analyzed by agarose gel electrophoresis. The presence of two fragments, 754 and 567 bp, was identified, and they were designated as 1/1 (750-bp band), 1/2 (754- and 567-bp bands), and 2/2 (567-bp band). The PCR method is considerably less time consuming than the conventional DNA genotyping using Southern blot analysis. To ensure that this new method identified the same PAI-1 genotypes as previously identified by Hind III restriction fragment length polymorphism (RFLP), samples were simultaneously genotyped by PCR and Southern blot analysis. Both methods identified the same Hind III genotypes in all the samples, confirming the reliability of this new PCR method for the rapid identification of the Hind III polymorphism in the human PAI-1 gene.     

Immunocapture UPPCR combined with DGGE for rapid detection of Shigella species

AIMS: To develop an immunocapture universal primer PCR (iUPPCR) combined with denaturing gradient gel electrophoresis (DGGE) and evaluate it as a method permitting rapid detection of Shigella species and their serotypes. METHODS AND RESULTS: This method amplifying the conserved regions of bacterial 16S rRNA genes of different species or serotypes of Shigella dysentery bacilli captured and enriched by polyvalent antibodies can detect and distinguish causative pathogens rapidly. Four serotypes from three Shigella species including Shigella dysenteriae serotype 1, Shigella boydii serotype 1, Shigella flexneri serotypes 1a and 3a were examined. CONCLUSION: Our approach could be adopted for not only axenic bacterial population but also mixed communities and achieve rapid detection of various bacteria from the same genus or species in one sample. SIGNIFICANCE AND IMPACT OF THE STUDY: The iUPPCR-DGGE method was shown to be more convenient than serotype-specific-antibody-based method of iUPPCR for Shigella species detection and it could be also applied to the quick detection for other kinds of pathogens with many serotypes.     

弗氏2a志贺氏菌2457T株YciD蛋白的融合表达和纯化

目的：原核表达重组弗氏2a志贺氏菌2457T株YciD蛋白,为其功能研究奠定基础。方法：用PCR方法从弗氏2a志贺氏菌2457T株染色体中扩增YciD蛋白编码序列,经过纯化、酶切后克隆到原核表达载体pET32a中,构建重组载体pET32a-yciD,转化大肠杆菌BL21（DE3）菌株获得工程菌株,对其表达和纯化条件进行优化;利用Western Blot检测融合蛋白的表达。结果：构建了YciD蛋白的融合表达载体,并在大肠杆菌中获得高效表达;经Ni-NTA亲和层析柱纯化获得了高纯度的YciD蛋白;Western Blot表明,此蛋白可与His标签抗体反应,表明获得了目的蛋白。结论：在原核表达系统中表达、纯化弗氏2a志贺氏菌2457T株YciD蛋白,为进一步对其进行功能研究奠定了基础。     

Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery

The Shigella bacteria cause bacillary dysentery, which remains a significant threat to public health. The genus status and species classification appear no longer valid, as compelling evidence indicates that Shigella, as well as enteroinvasive Escherichia coli, are derived from multiple origins of E.coli and form a single pathovar. Nevertheless, Shigella dysenteriae serotype 1 causes deadly epidemics but Shigella boydii is restricted to the Indian subcontinent, while Shigella flexneri and Shigella sonnei are prevalent in developing and developed countries respectively. To begin to explain these distinctive epidemiological and pathological features at the genome level, we have carried out comparative genomics on four representative strains. Each of the Shigella genomes includes a virulence plasmid that encodes conserved primary virulence determinants. The Shigella chromosomes share most of their genes with that of E.coli K12 strain MG1655, but each has over 200 pseudogenes, 300 approximately 700 copies of insertion sequence (IS) elements, and numerous deletions, insertions, translocations and inversions. There is extensive diversity of putative virulence genes, mostly acquired via bacteriophage-mediated lateral gene transfer. Hence, via convergent evolution involving gain and loss of functions, through bacteriophage-mediated gene acquisition, IS-mediated DNA rearrangements and formation of pseudogenes, the Shigella spp. became highly specific human pathogens with variable epidemiological and pathological features.     

Identification of Aeromonas hydrophila hybridization group 1 by PCR assays.

Synthetic oligonucleotide primers of 24 and 23 bases were used in a PCR assay to amplify a sequence of the lip gene, which encodes a thermostable extracellular lipase of Aeromonas hydrophila. A DNA fragment of approximately 760 bp was amplified from both sources, i.e., lysed A. hydrophila cells and isolated DNA. The amplified sequence was detected in ethidium bromide-stained agarose gels or by Southern blot analysis with an internal HindIII-BamHI 356-bp fragment as a hybridization probe. With A. hydrophila cells, the sensitivity of the PCR assay was < 10 CFU, and with the isolated target, the lower detection limit was 0.89 pg of DNA. Primer specificity for A. hydrophila was determined by the PCR assay with cells of 50 strains of bacteria, including most of the 14 currently recognized DNA hybridization groups of Aeromonas spp. as well as other human and environmental Aeromonas isolates. Detection of A. hydrophila by PCR amplification of DNA has great potential for rapid identification of this bacterium because it has proved to be highly specific.     

Occurrence and characteristics of the cytolysin A gene in Shigella strains and other members of the family Enterobacteriaceae

Cytolysin A (ClyA, HlyE, SheA) is a hemolytic pore-forming toxin found in Escherichia coli and Salmonella enterica serovars Typhi and Paratyphi A. In the present study, analysis of several Shigella strains revealed that they harbor only nonfunctional clyA gene copies that have been inactivated either by the integration of insertion sequence (IS) elements (Shigella dysenteriae, Shigella boydii, and Shigella sonnei strains) or by a frameshift mutation (Shigella flexneri). Shigella dysenteriae and S. boydii strains also exhibited IS-associated deletions at the clyA locus. PCR and Southern blot analyses as well as database searches indicated that clyA-related DNA sequences are completely absent in strains belonging to various other genera of the family Enterobacteriaceae. According to these data, ClyA may play a role only for a rather small subset of the enteric bacteria.     

Detecting low concentrations of Shigella sonnei in environmental water samples by PCR

Outbreaks of Shigella sonnei associated with contaminated water have been reported and methods for the simultaneous detection of Shigellae and enteroinvasive Escherichia coli in water samples have been developed with detection limits of 10(1)-10(2) CFU mL(-1) of water. Because 10(1)-10(2)Shigellae can cause disease, a more sensitive detection method as an addition to the existing methods for detection of Shigella sonnei in water samples is reported here. Initially, 33 Shigella sonnei and 72 non-Shigella sonnei isolates were tested and one primer pair was found capable of specifically amplifying a 369-bp insertion sequence 1 (IS1) fragment from all 33 Shigella sonnei isolates and one Shigella dysenteriae ATCC isolate by PCR. The detection method was developed, which included filtration of 50 mL of water through a membrane and application of PCR to the membrane using this primer pair. Environmental water samples with total bacterial numbers of 384-2.84 x 10(7) CFU L(-1) were collected and seeded with 13 Shigella sonnei and the Shigella dysenteriae ATCC isolates. Detection limits were determined as 1.7-24.7 and 270-8000 CFU per 50 mL of water, respectively, using this detection method.     

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.