viaB Gene Research Articles

Real-Time Loop-Mediated Isothermal Amplification (LAMP) Method for Quantitative Salmonella Typhi Detection Based on ViaB Gene

Background & Objective: There is a vast range of diseases caused by Salmonella including gastroenteritis, enteric fever, bacteraemia, and focal infection. Salmonella infection is usually transmitted to humans by consuming foods or water contaminated with animal or human waste. This study aims to design a quantitative-LAMP method for real-time detection of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of Typhoid fever. Materials & Methods: A new LAMP primer set was designed and used based on the ViaB gene to specifically detect S. Typhi. The genome of some Salmonella-related and non- Salmonella-related bacteria was subjected to the LAMP assay for detection of S. Typhi to evaluate its analytical specificity, its analytical sensitivity and limit of detection (LOD). Further, turbidity in tubes was measured by a turbidimeter, and then a standard curve was depicted by plotting (Tt) time threshold values against the logarithm of ViaB gene copy number and this standard curve helped the researchers a lot to obtain a quantitative method. Results: The Salmonella Typhi LAMP assay specifically assessed the ViaB gene. It was shown that the analytical sensitivity of the assay was 0.28 fg using agarose gel electrophoresis or amplification plots in Loop amp real-time turbidimeter system, whereas the amplification was 2.8 fg under direct observation of fluorescent color changes. In the proposed methods, the lower limit of detection (LLOQ) of the assays was ~1 and 8 copies of the ViaB gene, respectively. In the LAMP assay for the quantitative detection of Salmonella Typhi, the linear correlation (r2= 0.97) was calculated between the log copy number and Tt values. Conclusions: The Salmonella Typhi LAMP assay is a simple and accurate tool to detect the causative agent of Typhoid fever that may be designed for clinical laboratories.

Molecular Study about some Genes among Salmonella Typhi Isolates un Hilla / Iraq

In this study, (100) blood sample were collected from patient suspected with typhoid fever how were visits the Alhashimiya, Hilla Teaching Hospital and Morjan Teaching Hospital in Babylon province (Iraq) during the period November (2018) to February (2019). Identification was done by cultural and biochemical tests, and finally identification by Vitek2. Results showed that positive were (16) isolates of Salmonella enteric serovar typhi by Vitek2 (16%) from the(100) blood. Molecular study was accomplished first by the isolation of DNA from Salmonella typhi isolates, they used method had succeeded in extraction of genomic DNA from all isolates. Second by using PCR technique to detection four gene by using a specific PCR primer were done by comparison with allelic ladder which gave a (578bp) it was found that (flicA) gene present in (15) isolates (93.75%) of the positive sample. viaB gene was also detected in S. typhi sample and found that in (15) isolates (93.75%) of the positive sample which gave molecular length (438bp) band. Molecular detection of sipA gene in S. typhi present of this gene in all (16) isolated (100%) positive, which gave molecular length (1126bp). Molecular detection of sdiA gene was done for (16) isolated of S. typhi, and the results showed that (15) isolates (93.75%) the positive results for sdiA virulence were detected by the presence of (274bp) band compared with allelic ladder. This study was conducted to identify the presence of Salmonella enterica serovar typhi within five isolated bacterial samples. One genetic locus covering a particular coding portion within the sdiA gene, which is responsible on the encoding of cell-division regulatory protein, was selected for amplification

Molecular Characterization of the viaB Locus Encoding the Biosynthetic Machinery for Vi Capsule Formation in Salmonella Typhi

The Vi capsular polysaccharide (CPS) of Salmonella enterica serovar Typhi, the cause of human typhoid, is important for infectivity and virulence. The Vi biosynthetic machinery is encoded within the viaB locus composed of 10 genes involved in regulation of expression (tviA), polymer synthesis (tviB-tviE), and cell surface localization of the CPS (vexA-vexE). We cloned the viaB locus from S. Typhi and transposon insertion mutants of individual viaB genes were characterized in Escherichia coli DH5α. Phenotype analysis of viaB mutants revealed that tviB, tviC, tviD and tviE are involved in Vi polymer synthesis. Furthermore, expression of tviB-tviE in E. coli DH5α directed the synthesis of cytoplasmic Vi antigen. Mutants of the ABC transporter genes vexBC and the polysaccharide copolymerase gene vexD accumulated the Vi polymer within the cytoplasm and productivity in these mutants was greatly reduced. In contrast, de novo synthesis of Vi polymer in the export deficient vexA mutant was comparable to wild-type cells, with drastic effects on cell stability. VexE mutant cells exported the Vi, but the CPS was not retained at the cell surface. The secreted polymer of a vexE mutant had different physical characteristics compared to the wild-type Vi.

Quick PCR based diagnosis of typhoid using specific genetic markers

A quick multiplex PCR based detection method was developed for early diagnosis of typhoid using specific genetic markers of S. typhi. Primers of tyv gene, flag gene, viaB gene and ratA gene confirmed the specificity and sensitivity of the PCR. The serum samples of the suspected typhoid patients were taken directly for PCR without culturing and isolating genomic DNA. Overall diagnosis required 2 h which is the least time ever reported for a PCR based methods. The sensitivity of the method is up to 5 fg genomic DNA. The genetic markers are specific and the four pairs of primers give selective amplification and differentiate S. typhi from closely related S. typhimurium.

TheSalmonella entericaserovar Typhi Vi capsule and self-association pili share controls on expression

Salmonella enterica serovar Typhi uses type IVB pili to facilitate eukaryotic cell invasion. Here, we compare environmental and genetic controls on pil operon transcription with those regulating viaB genes required for Vi antigen expression. Transcription of pil occurs only in the late logarithmic and stationary phases of bacterial growth while viaB expression occurs in the logarithmic growth phase. Expression of both viaB and pil was, however, optimal at 100 mM NaCl, and mutations in envZ/ompR, rcsB/rcsC, (but not rcsA), tviA, ihfB or fis affected transcription of both viaB and pil DNA. As both Vi antigen and Type IVB pili facilitate serovar Typhi invasion of human monocytes, an overlap of production controls is logical. It appears that Vi antigen synthesis precedes pilus production.

Enhanced detection rate of typhoid fever in children in a periurban slum in Karachi, Pakistan using polymerase chain reaction technology

Objective. Yield of blood culture in clinically suspected cases of typhoid fever is low, whereas indirect serological diagnostic tests are unreliable. Hence, polymerase chain reaction (PCR)‐based detection of Salmonella enterica Serovar typhi was used as an aid for diagnosis of typhoid fever in addition to other diagnostic tests. Two periurban communities in Karachi were selected for an epidemiological study of typhoid fever. The aim of the study was to assess whether PCR increased the detection rate of typhoid fever in children in the community. Material and methods. Children aged 2 to 14 years presenting with fever lasting for three or more days were selected. PCR using Hashimoto's protocol based on ViaB gene sequence was used in addition to blood culture and other serological tests. Results. Of the 214 children included in the study, blood culture was found positive for S. enterica S. typhi in 26 (12.4%) cases, whereas 24 children (11.7%) were diagnosed as suffering from typhoid fever when the PCR‐based method was used. Both tests were positive in only 10 (4.9%) children. The number of children found positive for either test was 40. PCR increased the rate of detection of typhoid fever by 51%. Conclusion. The sensitivity, specificity, +ve and −ve predictive values of PCR in this study were 40%, 93%, 45% and 92%, respectively.

Development of PCR Primers for the Detection of Salmonella enterica Serovar Choleraesuis Based on the fliC Gene

Salmonella enterica serovar Choleraesuis may cause swine salmonellosis and human infection. Because the conventional method for detection of this Salmonella serovar may take 3 to 5 days, a PCR method for detection was evaluated. By comparing the sequence of the phase 1 flagellin (fliC) gene ofSalmonella Choleraesuis with that of other Salmonella serovars and of other bacteria species available in GenBank, two PCR primers (flinC-F and flinC-R) were designed. Using these primers, all 97 Salmonella Choleraesuis strains assayed generated the expected PCR product, with a molecular mass of 963 bp. Except for S. enterica Paratyphi C, Salmonella isolates other than Salmonella Choleraesuis and non-Salmonella isolates, including strains of Enterobacteriaceae, all generated negative PCR results. Salmonella Paratyphi C could be differentiated from Salmonella Choleraesuis through the use of primers designed from the viaB gene. When Salmonella Choleraesuis isolates from swine stool, pork, liver, feed, and human whole blood samples were assayed with a preenrichment step, as low as 1 CFU/g or ml of the original sample could be detected.

Role of IS1 in the conversion of virulence (Vi) antigen expression in Enterobacteriaceae.

When Escherichia coli HB101 harbors pWR127, a plasmid comprising the viaB gene from Citrobacter freundii WR7004 and the ColE1-derived pACKC1, the strain produces the virulence (Vi) antigen. Vi antigen expression is abolished (Vi- phenotype), however, when an IS1 or IS1-like DNA element inserts into the viaB region. To determine the sites of IS1 insertion, pWR127 DNAs extracted from 95 independently isolated Vi- strains were analyzed by digestion with the restriction endonuclease PstI and agarose gel electrophoresis. Ten insertion sites were found distributed non-randomly in an area of about 1.3 kb. Nine Vi+ strains (two Citrobacter, two E. coli, and five Salmonella strains), four of which contain pWR127, were then tested for the presence of IS1 by DNA-DNA hybridization. Of the nine strains, five were stable Vi+ and did not contain IS1. The other four which generated Vi- strains, contained IS1. When pRR134, a plasmid that contains IS1 was transferred into a stable Vi+ Salmonella typhimurium strain carrying pWR127 (OU5140), Vi- strains were produced from which pWR127 derivatives carrying IS1 inserts could be isolated. It appears, therefore, that the presence of an IS1 or IS1-like element in a strain is required for conversion of the Vi+ expression state to the Vi- expression state.

Construction of a recombinant oral vaccine against Salmonella typhi and Salmonella typhimurium

The viaB gene coding for the Vi antigen of Salmonella typhi Ty2 was subcloned into expression vector pYA248. The recombinant plasmid was termed SMM202 and transformed into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant. Recombinant S. typimurium Vi4072 had the ability to produce Vi capsular polysaccharide and also to invade and colonize the small intestine, mesenteric lymph nodes, and spleen of BALB/c mice. Mice orally immunized with Vi4072 developed serum and secretory antibody responses to the Vi antigen, as measured by a passive hemagglutination assay. Mice developed a delayed-type hypersensitivity following a footpad injection with Vi antigen after being sensitized orally with a suitable dose of Vi4072. Immunization of mice with Vi4072 afforded complete protection against fatal infection with virulent S. typhi Ty2. All data indicate that this route of antigen delivery is effective for stimulating antibody-mediated immunity and for inducing a cell-mediated immune response in BALB/c mice. Thus, S. typhimurium Vi4072 may serve as a vaccine for protection against typhoid fever and salmonellosis caused by S. typhimurium.

Development of a DNA probe to detect Salmonella typhi.

This study was undertaken to identify a DNA sequence that could be used to facilitate the diagnostic identification of Salmonella typhi, the causative agent of typhoid fever. All virulent S. typhi strains encode a relatively unique capsular antigen termed the virulence (Vi) antigen. Two distinct genetic loci, viaA and viaB, are involved in the synthesis of this antigen. The structural genes, located at viaB, were considered as a possible specific DNA probe. The viaB locus, contained in a recombinant cosmid, was subcloned to various plasmid vectors for this purpose. Selected viaB-region DNA fragments were then analyzed for specificity in DNA colony hybridization reactions with more than 170 strains representing a variety of enteric bacteria. An 8.6-kilobase EcoRI fragment was highly specific for the viaB gene region and was considered a good hybridization probe. This DNA probe should prove useful in rapid diagnostic assays set up to detect S. typhi in mixed bacterial samples (e.g., stools) within a few hours of specimen collection.

Genetic basis of Vi antigen expression in Salmonella paratyphi C.

Analysis of hybrids formed in a cross between a Salmonella paratyphi C Hfr and an S. typhimurium recipient indicated that the structural genetic determinants of the S. paratyphi C Vi antigen are located closely adjacent to the mel determinant, between this marker and purA. A similar location was indicated for the structural genetic determinants of the S. typhi Vi antigen (the viaB locus) by the results of a mating in which a hybrid S. typhimurium Hfr bearing the S. typhi viaB determinants was used to transfer these genes to an S. typhimurium recipient. Mating experiments with a Vi-antigen-expressing S. typhi Hfr and an S. typhimurium hybrid recipient expressing the Vi antigen of S. paratyphi C yielded no recombinants in which loss of Vi antigen expression occurred, indicating that the chromosomal locus occupied by the genetic determinants of the S. paratyphi C Vi antigen is the same one at which, in S. typhi, the viaB genes reside. Introduction of a mutant S. typhi viaA gene into an S. typhimurium hybrid expressing the Vi antigen, as the consequence of prior receipt of the S. paratyphi C viaB determinants, resulted in that hybrid's loss of Vi antigen expression, demonstrating that the viaA determinant plays a role in Vi antigen expression in S. paratyphi C, as well as in S. typhi. Although the percentages of coinheritance of the viaB and mel determinants in the mating experiments suggested that their linkage is sufficiently close to allow cotransduction by P22, attempts to accomplish this with lysates prepared on S. typhimurium hybrids expressing either S. typhi or S. paratyphi C viaB determinants were not successful.