Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis as a reliable evidence for suspected Shigella spp. outbreaks

Abstract Background Shigellosis remains a serious public health problem and an important cause of morbidity and mortality worldwide. The aim of this study was to characterize fliC and the genetic relatedness of Shigella spp. isolated during a one-year period from children in a suspected outbreak in Tehran, Iran. Methods and results Fifty Shigella spp. were isolated from 3779 stool samples of children with diarrhea (prevalence rate: 1.32%). Among the isolates, 92% were characterized as Shigella sonnei, while 6% and 2% were identified as S. flexneri and S. boydii, respectively. S. dysenteriae was not recovered from the patients. All isolates were negative for fliC except for Shigella standard strains. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) profiles allowed differentiating the 50 isolates into 5 ERIC types, which were grouped into five clusters (ET1-ET5). Computer-assisted clustering of the strains showed a high degree of similarity among the isolates. Conclusion In conclusion, given the clonal correlation of the Shigella strains isolated in this study and the lack of fliC among them, we propose that probably a single or limited fliC-defected Shigella clone spread and caused the outbreak.

Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.

Salmonella Enteritidis Flagella may Enhance Attachment and Invasion of Hen Ovarian Granulosa Cells and Induce Protective Immune Response in Egg-laying Hens.

Aims: To study the effect of flagellin on bacterial attachment and invasion of avian ovary cells in vitro by comparing the attachment and invasion of wild-type S. Enteritidis with nonmotile mutants. To assess the immunogenic properties of extracted flagellin against Salmonella Enteritidis experimental infection in laying hens. Methodology: Non-flagellated mutants for wild-type S. Enteritidis (phage type 8, 13A and 28) were produced by using a strain of S. Enteritidis, SA4502, which carried an fliC::Tn 10 to transfer fliC::Tn 10 insertion into the wild type strains using phage 22 (P22)-mediated transduction with selection for antibiotic resistance encoded within the mutant alleles. Granulosa cells were harvested from Single Comb White Leghorn hens between 18-45 weeks of age. Flagellin was purified from the studied bacterial cultures of Salmonella Enteritidis following reported methods. Laying hens were immunized with the flagellin with adjuvant Results: Non-motile mutants of S. Enteritidis phage wild types were analyzed to confirm the elimination of H1 flagellin synthesis. Wild-type and fliC mutant strains were assessed for their ability to adhere to hen's ovarian granulosa cells. The adherence of the mutant strain was reduced nearly ten-fold compared with that of the wild-type phage 8. Similarly, light microscopic observation of fixed cover slips from wild-type phage types and its mutant strain revealed fewer numbers of the bacterial mutants adhered to the cultured granulosa cell monolayer. Light microscopy revealed similar findings for mutant phage types 28 and 13 A when compared to the wild-type control. There was five folds rise in the egg yolk antibody during the 2-3 weeks post-immunization. No rise was detected in the egg yolk samples from the control hens injected with the placebo mixture without flagellin. Conclusion: It was concluded that Flagellin has an important role in the attachment and invasion of Salmonella Enteritidis to avian ovary cells and that it can be used as immunogenic components to induce a protective immune response in vaccinated hens against challenge infection with the wild type strains.

Identification of putative new Escherichia coli flagellar antigens from human origin using serology, PCR-RFlP and DNA sequencing methods

Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO) of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein) by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.

Characterization of a phase 1-d epitope on Salmonella typhi flagellin and its role in the serodiagnosis of typhoid fever.

A monoclonal antibody (MAb) directed against Salmonella typhi 52 kDa flagellin protein has been previously produced by our group. In this study, we have demonstrated that the epitope specific to the MAb is unique to phase 1-d. To map the epitope, plasmids encoding different regions of S. typhi flagellin gene were constructed. Analysis of protein produced from each recombinant plasmid indicated that the epitope specific to the MAb resided within amino acids 171-303 (region IV) of S. typhi flagellin protein. The recombinant region IV flagellin was used to develop an ELISA for the detection of IgM antibody to S. typhi in serum. In the hemoculture-positive typhoid group, the developed ELISA was positive in 77 of 92 cases. In patients with non-typhoidal Salmonella, gram-positive and gram-negative bacteria or dengue virus, the ELISA was negative in all 78 cases. Two from 116 healthy control subjects had positive reactions with the assay. The calculated sensitivity, specificity, positive and negative predictive values of the test were 83.7%, 99.0%, 97.5% and 92.8%, respectively. With such high validity together with the requirement of only a single serum specimen and one day for performing the test, the developed ELISA should become a valuable diagnostic test for typhoid fever.

Detection of IgM antibody against region IV flagellin of Salmonella paratyphi A.

Salmonella paratyphi A is a pathogenic bacterium that causes paratyphoid fever. The current laboratory diagnostic techniques are unsatisfactory. To improve diagnosis, a plasmid (pSK-8E) encoding phase 1 flagellin gene nucleotide position 452-890 from S. paratyphi A has been constructed. The recombinant protein expressed from the plasmid has been used to develop an indirect ELISA for IgM antibody detection. Sera from patients with hemoculture positive for S. paratyphi A, S. typhi, other gram-positive and gram-negative bacteria, and dengue hemorrhagic fever as well as from healthy control subjects were tested. Sensitivity, specificity, positive and negative predictive values of the test were 56.9%, 98.8%, 90.6% and 92.1%, respectively. Since the sensitivity was low, the explanation for this result was investigated. It was found that the sensitivity of the test could be increased to 83.3% if the sera were obtained 9-12 days after onset of fever. The sera obtained earlier or later gave only 33.3% and 66.6% sensitivity, respectively. This result suggests that the IgM antibody detection assay which we have developed is a valuable tool for diagnosis of S. paratyphi A infection when the serum samples are taken at the appropriate time.

Molecular cloning and expression of Salmonella paratyphi A 52 kDa specific protein gene.

Monoclonal antibodies (MAbs) specific to Salmonella paratyphi A have been established by our group in 1989. These MAbs were proven to be species-specific for 52 kDa protein of S. paratyphi A but the nature of this protein is unknown. However, our group have proved that the 52 kDa protein which is specific to S. typhi was flagellin. This present study has characterized the 52 kDa protein of S. paratyphi A and identified its encoded gene. The plasmid containing the specific 52 kDa antigen gene was cloned from the S. paratyphi A genome, herein designated pSKA-4. Partial nucleotide sequences from this clone was analysed by computer program and found to be phase 1-a flagellin gene of S. paratyphi A. In addition, the nucleotide sequence analysis from such clone also showed that the structural gene for phase 1 flagellin has amino acid sequences conserved at the terminal whereas the central region is variable among Salmonella spp. Therefore, the central portion of flagellin which highly polymorphic in amino acid sequences would be the most specific to S. paratyphi A, thus, should be used as specific antigen for developing specific diagnosis of S. paratyphi A infection. Using the PCR technique, an expression plasmid containing the antigen gene producing only the variable region in the central portion of flagellin from S. paratyphi A, namely pSKA-7, has been established. The recombinant protein produced by the established plasmid has a MW 33.5 kDa as detected by immunoblotting using specific MAbs. Further study by using this specific flagellin protein for immunodiagnosis of S. paratyphi A infection is being carried out in our laboratory.

Fusion protein of Salmonella typhi flagellin as antigen for diagnosis of typhoid fever.

We previously established the specific 52 kDa antigen of Salmonella typhi, detected by our monoclonal antibodies, which was a flagellin protein. Comparison of the nucleotide sequences of phase-1 flagellin of Salmonella species available through GenBank database showed high homology at both ends of the genes with lower degree of homology in the middle portion which contained the antigenically variable regions. Thus, proteins from the central regions of flagellin genes should be species specific and could be used as specific antigens for the immunodiagnostic tests. In this report, recombinant protein derived from the central region of S. typhi flagellin was produced as a fusion protein with glutathione-S-transferase. This fusion protein was used as specific S. typhi antigen for the immunodiagnostic test to detect IgM antibodies in sera using enzyme-linked immunosorbent assay. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of this test were 53.5, 98.0, 91.5, 82.1 and 92.4%, respectively.

Molecular cloning and expression of Salmonella typhi flagellin: characterization of 52 kDa specific antigen of S. typhi.

We previously reported monoclonal antibodies (MAbs) specific to S. typhi 52 kDa antigen which do not cross react with related protein antigens from 11 bacteria causing enteric fever and enteric fever-like illness. Using the combination of these specific MAbs and recombinant DNA technology, expression plasmids containing the antigen gene producing substantial amount of the S. typhi protein antigen have been established. Plasmid pSKM-T7 containing the specific 52 kDa antigen gene was cloned and the antigen expressed was detectable by immunoblotting using specific mAbs. The complete nucleotide sequence of this gene was compared with other bacterial sequences and found to be highly homologous with the flagellin gene H1-d of S. muenchen except in the hypervariable region in the central portion. The specific 52 kDa antigen of S. typhi detected by our MAbs is thus a flagellin.