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AIMS: Present study has explored the protective response of dual immunization using two different antigenic entities (i.e. flagellin epitope and cytolethal distending toxin subunit B (CdtB) protein) against lethal challenge of typhoidal serovars in a murine model. MAIN METHODS: In-vitro immunogenicity of flagellin epitope-BSA conjugate and CdtB protein was confirmed using Indirect ELISA of typhoid positive patients' sera. Further, both entities were administered intraperitoneally in mice individually or in combination, followed by lethal challenge of typhoidal Salmonellae. Various parameters were analysed such as bacterial burden, mice survival, histopathological analysis, cytokine analysis and immunophenotyping. Serum samples obtained from the immunized mice were used for passive immunization studies, wherein mice survival and mechanism of action of the generated antibodies was studied. KEY FINDINGS: Active immunization studies using the combination of both entities demonstrated improved mice survival after lethal challenge with typhoidal Salmonellae, reduced bacterial burden in organs, expression of immunophenotypic markers in splenocytes and restored tissue histoarchitecture. When used in combination, the effective doses of both the candidates reduced which may be attributed to multiprong approach used by the immune system to recognize Salmonella. Passive immunization studies further determined the protective efficacy of generated antibodies by different mechanisms such as complement mediated bactericidal action, swarming inhibition and increased phagocytic uptake. SIGNIFICANCE: Present study is the first phase of the proof-of-concept which may prove to be beneficial in developing an effective bi-functional vaccine candidate to render protection against both Vi-positive as well as Vi-negative Salmonella strains.
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Fiebre Tifoidea , Vacunas Tifoides-Paratifoides , Humanos , Animales , Ratones , Fiebre Tifoidea/prevención & control , Flagelina , Epítopos , Inmunización , Vacunación , SalmonellaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Typhoid fever caused by Salmonella enterica serovar Typhi (S.Typhi) continues to be a major problem, especially in developing countries. Due to the rapid emergence of multi-drug-resistant (MDR) strains, which limits the efficacy of conventional antibiotics as well as problems associated with the existing vaccines, efforts are being made to develop effective prophylactic agents. CdtB subunit of typhoid toxin was selected for assessing its vaccine potential due to its high conservation throughout the Typhi strains. In-vitro assessment of DNase activity of cloned and purified CdtB protein showed a significant decrease in the band intensity of DNA. The measure of metabolic activity and morphological alterations assessed using different cell lines in the presence of CdtB protein showed no significant signs of toxicity. These observations were further strengthened by cell cycle analysis, assessed by flow cytometry. Keeping these observations in mind, the immunoprotective potential of CdtB was assessed using S.Typhi induced mouse peritonitis model. A significant titer of IgG antibodies (>128000) against CdtB protein was recorded in the immunized mice by enzyme-linked immunosorbent assay (ELISA), which was also validated by immunoblotting. Active immunization with the protein protected 75% mice against a lethal dose of S.Typhi Ty2. The data indicated a significant (up to 5 log) reduction in the bacterial load in the spleen and liver of immunized-infected mice compared to control (unimmunized-infected) mice which might have resulted in the modulation of histoarchitecture of spleen and liver and the levels of cytokines (IL-6, TNF-α and IL-10) production; thereby indicating the effectiveness of the subunit. The observations deduced from the study give the proof of concept of immunogenic potential of protein. However, further studies involving the immunoreactivity of CdtB with the statistically significant number of sera samples obtained from the human patients would be helpful in establishing the relevance of CdtB protein in humans and for making the strategies to develop it as an effective vaccine candidate.
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Anticuerpos Antibacterianos/biosíntesis , Toxinas Bacterianas/administración & dosificación , Inmunoglobulina G/biosíntesis , Peritonitis/prevención & control , Salmonella typhi/efectos de los fármacos , Fiebre Tifoidea/prevención & control , Vacunas Tifoides-Paratifoides/administración & dosificación , Animales , Toxinas Bacterianas/inmunología , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inmunización/métodos , Inmunogenicidad Vacunal , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/microbiología , Ratones , Peritonitis/inmunología , Peritonitis/microbiología , Peritonitis/mortalidad , Salmonella typhi/inmunología , Salmonella typhi/patogenicidad , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/microbiología , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Fiebre Tifoidea/inmunología , Fiebre Tifoidea/microbiología , Fiebre Tifoidea/mortalidad , Vacunas Tifoides-Paratifoides/inmunologíaRESUMEN
We report a novel aptamer functionalized MoS2-rGO based electrochemical method for Vi polysaccharide antigen mediated detection of enteric fever. Herein, highly selective anti-Vi aptamers were screened from a pool of oligonucleotides using a microtitre based SELEX approach and characterized for its specificity and stability. The MoS2-rGO nanocomposite was synthesized using a liquid assisted exfoliation by taking optimum ratio of MoS2 and rGO. The nanocomposite presented synergistic effect owing to easy biomolecular functionalization and enhanced conductivity. The screened anti-Vi aptamers were embedded on the MoS2-rGO nanocomposite via thiol linkage to give a stable biointerface. The developed aptasensor was characterized and further evaluated for its performance with different concentrations of Vi antigen using ferrocene labeled boronic acid as an electroactive probe. The aptasensor responded linearly in the range between 0.1â¯ngâ¯mL-1 to 1000â¯ngâ¯mL-1with a detection limit of 100â¯pgâ¯mL-1, and did not show any cross-reactivity with other bacterial polysaccharides indicating high specificity. The applicability of the developed aptasensor was further validated in urine and sera specimens spiked with Vi antigen.
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Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Grafito/química , Nanocompuestos/química , Polisacáridos Bacterianos/sangre , Polisacáridos Bacterianos/orina , Salmonella typhi/aislamiento & purificación , Ácidos Borónicos/química , Disulfuros/química , Compuestos Ferrosos/química , Humanos , Límite de Detección , Metalocenos/química , Molibdeno/química , Nanocompuestos/ultraestructura , Polisacáridos Bacterianos/análisis , Fiebre Tifoidea/sangre , Fiebre Tifoidea/diagnóstico , Fiebre Tifoidea/microbiología , Fiebre Tifoidea/orinaRESUMEN
Bacterial persisters (defined as dormant, non-dividing cells with globally reduced metabolism) are the major cause of recurrent infections. As they neither grow nor die in presence of antibiotics, it is difficult to eradicate these cells using antibiotics, even at higher concentrations. Reports of metabolites (which help in waking up of these inactive cells) enabled eradication of bacterial persistence by aminoglycosides, suggest the new potential strategy to improve antibiotic therapy. Here we propose, mannitol enabled elimination of Salmonella persister cells by the nisin-antibiotic combination. For this, persister cells were developed and characterized for their typical properties such as non-replicative state and metabolic dormancy. Different carbon sources viz. glucose, glycerol, and mannitol were used, each as an adjunct to ampicillin for the eradication of persister cells. The maximum (but not complete) killing was observed with mannitol-ampicillin, out of all the combinations used. However, significant elimination (about 78%) could be observed, when nisin (an antimicrobial peptide) was used with ampicillin in presence of mannitol, which might have mediated the transfer of antibiotic-nisin combination at the same time when the cells tried to grab the carbon molecule. Further, the effectiveness of the trio was confirmed by flow cytometry. Overall, our findings highlight the potential of this trio-combination for developing it as an option for tackling Salmonella persister cells.
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A specific surface antigen, OmpD has been reported first time as a surface biomarker in the development of selective and sensitive immunosensor for detecting Salmonella typhimurium species. The OmpD surface antigen extraction was done from Salmonella typhimurium serovars, under the optimized growth conditions for its expression. Anti-OmpD antibodies were generated and used as detector probe in immunoassay format on graphene-graphene oxide (G-GO) modified screen printed carbon electrodes. The water samples were spiked with standard Salmonella typhimurium cells, and detection was done by measuring the change in impedimetric response of developed immunosensor to know the concentration of serovar Salmonella typhimurium. The developed immunosensor was able to specifically detect S. typhimurium in spiked water and juice samples with a sensitivity upto 10(1)CFUmL(-1), with high selectivity and very low cross-reactivity with other strains. This is the first report on the detection of Salmonella typhimurum species using a specific biomarker, OmpD. The developed technique could be very useful for the detection of nontyphoidal Salmonellosis and is also important from an epidemiological point of view.
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Anticuerpos Inmovilizados/química , Espectroscopía Dieléctrica/instrumentación , Jugos de Frutas y Vegetales/microbiología , Grafito/química , Porinas/análisis , Salmonella typhimurium/aislamiento & purificación , Microbiología del Agua , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Humanos , Inmunoensayo/instrumentación , Límite de Detección , Óxidos/química , Infecciones por Salmonella/microbiologíaRESUMEN
Combining synthetic macromolecules and biomolecular recognition units are promising in developing novel diagnostic and analysis techniques for detecting environmental and/or clinically important substances. Fluorescence resonance energy transfer (FRET) apta-immunosensor for explosive detection is reported using 2,4,6-trinitrotoluene (TNT) specific aptamer and antibodies tagged with respective FRET pair dyes in a sandwich immunoassay format. FITC-labeled aptamer was used as a binder molecule in the newly developed apta-immunoassay format where the recognition element was specific anti-TNT antibody labeled with rhodamine isothiocyanate. The newly developed sensing platform showed excellent sensitivity with a detection limit of the order of 0.4 nM presenting a promising candidate for routine screening of TNT in samples.
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Anticuerpos/química , Aptámeros de Nucleótidos/química , Transferencia Resonante de Energía de Fluorescencia , Trinitrotolueno/análisis , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
Specific nucleic acid aptamers using the microtiter plate based modified SELEX method against explosive trinitrotoluene are reported. Efficient partitioning of dsDNA was carried out using streptavidin labeled gold nanoprobes for the selection of specific aptamers. The selected binders having an affinity of ~10(-7) M were used in the newly developed electrochemical aptasensor, exhibiting a detection limit of around 1 ppb for trinitrotoluene.