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Rapid and highly efficient method for scarless mutagenesis within the Salmonella enterica chromosome.

ABSTRACT: Direct manipulation of bacterial chromosomes by recombination-based techniques has become increasingly important for both cognitive and applied research. Here we demonstrate, for the first time, the combination of the Red recombinase system with I-SceI endonuclease-based selection of successful recombinants after electroporation with short synthetic oligonucleotides. We show the generation of scarless gene knockouts as well as site-directed mutagenesis using the Salmonella virulence-associated two component signaling system PhoPQ. The presented approach is very versatile for generating in-frame deletions, point mutations or insertions within bacterial chromosomes.

SUBMITTER: Blank K

PROVIDER: S-EPMC3021506 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Rapid and highly efficient method for scarless mutagenesis within the Salmonella enterica chromosome.

Blank Kathrin K Hensel Michael M Gerlach Roman G RG

PloS one 20110114 1

Direct manipulation of bacterial chromosomes by recombination-based techniques has become increasingly important for both cognitive and applied research. Here we demonstrate, for the first time, the combination of the Red recombinase system with I-SceI endonuclease-based selection of successful recombinants after electroporation with short synthetic oligonucleotides. We show the generation of scarless gene knockouts as well as site-directed mutagenesis using the Salmonella virulence-associated t ...[more]

PMID: 21264289

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Project description:BackgroundSalmonella Typhi causes an estimated 21 million new cases of typhoid fever and 216,000 deaths every year. Blood culture is currently the gold standard for diagnosis of typhoid fever, but it is time-consuming and takes several days for isolation and identification of causative organisms. It is then too late to initiate proper antibiotic therapy. Serological tests have very low sensitivity and specificity, and no practical value in endemic areas. As early diagnosis of the disease and prompt treatment are essential for optimal management, especially in children, a rapid sensitive detection method for typhoid fever is urgently needed. Although PCR is sensitive and rapid, initial research indicated similar sensitivity to blood culture and lower specificity. We developed a fast and highly sensitive blood culture PCR method for detection of Salmonella Typhi, allowing same-day initiation of treatment after accurate diagnosis of typhoid.MethodsAn ox bile tryptone soy broth was optimized for blood culture, which allows the complete lysis of blood cells to release intracellular bacteria without inhibiting the growth of Salmonella Typhi. Using the optimised broth Salmonella Typhi bacteria in artificial blood samples were enriched in blood culture and then detected by a PCR targeting the fliC-d gene of Salmonella Typhi.ResultsTests demonstrated that 2.4% ox bile in blood culture not only lyzes blood cells completely within 1.5 hours so that the intracellular bacteria could be released, but also has no inhibiting effect on the growth of Salmonella Typhi. Three hour enrichment of Salmonella Typhi in tryptone soya broth containing 2.4% ox bile could increase the bacterial number from 0.75 CFU per millilitre of blood which is similar to clinical typhoid samples to the level which regular PCR can detect. The whole blood culture PCR assay takes less than 8 hours to complete rather than several days for conventional blood culture.ConclusionsThis novel blood culture PCR method is superior in speed and sensitivity to both conventional blood culture and PCR assays. Its use in clinical diagnosis may allow early detection of the causative organism and facilitate initiation of prompt treatment among patients with typhoid fever.

Project description:UNLABELLED:Nontyphoidal Salmonella enterica (NTS) infections are a major burden to global public health, as they lead to diseases ranging from gastroenteritis to systemic infections and there is currently no vaccine available. Here, we describe a highly effective component vaccine against S. enterica serovar Typhimurium in both gastroenteritis and systemic murine infection models. We devised an approach to generate supernatants of S. enterica serovar Typhimurium, an organism that is highly abundant in virulence factors. Immunization of mice with this supernatant resulted in dramatic protection against a challenge with serovar Typhimurium, showing increased survival in the systemic model and decreased intestinal pathology in the gastrointestinal model. Protection correlated with specific IgA and IgG levels in the serum and specific secretory IgA levels in the feces of immunized mice. Initial characterization of the protective antigens in the bacterial culture supernatants revealed a subset of antigens that exhibited remarkable stability, a highly desirable characteristic of an effective vaccine to be used under suboptimal environmental conditions in developing countries. We were able to purify a subset of the peptides present in the supernatants and show their potential for immunization of mice against serovar Typhimurium resulting in a decreased level of colonization. This component vaccine shows promise with regard to protecting against NTS, and further work should significantly help to establish vaccines against these prevalent infections. IMPORTANCE:Salmonella enterica infections other than typhoid and paratyphoid fever are a major global health burden, as they cause high morbidity and mortality worldwide. Strategies that prevent Salmonella-related diseases are greatly needed, and there is a significant push for the development of vaccines against nontyphoidal Salmonella enterica serovars. In this work, we describe an S. Typhimurium supernatant-derived vaccine that is effective in reducing bacterial colonization in mouse models of gastroenteritis as well as invasive disease. This is a component vaccine that shows high stability to heat, a feature that is important for use under suboptimal conditions, such as those found in sub-Saharan Africa.

Dataset Information

Rapid and highly efficient method for scarless mutagenesis within the Salmonella enterica chromosome.

Publications

Rapid and highly efficient method for scarless mutagenesis within the Salmonella enterica chromosome.

Similar Datasets

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