Web-based Investigation of Multistate Salmonellosis Outbreak

We investigated a large outbreak of Salmonella enterica serotype Javiana among attendees of the 2002 U.S. Transplant Games, including 1,500 organ transplant recipients. Web-based survey methods identified pre-diced tomatoes as the source of this outbreak, which highlights the utility of such investigative tools to cope with the changing epidemiology of foodborne diseases

Control of serine integrase recombination directionality by fusion with the directionality factor

Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The ‘reverse’ reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase–RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase–RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle

New Applications for Phage Integrases

Within the last twenty-five years bacteriophage integrases have rapidly risen to prominence as genetic tools for a wide range of applications from basic cloning to genome engineering. Serine integrases such as that from ϕC31 and its relatives have found an especially wide-range of applications within diverse micro-organisms right through to multi-cellular eukaryotes. Here we review the mechanisms of the two major families of integrases, the tyrosine and serine integrases, and the advantages and disadvantages of each type as they are applied in genome engineering and synthetic biology. In particular, we focus on the new areas of metabolic pathway construction and optimisation, bio-computing, heterologous expression and multiplexed assembly techniques. Integrases are versatile and efficient tools that can be used in conjunction with the various extant molecular biology tools to streamline the synthetic biology production line

HTLV-1 propels thymic human T cell development in “human immune system” Rag2-/- IL-2R γc-/- Mice

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that TaxHTLV-1 interferes with ß-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αβ T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a “Human Immune System” (HIS) Rag2-/-γc-/- mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2-/-γc-/- mice, mature single-positive (SP) CD4+ and CD8+ cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2-/-γc-/- animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1

Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing.

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices

Multiplex, Bead-Based Suspension Array for Molecular Determination of Common Salmonella Serogroups▿ †

We report the development and evaluation of a Salmonella O-group-specific Bio-Plex assay to detect the six most common serogroups in the United States (B, C1, C2, D, E, and O13) plus serotype Paratyphi A. The assay is based on rfb gene targets directly involved in O-antigen biosynthesis; it can be completed 45 min post-PCR amplification. The assay correctly and specifically identified 362 of 384 (94.3%) isolates tested in comparison to traditional serotyping. Seventeen isolates (4.4%) produced results consistent with what is known about the molecular basis for serotypes but different from the results of traditional serotyping, and five isolates (1.3%) generated false-negative results. Molecular determination of the serogroup for rough isolates was consistent with a common serotype in most instances, indicating that this approach has the potential to provide O-group information for isolates that do not express an O antigen. We also report the sequence of the O-antigen-encoding rfb gene cluster from Salmonella enterica serotype Poona (serogroup O13). Compared with other, previously characterized rfb regions, the O13 rfb gene cluster was most closely related to Escherichia coli O127 and O86. The O-group Bio-Plex assay described here provides an easy-to-use, high-throughput system for rapid detection of common Salmonella serogroups

Establishment of a Universal Size Standard Strain for Use with the PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocols: Converting the National Databases to the New Size Standard

The PulseNet National Database, established by the Centers for Disease Control and Prevention in 1996, consists of pulsed-field gel electrophoresis (PFGE) patterns obtained from isolates of food-borne pathogens (currently Escherichia coli O157:H7, Salmonella, Shigella, and Listeria) and textual information about the isolates. Electronic images and accompanying text are submitted from over 60 U.S. public health and food regulatory agency laboratories. The PFGE patterns are generated according to highly standardized PFGE protocols. Normalization and accurate comparison of gel images require the use of a well-characterized size standard in at least three lanes of each gel. Originally, a well-characterized strain of each organism was chosen as the reference standard for that particular database. The increasing number of databases, difficulty in identifying an organism-specific standard for each database, the increased range of band sizes generated by the use of additional restriction endonucleases, and the maintenance of many different organism-specific strains encouraged us to search for a more versatile and universal DNA size marker. A Salmonella serotype Braenderup strain (H9812) was chosen as the universal size standard. This strain was subjected to rigorous testing in our laboratories to ensure that it met the desired criteria, including coverage of a wide range of DNA fragment sizes, even distribution of bands, and stability of the PFGE pattern. The strategy used to convert and compare data generated by the new and old reference standards is described

Emergence of multidrug-resistant salmonella concord infections in europe and the united states in children adopted from ethiopia, 2003–2007

Background: Multidrug-resistant Salmonella serovar Concord infections have been reported from children adopted from Ethiopia. We interviewed patients, characterized the isolates, and gathered information about adoptions from Ethiopia to assess public health implications. Methods: Information about Salmonella Concord cases and adoptions were provided from Austria, Denmark, England (and Wales), Ireland, the Netherlands and the United States. Patients from Denmark and the United States were interviewed to determine the orphanages of origin; orphanages in Ethiopia were visited. Isolates were subtyped by pulsed-field gel electrophoresis and antimicrobial susceptibility; specific antimicrobial resistance genes were characterized. Results: Salmonella Concord was isolated from 78 persons from 2003 to 2007. Adoption status was known for 44 patients <= 3 years of age; 98% were adopted from Ethiopia. The children adopted from Ethiopia were from several orphanages; visited orphanages had poor hygiene and sanitation and frequent use of antimicrobial agents. The number of children adopted from Ethiopia in the participating countries increased 527% from 221 in 2003 to 1385 in 2007. Sixty-four Salmonella Concord isolates yielded 53 pulsed-field gel electrophoresis patterns including 6 patterns with >2 indistinguishable isolates; one isolate from an Ethiopia adoptee. Antimicrobial susceptibility was per-formed on 43 isolates; 81% were multidrug-resistant (>= 3 agents). Multidrug-resistant isolates were from Ethiopian adoptees and were resistant to third and fourth generation cephalosporins and 14% had decreased susceptibility to ciprofloxacin. Conclusions: Improved hygiene and sanitation and more appropriate use of antimicrobial agents are needed in orphanages in Ethiopia. Culturing of stool specimens of children adopted from Ethiopia and appropriate hygiene may prevent further disease transmission

Emergence of Multidrug-Resistant Salmonella Concord Infections in Europe and the United States in Children Adopted From Ethiopia, 2003-2007

Background: Multidrug-resistant Salmonella serovar Concord infections have been reported from children adopted from Ethiopia. We interviewed patients, characterized the isolates, and gathered information about adoptions from Ethiopia to assess public health implications. Methods: Information about Salmonella Concord cases and adoptions were provided from Austria, Denmark, England (and Wales), Ireland, the Netherlands and the United States. Patients from Denmark and the United States were interviewed to determine the orphanages of origin; orphanages in Ethiopia were visited. Isolates were subtyped by pulsed-field gel electrophoresis and antimicrobial susceptibility; specific antimicrobial resistance genes were characterized. Results: Salmonella Concord was isolated from 78 persons from 2003 to 2007. Adoption status was known for 44 patients <= 3 years of age; 98% were adopted from Ethiopia. The children adopted from Ethiopia were from several orphanages; visited orphanages had poor hygiene and sanitation and frequent use of antimicrobial agents. The number of children adopted from Ethiopia in the participating countries increased 527% from 221 in 2003 to 1385 in 2007. Sixty-four Salmonella Concord isolates yielded 53 pulsed-field gel electrophoresis patterns including 6 patterns with >2 indistinguishable isolates; one isolate from an Ethiopia adoptee. Antimicrobial susceptibility was per-formed on 43 isolates; 81% were multidrug-resistant (>= 3 agents). Multidrug-resistant isolates were from Ethiopian adoptees and were resistant to third and fourth generation cephalosporins and 14% had decreased susceptibility to ciprofloxacin. Conclusions: Improved hygiene and sanitation and more appropriate use of antimicrobial agents are needed in orphanages in Ethiopia. Culturing of stool specimens of children adopted from Ethiopia and appropriate hygiene may prevent further disease transmission