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Rat Jugular Catheter Model of Biofilm-Mediated Infection
Staphylococcus epidermidis is now recognized as the primary cause of nosocomial catheter-mediated infections. Bacteria may be introduced exogenously via contamination of the catheter hub or insertion site and endogenously from sepsis. The in vivo model described in this chapter examines the infection resulting from hematogenous seeding of jugular vein catheters. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

Generation of a Central Nervous System Catheter-Associated Infection in Mice with Staphylococcus epidermidis
Animal models are valuable tools for investigating the in vivo pathogenesis of Staphylococcus epidermidis infections. Here, we present the procedure for generating a central nervous system catheter-associated infection in a mouse, to model the central nervous system shunt infections that frequently complicate the treatment of hydrocephalus in humans. This model uses stereotactic guidance to place silicone catheters, pre-coated with S. epidermidis, into the lateral ventricles of mice. This results in a catheter-associated infection in the brain, with concomitant illness and inflammation. This animal model is a valuable tool...
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

A Mouse Model of Staphylococcus Catheter-Associated Biofilm Infection
Biofilms are adherent communities of bacteria contained within a complex matrix. Staphylococcal species are frequent etiological agents of device-associated biofilm infections in humans that are highly recalcitrant to antimicrobial therapy and alter host immune responses to facilitate bacterial persistence. Here we describe a mouse model of catheter-associated biofilm infection, which can be utilized to investigate the importance of various staphylococcal determinants on disease progression as well as the host immune response to staphylococcal biofilms. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

Mouse Model of Post-arthroplasty Staphylococcus epidermidis Joint Infection
Animal models are invaluable tools for translational research, allowing investigators to recapitulate observed clinical scenarios within the laboratory that share attributes with human disease. Here, we describe a mouse model of post-arthroplasty Staphylococcus epidermidis joint infection which mimics human disease and may be utilized to explore the complex series of events during staphylococcal implant-associated infections by identifying key immunological, bacterial, and/or therapeutic mechanisms relevant to these persistent infections. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

Bacteriophage Transduction in Staphylococcus epidermidis
The genetic manipulation of Staphylococcus epidermidis for molecular experimentation has long been an area of difficulty. Many of the traditional laboratory techniques for strain construction are laborious and hampered by poor efficiency. The ability to move chromosomal genetic markers and plasmids using bacteriophage transduction has greatly increased the speed and ease of S. epidermidis studies. These molecular genetic advances have advanced the S. epidermidis research field beyond a select few genetically tractable strains and facilitated investigations of clinically relevant isolates. (Source: Springer protocols feed b...
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

Rapid Quantitative and Qualitative Analysis of Biofilm Production by Staphylococcus epidermidis Under Static Growth Conditions
Rapid screening of biofilm forming capacity by Staphylococcus epidermidis is possible using in vitro assays with 96-well plates. This method first developed by Christensen et al. in 1985 is fast and does not require specialized instruments. Thus, laboratories with standard microbiology infrastructure and a 96-well plate reader can easily use this technique to generate data on the biofilm phenotypes of multiple S. epidermidis strains and clinical isolates. Furthermore, this method can be adapted to gain insights into biofilm regulation and the characteristics of biofilms produced by different S. epidermidis isolates. Althou...
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

Examination of Staphylococcus epidermidis Biofilms Using Flow-Cell Technology
A common in vitro method to study Staphylococcus epidermidis biofilm development is to allow the bacteria to attach and grow on a solid surface in the presence of a continuous flow of nutrients. Under these conditions, the bacteria progress through a series of developmental steps, ultimately forming a multicellular structure containing differentiated cell populations. The observation of the biofilm at various time-points throughout this process provides a glimpse of the temporal changes that occur. Furthermore, use of metabolic stains and fluorescent reporters provides insight into the physiologic and transcriptional chang...
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

Methods to Generate a Sequence-Defined Transposon Mutant Library in Staphylococcus epidermidis Strain 1457
Transposon mutant libraries are valuable resources to investigators studying bacterial species, including Staphylococcus epidermidis, which are difficult to genetically manipulate. Although sequence-defined transposon mutant libraries have been constructed in Staphylococcus aureus, no such library exists for S. epidermidis. Nevertheless, the study of Tn917-mediated mutations has been paramount in discovering unique aspects of S. epidermidis biology including initial adherence and accumulation during biofilm formation. Herein, we describe modifications to the methodology first described by Bae et al. to utilize the mariner-...
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

Use of Electroporation and Conjugative Mobilization for Genetic Manipulation of Staphylococcus epidermidis
To perform mechanistic studies on the biology of bacteria including metabolism, physiology, and pathogenesis, it is essential to possess the tools required for genetic manipulation. Introduction of plasmid DNA into Staphylococcus epidermidis for subsequent genetic manipulation, including allelic replacement and complementation experiments, is typically performed by either electroporation or conjugative mobilization. Herein, standard protocols and tips for the transfer of plasmid DNA to S. epidermidis by these two methods are provided. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

Isolation of Staphylococcus sp. RNA
Isolation of RNA (ribonucleic acid) is a valuable technique to study gene regulation and functional RNAs. It is important to obtain pure samples of RNA for downstream applications, while avoiding the negative effects of ribonucleases (RNases). Here we describe several methods of extracting RNA, with an emphasis on Staphylococcus RNA isolation, and measures that must be taken to ensure high-quality, high-yield RNA. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - November 18, 2013 Category: Infectious Diseases Source Type: news

Analyzing Caspase-1 Activation During Legionella pneumophila Infection in Macrophages
Caspase-1 is a critical factor in the innate immune response to Legionella pneumophila. The development of methods for analyzing caspase-1 activation pathways and downstream caspase-1-associated activities has helped in understanding the regulation of this protease and the signaling components involved. Here we outline methods for directly detecting active caspase-1, measuring caspase-1 activities and analyzing components involved in the regulation of caspase-1 during L. pneumophila infection in macrophages. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - February 1, 2013 Category: Infectious Diseases Source Type: news

Assessment of Legionella-Specific Immunity in Mice
Legionella pneumophila is the causative agent of the potentially fatal Legionnaires’ disease in humans. Mice have proved to be valuable model organisms to study the pathogenesis of this intracellular bacterium, as well as immune responses against it. In this chapter we describe a selection of mouse infection protocols to study the innate and adaptive immune responses raised after an infection with Legionella. Included are protocols for systemic and pulmonary infections, surgical collection of organs as well as determination of cell composition, cytokines, and antibody titers therein. Furthermore, we describe an immun...
Source: Springer protocols feed by Infectious Diseases - February 1, 2013 Category: Infectious Diseases Source Type: news

The Guinea Pig Model of Legionnaires’ Disease
The guinea pig pneumonia model mimics Legionnaires’ disease as seen in immunocompromised humans, with high untreated fatality rates and as such lends itself to studies of experimental chemotherapy. Guinea pig infection is also used to assess relative virulence of different Legionella bacterial strains, and has also been used to study host immune defenses. Here I describe the method used to produce Legionella sp. pneumonia in the guinea pig using the intratracheal infection technique. This method uses directly observed intratracheal injection of a bacterial suspension, requiring surgical exposure of the trachea. (Sour...
Source: Springer protocols feed by Infectious Diseases - February 1, 2013 Category: Infectious Diseases Source Type: news

Human Susceptibility to Legionnaires’ Disease
Legionella pneumophila is a facultative intracellular pathogen that is an important cause of pneumonia. Although host factors that may predispose to acquisition of Legionnaire’s Disease (LD) include comorbid illnesses (e.g., diabetes, chronic lung disease), age, male sex, and smoking, many individuals have no identifiable risk factors. Some studies suggest that genetic factors may enhance susceptibility to LD. In this chapter we discuss current techniques and scientific methods to identify genetic susceptibility factors. These genetic studies provide insight into the human immune response to intracellular pathogens a...
Source: Springer protocols feed by Infectious Diseases - February 1, 2013 Category: Infectious Diseases Source Type: news

cDNA Library Construction for Next-Generation Sequencing to Determine the Transcriptional Landscape of Legionella pneumophila
The adaptation of Legionella pneumophila to the different conditions it encounters in the environment and in the host is governed by a complex regulatory system. Current knowledge of these regulatory networks and the transcriptome responses of L. pneumophila is mainly based on microarray analysis and limited to transcriptional products of annotated protein-coding genes. The application of the Next-Generation Sequencing (NGS) technology allows now genome-wide strand-specific sequencing and accurate determination of all expressed regions of the genome to reveal the complete transcriptional network and the dynamic interplay o...
Source: Springer protocols feed by Infectious Diseases - February 1, 2013 Category: Infectious Diseases Source Type: news