Current protocols in microbiology

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  • ISSN
    1934-8533

Publications in this journal

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    ABSTRACT: Staphylococcus aureus is a facultative anaerobic Gram-positive coccus and a member of the normal skin flora, as well as that of the nasal passages of humans. However, S. aureus can also gain entry into the host and cause life-threatening infections or persist as disease foci that develop into suppurative abscesses. While genetically tractable, the manipulation of S. aureus remains challenging. This unit describes methods developed in our laboratory for gene disruption by allelic replacement and transposition. We also provide a protocol for bacteriophage-mediated transduction of mutants marked with selectable alleles and describe plasmid utilization for complementation studies. Curr. Protoc. Microbiol. 32:9C.3.1-9C.3.19. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 32:9C.3.1-9C.3.19.
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    ABSTRACT: Stenotrophomonas maltophilia is a ubiquitous soil bacterium that is increasingly recognized as an emerging nosocomial pathogen. This unit includes protocols for the in vitro growth and maintenance of S. maltophilia. Curr. Protoc. Microbiol. 32:6F.1.1-6F.1.6. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 32:6F.1.1-6.
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    ABSTRACT: Poliovirus (PV) is the prototypical picornavirus. It is a non-enveloped RNA virus with a small (∼7.5-kb) genome of positive polarity. It has long served as a model to study RNA virus biology, pathogenesis, and evolution. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro transcribed viral genomes into an appropriate host cell. PV infects many human and non-human primate cell lines including HeLa and HeLa S3 cells, and can grow to high titer in culture. Protocols for the production, propagation, quantification, and purification of PV are presented. Curr. Protoc. Microbiol. 29:15H.1.1-15H.1.27. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 05/2013; Chapter 15H:Unit15H.1.
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    ABSTRACT: Poliovirus (PV) is the prototypical picornavirus. It is a non-enveloped RNA virus with a small (∼7.5-kb) genome of positive polarity. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro-transcribed viral genomes into an appropriate host cell. The ease of genetic studies in poliovirus is a primary reason that it has long served as a model to study RNA virus biology, pathogenesis, and evolution. Protocols for the generation and characterization of PV mutants are presented. Curr. Protoc. Microbiol. 29:15H.2.1-15H.2.32. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 05/2013; Chapter 15:Unit15H.2.
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    ABSTRACT: The incorporation of a fluorescent reporter gene into a replication-competent influenza A virus (IAV) has made it possible to trace IAV infection in vivo. This protocol describes the process of inserting a green fluorescent protein (GFP) reporter into the IAV genome using the established reverse genetics system. The strategy begins with the reorganization of segment eight of the IAV genome, during which the open reading frames of nonstructural protein 1 (NS1) and the nuclear export protein (NEP) are separated to allow for GFP fusion to the NS1 protein. The NS1, GFP, and NEP open reading frames (ORF) are then cloned into the IAV rescue system backbone. Upon construction of the GFP-encoding segment eight rescue plasmid, recombinant NS1-GFP influenza virus can be rescued via co-transfection with the remaining seven rescue plasmids. The generated NS1-GFP IAV can subsequently be used to visualize infected cells, both in vitro and in vivo. Curr. Protoc. Microbiol. 29:15G.4.1-15G.4.16. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 05/2013; Chapter 15:Unit15G.4.
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    ABSTRACT: Ocular gene therapy is a fast-growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged and easily accessible, and direct viral delivery results primarily in local infection. Because the eye is not a vital organ, mutations in eye-specific genes tend to be more common. To date, over 40 eye-specific genes have been identified harboring mutations that lead to blindness. Gene therapy with recombinant adeno-associated virus (rAAV) holds the promise to treat patients with such mutations. However, proof-of-concept and safety evaluation for gene therapy remains to be established for most of these diseases. This unit describes the in vivo delivery of genes to the mouse eye by rAAV-mediated gene transfer and plasmid DNA electroporation. Advantages and limitations of these methods are discussed, and detailed protocols for gene delivery, required materials, and subsequent tissue processing methods are described. Curr. Protoc. Microbiol. 28:14D.4.1-14D.4.32. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 02/2013; Chapter 14:Unit14D.4.
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    ABSTRACT: Staphylococcus aureus is an important bacterial pathogen in the hospital and community settings, especially Staphylococcus aureus clones that exhibit methicillin-resistance (MRSA). Many strains of S. aureus are utilized in the laboratory, underscoring the genetic differences inherent in clinical isolates. S. aureus grows quickly at 37°C with aeration in rich media (e.g., BHI) and exhibits a preference for glycolytic carbon sources. Furthermore, S. aureus has a gold pigmentation, exhibits β-hemolysis, and is catalase and coagulase positive. The four basic laboratory protocols presented in this unit describe how to culture S. aureus on liquid and solid media, how to identify S. aureus strains as methicillin resistant, and how to generate a freezer stock of S. aureus for long-term storage. Curr. Protoc. Microbiol. 28:9C.2.1-9C.2.14. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 02/2013; Chapter 9:Unit9C.2.
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    ABSTRACT: Staphylococcus aureus is a facultative anaerobic Gram-positive coccus and a member of the normal skin flora as well as the nasal passages of humans. S. aureus is also the etiological agent of suppurative abscesses, as first described by Sir Alexander Ogston in 1880. Ever since, studies on S. aureus have focused on the complex battery of virulence factors and regulators that allow for its swift transition between commensalism and pathogenic states and escape from host immune defenses. The success of this pathogen is further evidenced by its ability to acquire antibiotic resistance traits through mechanisms that often remain poorly understood. Curr. Protoc. Microbiol. 28:9C.1.1-9C.1.9. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 02/2013; Chapter 9:Unit9C.1.
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    ABSTRACT: Bacteria within arthropods can be identified using culture-independent methods. This unit describes protocols for surface sterilization of arthropods, DNA extraction of whole bodies and tissues, touchdown PCR amplification using 16S rDNA general bacteria primers, and profiling the bacterial community using denaturing gradient gel electrophoresis. Curr. Protoc. Microbiol. 28:1E.6.1-1E.6.14. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 02/2013; Chapter 1:Unit1E.6.
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    ABSTRACT: Streptococcus pyogenes (the Group A Streptococcus, GAS) is a Gram-positive bacterium responsible for a wide spectrum of diseases ranging from mild superficial infections (pharyngitis, impetigo) to severe, often life-threatening invasive diseases (necrotizing fasciitis, streptococcal toxic shock syndrome) in humans. This unit describes molecular techniques for the genetic manipulation of S. pyogenes with detailed protocols for transformation, gene disruption, allelic exchange, transposon mutagenesis, and genetic complementation. Curr. Protoc. Microbiol. 30:9D.3.1-9D.3.29. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2013; 30:9D.3.1-9D.3.29.
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    ABSTRACT: Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) provides a means to investigate RNA structure with better resolution and higher throughput than has been possible with traditional methods. We present several protocols, which are based on a variety of previously published methods and were adapted and optimized for the analysis of poliovirus RNA in the Andino laboratory. These include methods for nondenaturing RNA extraction, RNA modification and primer extension, and data processing in ShapeFinder. Curr. Protoc. Microbiol. 30:15H.3.1-15H.3.12. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2013; 30:15H.3.1-15H.3.12.
  • Current protocols in microbiology 01/2013; Chapter 14:Unit14D.6..
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    ABSTRACT: Listeria monocytogenes causes foodborne disease in humans that ranges in severity from mild, self-limiting gastroenteritis to life-threatening systemic infections of the blood, brain, or placenta. The most commonly used animal model of listeriosis is intravenous infection of mice. This systemic model is highly reproducible, and thus, useful for studying cell-mediated immune responses against an intracellular bacterial pathogen, but it completely bypasses the gastrointestinal phase of L. monocytogenes infection. Intragastric inoculation of L. monocytogenes produces more variable results and may cause direct bloodstream invasion in some animals. The foodborne transmission model described here does not require specialized skills to perform and results in infections that more closely mimic human disease. This natural feeding model can be used to study both the host- and pathogen-derived factors that govern susceptibility or resistance to orally acquired L. monocytogenes. Curr. Protoc. Microbiol. 31:9B.3.1-9B.3.16. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2013; 31:9B.3.1-9B.3.16.
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    ABSTRACT: Virus-like particles (VLPs) are large particles, the size of viruses, composed of repeating structures that mimic those of infectious virus. Since their structures are similar to that of viruses, they have been used to study the mechanisms of virus assembly. They are also in development for delivery of molecules to cells and in studies of the immunogenicity of particle-associated antigens. However, they have been most widely used for development of vaccines and vaccine candidates. VLPs can form upon the expression of the structural proteins of many different viruses. This chapter describes the generation and purification of VLPs formed with the structural proteins, M, NP, F, and HN proteins, of Newcastle disease virus (NDV). Newcastle disease virus-like particles (ND VLPs) have also been developed as a platform for assembly into VLPs of glycoproteins from other viruses. This chapter describes the methods for this use of ND VLPs. Curr. Protoc. Microbiol. 30:18.2.1-18.2.21. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2013; 30:18.2.1-18.2.21.
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    ABSTRACT: DNA immunization was discovered in early 1990s, and its use has been expanded from vaccine studies to a broader range of biomedical research areas, such as the generation of high-quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation, and gene gun. In addition, several common considerations related to DNA immunization are discussed. Curr. Protoc. Microbiol. 31:18.3.1-18.3.24. ©2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2013; 31:18.3.1-18.3.24.
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    ABSTRACT: Streptococcus pyogenes is a Gram-positive bacterium that strictly infects humans. It is the causative agent of a broad spectrum of diseases accounting for millions of infections and at least 517,000 deaths each year worldwide. It is a nutritionally fastidious organism that ferments sugars to produce lactic acid and has strict requirements for growth. To aid in the study of this organism, this unit describes the growth and maintenance of S. pyogenes. Curr. Protoc. Microbiol. 30:9D.2.1-9D.2.13. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2013; 30:9D.2.1-9D.2.13.
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    ABSTRACT: Metronidazole and vancomycin remain the front-line therapies for most Clostridium difficile infections (CDI). However, recurrent CDI occurs in ∼25% of patients, causing significant morbidity and mortality and healthcare costs. For this population, traditional antibiotic therapies fail and new treatment options are greatly needed. The US Food and Drug Administration recently approved fidaxomicin for CDI treatment. This narrow-spectrum antibiotic preserves the normal gut microbiota and shows promise as a treatment for severe and recurrent CDI. Monoclonal antibodies and vaccines directed against toxin are currently in clinical trials and represent alternative, non-antibiotic therapies. Less traditional therapeutic interventions include bacteriotherapy with non-toxigenic C. difficile and fecal transplant. This commentary will provide an overview of current and forthcoming CDI therapies. Curr. Protoc. Microbiol. 30:9A.3.1-9A.3.9. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2013; 30:9A.3.1-9.

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