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Current Protocols in Microbiology

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https://www.readbyqxmd.com/read/28800159/mouse-models-of-acinetobacter-baumannii-infection
#1
Greg Harris, Rhonda KuoLee, H Howard Xu, Wangxue Chen
This unit describes basic protocols for infecting mice through intranasal and intraperitoneal routes with Acinetobacter baumannii to induce associated pneumonia and sepsis, the two most common manifestations of clinical infections with this pathogen. By selecting the appropriate protocols and bacterial strains of different virulence, these mouse models provide an opportunity to study the infection pathogenesis and host-immune responses, and to evaluate the efficacies of prophylactic and therapeutic anti-A. baumannii candidates...
August 11, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28800158/using-kbase-to-assemble-and-annotate-prokaryotic-genomes
#2
Benjamin Allen, Meghan Drake, Nomi Harris, Tarah Sullivan
The DOE Systems Biology Knowledgebase (KBase, http://kbase.us/) is an open-access bioinformatics software and data platform for analyzing plants, microbes, and their communities. KBase enables scientists to create, execute, collaborate on, and share reproducible analyses of their biological data in the context of public data and private collaborator data. For microbiologists researching prokaryotes, KBase offers analysis tools for performing quality control and assessment of Next-Generation Sequencing reads, de novo assembly, genome annotation, and tools for analyzing structural and functional features of genomes...
August 11, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28800157/generating-and-maintaining-transgenic-cryptosporidium-parvum-parasites
#3
Mattie C Pawlowic, Sumiti Vinayak, Adam Sateriale, Carrie F Brooks, Boris Striepen
The apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease and an important contributor to overall global child mortality. We currently lack effective treatment and immune prophylaxis. Recent advances now permit genetic modification of this important pathogen. We expect this to produce rapid advances in fundamental as well as translational research on cryptosporidiosis. Here we outline genetic engineering for Cryptosporidium in sufficient detail to establish transfection in any laboratory that requires access to this key technology...
August 11, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28800156/quantitative-dextran-trafficking-to-the-coxiella-burnetii-parasitophorous-vacuole
#4
Seth Winfree, Stacey D Gilk
The gram-negative bacterium Coxiella burnetii causes human Q fever, a disease characterized by a debilitating flu-like illness in acute cases and endocarditis in chronic patients. An obligate intracellular pathogen, Coxiella burnetii survives within a large, lysosome-like vacuole inside the host cell. A unique feature of the Coxiella parasitophorous vacuole (PV) is high levels of fusion with the host endocytic pathway, with PV-endosome fusion critical for Coxiella survival within the host cell. This unit describes quantitating PV-endosome fusion by measuring delivery of the fluid phase endosome marker dextran to the PV using live cell imaging...
August 11, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28800155/epigenetic-analysis-of-sv40-minichromosomes
#5
Lata Balakrishnan, Barry Milavetz
Simian virus 40 (SV40) is one of the best-characterized members of the polyomavirus family of small DNA tumor viruses. It has a small genome of 5243 bp and utilizes cellular proteins for its molecular biology, with the exception of the T-antigen protein, which is coded by the virus and is involved in regulating transcription and directing replication. Importantly, SV40 exists as chromatin in both the virus particle and intracellular minichromosomes. These facts, combined with high yields of virus and minichromosomes following infection and ease of manipulation, have made SV40 an extremely useful model to study all aspects of eukaryotic molecular biology...
August 11, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28510364/lyophilization-of-bdellovibrio-bacteriovorus-109j-for-long-term-storage
#6
Mélanie J Boileau, Rinosh Mani, Kenneth D Clinkenbeard
Bdellovibrio bacteriovorus 109J is a Gram-negative predatory bacterium with obligate host dependency on other Gram-negative bacteria. This bacteriolytic predator collides with, enters, and establishes growth within the prey (host) periplasm, eventually lysing the prey cell wall to release fresh, motile B. bacteriovorus progeny. Laboratory maintenance of B. bacteriovorus has been previously described by other investigators. The protocols included in this unit deal with the technique required to lyophilize or freeze dry host-dependent B...
May 16, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28510363/purification-toxoplasma-gondii-tissue-cysts-using-percoll-gradients
#7
Elizabeth A Watts, Animesh Dhara, Anthony P Sinai
The protozoan parasite Toxoplasma gondii is capable of infecting all warm-blooded animals and humans. Infectious, transmissible forms of the parasite include oocysts produced by the sexual cycle within the definitive feline host and tissue cysts that form Toxoplasma in the central nervous system and muscle during the asexual cycle within all chronically infected warm-blooded hosts. These tissue cysts are populated with slow-growing bradyzoites, which until recently have been thought to be dormant entities in the context of immune sufficiency...
May 16, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28510362/burkholderia-thailandensis-genetic-manipulation
#8
Erin C Garcia
Burkholderia thailandensis is a Gram-negative bacterium endemic to Southeast Asian and northern Australian soils. It is non-pathogenic; therefore, it is commonly used as a model organism for the related human pathogens Burkholderia mallei and Burkholderia pseudomallei. B. thailandensis is relatively easily genetically manipulated and a variety of robust genetic tools can be used in this organism. This unit describes protocols for conjugation, natural transformation, mini-Tn7 insertion, and allelic exchange in B...
May 16, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28510361/chlamydia-trachomatis-transformation-and-allelic-exchange-mutagenesis
#9
Konrad E Mueller, Katerina Wolf, Kenneth A Fields
Gene inactivation is essential for forward and reverse genetic approaches to establish protein function. Techniques such as insertion or chemical mutagenesis have been developed to mutagenize chlamydiae via targeted or random mutagenesis, respectively. Both of these approaches require transformation of chlamydiae to either introduce insertion elements or complement mutants. We have recently developed a targeted mutagenesis strategy, fluorescence-reported allelic exchange mutagenesis (FRAEM), to delete Chlamydia trachomatis L2 genes...
May 16, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28510360/analysis-of-human-papillomavirus-genome-replication-using-two-and-three-dimensional-agarose-gel-electrophoresis
#10
Liisi Henno, Eva-Maria Tombak, Jelizaveta Geimanen, Marit Orav, Ene Ustav, Mart Ustav
This unit includes the necessary information to conduct neutral/neutral and neutral/alkaline two-dimensional and neutral/neutral/alkaline three-dimensional agarose gel electrophoresis. The methodology has been optimized over the years to gain a better outcome from the hard-to-interpret signals of human papilloma virus replication intermediates obtained from two- and three-dimensional agarose gels. Examples of typical results and interpretation of replication intermediate patterns are included, and the outcomes of multiple-dimension assays are assessed using previously published experimental data...
May 16, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28510359/isolation-culture-and-cryopreservation-of-sarcocystis-species
#11
S K Verma, D S Lindsay, M E Grigg, J P Dubey
More than 200 valid Sarcocystis species have been described in the parasitological literature. The developmental life cycle in the intermediate host and definitive host has only been described for a few species. Sarcocystis parasites are common pathogens infecting a wide range of animals, including humans, and this unit reviews the methods used for isolating infective stages of the parasite from both definitive and intermediate host(s), as well as methods used to initiate cultures from sporocysts and merozoites and for cryopreservation of various Sarcocystis spp...
May 16, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28166387/toxoplasma-gondii-laboratory-maintenance-and-growth
#12
Asis Khan, Michael E Grigg
Toxoplasma gondii is a highly successful apicomplexan protozoan capable of infecting any warm-blooded animal worldwide. In humans, Toxoplasma infections are life-long, with approximately one-third of the world's population chronically infected. Although normally controlled by the host immune system, T. gondii infection can lead to a variety of clinical outcomes in individuals with immature or suppressed immune systems. After penetrating the intestine, parasites rapidly disseminate throughout the body and stimulate production of the cytokines interleukin (IL)-12, IL-18, and interferon (IFN)-γ by immune cells...
February 6, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28166386/actinomadura-species-laboratory-maintenance-and-ribosome-engineering
#13
Dipesh Dhakal, Nguyen Thanh Chung, Vijay Rayamajhi, Jae Kyung Sohng
Actinomadura spp. are aerobic, Gram-positive, catalase-positive, non-acid fast, non-motile actinomycetes. Some species of Actinomadura are associated with opportunistic infections in humans. However, many bioactive compounds with pharmaceutical applications can be isolated from various Actinomadura spp. This unit includes general protocols for the laboratory maintenance of Actinomadura spp., including growth in liquid medium, growth on solid agar, long-term storage, and generation of a higher producing strain by ribosome engineering...
February 6, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28166385/virus-hunting-discovery-of-new-episomal-circular-viruses-by-rolling-circle-techniques
#14
Bert Vanmechelen, Annabel Rector, Piet Maes
Many methods for the discovery of novel viruses are based on amplification of the virus using consensus or degenerate PCR primers. A downside of this approach is that it requires prior knowledge of the viral nucleotide sequence to be applicable. Presented in this unit is a method for the sequence-independent amplification of circular viral genomes that is based on the rolling-circle mechanism used by certain viruses in their natural replication cycle. The amplification of the virus of interest is coupled to the isolation of the viral genome by gel extraction following a restriction digestion...
February 6, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28166384/cryptosporidium-identification-and-genetic-typing
#15
Jan Šlapeta
Cryptosporidium spp. are obligate protozoan parasites of the gastrointestinal tract of vertebrates, including humans. In the majority of human cases, the diarrheal disease cryptosporidiosis is caused by either the human-adapted species Cryptosporidium hominis or the zoonotic Cryptosporidium parvum 'bovine genotype' (also known as Cryptosporidium pestis). The infectious stage, environmentally resilient Cryptosporidium oocysts, are shed by the infected host. Cryptosporidium parasites are transmitted by the fecal-oral route and are one of the major water-borne pathogens...
February 6, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28166383/efficient-genome-editing-in-the-oomycete-phytophthora-sojae-using-crispr-cas9
#16
Yufeng Fang, Linkai Cui, Biao Gu, Felipe Arredondo, Brett M Tyler
Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has been unattainable in oomycetes, because of their extremely low rate of homologous recombination. The recent implementation of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system in the soybean pathogen Phytophthora sojae, an experimental model for oomycetes, has opened up a powerful new research capability for the oomycete community...
February 6, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28166382/saccharopolyspora-species-laboratory-maintenance-and-enhanced-production-of-secondary-metabolites
#17
Dipesh Dhakal, Anaya Raj Pokhrel, Amit Kumar Jha, Nguyen Huy Thuan, Jae Kyung Sohng
Saccharopolyspora spp. are aerobic, Gram-positive, non-acid-fast, and non-motile actinomycetes. Various species of the genus Saccharopolyspora have been reported with an ability to produce various bioactive compounds for pharmaceutical and agricultural uses. This unit includes general protocols for the laboratory maintenance of Saccharopolyspora species, including growth in liquid medium, growth on solid agar, long-term storage, and generation of a higher producer strain by mutagenesis. Saccharopolyspora spinosa ATCC 49460 is used as a prototype for explaining the considerations for efficient laboratory maintenance of Saccharopolyspora spp...
February 6, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/27858973/detection-and-genotyping-of-human-papillomaviruses-from-archival-formalin-fixed-tissue-samples
#18
Koenraad Van Doorslaer, Zigui Chen, Alison A McBride
Pathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting virus...
November 18, 2016: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/27858972/a-simple-and-low-cost-procedure-for-growing-geobacter-sulfurreducens-cell-cultures-and-biofilms-in-bioelectrochemical-systems
#19
J Patrick O'Brien, Nikhil S Malvankar
Anaerobic microorganisms play a central role in several environmental processes and regulate global biogeochemical cycling of nutrients and minerals. Many anaerobic microorganisms are important for the production of bioenergy and biofuels. However, the major hurdle in studying anaerobic microorganisms in the laboratory is the requirement for sophisticated and expensive gassing stations and glove boxes to create and maintain the anaerobic environment. This appendix presents a simple design for a gassing station that can be used readily by an inexperienced investigator for cultivation of anaerobic microorganisms...
November 18, 2016: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/27858971/molecular-identification-and-subtype-analysis-of-blastocystis
#20
C Rune Stensvold, C Graham Clark
Several typing methods have been used in studies aiming to unravel the molecular epidemiology of Blastocystis, which is one of the most common intestinal parasites in human and many non-human hosts. Such studies have the potential to add to knowledge on Blastocystis transmission, host specificity, phylogeography, and clinical and public health significance, but rely on robust, standardized methods by which data can be generated and compared directly between studies. One of the most used methods is "barcoding,", which involves single-round PCR amplification and sequencing of partial small subunit ribosomal RNA genes of the parasites...
November 18, 2016: Current Protocols in Microbiology
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