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Current Protocols in Microbiology

Catherine J Redmond, Haiqing Fu, Mirit I Aladjem, Alison A McBride
Human papillomaviruses (HPVs) are frequently integrated in HPV-associated cancers. HPV genomes can be integrated in three patterns: A single integrated HPV genome (type I), multiple, tandemly integrated HPV genomes (type II), and multiple, tandemly integrated HPV genomes interspersed with host DNA (type III). Analysis of the organization of type II and type III integration sites is complicated by their repetitive nature, as sequences of individual repeats are difficult to distinguish from each other. This article presents a method for directly visualizing HPV integration sites using molecular combing combined with fluorescent in situ hybridization, also known as fiber-FISH...
August 20, 2018: Current Protocols in Microbiology
Robert T Todd, Ann L Braverman, Anna Selmecki
Ploidy, the number of sets of homologous chromosomes in a cell, can alter cellular physiology, gene regulation, and the spectrum of acquired mutations. Advances in single-cell flow cytometry have greatly improved the understanding of how genome size contributes to diverse biological processes including speciation, adaptation, pathogenesis, and tumorigenesis. For example, fungal pathogens can undergo whole genome duplications during infection of the human host and during acquisition of antifungal drug resistance...
August 2018: Current Protocols in Microbiology
Kwang-Woo Jung, Kyung-Tae Lee, Yee-Seul So, Yong-Sun Bahn
Cryptococcus neoformans is an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals and is responsible for more than 1,000,000 infections and 600,000 deaths annually worldwide. Nevertheless, anti-cryptococcal therapeutic options are limited, mainly because of the similarity between fungal and human cellular structures. Owing to advances in genetic and molecular techniques and bioinformatics in the past decade, C. neoformans, belonging to the phylum basidiomycota, is now a major pathogenic fungal model system...
August 2018: Current Protocols in Microbiology
Megha Gulati, Matthew B Lohse, Craig L Ennis, Ruth E Gonzalez, Austin M Perry, Priyanka Bapat, Ashley Valle Arevalo, Diana L Rodriguez, Clarissa J Nobile
Candida albicans is a normal member of the human microbiota that asymptomatically colonizes healthy individuals, however it is also an opportunistic pathogen that can cause severe infections, especially in immunocompromised individuals. The medical impact of C. albicans depends, in part, on its ability to form biofilms, communities of adhered cells encased in an extracellular matrix. Biofilms can form on both biotic and abiotic surfaces, such as tissues and implanted medical devices. Once formed, biofilms are highly resistant to antifungal agents and the host immune system, and can act as a protected reservoir to seed disseminated infections...
August 2018: Current Protocols in Microbiology
Benjamin J Koestler, Cara M Ward, Shelley M Payne
Shigella is an enteroinvasive human pathogen that infects the colonic epithelium and causes Shigellosis, an infectious diarrheal disease. There is no vaccine for the prevention or treatment of Shigellosis and antibiotic-resistant strains of Shigella are increasing, emphasizing the need for a deeper understanding of Shigella pathogenesis in order to design effective antimicrobial therapies. Small animal models do not recapitulate Shigellosis, therefore tissue-cultured cells have served as model systems to study Shigella pathogenesis...
August 2018: Current Protocols in Microbiology
Savannah E Sanchez, Eduardo Vallejo-Esquerra, Anders Omsland
Coxiella burnetii is a highly infectious obligate intracellular bacterium and the etiological agent of the zoonosis Query (Q) fever. This Gram-negative gamma-proteobacterium has adapted to replicate within a specialized compartment in mammalian phagocytic cells, known as the Coxiella-containing vacuole (CCV). Knowledge of critical characteristics of the CCV microenvironment (e.g., luminal pH), analysis of the C. burnetii genome sequence, and strategic metabolic profiling have provided the basis for determining the physicochemical and nutritional conditions necessary to support axenic replication of C...
August 2018: Current Protocols in Microbiology
Nina M Poole, Anubama Rajan, Anthony W Maresso
Adherence, invasion, and translocation to and through the intestinal epithelium are important drivers of disease for many enteric bacteria. However, most work has been limited to transformed intestinal cell lines or murine models that often do not faithfully recapitulate key elements associated with human disease. The recent technological advances in organotypic tissue and cell culture are providing unparalleled access to systems with human physiology and complexity. Human intestinal enteroids (HIEs), derived from patient biopsy or surgical specimens of intestinal tissues, are organotypic cultures now being adapted to the study of enteric infections...
August 2018: Current Protocols in Microbiology
Stephanie K Lathrop, Kendal G Cooper, Kelsey A Binder, Tregei Starr, Veena Mampilli, Corrella S Detweiler, Olivia Steele-Mortimer
The successful infection of macrophages by non-typhoidal serovars of Salmonella enterica is likely essential to the establishment of the systemic disease they sometimes cause in susceptible human populations. However, the interactions between Salmonella and human macrophages are not widely studied, with mouse macrophages being a much more common model system. Fundamental differences between mouse and human macrophages make this less than ideal. Additionally, the inability of human macrophage-like cell lines to replicate some properties of primary macrophages makes the use of primary cells desirable...
August 2018: Current Protocols in Microbiology
Sandeep Vellanki, Maria Isabel Navarro-Mendoza, Alexis Garcia, Laura Murcia, Carlos Perez-Arques, Victoriano Garre, Francisco E Nicolas, Soo Chan Lee
Mucor circinelloides is a fungus that belongs to the order Mucorales. It grows as mold in the environment and can cause mucormycosis, a potentially fatal infection in immunocompromised patients. M. circinelloides is a biodiesel producer and serves as a model organism for studying several biological processes, such as light responses and RNA interference-mediated gene silencing. Over the past decade, the increasing number of molecular tools has also allowed us to manipulate the genome of this fungus. This article outlines the fundamental protocols for the in vitro growth, maintenance, and genetic manipulation of M...
May 2018: Current Protocols in Microbiology
Tanel Punga, Sibel Ciftci, Mats Nilsson, Tomasz Krzywkowski
Infection by DNA viruses such as human adenoviruses (HAdVs) causes a high-level accumulation of viral DNA and mRNA in the cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not inevitably reflect the abundance in individual cells. As the vast majority of virus infection studies is carried out using standard experimental procedures with heterogeneous cell populations, there is a need for a method allowing simultaneous detection and quantitative analysis of viral genome accumulation and gene expression in individual infected cells within a population...
May 2018: Current Protocols in Microbiology
Shin-Hee Kim, Siba K Samal
Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and pathogenesis of NDV. Furthermore, NDV has modular genome and accommodates insertion of a foreign gene as a transcriptional unit, thus enabling NDV as a vaccine vector against diseases of humans and animals. Avirulent NDV strains (e...
February 22, 2018: Current Protocols in Microbiology
Lindsey O'Neal, Tanmoy Mukherjee, Gladys Alexandre
Bacteria of the genus A. brasilense are motile and capable of chemotaxis and aerotaxis (taxis in gradient of oxygen) using a single polar flagellum that propels the cells in aqueous environments. Responses to attractants and repellents have been described and spatial gradient assays that permit the visualization of these responses are detailed in this unit. These assays are simple and can be readily implemented with minimum set ups. © 2018 by John Wiley & Sons, Inc.
February 22, 2018: Current Protocols in Microbiology
Osama S Mahdi, Nathan A Fisher
Paenibacillus larvae is a Gram-positive, spore-forming bacterium and the causative agent of American foulbrood disease (AFB), a highly contagious, fatal disease affecting managed honeybee (Apis mellifera) colonies. As the etiological agent of American foulbrood disease, P. larvae is the most economically significant bacterial pathogen infecting honeybees. This unit includes protocols for the in vitro growth and laboratory maintenance of P. larvae. © 2018 by John Wiley & Sons, Inc.
February 22, 2018: Current Protocols in Microbiology
Osama S Mahdi, Nathan A Fisher
Endospores are metabolically dormant cells formed by a variety of Gram-positive bacteria within the phylum Firmicutes in response to nutrient limiting or otherwise unfavorable growth conditions. American foulbrood disease (AFB) is a serious disease of honeybees that is caused by the introduction of Paenibacillus larvae endospores into a honeybee colony. Progression to fulminant disease and eventual collapse of the colony requires multiple rounds of endospore germination, vegetative replication, endospore formation, and subsequent spread within the colony...
February 22, 2018: Current Protocols in Microbiology
Shaoli Lin, Liping Yang, Yan-Jin Zhang
Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal-oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV strains infecting humans mainly belong to four genotypes. Genotypes 1 and 2 are restricted to human infection, while genotypes 3 and 4 are zoonotic. HEV genotype 3 (HEV-3) can cause both acute and chronic liver diseases. Several cell lines (mainly hepatocytes) have been developed for HEV propagation and biological study...
February 22, 2018: Current Protocols in Microbiology
Ekaterina G Viktorova, Sunil Khattar, Siba Samal, George A Belov
Poliovirus is a prototype member of the Enterovirus genus of the Picornaviridae family of small positive strand RNA viruses, which include important human and animal pathogens. Quantitative assessment of viral replication is very important for investigation of the virus biology and the development of anti-viral strategies. The poliovirus genome structure allows replacement of structural genes with a reporter protein, such as a luciferase or a fluorescent protein, whose signals can be detected and quantified in vivo, thus permitting observation of replication kinetics in live cells...
February 22, 2018: Current Protocols in Microbiology
Zexu Ma, Liping Yang, Yan-Jin Zhang
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirale. PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a genome around 15 kb in length. For propagation of PRRSV in vitro, the MARC-145 cell line is the most often used in a laboratory setting. Infectious cDNA clones of many PRRSV strains have been established, from which these viruses can be recovered. PRRSV titration is generally done in MARC-145 cells. PRRSV RNA copy numbers can be assessed by reverse transcription and real-time PCR...
February 22, 2018: Current Protocols in Microbiology
Daniel K Howe, Michelle Yeargan, Landon Simpson, Sriveny Dangoudoubiyam
Sarcocystis neurona is a member of the important phylum Apicomplexa and the primary cause of equine protozoal myeloencephalitis (EPM). Moreover, S. neurona is the best-studied species in the genus Sarcocystis, one of the most successful parasite taxa, as virtually all vertebrate animals may be infected by at least one species. Consequently, scientific investigation of S. neurona will aid in the control of EPM and neurologic disease in sea mammals, while also improving our understanding of a prominent branch on the apicomplexan phylogenetic tree...
February 22, 2018: Current Protocols in Microbiology
Gladys Alexandre
Bacteria of the genus Azospirillum comprise 15 species to date, with A. brasilense the best studied species in the genus. Azospirillum are soil bacteria able to promote the growth of plants from 113 species spanning 35 botanical families. These non-pathogenic and beneficial bacteria are ubiquitous in soils and inhabit the roots of diverse plants. These bacteria are microaerophilic, able to fix nitrogen under free-living conditions, motile, and able to navigate in gradients of various chemicals, including oxygen...
November 9, 2017: Current Protocols in Microbiology
Matthieu J Miossec, Sandro L Valenzuela, Katterinne N Mendez, Eduardo Castro-Nallar
As the field of microbiomics advances, the burden of computational work that scientists need to perform in order to extract biological insight has grown accordingly. Likewise, while human microbiome analyses are increasingly shifting toward a greater integration of various high-throughput data types, a core number of methods form the basis of nearly every study. In this unit, we present step-by-step protocols for five core stages of human microbiome research. The protocols presented in this unit provide a base case for human microbiome analysis, complete with sufficient detail for researchers to tailor certain aspects of the protocols to the specificities of their data...
November 9, 2017: Current Protocols in Microbiology
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