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Current Protocols in Microbiology

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
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
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
C Graham Clark, C Rune Stensvold
Blastocystis is an intestinal parasite that is very easily isolated in culture from fresh stool samples. In fact, the parasite grows so readily in culture that short-term in vitro culture is sometimes used as a diagnostic tool in the absence of DNA-based methods. While axenizing Blastocystis cultures remains a significant challenge, the parasite can be propagated for several months in the presence of metabolically active bacteria (xenic culture). Hence, culture can be used for maintaining live Blastocystis strain libraries...
November 18, 2016: Current Protocols in Microbiology
Darrell S Agbulos, Larissa Barelli, Bryan V Giordano, Fiona F Hunter
Zika virus (ZIKV), belonging to the family Flaviviridae, genus Flavivirus, is an arthropod-borne virus that was first discovered from the Zika forest in Uganda in 1947. Recent outbreaks in South America have linked ZIKV to cases of microcephaly and Guillain-Barré syndrome in humans. With the increased interest in ZIKV, protocols must be established to facilitate proper research. Here we describe the laboratory techniques required to quantify, propagate, and store ZIVK. We also review the proper safety protocol for the handling of ZIKV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention...
November 18, 2016: Current Protocols in Microbiology
Erin C Garcia, Peggy A Cotter
Burkholderia thailandensis is a nonpathogenic Gram-negative bacterium found in tropical soils. Closely related to several human pathogens, its ease of genetic manipulation, rapid growth in the laboratory, and low virulence make B. thailandensis a commonly used model organism. This unit describes the fundamental protocols for in vitro growth and maintenance of B. thailandensis in the laboratory. © 2016 by John Wiley & Sons, Inc.
August 12, 2016: Current Protocols in Microbiology
Rachael Melton-Kreft, Tracy Spirk
Nucleic acids can be obtained in numerous ways from clinical specimens; however, the quality of the nucleic acid is only as good as the sampling and isolation protocol. While nucleic acids may be extracted they may not be representative of the original source. Large areas of tissue and explanted hardware must be successfully surveyed to reflect the overall clinical picture. Once good sampling technique has been established, successful bacterial nucleic acid isolation is essential. Clinical samples may be difficult to process because of the presence of scar tissue, bone, implants, and bacterial biofilms...
February 8, 2016: Current Protocols in Microbiology
Thomas Günther, Juliane M Theiss, Nicole Fischer, Adam Grundhoff
Complex regulation of viral transcription patterns and DNA replication levels is a feature of many DNA viruses. This is especially true for those viruses which establish latent or persistent infections (e.g., herpesviruses, papillomaviruses, polyomaviruses, or adenovirus), as long-term persistence often requires adaptation of gene expression programs and/or replication levels to the cellular milieu. A key factor in the control of such processes is the establishment of a specific chromatin state on promoters or replication origins, which in turn will determine whether or not the underlying DNA is accessible for other factors that mediate downstream processes...
February 8, 2016: Current Protocols in Microbiology
Cynthia A Danquah, Arundhati Maitra, Simon Gibbons, Jane Faull, Sanjib Bhakta
Antibiotic resistance is one of the major threats to global health and well-being. The past decade has seen an alarming rise in the evolution and spread of drug-resistant strains of pathogenic microbes. The emergence of extensively drug resistant (XDR) strains of Mycobacterium tuberculosis and antimicrobial resistance among the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species) as well as fungal pathogens (such as certain species of Candida, Aspergillus, Cryptococcus, and Trichophyton) poses a significant 21st century scientific challenge...
February 8, 2016: Current Protocols in Microbiology
Xiaohong Wang, Zhi-Ming Zheng
Papillomaviruses are a family of small, non-enveloped DNA tumor viruses. Knowing a complete transcription map of each papillomavirus genome can provide guidance for various papillomavirus studies. This unit provides detailed protocols to construct a transcription map of human papillomavirus type 18. The same approach can be easily adapted to other transcription map studies of any other papillomavirus genotype due to the high degree of conservation in genome structure, organization, and gene expression among papillomaviruses...
February 8, 2016: Current Protocols in Microbiology
Dipesh Dhakal, Amit Kumar Jha, Anaya Pokhrel, Anil Shrestha, Jae Kyung Sohng
Nocardia spp. are aerobic, Gram-positive, catalase positive, and non-motile actinomycetes. They are associated with human infections. However, some species produce important natural products, degrade toxic chemicals, and are involved in biotransformation of valuable products. The lack of robust genetic tools has hindered detailed studies and advanced research. This unit describes the major genetic engineering approaches using Nocardia sp. CS682 as a prototype. These methods will certainly help in understanding the basis of their pathogenicity as well as biosynthetic and biotransforming abilities...
February 8, 2016: Current Protocols in Microbiology
Michael E Woodman, Christina R Savage, William K Arnold, Brian Stevenson
This protocol describes an efficient method for screening intact bacteria for the presence of desired DNA sequences using the polymerase chain reaction (PCR). This method is commonly referred to as colony PCR. © 2016 by John Wiley & Sons, Inc.
2016: Current Protocols in Microbiology
Erna Geessien Kroon, Jônatas Santos Abrahão, Giliane de Souza Trindade, Graziele Pereira Oliveira, Ana Paula Moreira Franco Luiz, Galileu Barbosa Costa, Mauricio Teixeira Lima, Rafael Silva Calixto, Danilo Bretas de Oliveira, Betânia Paiva Drumond
Natural infections of Vaccinia virus (VACV)-the prototype species of the Orthopoxvirus genus, from the family Poxviridae and subfamily Chordopoxvirinae-cause an occupational emergent zoonotic disease that is primarily associated with the handling of infected dairy cattle. In humans, VACV infection is characterized by skin lesions, primarily on the hands, and accompanied by systemic symptoms such as fever, myalgia, headache, and lymphadenopathy. The diagnosis of VACV is usually performed according to the methods described for other orthopoxviruses...
2016: Current Protocols in Microbiology
Brandee L Stone, Catherine A Brissette
Borrelia miyamotoi is a relapsing fever tick-borne pathogen found in Ixodes spp. (hard) ticks. In vitro culturing has proven difficult despite initial reports of cultures maintained in Barbour-Stoenner-Kelly-II (BSK-II) medium. The ability to culture in vitro opens many avenues for investigating the genetics and physiology of bacterial species. This unit describes methods for the maintenance and cultivation of B. miyamotoi in liquid medium. © 2016 by John Wiley & Sons, Inc.
2016: Current Protocols in Microbiology
Julia A Maresca, Jessica L Keffer, Kelsey J Miller
Ion-pumping rhodopsins transfer ions across the microbial cell membrane in a light-dependent fashion. As the rate of biochemical characterization of microbial rhodopsins begins to catch up to the rate of microbial rhodopsin identification in environmental and genomic sequence data sets, in vitro analysis of their light-absorbing properties and in vivo analysis of ion pumping will remain critical to characterizing these proteins. As we learn more about the variety of physiological roles performed by microbial rhodopsins in different cell types and environments, observing the localization patterns of the rhodopsins and/or quantifying the number of rhodopsin-bearing cells in natural environments will become more important...
2016: Current Protocols in Microbiology
Yanping Ma, Poching Liu, Vladimir Majerciak, Jun Zhu, Zhi-Ming Zheng
Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is etiologically linked to the development of several malignancies, mainly Kaposi's sarcoma. Expressed as an early viral protein, KSHV ORF57 is essential for lytic replication and virion production. ORF57 selectively binds to a subset of viral RNA and affects nearly all aspects of viral RNA processing. To globally identify all viral and host RNA associated with KSHV ORF57 in the infected cells, we have utilized UV cross-linking and immunoprecipitation (CLIP) of KSHV ORF57 combined with high-throughput RNA sequencing (CLIP-seq) to identify ORF57-binding RNA in BCBL-1 cells at genome-wide level...
2016: Current Protocols in Microbiology
Jônatas Santos Abrahão, Graziele Pereira Oliveira, Lorena Christine Ferreira da Silva, Ludmila Karen Dos Santos Silva, Erna Geessien Kroon, Bernard La Scola
The aim of this protocol is to describe the replication, purification, and titration of mimiviruses. These viruses belong to the Mimiviridae family, the first member of which was isolated in 1992 from a cooling tower water sample collected during an outbreak of pneumonia in a hospital in Bradford, England. In recent years, several new mimiviruses have been isolated from different environmental conditions. These giant viruses are easily replicated in amoeba of the Acanthamoeba genus, its natural host. Mimiviruses present peculiar features that make them unique viruses, such as the particle and genome size and the genome's complexity...
2016: Current Protocols in Microbiology
Ting Ni, Vladimir Majerciak, Zhi-Ming Zheng, Jun Zhu
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncovirus linked to the development of several malignancies in immunocompromised patients. Like other herpesviruses, KSHV has a large DNA genome encoding more than 100 distinct gene products. Despite being transcribed and processed by cellular machinery, the structure and organization of KSHV genes in the virus genome differ from what is observed in cellular genes from the human genome. A typical feature of KSHV expression is the production of polycistronic transcripts initiated from different promoters but sharing the same polyadenylation site (pA site)...
2016: Current Protocols in Microbiology
Denis Lee, Kathryn Norby, Mitchell Hayes, Ya-Fang Chiu, Bill Sugden, Paul F Lambert
Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a consequence of integration of HPV DNA into the host genome. Such cancers represent "dead ends" for the virus as integration disrupts the viral genome and because the cancers are defective in normal epithelial differentiation, which is required for production of progeny papillomavirus...
2016: Current Protocols in Microbiology
Heather Griffin, John Doorbar
Molecular events during the papillomavirus life cycle can be mapped in infected tissue biopsies using antibodies to viral and cellular gene products, or by in situ hybridization approaches that detect viral DNA or viral transcription products. For proteins, ease of immunodetection depends on antibody specificity and antigen availability. Epitopes in formalin-fixed paraffin-embedded (FFPE) samples are often masked by crosslinking and must be exposed for immunodetection. RNA in FFPE material is often degraded, and such tissue must be handled carefully to optimize detection...
2016: Current Protocols in Microbiology
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