DNA sequencing of fungi in a microbiology laboratory

The identification of fungi in a general microbiology laboratory using traditional techniques has always been problematic for several reasons. These include the requirement for trained personnel to identify the fungi, as well as the fact that many fungi take much longer than bacteria to grow, making identification of these organisms in a timely fashion difficult. An additional complication is that some fungi do not show characteristic structures, making identification by traditional techniques difficult, if not impossible. DNA sequencing for the identification of fungal cultures can be easily established in a laboratory that is set up for bacterial DNA sequencing. This technology, when complemented with appropriate levels of training in classical fungal identification, will extend the mycological capabilities of a general microbiology laboratory.

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MALDI-TOF MS and its requirements for fungal identification.

Trends in the systematics of bacteria and fungi ◽

10.1079/9781789244984.0119 ◽

2021 ◽

pp. 119-140

Author(s):

Cledir Santos ◽

Paula Galeano ◽

Reginaldo Lima Neto ◽

Manoel Marques Evangelista Oliveira ◽

Nelson Lima

Keyword(s):

Sample Preparation ◽

Black Box ◽

Data Sets ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Fungal Identification ◽

Identification Of Fungi ◽

Tof Ms

Abstract Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is now used as a routine technique for the fast and reliable identification of fungi at the species level and, currently, it represents an important phenotypic methodology based on proteomic profiles. The main limitations to MALDI-TOF MS for fungal identification are related to sample quality (e.g. quality of biological material such as rigidity or pigmentation of cell walls), sample preparation (e.g. the myriad of sample preparation methodologies that deliver different data sets to different MALDI-TOF MS databases) and the databases themselves (e.g. the 'black-box' commercial databases). This chapter presents an overview and discussion of the use of MALDI-TOF MS for fungal identification. The major known limitations of the technique for fungal taxonomy, and how to overcome these, are also discussed.

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André Boivin: A pioneer in endotoxin research and an amazing visionary during the birth of molecular biology

Innate Immunity ◽

10.1177/1753425919842307 ◽

2019 ◽

Vol 26 (3) ◽

pp. 165-171

Author(s):

Jean-Marc Cavaillon

Keyword(s):

Molecular Biology ◽

Premature Death ◽

Biological Chemistry ◽

Scientific Contribution ◽

Bacterial Dna ◽

Dna And Rna ◽

Assistant Director ◽

Pyrimidine Bases ◽

Carbon Molecules ◽

Set Up

André Boivin (1895–1949) started his career in Marseille as a biochemist. Soon after the discovery of insulin, he worked on its purification, allowing for the treatment of local patients. He later moved to Strasbourg and set-up a microtitration technique of small carbon molecules and a method for quantifying purine and pyrimidine bases. His main scientific contribution occurred in Bucharest, where he was recruited to organize the teaching of medicinal chemistry. Together with Ion and Lydia Mesrobeanu, at the Cantacuzene Institute, they were the first to characterize the biochemical nature of endotoxins, which he termed the “glucido-lipidic antigen.” After joining the Institut Pasteur annex near Paris, he worked with Gaston Ramon pursuing his research on smooth and rough LPS. Additionally, with Albert Delaunay, he researched the formation of exotoxins and antibodies (Abs). He was nominated assistant-director of the Institut Pasteur in 1940. He initiated research on bacterial DNA and RNA, and was the first to hypothesize on how RNA fits into gene function. In 1947 he moved for a second time to Strasbourg, accepting a position as a Professor of Biological Chemistry. After his premature death at the age of 54, the French academies mourned his loss and recognized him as one of their outstanding masters of biochemistry, microbiology, immunology, and molecular biology.

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An investigation of PIK3CA mutations in isolated macrodactyly

Journal of Hand Surgery (European Volume) ◽

10.1177/1753193418770366 ◽

2018 ◽

Vol 43 (7) ◽

pp. 756-760 ◽

Cited By ~ 12

Author(s):

Jingheng Wu ◽

Wei Tian ◽

Guanglei Tian ◽

Kelli Sumner ◽

Douglas T. Hutchinson ◽

...

Keyword(s):

Dna Sequencing ◽

Molecular Mechanism ◽

Mutation Analysis ◽

Detection Rate ◽

Mutation Detection ◽

Pik3ca Mutation ◽

Mutation Detection Rate ◽

Pik3ca Mutations ◽

Diagnosis And Classification ◽

Set Up

Somatic PIK3CA mutations may relate to pathogenesis of isolated macrodactyly. We set up to test the association between PIK3CA mutations with isolated macrodactyly in order to establish a more accurate and molecular mechanism-based diagnosis and classification. DNA extracted from affected tissues in 12 individuals with isolated macrodactyly was tested for PIK3CA mutation using targeted Sanger DNA sequencing. Ten patients had macrodactyly in the foot and two in the hand. Nine of the 12 patients were found to carry a low-level, mosaic PIK3CA mutation. The mutations identified, p.His1047Arg, p.His1047Leu, p.Glu545Lys, and p.Glu542Lys, are codons frequently mutated in cancers. Among all tissues tested, adipose had the highest mutation detection rate, followed by nerve and skin. Our results indicate that a high proportion of isolated macrodactyly patients carry a pathogenic PIK3CA mutation. Affected adipose, nerve and skin tissues are ideal for PIK3CA mutation analysis.

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THE NET ENVIRONMENTAL BENEFIT OF A SUCCESSFUL DISPERSANT OPERATION AT THE SEA EMPRESS INCIDENT

International Oil Spill Conference Proceedings ◽

10.7901/2169-3358-1997-1-185 ◽

1997 ◽

Vol 1997 (1) ◽

pp. 185-194 ◽

Cited By ~ 8

Author(s):

Tim Lunel ◽

Jan Rusin ◽

Nick Bailey ◽

Chris Halliwell ◽

Louise Davies

Keyword(s):

Water Column ◽

Monitoring Program ◽

Environmental Benefit ◽

Sea Birds ◽

Sufficient Detail ◽

Timely Fashion ◽

Spill Site ◽

Set Up

ABSTRACT The monitoring of oil concentrations in the water column during a dispersant operation carried out at the Sea Empress incident has clearly demonstrated that, although a monitoring program should not be viewed as a prerequisite to response (since the spill site may be too remote to allow monitoring to be set up for the start of the response operation), if monitoring is planned as part of the response, it enables operations to be executed efficiently. The estimates put forward in this paper indicate that the targeted use of dispersants probably prevented 57,000 to 110,000 tons of emulsion from impacting the shoreline and potentially resulting in greatly increased impacts on sea birds, coastal waders, intertidal vertebrates and invertebrates, and amenity areas. The paper shows that these benefits outweighed the potential disadvantages associated with elevated oil concentrations in the water column. The Sea Empress is the first incident that has been monitored in a timely fashion in sufficient detail. The monitoring program demonstrated that the use of dispersants resulted in a net environmental benefit at the Sea Empress incident.

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Validation of a DNA Sequencing Assay for Fungal Identification Directly from Human Tissue Specimens

American Journal of Clinical Pathology ◽

10.1093/ajcp/140.suppl1.048 ◽

2013 ◽

Vol 140 (suppl 1) ◽

pp. A048-A048

Author(s):

Hongliu Sun ◽

Kenneth L. Muldrew

Keyword(s):

Dna Sequencing ◽

Human Tissue ◽

Fungal Identification ◽

Tissue Specimens

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Fungal Genomics in Dermatology

Journal of Mycology and Infection ◽

10.17966/jmi.2019.24.2.37 ◽

2019 ◽

pp. 37-44

Author(s):

Young Bok Lee ◽

Soo Young Lee ◽

Ji Min Seo ◽

Min Ji Kang ◽

Dong Soo Yu

Keyword(s):

Large Scale ◽

Ecological Studies ◽

Fungal Genomics ◽

Emerging Trends ◽

Fungal Identification ◽

Identification Of Fungi ◽

Bioinformatic Approaches ◽

Fungal Genomes ◽

Basic Concepts ◽

Generation Sequencing

To date, hundreds of fungal genomes have been sequenced, and many more are underway. Recently developed cutting-edge techniques generate very large amounts of data, and the field of fungal genomics in dermatology has consequently evolved substantially. Methodological improvements have broadened the scope of large-scale ecological studies in dermatology, including biodiversity assessments and genomic identification of fungi. Here, we aimed to provide a brief introduction to bioinformatic approaches to fungal genomics in the field of dermatology. We described the history and basic concepts of fungal genomics and presented sequencing-based techniques for fungal identification, including a list of the revised taxa of dermatophytes, as determined by current phylogenetic analysis. Finally, we discussed the emerging trends in fungal genomics in dermatology, such as next-generation sequencing.

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Incidence, bacteriological profile and risk factor analysis of neonatal sepsis in a peri urban set up of North India

International Journal of Contemporary Pediatrics ◽

10.18203/2349-3291.ijcp20202146 ◽

2020 ◽

Vol 7 (6) ◽

pp. 1349

Author(s):

Shalini Upadhyay ◽

Anita Chakravarti ◽

Tanisha Bharara ◽

Priti Agarwal

Keyword(s):

Risk Factors ◽

Neonatal Sepsis ◽

Neonatal Intensive Care ◽

Blood Cultures ◽

North India ◽

Microbiology Laboratory ◽

Coagulase Negative Staphylococci ◽

Bacteriological Profile ◽

Increased Risk ◽

Set Up

Background: The high incidence and antimicrobial resistance among the pathogens causing neonatal sepsis is alarming. In addition to substantial immediate mortality, survivors of infections in the neonatal period are at increased risk of long-term disability. The present study was conducted to know the bacteriological profile and risk factors associated with culture proven neonatal sepsis in a peri urban population.Methods: This study was conducted over a period of 4 year (2015-2019). On clinical suspicion, blood culture specimens were sent to microbiology laboratory. The organisms isolated from blood cultures were identified and tested for antimicrobial susceptibility. As part of infection control practices, environmental samples from the neonatal intensive care units were tested.Results: Of 907 blood cultures of neonates received in the microbiology laboratory, 20.7% were culture positive. Majority of the episodes occurred at or before 72 hours of life (81.4%). 54.3% were Gram positive cocci including Coagulase negative Staphylococci, Staphylococcus aureus and Enterococcus spp. The common Gram-negative pathogens included Escherichia coli, Klebsiella spp. and Pseudomonas spp. Common risk factors involved were preterm birth, low birth weight, premature rupture of membrane, prolonged labour and iatrogenic causes.Conclusions: The early signs of sepsis are often subtle and nonspecific. Therefore, a high index of suspicion is needed for early diagnosis. Rapid, reliable detection and appropriate case management can save lives of many new-borns.

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Benzo-homologated nucleobases in a nanotube-electrode set-up for DNA sequencing

Nanotechnology ◽

10.1088/0957-4484/18/42/424019 ◽

2007 ◽

Vol 18 (42) ◽

pp. 424019 ◽

Cited By ~ 7

Author(s):

Miguel Fuentes-Cabrera ◽

Vincent Meunier ◽

Bobby G Sumpter

Keyword(s):

Dna Sequencing ◽

Set Up

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Comparative Study of Metagenomics and Metatranscriptomics to Reveal Microbiomes in Overwintering Pepper Fruits

International Journal of Molecular Sciences ◽

10.3390/ijms22126202 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6202

Author(s):

Yeonhwa Jo ◽

Chang-Gi Back ◽

Kook-Hyung Kim ◽

Hyosub Chu ◽

Jeong Hun Lee ◽

...

Keyword(s):

Dna Sequencing ◽

Analytical Methods ◽

Species Level ◽

Dna Viruses ◽

Mrna Sequencing ◽

Microbial Species ◽

Genome Data ◽

Identification Of Fungi ◽

Identification Of Bacteria ◽

Fungal Database

Red pepper (Capsicum annuum, L.), is one of the most important spice plants in Korea. Overwintering pepper fruits are a reservoir of various microbial pepper diseases. Here, we conducted metagenomics (DNA sequencing) and metatranscriptomics (RNA sequencing) using samples collected from three different fields. We compared two different library types and three different analytical methods for the identification of microbiomes in overwintering pepper fruits. Our results demonstrated that DNA sequencing might be useful for the identification of bacteria and DNA viruses such as bacteriophages, while mRNA sequencing might be beneficial for the identification of fungi and RNA viruses. Among three analytical methods, KRAKEN2 with raw data reads (KRAKEN2_R) might be superior for the identification of microbial species to other analytical methods. However, some microbial species with a low number of reads were wrongly assigned at the species level by KRAKEN2_R. Moreover, we found that the databases for bacteria and viruses were better established as compared to the fungal database with limited genome data. In summary, we carefully suggest that different library types and analytical methods with proper databases should be applied for the purpose of microbiome study.

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