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Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging

BACKGROUND: Tissue samples should be fixed and permanently stabilized as soon as possible ex-vivo to avoid variations in proteomic content. Tissues collected from studies involving infectious microorganisms, must face the additional challenge of pathogen inactivation before downstream proteomic anal...

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Autores principales: Cazares, Lisa H, Van Tongeren, Sean A, Costantino, Julie, Kenny, Tara, Garza, Nicole L, Donnelly, Ginger, Lane, Douglas, Panchal, Rekha G, Bavari, Sina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429342/
https://www.ncbi.nlm.nih.gov/pubmed/25966989
http://dx.doi.org/10.1186/s12866-015-0431-7
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author Cazares, Lisa H
Van Tongeren, Sean A
Costantino, Julie
Kenny, Tara
Garza, Nicole L
Donnelly, Ginger
Lane, Douglas
Panchal, Rekha G
Bavari, Sina
author_facet Cazares, Lisa H
Van Tongeren, Sean A
Costantino, Julie
Kenny, Tara
Garza, Nicole L
Donnelly, Ginger
Lane, Douglas
Panchal, Rekha G
Bavari, Sina
author_sort Cazares, Lisa H
collection PubMed
description BACKGROUND: Tissue samples should be fixed and permanently stabilized as soon as possible ex-vivo to avoid variations in proteomic content. Tissues collected from studies involving infectious microorganisms, must face the additional challenge of pathogen inactivation before downstream proteomic analysis can be safely performed. Heat fixation using the Denator Stabilizor System (Gothenburg, Sweden) utilizes conductive heating, under a mild vacuum, to rapidly eliminate enzymatic degradation in tissue samples. Although many studies have reported on the ability of this method to stop proteolytic degradation and other sample changes immediately and permanently, pathogen inactivation has not been studied. RESULTS: We examined the ability of the heat fixation workflow to inactivate bacterial and viral pathogens and the suitability of this tissue for Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI-MSI). Mice were infected with viral or bacterial pathogens representing two strains of Venezuelan Equine Encephalitis virus (VEEV) and two strains of Burkholderia. Additionally, a tissue mimetic model was employed using Escherichia, Klebsiella and Acinetobacter isolates. Infected tissue samples harvested from each animal or mimetic model were sectioned in half. One half was heat fixed and the other remained untreated. Lysates from each sample were checked for organism viability by performing plaque (infectivity) assays or plating on nutrient agar for colony forming unit (CFU) calculation. Untreated infected control tissue demonstrated the presence of each viable pathogen by positive plaque or colony formation, whereas heat fixation resulted in complete inactivation of both the viral and bacterial pathogens. MALDI-MSI images produced from heat fixed tissue were reflective of molecular distributions within brain, spleen and lung tissue structures. CONCLUSIONS: We conclude that heat fixation inactivates viral and bacterial pathogens and is compatible with proteomic analysis by MALDI-MSI. This treatment will enable the use of infected tissue from studies performed in bio-safety level 3 laboratories with VEEV and Burkholderia to be safely used for proteomic, small molecule drug detection, and imaging mass spectrometry analysis.
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spelling pubmed-44293422015-05-14 Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging Cazares, Lisa H Van Tongeren, Sean A Costantino, Julie Kenny, Tara Garza, Nicole L Donnelly, Ginger Lane, Douglas Panchal, Rekha G Bavari, Sina BMC Microbiol Methodology Article BACKGROUND: Tissue samples should be fixed and permanently stabilized as soon as possible ex-vivo to avoid variations in proteomic content. Tissues collected from studies involving infectious microorganisms, must face the additional challenge of pathogen inactivation before downstream proteomic analysis can be safely performed. Heat fixation using the Denator Stabilizor System (Gothenburg, Sweden) utilizes conductive heating, under a mild vacuum, to rapidly eliminate enzymatic degradation in tissue samples. Although many studies have reported on the ability of this method to stop proteolytic degradation and other sample changes immediately and permanently, pathogen inactivation has not been studied. RESULTS: We examined the ability of the heat fixation workflow to inactivate bacterial and viral pathogens and the suitability of this tissue for Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI-MSI). Mice were infected with viral or bacterial pathogens representing two strains of Venezuelan Equine Encephalitis virus (VEEV) and two strains of Burkholderia. Additionally, a tissue mimetic model was employed using Escherichia, Klebsiella and Acinetobacter isolates. Infected tissue samples harvested from each animal or mimetic model were sectioned in half. One half was heat fixed and the other remained untreated. Lysates from each sample were checked for organism viability by performing plaque (infectivity) assays or plating on nutrient agar for colony forming unit (CFU) calculation. Untreated infected control tissue demonstrated the presence of each viable pathogen by positive plaque or colony formation, whereas heat fixation resulted in complete inactivation of both the viral and bacterial pathogens. MALDI-MSI images produced from heat fixed tissue were reflective of molecular distributions within brain, spleen and lung tissue structures. CONCLUSIONS: We conclude that heat fixation inactivates viral and bacterial pathogens and is compatible with proteomic analysis by MALDI-MSI. This treatment will enable the use of infected tissue from studies performed in bio-safety level 3 laboratories with VEEV and Burkholderia to be safely used for proteomic, small molecule drug detection, and imaging mass spectrometry analysis. BioMed Central 2015-05-13 /pmc/articles/PMC4429342/ /pubmed/25966989 http://dx.doi.org/10.1186/s12866-015-0431-7 Text en © Cazares et al.; licensee BioMed Central. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Cazares, Lisa H
Van Tongeren, Sean A
Costantino, Julie
Kenny, Tara
Garza, Nicole L
Donnelly, Ginger
Lane, Douglas
Panchal, Rekha G
Bavari, Sina
Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging
title Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging
title_full Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging
title_fullStr Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging
title_full_unstemmed Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging
title_short Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging
title_sort heat fixation inactivates viral and bacterial pathogens and is compatible with downstream maldi mass spectrometry tissue imaging
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429342/
https://www.ncbi.nlm.nih.gov/pubmed/25966989
http://dx.doi.org/10.1186/s12866-015-0431-7
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