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

BMC Microbiology

Sina Bavari¹  Rekha G Panchal¹  Douglas Lane¹  Ginger Donnelly¹  Nicole L Garza¹  Tara Kenny¹  Julie Costantino¹  Sean A Van Tongeren¹  Lisa H Cazares¹ 

[1]Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA

关键词: Tissue imaging;    Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS);    Microbial inactivation;    Proteomics;    Tissue fixation;   

Others  :  1212042
DOI  :  10.1186/s12866-015-0431-7

 received in 2014-08-11, accepted in 2015-04-23,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Cazares et al.; licensee BioMed Central.

【 图 表 】

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