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The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients

BACKGROUND: The application of molecular based diagnostics in sepsis has had limited success to date. Molecular community profiling methods have indicated that polymicrobial infections are more common than suggested by standard clinical culture. A molecular profiling approach was developed to invest...

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Autores principales: Faria, MMP, Conly, JM, Surette, MG
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609058/
https://www.ncbi.nlm.nih.gov/pubmed/26474751
http://dx.doi.org/10.1186/s12866-015-0557-7
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author Faria, MMP
Conly, JM
Surette, MG
author_facet Faria, MMP
Conly, JM
Surette, MG
author_sort Faria, MMP
collection PubMed
description BACKGROUND: The application of molecular based diagnostics in sepsis has had limited success to date. Molecular community profiling methods have indicated that polymicrobial infections are more common than suggested by standard clinical culture. A molecular profiling approach was developed to investigate the propensity for polymicrobial infections in patients predicted to have bacterial sepsis. RESULTS: Disruption of blood cells with saponin and hypotonic shock enabled the recovery of microbial cells with no significant changes in microbial growth when compared to CFU/ml values immediately prior to the addition of saponin. DNA extraction included a cell-wall digestion step with both lysozyme and mutanolysin, which increased the recovery of terminal restriction fragments by 2.4 fold from diverse organisms. Efficiencies of recovery and limits of detection using Illumina sequencing of the 16S rRNA V3 region were determined for both viable cells and DNA using mock bacterial communities inoculated into whole blood. Bacteria from pre-defined communities could be recovered following lysis and removal of host cells with > 97 % recovery of total DNA present. Applying the molecular profiling methodology to three septic patients in the intensive care unit revealed microbial DNA from blood had consistent alignment with cultured organisms from the primary infection site providing evidence for a bloodstream infection in the absence of a clinical lab positive blood culture result in two of the three cases. In addition, the molecular profiling indicated greater diversity was present in the primary infection sample when compared to clinical diagnostic culture. CONCLUSIONS: A method for analyzing bacterial DNA from whole blood was developed in order to characterize the bacterial DNA profile of sepsis infections. Preliminary results indicated that sepsis infections were polymicrobial in nature with the bacterial DNA recovered suggesting a more complex etiology when compared to blood culture data.
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spelling pubmed-46090582015-10-18 The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients Faria, MMP Conly, JM Surette, MG BMC Microbiol Methodology Article BACKGROUND: The application of molecular based diagnostics in sepsis has had limited success to date. Molecular community profiling methods have indicated that polymicrobial infections are more common than suggested by standard clinical culture. A molecular profiling approach was developed to investigate the propensity for polymicrobial infections in patients predicted to have bacterial sepsis. RESULTS: Disruption of blood cells with saponin and hypotonic shock enabled the recovery of microbial cells with no significant changes in microbial growth when compared to CFU/ml values immediately prior to the addition of saponin. DNA extraction included a cell-wall digestion step with both lysozyme and mutanolysin, which increased the recovery of terminal restriction fragments by 2.4 fold from diverse organisms. Efficiencies of recovery and limits of detection using Illumina sequencing of the 16S rRNA V3 region were determined for both viable cells and DNA using mock bacterial communities inoculated into whole blood. Bacteria from pre-defined communities could be recovered following lysis and removal of host cells with > 97 % recovery of total DNA present. Applying the molecular profiling methodology to three septic patients in the intensive care unit revealed microbial DNA from blood had consistent alignment with cultured organisms from the primary infection site providing evidence for a bloodstream infection in the absence of a clinical lab positive blood culture result in two of the three cases. In addition, the molecular profiling indicated greater diversity was present in the primary infection sample when compared to clinical diagnostic culture. CONCLUSIONS: A method for analyzing bacterial DNA from whole blood was developed in order to characterize the bacterial DNA profile of sepsis infections. Preliminary results indicated that sepsis infections were polymicrobial in nature with the bacterial DNA recovered suggesting a more complex etiology when compared to blood culture data. BioMed Central 2015-10-16 /pmc/articles/PMC4609058/ /pubmed/26474751 http://dx.doi.org/10.1186/s12866-015-0557-7 Text en © Faria et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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
Faria, MMP
Conly, JM
Surette, MG
The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients
title The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients
title_full The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients
title_fullStr The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients
title_full_unstemmed The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients
title_short The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients
title_sort development and application of a molecular community profiling strategy to identify polymicrobial bacterial dna in the whole blood of septic patients
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609058/
https://www.ncbi.nlm.nih.gov/pubmed/26474751
http://dx.doi.org/10.1186/s12866-015-0557-7
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