Cargando…

Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter

The variety of applied antibiotics in animal and human medicine results in the release, development, and spread of relevant numbers of antibiotic resistance genes (ARGs) in the environment. The majority of ARGs are present in intracellular forms (in bacteria). Neglected aspects are extracellular var...

Descripción completa

Detalles Bibliográficos
Autores principales: Nischwitz, Volker, Stelmaszyk, Lara, Piel, Sandra, Tiehm, Andreas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9274009/
https://www.ncbi.nlm.nih.gov/pubmed/35836676
http://dx.doi.org/10.3389/fchem.2022.919442
_version_ 1784745222905790464
author Nischwitz, Volker
Stelmaszyk, Lara
Piel, Sandra
Tiehm, Andreas
author_facet Nischwitz, Volker
Stelmaszyk, Lara
Piel, Sandra
Tiehm, Andreas
author_sort Nischwitz, Volker
collection PubMed
description The variety of applied antibiotics in animal and human medicine results in the release, development, and spread of relevant numbers of antibiotic resistance genes (ARGs) in the environment. The majority of ARGs are present in intracellular forms (in bacteria). Neglected aspects are extracellular variants of ARGs (eARGs) and their fragments, which have been detected in surface-water samples and sediments. The stability of eARGs is expected to be low; however, binding to particulate matter is likely to improve their stability and also affect their transport and dissemination behavior. Few studies have investigated DNA particle interactions, mostly via indirect characterization of adduct formation in model systems but not in real environmental matrices. Therefore, our study aims at a novel approach for direct characterization of desoxyribonucleic acid (DNA) particle interactions using both cascade filtration and field-flow fractionation. Cascade filtration with quantitative polymerase chain reaction (qPCR) detection indicated retention of ARGs on filters with much larger pore sizes supporting the hypothesis of ARG-particle interactions. However, artifacts from membrane clogging or DNA–membrane interaction cannot be excluded. Consequently, asymmetric flow field-flow fractionation was investigated as an alternative separation technique with the advantage of particle separation in a thin channel, reducing the risk of artifacts. The key method parameters, membrane composition, molecular weight cut off, and carrier composition, were systematically investigated using a calf-thymus DNA-spiked surface-water sample as a model. The results clearly showed a shift in the elution time of clay particles suggesting the presence of DNA–clay adducts. Multi-element detection by inductively coupled plasma mass spectrometry (ICP-MS) enabled monitoring of clay via the Al, Fe, and Si signals and DNA via the P signal. Matching peak profiles for the new fraction in the fractograms of the ARG and DNA-spiked water sample support adduct formation. Further evidence was provided by a novel post-channel filtration approach for the separation of free DNA from DNA–clay adducts.
format Online
Article
Text
id pubmed-9274009
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-92740092022-07-13 Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter Nischwitz, Volker Stelmaszyk, Lara Piel, Sandra Tiehm, Andreas Front Chem Chemistry The variety of applied antibiotics in animal and human medicine results in the release, development, and spread of relevant numbers of antibiotic resistance genes (ARGs) in the environment. The majority of ARGs are present in intracellular forms (in bacteria). Neglected aspects are extracellular variants of ARGs (eARGs) and their fragments, which have been detected in surface-water samples and sediments. The stability of eARGs is expected to be low; however, binding to particulate matter is likely to improve their stability and also affect their transport and dissemination behavior. Few studies have investigated DNA particle interactions, mostly via indirect characterization of adduct formation in model systems but not in real environmental matrices. Therefore, our study aims at a novel approach for direct characterization of desoxyribonucleic acid (DNA) particle interactions using both cascade filtration and field-flow fractionation. Cascade filtration with quantitative polymerase chain reaction (qPCR) detection indicated retention of ARGs on filters with much larger pore sizes supporting the hypothesis of ARG-particle interactions. However, artifacts from membrane clogging or DNA–membrane interaction cannot be excluded. Consequently, asymmetric flow field-flow fractionation was investigated as an alternative separation technique with the advantage of particle separation in a thin channel, reducing the risk of artifacts. The key method parameters, membrane composition, molecular weight cut off, and carrier composition, were systematically investigated using a calf-thymus DNA-spiked surface-water sample as a model. The results clearly showed a shift in the elution time of clay particles suggesting the presence of DNA–clay adducts. Multi-element detection by inductively coupled plasma mass spectrometry (ICP-MS) enabled monitoring of clay via the Al, Fe, and Si signals and DNA via the P signal. Matching peak profiles for the new fraction in the fractograms of the ARG and DNA-spiked water sample support adduct formation. Further evidence was provided by a novel post-channel filtration approach for the separation of free DNA from DNA–clay adducts. Frontiers Media S.A. 2022-06-28 /pmc/articles/PMC9274009/ /pubmed/35836676 http://dx.doi.org/10.3389/fchem.2022.919442 Text en Copyright © 2022 Nischwitz, Stelmaszyk, Piel and Tiehm. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Chemistry
Nischwitz, Volker
Stelmaszyk, Lara
Piel, Sandra
Tiehm, Andreas
Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter
title Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter
title_full Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter
title_fullStr Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter
title_full_unstemmed Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter
title_short Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter
title_sort cascade filtration with pcr detection and field-flow-fractionation online with icp-ms for the characterization of dna interaction with suspended particulate matter
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9274009/
https://www.ncbi.nlm.nih.gov/pubmed/35836676
http://dx.doi.org/10.3389/fchem.2022.919442
work_keys_str_mv AT nischwitzvolker cascadefiltrationwithpcrdetectionandfieldflowfractionationonlinewithicpmsforthecharacterizationofdnainteractionwithsuspendedparticulatematter
AT stelmaszyklara cascadefiltrationwithpcrdetectionandfieldflowfractionationonlinewithicpmsforthecharacterizationofdnainteractionwithsuspendedparticulatematter
AT pielsandra cascadefiltrationwithpcrdetectionandfieldflowfractionationonlinewithicpmsforthecharacterizationofdnainteractionwithsuspendedparticulatematter
AT tiehmandreas cascadefiltrationwithpcrdetectionandfieldflowfractionationonlinewithicpmsforthecharacterizationofdnainteractionwithsuspendedparticulatematter