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Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification

Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirect...

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Autores principales: Thieme, Lara, Hartung, Anita, Tramm, Kristina, Graf, Julia, Spott, Riccardo, Makarewicz, Oliwia, Pletz, Mathias W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428173/
https://www.ncbi.nlm.nih.gov/pubmed/34512560
http://dx.doi.org/10.3389/fmicb.2021.631248
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author Thieme, Lara
Hartung, Anita
Tramm, Kristina
Graf, Julia
Spott, Riccardo
Makarewicz, Oliwia
Pletz, Mathias W.
author_facet Thieme, Lara
Hartung, Anita
Tramm, Kristina
Graf, Julia
Spott, Riccardo
Makarewicz, Oliwia
Pletz, Mathias W.
author_sort Thieme, Lara
collection PubMed
description Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds.
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spelling pubmed-84281732021-09-10 Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification Thieme, Lara Hartung, Anita Tramm, Kristina Graf, Julia Spott, Riccardo Makarewicz, Oliwia Pletz, Mathias W. Front Microbiol Microbiology Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds. Frontiers Media S.A. 2021-08-26 /pmc/articles/PMC8428173/ /pubmed/34512560 http://dx.doi.org/10.3389/fmicb.2021.631248 Text en Copyright © 2021 Thieme, Hartung, Tramm, Graf, Spott, Makarewicz and Pletz. 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 Microbiology
Thieme, Lara
Hartung, Anita
Tramm, Kristina
Graf, Julia
Spott, Riccardo
Makarewicz, Oliwia
Pletz, Mathias W.
Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification
title Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification
title_full Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification
title_fullStr Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification
title_full_unstemmed Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification
title_short Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification
title_sort adaptation of the start-growth-time method for high-throughput biofilm quantification
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428173/
https://www.ncbi.nlm.nih.gov/pubmed/34512560
http://dx.doi.org/10.3389/fmicb.2021.631248
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