A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms
Pathogenic bacteria form biofilms during infection, and polymicrobial biofilms are the most frequent manifestation. Biofilm attachment, maturation, and/or antibiotic sensitivity are mainly evaluated with microtiter plate assays, in which bacteria are stained to enable the quantification of the bioma...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-09-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/11/9/2244 |
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| author | Víctor Campo-Pérez Júlia Alcàcer-Almansa Esther Julián Eduard Torrents |
| author_facet | Víctor Campo-Pérez Júlia Alcàcer-Almansa Esther Julián Eduard Torrents |
| author_sort | Víctor Campo-Pérez |
| collection | DOAJ |
| description | Pathogenic bacteria form biofilms during infection, and polymicrobial biofilms are the most frequent manifestation. Biofilm attachment, maturation, and/or antibiotic sensitivity are mainly evaluated with microtiter plate assays, in which bacteria are stained to enable the quantification of the biomass by optical absorbance or fluorescence emission. However, using these methods to distinguish different species in dual-species or polymicrobial biofilms is currently impossible. Colony-forming unit counts from homogenized dual-species biofilms on selective agar medium allow species differentiation but are time-consuming for a high-throughput screening. Thus, reliable, feasible, and fast methods are urgently needed to study the behavior of polymicrobial and dual-species communities. This study shows that <i>Pseudomonas aeruginosa</i> and <i>Burkholderia cenocepacia</i> strains expressing specific fluorescent or bioluminescent proteins permit the more efficient study of dual-species biofilms compared to other methods that rely on measuring the total biomass. Combining fluorescence and bioluminescence measurements allows an independent analysis of the different microbial species within the biofilm, indicating the degree of presence of each one over time during a dual-species biofilm growth. The quantitative strategies developed in this work are reproducible and recommended for dual-species biofilm studies with high-throughput microtiter plate approaches using strains that can constitutively express fluorescent or bioluminescent proteins. |
| first_indexed | 2024-03-10T22:26:48Z |
| format | Article |
| id | doaj.art-2a80aed03b60437a8e31cacc7925fe0f |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T22:26:48Z |
| publishDate | 2023-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-2a80aed03b60437a8e31cacc7925fe0f2023-11-19T12:02:38ZengMDPI AGMicroorganisms2076-26072023-09-01119224410.3390/microorganisms11092244A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species BiofilmsVíctor Campo-Pérez0Júlia Alcàcer-Almansa1Esther Julián2Eduard Torrents3Bacterial Infections and Antimicrobial Therapies Group, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 15-21, 08028 Barcelona, SpainBacterial Infections and Antimicrobial Therapies Group, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 15-21, 08028 Barcelona, SpainDepartament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Barcelona, SpainBacterial Infections and Antimicrobial Therapies Group, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 15-21, 08028 Barcelona, SpainPathogenic bacteria form biofilms during infection, and polymicrobial biofilms are the most frequent manifestation. Biofilm attachment, maturation, and/or antibiotic sensitivity are mainly evaluated with microtiter plate assays, in which bacteria are stained to enable the quantification of the biomass by optical absorbance or fluorescence emission. However, using these methods to distinguish different species in dual-species or polymicrobial biofilms is currently impossible. Colony-forming unit counts from homogenized dual-species biofilms on selective agar medium allow species differentiation but are time-consuming for a high-throughput screening. Thus, reliable, feasible, and fast methods are urgently needed to study the behavior of polymicrobial and dual-species communities. This study shows that <i>Pseudomonas aeruginosa</i> and <i>Burkholderia cenocepacia</i> strains expressing specific fluorescent or bioluminescent proteins permit the more efficient study of dual-species biofilms compared to other methods that rely on measuring the total biomass. Combining fluorescence and bioluminescence measurements allows an independent analysis of the different microbial species within the biofilm, indicating the degree of presence of each one over time during a dual-species biofilm growth. The quantitative strategies developed in this work are reproducible and recommended for dual-species biofilm studies with high-throughput microtiter plate approaches using strains that can constitutively express fluorescent or bioluminescent proteins.https://www.mdpi.com/2076-2607/11/9/2244crystal violetbiomass quantification<i>Pseudomonas aeruginosa</i><i>Burkholderia cenocepacia</i>dual-species biofilms |
| spellingShingle | Víctor Campo-Pérez Júlia Alcàcer-Almansa Esther Julián Eduard Torrents A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms Microorganisms crystal violet biomass quantification <i>Pseudomonas aeruginosa</i> <i>Burkholderia cenocepacia</i> dual-species biofilms |
| title | A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms |
| title_full | A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms |
| title_fullStr | A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms |
| title_full_unstemmed | A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms |
| title_short | A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms |
| title_sort | high throughput microtiter plate screening assay to quantify and differentiate species in dual species biofilms |
| topic | crystal violet biomass quantification <i>Pseudomonas aeruginosa</i> <i>Burkholderia cenocepacia</i> dual-species biofilms |
| url | https://www.mdpi.com/2076-2607/11/9/2244 |
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