177fb0a58a231dcfe77b891597608cb6 microorganisms-11-02244.pdf c78eebf827a896d43eb419a4fef3f45369fd32cf microorganisms-11-02244.pdf 79b42c9d4e7788ae549559ff5428020a1240f3d614a699abb8a77ee49bfa7d65 microorganisms-11-02244.pdf Title: A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms Subject: 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 Pseudomonas aeruginosa and Burkholderia cenocepacia 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. Keywords: crystal violet; biomass quantification; Pseudomonas aeruginosa; Burkholderia cenocepacia; dual-species biofilms Author: Víctor Campo-Pérez, Júlia Alcàcer-Almansa, Esther Julián and Eduard Torrents Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Sep 6 11:06:33 2023 CEST ModDate: Wed Sep 6 11:28:48 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 12 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4624354 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- DDRAXE+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 LWJOET+VnURWPalladioL Type 1 Custom yes yes no 15 0 BPRBYT+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 IGYYYR+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 DYSTJV+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 SIXRFS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 46 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 71 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 75 0 NJBNHO+PalatinoLinotype TrueType MacRoman yes yes no 92 0 NJBOLH+PalatinoLinotype,Italic TrueType MacRoman yes yes no 95 0 NJBMNJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 98 0 NJBMNK+PalatinoLinotype CID TrueType Identity-H yes yes yes 101 0 NJBMNL+PalatinoLinotype TrueType WinAnsi yes yes no 107 0 NJBMON+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 110 0 NJBNHO+PalatinoLinotype TrueType MacRoman yes yes no 127 0 NJBMNJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 130 0 NJBMNK+PalatinoLinotype CID TrueType Identity-H yes yes yes 133 0 NJBMNL+PalatinoLinotype TrueType WinAnsi yes yes no 139 0 NJBMON+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 142 0 NJBNHO+PalatinoLinotype TrueType MacRoman yes yes no 174 0 NJBMNJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 177 0 NJBMNK+PalatinoLinotype CID TrueType Identity-H yes yes yes 180 0 NJBMNL+PalatinoLinotype TrueType WinAnsi yes yes no 186 0 NJBMON+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 189 0 NJBNHO+PalatinoLinotype TrueType MacRoman yes yes no 230 0 NJBMNJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 233 0 NJBMNK+PalatinoLinotype CID TrueType Identity-H yes yes yes 236 0 NJBMNL+PalatinoLinotype TrueType WinAnsi yes yes no 242 0 NJBMON+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 245 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-10-27 02:24:51 CEST RepresentationInformation: microorganisms-11-02244.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-10-20 01:34:37 CEST Size: 4624354 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 408 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Bacterial Strains and Culture Conditions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Plasmid Construction and B. cenocepacia Transformation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Plasmid Maintenance Depending on Antibiotic Selection Pressure in the B. cenocepacia Transformed Strain Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Development of a Microtiter Plate Screen for Dual-Species Biofilm Biomass Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Colony-Forming Unit Counts from Biofilms Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Fluorescence Microscopy to Visualize Biofilm Formation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Info: Title: A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms Author: Víctor Campo-Pérez, Júlia Alcàcer-Almansa, Esther Julián and Eduard Torrents Subject: 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 Pseudomonas aeruginosa and Burkholderia cenocepacia 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. 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