A Novel Computerized Cell Count Algorithm for Biofilm Analysis

Klinger-Strobel, Mareike; Suesse, Herbert; Fischer, Dagmar; Pletz, Mathias W.; Makarewicz, Oliwia

doi:10.1371/journal.pone.0154937

Cited by 30 publications

(28 citation statements)

References 22 publications

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“…The biofilms were stained using LIVE/DEAD BacLight Bacterial Viability Kit for microscopy (Life Technologies GmbH) according to manufacturer’s protocol. Stained biofilms were analyzed under vital conditions using an inverted confocal laser scanning microscope (CLSM) Carl Zeiss LSM 780 (Carl Zeiss AG) at green (522 nm) and red (635 nm) filters, respectively, using laser excitation at 490 nm as described previously ( Klinger-Strobel et al, 2016 ; Baidamshina et al, 2017 ). An area of approximately 100 μm (X) × 100 μm (Y) was screened in 1 μm Z-intervals (Z-stack).…”

Section: Methodsmentioning

confidence: 99%

Antimicrobial Effects of Sulfonyl Derivative of 2(5H)-Furanone against Planktonic and Biofilm Associated Methicillin-Resistant and -Susceptible Staphylococcus aureus

et al. 2017

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The gram-positive opportunistic bacterium Staphylococcus aureus is one of the most common causatives of a variety of diseases including skin and skin structure infection or nosocomial catheter-associated infections. The biofilm formation that is an important virulence factor of this microorganism renders the antibiotic therapy ineffective, because biofilm-embedded bacteria exhibit strongly increased tolerance to antimicrobials. Here, we describe a novel 3-chloro-5(S)-[(1R,2S,5R)-2-isopropyl-5-methylcyclohexyloxy]-4-[4-methylphenylsulfonyl]-2(5H)-furanone (F105), possessing a sulfonyl group and l-menthol moiety. Minimal inhibitory and bactericidal concentration values (MIC and MBC) of F105 were 10 and 40 mg/L, respectively, suggesting F105 biocidal properties. F105 exhibits pronounced activity against biofilm-embedded S. aureus and increases the efficacy of aminoglycosides (amikacin, gentamicin, and kanamycin) and benzalkonium chloride with fractional inhibitory concentration index values of 0.33-0.44 and 0.29, respectively, suggesting an alternative external treatment option, e.g., for wound infections. Moreover, low concentrations (0.5-1.3 mg/L) of F105 reduced the MICs of these antimicrobials twofold. By using confocal laser scanning microscopy and CFU counting, we show explicitly that F105 also restores the antimicrobial activity of gentamicin and ampicillin against S. aureus biofilms by several orders of magnitude. Biofilm structures were not destroyed but sterilized, with embedded cells being almost completely killed at twofold MBC. While F105 is quite toxic (CC 50 /MBC ratio 0.2), our data suggest that the F105 chemotype might be a promising starting point for the development of complex topical agents for combined anti-staphylococcal biofilm-therapies restoring the efficacy of some antibiotics against difficult to treat S. aureus biofilm.

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Section: Methodsmentioning

confidence: 99%

Antimicrobial Effects of Sulfonyl Derivative of 2(5H)-Furanone against Planktonic and Biofilm Associated Methicillin-Resistant and -Susceptible Staphylococcus aureus

et al. 2017

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“…In addition, it is well known that sub-MIC levels of antibiotic can dramatically alter gene expression profiles in bacteria (37)(38)(39), indicating that biofilm enhancement may arise from a complex combination of multiple factors. Finally, we note that live-dead cell staining results should be interpreted with some caution, because uptake of various stains may be variable (40)(41)(42)(43)(44). Nevertheless, our results are promising because they suggest that, at least under the experimental conditions assessed here, a simple conceptual (and mathematical) model is sufficient to describe and to predict the primary effects of drug exposure.…”

Section: Discussionmentioning

confidence: 65%

Interplay between antibiotic efficacy and drug-induced lysis underlies enhanced biofilm formation at subinhibitory drug concentrations

Hallinen

Wood

2017

Preprint

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Subinhibitory concentrations of antibiotics have been shown to enhance biofilm formation in multiple bacterial species. While antibiotic exposure has been associated with modulated expression of many biofilm-related genes, the mechanisms of drug-induced biofilm formation remain a focus of ongoing research efforts and may vary significantly across species. In this work, we investigate antibiotic-induced biofilm formation in Enterococcus faecalis, a leading cause of nosocomial infections. We show that biofilm formation is enhanced by subinhibitory concentrations of cell wall synthesis inhibitors but not by inhibitors of protein, DNA, folic acid, or RNA synthesis. Furthermore, enhanced biofilm is associated with increased cell lysis, increases in extracellular DNA (eDNA) levels, and increases in the density of living cells in the biofilm. In addition, we observe similar enhancement of biofilm formation when cells are treated with nonantibiotic surfactants that induce cell lysis. These findings suggest that antibiotic-induced biofilm formation is governed by a trade-off between drug toxicity and the beneficial effects of cell lysis. To understand this trade-off, we developed a simple mathematical model that predicts changes in antibiotic-induced biofilm formation due to external perturbations, and we verified these predictions experimentally. Specifically, we demonstrate that perturbations that reduce eDNA (DNase treatment) or decrease the number of living cells in the planktonic phase (a second antibiotic) decrease biofilm induction, while chemical inhibitors of cell lysis increase relative biofilm induction and shift the peak to higher antibiotic concentrations. Overall, our results offer experimental evidence linking cell wall synthesis inhibitors, cell lysis, increased eDNA levels, and biofilm formation in E. faecalis while also providing a predictive quantitative model that sheds light on the interplay between cell lysis and antibiotic efficacy in developing biofilms.KEYWORDS Enterococcus, biofilms, mathematical modeling, subinhibitory antibiotics B iofilms are dense, surface-associated, microbial communities that play important roles in infectious diseases and a range of device-related clinical infections (1, 2). Biofilms exhibit a fascinating range of community behavior (3), including long-range metabolic codependence (4) and electrical signaling (5-7), phenotypic phase variation (8) and spatial heterogeneity (9), strong ecological competition (10), and multiple types of cooperative behavior, including collective resistance to antimicrobial therapy (11-13). The biofilm response to antibiotics has been a topic of particular interest, with biofilms across species showing dramatically increased resistance to antibiotics relative to planktonic cells. However, a number of studies have shown, somewhat counterintuitively, that exposure to sublethal doses of antibiotics may enhance biofilm formation in a wide range of species (14-16). While antibiotic-mediated biofilm induction has been associated with modul...

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“…In comparison with some other freely available image processing and analysis software [18-20], our image-processing procedure is semi-automatic: settings are modified by manually adjusting the analysis parameters and each step is controlled with an expert to evaluate the analysis quality. Being optimized once for a first image in a series the software analyzes other images automatically over coffee-break.…”

Section: Resultsmentioning

confidence: 99%

“…While the detection methods fail under cell overlapping conditions, the statistical assessment methods are unable to differentiate between various types of cells. Among few exceptions that largely overcome the above limitations, a very recently designed software tool for quantification of live/dead cells in a biofilm based on a series of image transformation could be mentioned [18].…”

Section: Introductionmentioning

confidence: 99%

Fast and Simple Tool for the Quantification of Biofilm-Embedded Cells Sub-Populations from Fluorescent Microscopic Images

Bogachev

Markelov

Trizna

et al. 2017

Preprint

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27Fluorescent staining is a common tool for both quantitative and qualitative assessment of pro-and 28 eukaryotic cells sub-population fractions by using microscopy and flow cytometry. However, direct cell 29 counting by flow cytometry is often limited, for example when working with cells rigidly adhered 30 either to each other or to external surfaces like in bacterial biofilms or adherent cell lines and tissue 31samples. An alternative approach is provided by using fluorescent microscopy and confocal laser 32 scanning microscopy (CLSM), which enables the evaluation of fractions of cells subpopulations in a 33 given sample. To facilitate the quantitative assessment of cell fractions in microphotographs, we 34 suggest a simple two-step algorithm that combines the cell selection based and the statistical 35 approaches. Based on a series of experimental measurements performed on bacterial and eukaryotic 36 cells under various measurement conditions, we show explicitly that the suggested approach effectively 37 accounts for the fractions of different cell sub-populations (like the live/dead staining in our samples) 38 in all studied cases that are in good agreement with manual cell counting on microphotographs and 39 flow cytometry data. This algorithm is implemented as a simple software tool that includes an intuitive 40 and user-friendly graphical interface for the initial adjustment of algorithm parameters to the 41 microscopic imaging conditions as well as for the sequential analysis of homogeneous series of similar 42 microscopic images without further user intervention. The software tool entitled BioFilmAnalyzer is 43 freely available online at

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A Novel Computerized Cell Count Algorithm for Biofilm Analysis

Cited by 30 publications

References 22 publications

Antimicrobial Effects of Sulfonyl Derivative of 2(5H)-Furanone against Planktonic and Biofilm Associated Methicillin-Resistant and -Susceptible Staphylococcus aureus

Antimicrobial Effects of Sulfonyl Derivative of 2(5H)-Furanone against Planktonic and Biofilm Associated Methicillin-Resistant and -Susceptible Staphylococcus aureus

Interplay between antibiotic efficacy and drug-induced lysis underlies enhanced biofilm formation at subinhibitory drug concentrations

Fast and Simple Tool for the Quantification of Biofilm-Embedded Cells Sub-Populations from Fluorescent Microscopic Images

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