Quantitative image analysis of microbial communities with BiofilmQ

Hartmann, Raimo; Jeckel, Hannah; Jelli, Eric; Singh, Praveen; Vaidya, Sanika; Bayer, Miriam; Rode, D; Vidakovic, Lucia; Díaz-Pascual, Francisco; Fong, Jiunn C. N.; Dragoš, Anna; Lamprecht, Olga; Thöming, Janne G.; Netter, Niklas; Häußler, Susanne; Nadell, Carey D.; Sourjik, Victor; Kovács, Ákos T.; Yildiz, Fitnat H.; Drescher, Knut

doi:10.1038/s41564-020-00817-4

Cited by 197 publications

(185 citation statements)

References 34 publications

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“…Detection and quantification of fluorescent foci. Detection and quantification of foci was performed by first segmenting the images using the software BiofilmQ 94 . After segmentation, fluorescent foci were detected using a customized script within BiofilmQ v0.1.3.…”

Section: Methodsmentioning

confidence: 99%

Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH

et al. 2021

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Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. Here we report that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. We show that in Yersinia enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.

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Section: Methodsmentioning

confidence: 99%

Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH

et al. 2021

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“…Over the past two decades, many researchers have attempted to tackle the problem of automated analysis of biofilm micrographs, including developing commercial and free software tools 29 – 31 . Depending on the size of the cells, the quality and resolution of the image, and thickness or density of the biofilm, two approaches to automated quantification can be taken: (1) by detecting and counting discrete objects or cells or (2) by making assumptions regarding the manner in which the properties of the entire image relate to the biofilm characteristics.…”

Section: Discussionmentioning

confidence: 99%

“…Some of the more frequently used software packages developed specifically for biofilm analysis, such as COMSTAT and BiofilmQ, have an easy to use graphical user interface that make them popular for use. However, one main drawback of both of these packages is that they do not have options to apply filters and morphological operators as with those used in the current study 29 , 31 , which allow for accurate detection of the bacteria in the image whilst neglecting EPS and non-specific staining, commonly found in biofilms. In addition, these software packages rely on the user deciding if pre-processing of the images is necessary, deciding which operators to apply and implementing any pre-processing, which is difficult for those with no prior knowledge of image analysis.…”

Section: Discussionmentioning

confidence: 99%

“…While more robust segmentation protocols have been reported, they are not always accessible or reproducible if the method lacks detail and they may be particularly challenging to implement for non-experts. Many studies use bespoke software such as Imaris 25 , COMSTAT 29 , PHLIP 30 and most recently BiofilmQ 31 . These can make CLSM micrograph analysis easier to navigate through a user-interface.…”

Section: Introductionmentioning

confidence: 99%

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Biofilm viability checker: An open-source tool for automated biofilm viability analysis from confocal microscopy images

Mountcastle

Vyas

Villapún

et al. 2021

npj Biofilms Microbiomes

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Quantifying biofilm formation on surfaces is challenging because traditional microbiological methods, such as total colony-forming units (CFUs), often rely on manual counting. These are laborious, resource intensive techniques, more susceptible to human error. Confocal laser scanning microscopy (CLSM) is a high-resolution technique that allows 3D visualisation of biofilm architecture. In combination with a live/dead stain, it can be used to quantify biofilm viability on both transparent and opaque surfaces. However, there is little consensus on the appropriate methodology to apply in confocal micrograph processing. In this study, we report the development of an image analysis approach to repeatably quantify biofilm viability and surface coverage. We also demonstrate its use for a range of bacterial species and translational applications. This protocol has been created with ease of use and accessibility in mind, to enable researchers who do not specialise in computational techniques to be confident in applying these methods to analyse biofilm micrographs. Furthermore, the simplicity of the method enables the user to adapt it for their bespoke needs. Validation experiments demonstrate the automated analysis is robust and accurate across a range of bacterial species and an improvement on traditional microbiological analysis. Furthermore, application to translational case studies show the automated method is a reliable measurement of biomass and cell viability. This approach will ensure image analysis is an accessible option for those in the microbiology and biomaterials field, improve current detection approaches and ultimately support the development of novel strategies for preventing biofilm formation by ensuring comparability across studies.

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“…Fortunately, over the last decade, methods of empirical lineage tracking have strongly improved through advances in single-cell microscopy [126][127][128], image analysis [129], singlecell sequencing [51,130,131], and genome editing [132,133], thereby opening up the possibility of studying primitive cell collectives across a wide range of organisms [126]. For example, the stochastic and combinatorial expression of fluorescent proteins makes it possible to trace up to approximately 100 lineages without interrupting the spatial configuration of cells [134].…”

Section: Plos Biologymentioning

confidence: 99%

Cryptic surface-associated multicellularity emerges through cell adhesion and its regulation

Gestel

Wagner

2021

PLoS Biol

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The repeated evolution of multicellularity leads to a wide diversity of organisms, many of which are sessile, including land plants, many fungi, and colonial animals. Sessile organisms adhere to a surface for most of their lives, where they grow and compete for space. Despite the prevalence of surface-associated multicellularity, little is known about its evolutionary origin. Here, we introduce a novel theoretical approach, based on spatial lineage tracking of cells, to study this origin. We show that multicellularity can rapidly evolve from 2 widespread cellular properties: cell adhesion and the regulatory control of adhesion. By evolving adhesion, cells attach to a surface, where they spontaneously give rise to primitive cell collectives that differ in size, life span, and mode of propagation. Selection in favor of large collectives increases the fraction of adhesive cells until a surface becomes fully occupied. Through kin recognition, collectives then evolve a central-peripheral polarity in cell adhesion that supports a division of labor between cells and profoundly impacts growth. Despite this spatial organization, nascent collectives remain cryptic, lack well-defined boundaries, and would require experimental lineage tracking technologies for their identification. Our results suggest that cryptic multicellularity could readily evolve and originate well before multicellular individuals become morphologically evident.

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Quantitative image analysis of microbial communities with BiofilmQ

Cited by 197 publications

References 34 publications

Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH

Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH

Biofilm viability checker: An open-source tool for automated biofilm viability analysis from confocal microscopy images

Cryptic surface-associated multicellularity emerges through cell adhesion and its regulation

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