scholarly journals Efficient microbial colony growth dynamics quantification with ColTapp, an automated image analysis application

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Julian Bär ◽  
Mathilde Boumasmoud ◽  
Roger D. Kouyos ◽  
Annelies S. Zinkernagel ◽  
Clément Vulin
Keyword(s):  
Growth Dynamics ◽  
Time Lapse ◽  
Colony Growth ◽  
Lag Time ◽  
Automated Image Analysis ◽  
Clinical Samples ◽  
Clinical Settings ◽  
Bacterial Colony ◽  
Analysis Application ◽  
Population Survival

Abstract Populations of genetically identical bacteria are phenotypically heterogeneous, giving rise to population functionalities that would not be possible in homogeneous populations. For instance, a proportion of non-dividing bacteria could persist through antibiotic challenges and secure population survival. This heterogeneity can be studied in complex environmental or clinical samples by spreading the bacteria on agar plates and monitoring time to growth resumption in order to infer their metabolic state distribution. We present ColTapp, the Colony Time-lapse application for bacterial colony growth quantification. Its intuitive graphical user interface allows users to analyze time-lapse images of agar plates to monitor size, color and morphology of colonies. Additionally, images at isolated timepoints can be used to estimate lag time. Using ColTapp, we analyze a dataset of Staphylococcus aureus time-lapse images including populations with heterogeneous lag time. Colonies on dense plates reach saturation early, leading to overestimation of lag time from isolated images. We show that this bias can be corrected by taking into account the area available to each colony on the plate. We envision that in clinical settings, improved analysis of colony growth dynamics may help treatment decisions oriented towards personalized antibiotic therapies.

scholarly journals ColTapp, an automated image analysis application for efficient microbial colony growth dynamics quantification

2020 ◽  
Author(s):  
Julian Bär ◽  
Mathilde Boumasmoud ◽  
Roger Kouyos ◽  
Annelies S. Zinkernagel ◽  
Clément Vulin
Keyword(s):  
Growth Dynamics ◽  
Time Estimation ◽  
Time Lapse ◽  
Colony Growth ◽  
Lag Time ◽  
Phenotypic Heterogeneity ◽  
Automated Image Analysis ◽  
Bacterial Colony ◽  
Staphyloccocus Aureus ◽  
Analysis Application

AbstractPhenotypic heterogeneity occurs in a population of genetically identical bacteria due to stochastic molecular fluctuations and environmental variations. In extreme cases of phenotypic heterogeneity, a fraction of the bacterial population enters dormancy, and these metabolically inactive or non-dividing bacteria persist through most antibiotic challenges. These subpopulations of persister cells are difficult to study in patient samples. However, the proportion of persisters in a sample can be accessed by physically separating bacteria on a plate measuring the time until colonies become visible as dormant bacteria resume growth later than their active counterparts and form smaller colonies.Here, we present ColTapp (Colony Time-lapse app), an application dedicated to bacterial colony growth quantification, freely available for download together with its MATLAB source code or as a MacOS/Windows executable. ColTapp’s intuitive graphical user interface allows users without prior coding knowledge to analyze endpoint or time-lapse images of colonies on agar plates. Colonies are detected automatically, and their radius can be tracked over time. Downstream analyses to derive colony lag time and growth rate are implemented.We demonstrate here the applicability of ColTapp on a dataset of Staphyloccocus aureus colony time-lapse images. Colonies on dense plates reached saturation early, biasing lag time estimation from endpoint images. This bias can be reduced by considering the area available to each colony on a plate.By facilitating the analysis of colony growth dynamics in clinical settings, this application will enable a new type of diagnostics, oriented towards personalized antibiotic therapies.

scholarly journals Author Correction: Efficient microbial colony growth dynamics quantification with ColTapp, an automated image analysis application

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Julian Bär ◽  
Mathilde Boumasmoud ◽  
Roger D. Kouyos ◽  
Annelies S. Zinkernagel ◽  
Clément Vulin
Keyword(s):  
Image Analysis ◽  
Growth Dynamics ◽  
Colony Growth ◽  
Automated Image Analysis ◽  
Analysis Application

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

scholarly journals Quantification of within patient Staphylococcus aureus phenotypic heterogeneity as a proxy for presence of persisters across clinical presentations

2021 ◽  
Author(s):  
Julian Baer ◽  
Mathilde Boumasmoud ◽  
Srikanth Mairpady Shambat ◽  
Cl&eacutement Vulin ◽  
Markus Huemer ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Colony Size ◽  
Genetic Resistance ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Colony Growth ◽  
Phenotypic Heterogeneity ◽  
Growth Delay ◽  
Size Heterogeneity

Background: Difficult-to-treat infections caused by antibiotic susceptible strains have been linked with the occurrence of persisters. Persisters are a subpopulation of dormant bacteria that tolerate antibiotic exposure despite lacking genetic resistance. They can be identified phenotypically upon plating on nutrient agar because of their altered growth dynamics, resulting in colony size heterogeneity. The occurrence of within-patient bacterial phenotypic heterogeneity in various infections and clinical determinants of persister formation remain unknown. Methods: We plated bacteria derived from 132 patient-samples of difficult-to-treat infections directly on nutrient-rich agar and monitored colony growth by time lapse imaging. Of these, we retained 36 Staphylococcus aureus mono-cultures for further analysis. We investigated clinical factors potentially associated with increased colony growth-delay with regression analyses. Additionally, we corroborated the clinical findings using in vitro grown static biofilms, exposed to distinct antibiotics. Results: The extent of phenotypic heterogeneity of patient-derived S. aureus varied substantially between patients. Increased heterogeneity coincided with increased median growth-delay. Multivariable regression showed that rifampicin treatment was significantly associated with increased median growth-delay. S. aureus grown in biofilms and exposed to high concentrations of rifampicin or a combination of rifampicin with either clindamycin or levofloxacin exhibited prolonged growth-delay, correlating with a strain-dependent increase in antibiotic tolerance. Conclusion: Upon direct cultivation on nutrient-rich agar, S. aureus from difficult to treat infections commonly exhibited colony size heterogeneity. This was due to heterogeneous delays in growth resumption, with delays larger than two days in the most extreme cases. Since bacteria in a dormant state are tolerant to antibiotics, the observation of large growth-delays might have direct clinical implications. Future studies are needed to assess the potential of bacterial phenotypic heterogeneity quantification for staphylococcal infections prognosis.

scholarly journals Development of single-cell-level microfluidic technology for long-term growth visualization of living cultures of Mycobacterium smegmatis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Han Wang ◽  
Gloria M. Conover ◽  
Song-I Han ◽  
James C. Sacchettini ◽  
Arum Han
Keyword(s):  
Drug Treatment ◽  
Single Cell ◽  
Mycobacterium Smegmatis ◽  
Culture Media ◽  
Low Cost ◽  
Bacterial Pathogen ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Phenotype

AbstractAnalysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

scholarly journals Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells

2013 ◽  
Vol 79 (7) ◽  
pp. 2294-2301 ◽  
Author(s):  
Konstantinos P. Koutsoumanis ◽  
Alexandra Lianou
Keyword(s):  
Kinetic Parameters ◽  
Single Cells ◽  
Growth Curves ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Initial Population ◽  
Bacterial Cells ◽  
Stochastic Growth ◽  
Bacterial Populations ◽  
Content Type

ABSTRACTConventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells ofSalmonella entericaserotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μmax) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (N0). For bacterial populations withN0of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.

Validation of a new image analysis procedure for quantifying filamentous bacteria in activated sludge

2014 ◽  
Vol 70 (6) ◽  
pp. 955-963 ◽  
Author(s):  
Ewa Liwarska-Bizukojc ◽  
Marcin Bizukojc ◽  
Olga Andrzejczak
Keyword(s):  
Image Processing ◽  
Image Analysis ◽  
Activated Sludge ◽  
Analysis Procedure ◽  
Automated Image Analysis ◽  
Filamentous Bacteria ◽  
Image Processing And Analysis ◽  
Manual Counting ◽  
Analysis Application ◽  
Activated Sludge Systems

Quantification of filamentous bacteria in activated sludge systems can be made by manual counting under a microscope or by the application of various automated image analysis procedures. The latter has been significantly developed in the last two decades. In this work a new method based upon automated image analysis techniques was elaborated and presented. It consisted of three stages: (a) Neisser staining, (b) grabbing of microscopic images, and (c) digital image processing and analysis. This automated image analysis procedure possessed the features of novelty. It simultaneously delivered data about aggregates and filaments in an individual calculation routine, which is seldom met in the procedures described in the literature so far. What is more important, the macroprogram performing image processing and calculation of morphological parameters was written in the same software which was used for grabbing of images. Previously published procedures required using two different types of software, one for image grabbing and another one for image processing and analysis. Application of this new procedure for the quantification of filamentous bacteria in the full-scale as well as laboratory activated sludge systems proved that it was simple, fast and delivered reliable results.

scholarly journals Cell-to-cell heterogeneity in Escherichia coli stress response originates from pulsatile expression and growth

2020 ◽  
Author(s):  
Nadia M. V. Sampaio ◽  
Caroline M. Blassick ◽  
Jean-Baptiste Lugagne ◽  
Mary J. Dunlop
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Stress Response ◽  
Single Cell ◽  
Phenotypic Variability ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Heterogeneity ◽  
Temporal Expression ◽  
Functional Consequences

AbstractCell-to-cell heterogeneity in gene expression and growth can have critical functional consequences, such as determining whether individual bacteria survive or die following stress. Although phenotypic variability is well documented, the dynamics that underlie it are often unknown. This information is critical because dramatically different outcomes can arise from gradual versus rapid changes in expression and growth. Using single-cell time-lapse microscopy, we measured the temporal expression of a suite of stress response reporters in Escherichia coli, while simultaneously monitoring growth rate. In conditions without stress, we found widespread examples of pulsatile expression. Single-cell growth rates were often anti-correlated with gene expression, with changes in growth preceding changes in expression. These pulsatile dynamics have functional consequences, which we demonstrate by measuring survival after challenging cells with the antibiotic ciprofloxacin. Our results suggest that pulsatile expression and growth dynamics are common in stress response networks and can have direct consequences for survival.

scholarly journals The Face of Hypervirulent Klebsiella Pneumoniae (hvKp) Isolated from Clinical Samples of Two Iranian Teaching Hospitals

2021 ◽  
Author(s):  
Rahimeh Sanikhani ◽  
Mohammad Moeinirad ◽  
Hamid Solgi ◽  
Azar Haddadi ◽  
Fereshteh Shahcheraghi ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Klebsiella Pneumoniae ◽  
Multidrug Resistant ◽  
Teaching Hospitals ◽  
Clinical Samples ◽  
Clinical Settings ◽  
Urgent Problem ◽  
Clonal Relatedness ◽  
The Face ◽  
Global Concern

Abstract Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a pathogen of global concern. In this study, both phenotypic and genotypic tests were used to detect hvKp. Antimicrobial resistance profiles and clonal relatedness of clinical isolates were also determined. We found that 62.6% of the isolates were tellurite resistant and among them iucA or iutA or peg344 as hvKp molecular markers, were positive. The blaSHV (81.4%), followed by blaCTX−M15 (75.5%) and blaTEM (67.6%), blaOXA−48 (33.7%), blaNDM−1 (32.3%) were detected, while blaKPC−1 was not present in any hvKp isolates. It was found that the majority of hvKp isolates belonged to capsular serotype K20 and ompK36 group C, which is related to CG23 (e.g. ST23). A high percentage of multidrug-resistant hvKp (MDR-hvKp) and high resistance to imipenem (66%) indicated that there is an urgent problem that should be addressed in the clinical settings.

scholarly journals Plate reader microrheology

2020 ◽  
Author(s):  
Robert F. Hawkins ◽  
Gregg A. Duncan
Keyword(s):  
Particle Tracking ◽  
Fluorescence Polarization ◽  
Rotational Diffusion ◽  
Polymeric Materials ◽  
Soft Materials ◽  
Clinical Samples ◽  
Clinical Settings ◽  
Particle Tracking Microrheology ◽  
Plate Reader ◽  
Potential Applications

AbstractIn this work, we report the development of a simplified microrheological method that can be used to rapidly study soft materials. This approach uses fluorescence polarization and a plate reader format to measure the rotational diffusion of nanoparticles within a sample of interest. We show that this measurement is sensitive to viscosity-dependent changes in polymeric soft materials and is correlated with particle tracking microrheology, a previously validated measure of microrheology. Using these fluorescence polarization-based measurements, we describe formalism that enables reasonable estimation of viscosity in polymeric materials after accounting for length-scale dependent effects of the polymer environment on the nanoparticle rotational diffusion. The use of a plate reader format allows this approach to be higher throughput, less technically challenging, and more widely accessible than standard macro- and microrheological methods, making it available to non-experts. This approach has potential applications in academic and industry settings where conventional rheological equipment may not be available, as well as in clinical settings to rapidly characterize human clinical samples.

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