Reconstructing the Forest of Lineage Trees of Diverse Bacterial Communities Using Bio-inspired Image Analysis

Cell segmentation and tracking allow us to extract a plethora of cell attributes from bacterial time-lapse cell movies, thus promoting computational modeling and simulation of biological processes down to the single-cell level. However, to analyze successfully complex cell movies, imaging multiple interacting bacterial clones as they grow and merge to generate overcrowded bacterial communities with thousands of cells in the field of view, segmentation results should be near perfect to warrant good tracking results. We introduce here a fully automated closed-loop bio-inspired computational strategy that exploits prior knowledge about the expected structure of a colony's lineage tree to locate and correct segmentation errors in analyzed movie frames. We show that this correction strategy is effective, resulting in improved cell tracking and consequently trustworthy deep colony lineage trees. Our image analysis approach has the unique capability to keep tracking cells even after clonal subpopulations merge in the movie. This enables the reconstruction of the complete Forest of Lineage Trees (FLT) representation of evolving multi-clonal bacterial communities. Moreover, the percentage of valid cell trajectories extracted from the image analysis almost doubles after segmentation correction. This plethora of trustworthy data extracted from a complex cell movie analysis enables single-cell analytics as a tool for addressing compelling questions for human health, such as understanding the role of single-cell stochasticity in antibiotics resistance without losing site of the inter-cellular interactions and microenvironment effects that may shape it

Variability in the response of salmonella and listeria strains to different strategies for inactivation

[ENG] The last EFSA report, notified a total of 5146 food outbreaks in the European Union in 2018. The food industry plays an important role in the food chain and has to adapt and reinvent itself when consumers place certain demands on products. Food safety is the first issue to take into account. There is already a large variety of microbiological species that are capable of developing in food and the safety of food makes it questionable whether or not the product is suitable for consumption. Listeria monocytogenes and Salmonella spp. are characterised by being two ubiquitous microbial species capable of living in borderline conditions of pH, water activity and sodium chloride concentrations. Another circumstance of these two microorganisms is the variability between their different strains and the capacity they have to recover from the different stresses to which they are subjected. For these reasons, it is of utmost importance to know their response to stress in order to control their inactivation and/or development in any situation. This knowledge will enable to efficiently apply technologies and treatments that are capable of inactivating or inhibiting growth of microorganisms, so to prevent or, at least, reduce food contamination. So, in this thesis, the main objective was to study the application of different strategies for the inactivation of L monocytogenes and Salmonella that could reduce the intensity of the treatments, while ensuring food safety. In this thesis, the effect that different inactivation strategies have on L. monocytogenes and Salmonella spp. has been studied. Firstly, the heat resistance of four L. monocytogenes strains -Scott A, CECT 4031, CECT 4032 and 12MOB052- in three different matrices -buffered peptone water (BPW), pH 7 Mcllvaine phosphate citrate buffer and a last one of food origin; semi- skimmed milk- was studied. Under isothermal conditions, there was no between-strains and between-media variability in the resistance. However, when this experiment was carried out under dynamic conditions, some strains were able to develop acclimation to stress, leading to important differences in resistance. Between-strains, the CECT 4031 increased its D-value by 10, with CECT 4032 being the least acclimated and between-media, peptone water buffer and semi- skimmed milk were the mediums where most acclimation was found. These results are of great importance, as they highlight that some mechanisms of heat resistance may not be detected when conditions, such as acclimation.Another part of the research was carried out to evaluate the response to heat when Salmonella Senftenberg and S. Enteritidis - the treatment applied is under isothermal conditions, but they show up under dynamic heating were exposed to a previous acid shock. Both serovars of Salmonella were grown at pH 4.5 and subsequently exposed to heat treatment under isothermal conditions at four different temperatures. The heating medium used was peptone water at pH 4.5 and 7.0. The results showed that both serovars had a different response to a heat treatment after the exposure to acid. On the one hand, S. Senftenberg reduced its heat resistance when subjected to a previous acid shock, while in S. Enteritidis its heat resistance increased. Again, these results bring out the inherent variability in microorganisms, in this case related to a stress response mechanisms. Finally, the effectiveness of the combination of applying electrical pulses (PEF) and oregano essential oil to L. monocytogenes was determined. The medium used was a Mcllvaine buffer at pH 7 with a conductivity of 6 μS/cm. On the one hand, the efficiency of the electric field was evaluated between 5 and 20 kV/cm and it was found that after 60 pulses at 20 kV/cm field strength, 1 Hz frequency and 20 μs pulse width, an inactivation of 2.01 log10 cycles was achieved, while to achieve a similar level of inactivation at 15 kV/cm, 300 pulses were required. Then, PEF treatments were then applied (20 kV/cm field strength, 60 pulses, 1 Hz frequency and 20 μs pulse width) after having exposed the cells to the minimum inhibitory concentration (MIC) of the oregano essential oil (2000 ppm) and these results showed no significant differences (p>0.05) with respect to the PEF treatments applied alone. However, when the same PEF treatment as above was applied first and then the surviving microorganisms were resuspended on the MIC of oregano essential oil, significant differences (p0,05) con respecto a los tratamientos PEAV aplicados solos. Sin embargo, cuando se aplicó primero el mismo tratamiento PEAV que el anterior y luego se resuspendió a los microorganismos sobrevivientes en la CIM del aceite esencial de orégano, se encontraron diferencias significativas (p<0,05) con respecto al FEM aplicado solo y al PEAV después del orégano. También se encontró que al reducir a 1/16 la CIM (125 ppm) el efecto fue el mismo que si aplicáramos la CIM. Para concluir, esta tesis ha investigado cómo pueden combinarse diferentes estrategias de inactivación microbiana para conseguir un alimento seguro con un tratamiento más suave, que en un solo tratamiento convencional. También ha ayudado a abordar las predicciones para futuros estudios y como contribución a la industria alimentaria mejorará la cuantificación de la evaluación de riesgos microbiológicos.Escuela Internacional de Doctorado de la Universidad Politécnica de CartagenaUniversidad Politécnica de CartagenaPrograma de Doctorado de Técnicas avanzadas en investigación y desarrollo agrario y alimentari

Image analysis driven single-cell analytics for systems microbiology

Background: Time-lapse microscopy is an essential tool for capturing and correlating bacterial morphology and gene expression dynamics at single-cell resolution. However state-of-the-art computational methods are limited in terms of the complexity of cell movies that they can analyze and lack of automation. The proposed Bacterial image analysis driven Single Cell Analytics (BaSCA) computational pipeline addresses these limitations thus enabling high throughput systems microbiology. Results: BaSCA can segment and track multiple bacterial colonies and single-cells, as they grow and divide over time (cell segmentation and lineage tree construction) to give rise to dense communities with thousands of interacting cells in the field of view. It combines advanced image processing and machine learning methods to deliver very accurate bacterial cell segmentation and tracking (F-measure over 95%) even when processing images of imperfect quality with several overcrowded colonies in the field of view. In addition, BaSCA extracts on the fly a plethora of single-cell properties, which get organized into a database summarizing the analysis of the cell movie. We present alternative ways to analyze and visually explore the spatiotemporal evolution of single-cell properties in order to understand trends and epigenetic effects across cell generations. The robustness of BaSCA is demonstrated across different imaging modalities and microscopy types. Conclusions: BaSCA can be used to analyze accurately and efficiently cell movies both at a high resolution (single-cell level) and at a large scale (communities with many dense colonies) as needed to shed light on e.g. how bacterial community effects and epigenetic information transfer play a role on important phenomena for human health, such as biofilm formation, persisters&apos; emergence etc. Moreover, it enables studying the role of single-cell stochasticity without losing sight of community effects that may drive it. © 2017 The Author(s)

Additional file 8: of Image analysis driven single-cell analytics for systems microbiology

SalPhase time-lapse movie ground truth table. (XLSX 51 kb

Additional file 7: of Image analysis driven single-cell analytics for systems microbiology

Contains for each dataset the segmentation results of each method (.tif images) and corresponding parameterization files (.mat files). (ZIP 62299 kb