Biology subjects

Rubbens, P.

Publications and source records attributed to Rubbens, P..

7 recordsLinked to original sources

bioRxiv2022

Real-Time Flow Cytometry to assess qualitative and quantitative responses of oral pathobionts during exposure to antiseptics

Antiseptics are widely used in oral healthcare to prevent or treat oral diseases, such as gingivitis and periodontitis. However, the incidence of bacteria being tolerant to standard antiseptics has sharply increased over the last few years. This stresses the urgency for surveillance against tolerant organisms, as well as the discovery of novel antimicrobials. Traditionally, susceptibility to antimicrobials is assessed by broth micro-dilution or disc diffusion assays, both of which are time-consuming, labor-intensive and provide limited information on the mode of action of the antimicrobials. The above-mentioned limitations highlight the need for the development of new methods to monitor and further understand antimicrobial susceptibility. Here, we used real-time flow cytometry, combined with membrane permeability staining, as a quick and sensitive technology to study the quantitative and qualitative response of two oral pathobionts to different concentrations of chlorhexidine, cetylpyridinium chloride or triclosan. Apart from the real-time monitoring of cell damage, we further applied a phenotypic fingerprint method to differentiate between the bacterial subpopulations that arose due to treatment. We quantified the pathobiont damage rate of different antiseptics at different concentrations within 15 minutes of exposure and identified the conditions under which the bacteria were most susceptible. Moreover, we detected species-specific and treatment-specific phenotypic subpopulations. This proves that real-time flow cytometry can provide information on the susceptibility of different microorganisms in a short time frame while differentiating between antiseptics and thus could be a valuable tool in the discovery of novel antimicrobial compounds while at the same time deciphering their mode of action.

microbiology↗

bioRxiv2021

Predicting the presence and abundance of bacterial taxa in environmental communities through flow cytometric fingerprinting

Microbiome management research and applications rely on temporally-resolved measurements of community composition. Current technologies to assess community composition either make use of cultivation or sequencing of genomic material, which can become time consuming and/or laborious in case high-throughput measurements are required. Here, using data from a shrimp hatchery as an economically relevant case study, we combined 16S rRNA gene amplicon sequencing and flow cytometry data to develop a computational workflow that allows the prediction of taxon abundances based on flow cytometry measurements. The first stage of our pipeline consists of a classifier to predict the presence or absence of the taxon of interest, with yields an average accuracy of 88.13{+/-}4.78 % across the top 50 OTUs of our dataset. In the second stage, this classifier was combined with a regression model to predict the relative abundances of the taxon of interest, which yields an average R2 of 0.35{+/-}0.24 across the top 50 OTUs of our dataset. Application of the models on flow cytometry time series data showed that the generated models can predict the temporal dynamics of a large fraction of the investigated taxa. Using cell-sorting we validated that the model correctly associates taxa to regions in the cytometric fingerprint where they are detected using 16S rRNA gene amplicon sequencing. Finally, we applied the approach of our pipeline on two other datasets of microbial ecosystems. This pipeline represents an addition to the expanding toolbox for flow cytometry-based monitoring of bacterial communities and complements the current plating- and marker gene-based methods. ImportanceMonitoring of microbial community composition is crucial for both microbiome management research and applications. Existing technologies, such as plating and amplicon sequencing, can become laborious and expensive when high-throughput measurements are required. Over the recent years, flow cytometry-based measurements of community diversity have been shown to correlate well to those derived from 16S rRNA gene amplicon sequencing in several aquatic ecosystems, suggesting there is a link between the taxonomic community composition and phenotypic properties as derived through flow cytometry. Here, we further integrated 16S rRNA gene amplicon sequencing and flow cytometry survey data in order to construct models that enable the prediction of both the presence and the abundance of individual bacterial taxa in mixed communities using flow cytometric fingerprinting. The developed pipeline holds great potential to be integrated in routine monitoring schemes and early warning systems for biotechnological applications.

microbiology↗

bioRxiv2019

Cytometric fingerprints of gut microbiota predict Crohn’s disease state

Variations in the gut microbiome have been associated with changes in health state such as Crohns disease. Most surveys characterize the microbiome through analysis of the 16S rRNA gene. An alternative technology that can be used is flow cytometry. In this report we analyzed a disease cohort that has been characterized by both technologies. Changes in microbial community structure are reflected in both types of data. We demonstrate that cytometric fingerprints can be used as a diagnostic tool in order to classify samples according to Crohns disease state. These results highlight the potential of flow cytometry to perform rapid diagnostics of microbiome-associated diseases.

microbiology↗

bioRxiv2019

PhenoGMM: Gaussian mixture modelling of microbial cytometry data enables efficient predictions of biodiversity

Microbial flow cytometry allows to rapidly characterize microbial communities. Recent research has demonstrated a moderate to strong connection between the cytometric diversity and taxonomic diversity based on 16S rRNA gene amplicon sequencing data. This creates the opportunity to integrate both types of data to study and predict the microbial community diversity in an automated and efficient way. However, microbial flow cytometry data results in a number of unique challenges that need to be addressed. The results of our work are threefold: i) We expand current microbial cytometry fingerprinting approaches by proposing and validating a model-based fingerprinting approach based upon Gaussian Mixture Models, which we called PhenoGMM. ii) We show that microbial diversity can be rapidly estimated by PhenoGMM. In combination with a supervised machine learning model, diversity estimations based on 16S rRNA gene amplicon sequencing data can be predicted. iii) We evaluate our method extensively by using multiple datasets from different ecosystems and compare its predictive power with a generic binning fingerprinting approach that is commonly used in microbial flow cytometry. These results demonstrate the strong connection between the genetic make-up of a microbial community and its phenotypic properties as measured by flow cytometry. Our workflow facilitates the study of microbial diversity and community dynamics using flow cytometry in a fast and quantitative way. ImportanceMicroorganisms are vital components in various ecoystems on Earth. In order to investigate the microbial diversity, researchers have largely relied on the analysis of 16S rRNA gene sequences from DNA. Flow cytometry has been proposed as an alternative technique to characterize microbial community diversity and dynamics. It is an optical technique, able to rapidly characterize a number of phenotypic properties of individual cells. So-called fingerprinting techniques are needed in order to describe microbial community diversity and dynamics based on flow cytometry data. In this work, we propose a more advanced fingerprinting strategy based on Gaussian Mixture Models. When samples have been analyzed by both flow cytometry and 16S rRNA gene amplicon sequencing, we show that supervised machine learning models can be used to find the relationship between the two types of data. We evaluate our workflow on datasets from different ecosystems, illustrating its general applicability for the analysisof microbial flow cytometry data. PhenoGMM facilitates the rapid characterization and predictive modelling of microbial diversity using flow cytometry.

microbiology↗

bioRxiv2019

Characterizing phenotypic heterogeneity in isogenic bacterial populations using flow cytometry and Raman spectroscopy

Investigating phenotypic heterogeneity can help to better understand and manage microbial communities. However, characterizing phenotypic heterogeneity remains a challenge, as there is no standardized analysis framework. Several optical tools are available, which often describe properties of the individual cell. In this work, we compare Raman spectroscopy and flow cytometry to study phenotypic heterogeneity in bacterial populations. The growth phase of E. coli populations was characterized using both technologies. Our findings show that flow cytometry detects and quantifies shifts in phenotypic heterogeneity at the population level due to its high-throughput nature. Raman spectroscopy, on the other hand, offers a much higher resolution at the single-cell level (i.e. more biochemical information is recorded). Therefore, it is capable of identifying distinct phenotypic populations when coupled with standardized data analysis. In addition, it provides information about biomolecules that are present, which can be linked to cell functionality. We propose an automated workflow to distinguish between bacterial phenotypic populations using Raman spectroscopy and validated this approach with an external dataset. We recommend to apply flow cytometry to characterize phenotypic heterogeneity at the population level, and Raman spectroscopy to perform a more in-depth analysis of heterogeneity at the single-cell level. ImportanceSingle-cell techniques are frequently applied tools to study phenotypic characteristics of bacterial populations. As flow cytometry and Raman spectroscopy gain popularity in the field, there is a need to understand their advantages and limitations, as well as to create a more standardized data analysis framework. Our work shows that flow cytometry allows to study and quantify shifts at the bacterial population level, but since its resolution is limited for microbial purposes, distinct phenotypic populations cannot be distinguished at the single-cell level. Raman spectroscopy, combined with appropriate data analysis, has sufficient resolving power at the single-cell level, enabling the identification of distinct phenotypic populations. As regions in a Raman spectrum are associated with specific (bio)molecules, it is possible to link these to the cell state and/or its function.

bioengineering↗

bioRxiv2018

Coculturing bacteria leads to reduced phenotypic heterogeneities

Isogenic bacterial populations are known to exhibit phenotypic heterogeneity at the single cell level. Because of difficulties in assessing the phenotypic heterogeneity of a single taxon in a mixed community, the importance of this deeper level of organisation remains relatively unknown for natural communities. In this study, we have used membrane-based microcosms that allow the probing of the phenotypic heterogeneity of a single taxon while interacting with a synthetic or natural community. Individual taxa were studied under axenic conditions, as members of a coculture with physical separation, and as a mixed culture. Phenotypic heterogeneity was assessed through both flow cytometry and Raman spectroscopy. Using this setup, we investigated the effect of microbial interactions on the individual phenotypic heterogeneities of two interacting drinking water isolates. We have demonstrated that interactions between these bacteria lead to an adjustment of their individual phenotypic diversities, and that this adjustment is conditional on the bacterial taxon.\n\nImportanceLaboratory studies have shown the impact of phenotypic heterogeneity on the survival and functionality of isogenic populations. As phenotypic heterogeneity is known to play an important role in pathogenicity and virulence, antibiotics resistance, biotechnological applications and ecosystem properties, it is crucial to understand its influencing factors. An unanswered question is whether bacteria in mixed communities influence the phenotypic heterogeneity of their community partners. We found that coculturing bacteria leads to a reduction in their individual phenotypic heterogeneities, which led us to the hypothesis that the individual phenotypic diversity of a taxon is dependent on the community composition.

microbiology↗

bioRxiv2018

Using machine learning to associate bacterial taxa with functional groups through flow cytometry, 16S rRNA gene sequencing, and productivity data

High-(HNA) and low-nucleic acid (LNA) bacteria are two operational groups identified by flow cytometry (FCM) in aquatic systems. HNA cell density often correlates strongly with heterotrophic production, while LNA cell density does not. However, which taxa are specifically associated with these groups, and by extension, productivity has remained elusive. Here, we addressed this knowledge gap by using a machine learning-based variable selection approach that integrated FCM and 16S rRNA gene sequencing data collected from 14 freshwater lakes spanning a broad range in physicochemical conditions. There was a strong association between bacterial heterotrophic production and HNA absolute cell abundances (R2 = 0.65), but not with the more abundant LNA cells. This solidifies findings, mainly from marine systems, that HNA and LNA could be considered separate functional groups, the former contributing a disproportionately large share of carbon cycling. Taxa selected by the models could predict HNA and LNA absolute cell abundances at all taxonomic levels, with the highest performance at the OTU level. Selected OTUs ranged from low to high relative abundance and were mostly lake system-specific (89.5%-99.2%). A subset of selected OTUs was associated with both LNA and HNA groups (12.5%-33.3%) suggesting either phenotypic plasticity or within-OTU genetic and physiological heterogeneity. These findings may lead to the identification of systems-specific putative ecological indicators for heterotrophic productivity. Generally, our approach allows for the association of OTUs with specific functional groups in diverse ecosystems in order to improve our understanding of (microbial) biodiversity-ecosystem functioning relationships. ImportanceA major goal in microbial ecology is to understand how microbial community structure influences ecosystem functioning. Research is limited by the ability to readily culture most bacteria present in the environment and the difference in bacterial physiology in situ compared to in laboratory culture. Various methods to directly associate bacterial taxa to functional groups in the environment are being developed. In this study, we applied machine learning methods to relate taxonomic data obtained from marker gene surveys to functional groups identified by flow cytometry. This allowed us to identify the taxa that are associated with heterotrophic productivity in freshwater lakes and indicated that the key contributors were highly system-specific, regularly rare members of the community, and that some could switch between being low and high contributors. Our approach provides a promising framework to identify taxa that contribute to ecosystem functioning and can be further developed to explore microbial contributions beyond heterotrophic production.

ecology↗