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 make use of either 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 yielded an average accuracy of 88.13% ± 4.78% across the top 50 operational taxonomic units (OTUs) of our data set. In the second stage, this classifier was combined with a regression model to predict the relative abundances of the taxon of interest, which yielded an average R2 of 0.35 ± 0.24 across the top 50 OTUs of our data set. Application of the models to 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 to two other data sets 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. IMPORTANCE Monitoring 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. In recent years, flow cytometry-based measurements of community diversity have been shown to correlate well with those derived from 16S rRNA gene amplicon sequencing in several aquatic ecosystems, suggesting that 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 abundances of individual bacterial taxa in mixed communities using flow cytometric fingerprinting. The developed pipeline holds great potential to be integrated into routine monitoring schemes and early warning systems for biotechnological applications.

PhenoGMM: Gaussian Mixture Modeling of Cytometry Data Quantifies Changes in Microbial Community Structure.

Microbial flow cytometry can rapidly characterize the status of microbial communities. Upon measurement, large amounts of quantitative single-cell data are generated, which need to be analyzed appropriately. Cytometric fingerprinting approaches are often used for this purpose. Traditional approaches either require a manual annotation of regions of interest, do not fully consider the multivariate characteristics of the data, or result in many community-describing variables. To address these shortcomings, we propose an automated model-based fingerprinting approach based on Gaussian mixture models, which we call PhenoGMM. The method successfully quantifies changes in microbial community structure based on flow cytometry data, which can be expressed in terms of cytometric diversity. We evaluate the performance of PhenoGMM using data sets from both synthetic and natural ecosystems and compare the method with a generic binning fingerprinting approach. PhenoGMM supports the rapid and quantitative screening of microbial community structure and dynamics.IMPORTANCE Microorganisms are vital components in various ecosystems 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 technology to characterize microbial community diversity and dynamics. The technology enables a fast measurement of optical 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. We evaluated our workflow on data sets from both synthetic and natural ecosystems, illustrating its general applicability for the analysis of microbial flow cytometry data. PhenoGMM supports a rapid and quantitative analysis of microbial community structure using flow cytometry.

Computational Analysis of Microbial Flow Cytometry Data.

Flow cytometry is an important technology for the study of microbial communities. It grants the ability to rapidly generate phenotypic single-cell data that are both quantitative, multivariate and of high temporal resolution. The complexity and amount of data necessitate an objective and streamlined data processing workflow that extends beyond commercial instrument software. No full overview of the necessary steps regarding the computational analysis of microbial flow cytometry data currently exists. In this review, we provide an overview of the full data analysis pipeline, ranging from measurement to data interpretation, tailored toward studies in microbial ecology. At every step, we highlight computational methods that are potentially useful, for which we provide a short nontechnical description. We place this overview in the context of a number of open challenges to the field and offer further motivation for the use of standardized flow cytometry in microbial ecology research.

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 Crohn's disease (CD). 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 reanalyzed 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 CD state. These results highlight the potential of flow cytometry to perform rapid diagnostics of microbiome-associated diseases.

Fast Pathogen Identification Using Single-Cell Matrix-Assisted Laser Desorption/Ionization-Aerosol Time-of-Flight Mass Spectrometry Data and Deep Learning Methods.

In diagnostics of infectious diseases, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) can be applied for the identification of pathogenic microorganisms. However, to achieve a trustworthy identification from MALDI-TOF MS data, a significant amount of biomass should be considered. The bacterial load that potentially occurs in a sample is therefore routinely amplified by culturing, which is a time-consuming procedure. In this paper, we show that culturing can be avoided by conducting MALDI-TOF MS on individual bacterial cells. This results in a more rapid identification of species with an acceptable accuracy. We propose a deep learning architecture to analyze the data and compare its performance with traditional supervised machine learning algorithms. We illustrate our workflow on a large data set that contains bacterial species related to urinary tract infections. Overall we obtain accuracies up to 85% in discriminating five different species.

Discriminating Bacterial Phenotypes at the Population and Single-Cell Level: A Comparison of Flow Cytometry and Raman Spectroscopy Fingerprinting.

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, such as flow cytometry and Raman spectroscopy, which describe optical properties of the individual cell. In this work, we compare Raman spectroscopy and flow cytometry to study phenotypic heterogeneity in bacterial populations. The growth stages of three replicate Escherichia coli populations were 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 can identify distinct phenotypic populations when coupled with analyses tailored toward single-cell data. In addition, it provides information about biomolecules that are present, which can be linked to cell functionality. We propose a computational workflow to distinguish between bacterial phenotypic populations using Raman spectroscopy and validated this approach with an external data set. We recommend using flow cytometry to quantify phenotypic heterogeneity at the population level, and Raman spectroscopy to perform a more in-depth analysis of heterogeneity at the single-cell level. © 2019 International Society for Advancement of Cytometry.

Randomized Lasso Links Microbial Taxa with Aquatic Functional Groups Inferred from Flow Cytometry.

High-nucleic-acid (HNA) and low-nucleic-acid (LNA) bacteria are two operational groups identified by flow cytometry (FCM) in aquatic systems. A number of reports have shown that HNA cell density 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 (R 2 = 0.65), but not with the more abundant LNA cells. This solidifies findings, mainly from marine systems, that HNA and LNA bacteria 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. Selected operational taxonomic units (OTUs) ranged from low to high relative abundance and were mostly lake system specific (89.5% to 99.2%). A subset of selected OTUs was associated with both LNA and HNA groups (12.5% to 33.3%), suggesting either phenotypic plasticity or within-OTU genetic and physiological heterogeneity. These findings may lead to the identification of system-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.IMPORTANCE A major goal in microbial ecology is to understand how microbial community structure influences ecosystem functioning. 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 possibly 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.

Learning Single-Cell Distances from Cytometry Data.

Recent years have seen an increased interest in employing data analysis techniques for the automated identification of cell populations in the field of cytometry. These techniques highly depend on the use of a distance metric, a function that quantifies the distances between single-cell measurements. In most cases, researchers simply use the Euclidean distance metric. In this article, we exploit the availability of single-cell labels to find an optimal Mahalanobis distance metric derived from the data. We show that such a Mahalanobis distance metric results in an improved identification of cell populations compared with the Euclidean distance metric. Once determined, it can be used for the analysis of multiple samples that were measured under the same experimental setup. We illustrate this approach for cytometry data from two different origins, that is, flow cytometry applied to microbial cells and mass cytometry for the analysis of human blood cells. We also illustrate that such a distance metric results in an improved identification of cell populations when clustering methods are employed. Generally, these results imply that the performance of data analysis techniques can be improved by using a more advanced distance metric. © 2019 International Society for Advancement of Cytometry.

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 organization 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. Through flow cytometry we have demonstrated that interactions between these bacteria lead to a reduction of their individual phenotypic diversities and that this adjustment is conditional on the bacterial taxon. Single-cell Raman spectroscopy confirmed a taxon-dependent phenotypic shift due to the interaction. In conclusion, our data suggest that bacterial interactions may be a general driver of phenotypic heterogeneity in mixed microbial populations.IMPORTANCE Laboratory studies have shown the impact of phenotypic heterogeneity on the survival and functionality of isogenic populations. Because phenotypic heterogeneity plays an important role in pathogenicity and virulence, antibiotic 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.

Detection of microbial disturbances in a drinking water microbial community through continuous acquisition and advanced analysis of flow cytometry data.

Detecting disturbances in microbial communities is an important aspect of managing natural and engineered microbial communities. Here, we implemented a custom-built continuous staining device in combination with real-time flow cytometry (RT-FCM) data acquisition, which, combined with advanced FCM fingerprinting methods, presents a powerful new approach to track and quantify disturbances in aquatic microbial communities. Through this new approach we were able to resolve various natural community and single-species microbial contaminations in a flow-through drinking water reactor. Next to conventional FCM metrics, we applied metrics from a recently developed fingerprinting technique in order to gain additional insight into the microbial dynamics during these contamination events. Importantly, we found that multiple community FCM metrics based on different statistical approaches were required to fully characterize all contaminations. Furthermore we found that for accurate cell concentration measurements and accurate inference from the FCM metrics (coefficient of variation ≤ 5%), at least 1000 cells should be measured, which makes the achievable temporal resolution a function of the prevalent bacterial concentration in the system-of-interest. The integrated RT-FCM acquisition and analysis approach presented herein provides a considerable improvement in the temporal resolution by which microbial disturbances can be observed and simultaneously provides a multi-faceted toolset to characterize such disturbances.

Label-free Raman characterization of bacteria calls for standardized procedures.

Raman spectroscopy has gained relevance in single-cell microbiology for its ability to detect bacterial (sub)populations in a non-destructive and label-free way. However, the Raman spectrum of a bacterium can be heavily affected by abiotic factors, which may influence the interpretation of experimental results. Additionally, there is no publicly available standard for the annotation of metadata describing sample preparation and acquisition of Raman spectra. This article explores the importance of sample manipulations when measuring bacterial subpopulations using Raman spectroscopy. Based on the results of this study and previous findings in literature we propose a Raman metadata standard that incorporates the minimum information that is required to be reported in order to correctly interpret data from Raman spectroscopy experiments. Its aim is twofold: 1) mitigate technical noise due to sample preparation and manipulation and 2) improve reproducibility in Raman spectroscopy experiments studying microbial communities.

Stripping flow cytometry: How many detectors do we need for bacterial identification?

Multicolor approaches are challenging for microbial flow cytometry; as flow cytometers are mainly developed for biomedical applications, modern instruments contain more detectors than needed. Some of these additional fluorescence detectors measure biological information due to spectral overlap, yet the extent to which this information is relevant for the identification of bacterial populations is ambiguous. In this paper we characterize the usefulness of these additional detectors. We propose a data-driven detector selection method to select the smallest subset of detectors that will optimally discriminate between bacterial populations. Using a detector elimination strategy, we show that one or more detectors can be removed without loss of resolving power. A number of additional detectors are included in the final subset, which help to improve the identification of bacterial populations. Experimental data were retrieved from two types of modern cytometers with different configurations. The method reveals a clear ordering of detector importances, which depends on the instrument from which the data were retrieved. In addition, we were able to pinpoint unexpected behavior of SYBR Green I in the red spectrum. As the field of microbial flow cytometry is maturing, these results motivate the construction of a different kind of cytometric instruments for microbiologists, for which the number of detectors is reduced, but tailored toward the characteristics of microbial experiments. © 2017 International Society for Advancement of Cytometry.

Flow Cytometric Single-Cell Identification of Populations in Synthetic Bacterial Communities.

Bacterial cells can be characterized in terms of their cell properties using flow cytometry. Flow cytometry is able to deliver multiparametric measurements of up to 50,000 cells per second. However, there has not yet been a thorough survey concerning the identification of the population to which bacterial single cells belong based on flow cytometry data. This paper not only aims to assess the quality of flow cytometry data when measuring bacterial populations, but also suggests an alternative approach for analyzing synthetic microbial communities. We created so-called in silico communities, which allow us to explore the possibilities of bacterial flow cytometry data using supervised machine learning techniques. We can identify single cells with an accuracy >90% for more than half of the communities consisting out of two bacterial populations. In order to assess to what extent an in silico community is representative for its synthetic counterpart, we created so-called abundance gradients, a combination of synthetic (i.e., in vitro) communities containing two bacterial populations in varying abundances. By showing that we are able to retrieve an abundance gradient using a combination of in silico communities and supervised machine learning techniques, we argue that in silico communities form a viable representation for synthetic bacterial communities, opening up new opportunities for the analysis of synthetic communities and bacterial flow cytometry data in general.

Absolute quantification of microbial taxon abundances.

High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys.