Biology subjects

Finkel, S. E.

Publications and source records attributed to Finkel, S. E..

4 recordsLinked to original sources

bioRxiv2023

Benchmarking A Novel Quantitative PCR-based Microbiome Profiling Platform Against Sequencing-based Methods

BackgroundPCR-based diagnostics, predominantly utilized for pathogen detection, have faced challenges in broader microbial profiling due to disparities in genomic data availability. This study addresses this limitation by exploiting the surge in the number of microbial genomes, facilitated by advancements in next-generation sequencing (NGS) and metagenomic-assembled genomes. The primary aim was to develop and validate quantitative PCR (qPCR) assays for a wide range of gut commensals, traditionally overlooked due to inadequate genomic information. We sought to compare the efficacy of these qPCR assays against established NGS microbiome profiling methodologies - 16S amplicon and metagenomic sequencing. MethodsWe designed 110 species-specific qPCR assays for gut commensals using a novel proprietary in silico pipeline and validated the assays against stool samples from three healthy donors. The quantitative microbiome profiles were compared to taxonomic profiles generated by standard bioinformatic approaches for 16S amplicon and metagenomic sequencing. 16S amplicons were analyzed as amplicon sequence variants produced by DADA2 and metagenomic sequences were analyzed by multiple iterations of MetaPhlAn (versions 2, 3, and 4) and Kraken2/Bracken paired with two different genomic databases. The qPCR assays were assessed for their ability to detect low abundance microbes and their correlation with NGS results, focusing on taxonomic resolution and limits of quantification. ResultsThe qPCR assays demonstrated high concordance with advanced metagenomic and the ineffectiveness of 16S amplicon methods to achieve species-level assignments. qPCR microbiome profiles were more highly correlated with the most current bioinformatic methods than the bioinformatics methods were to each other. The profile comparisons also highlight how the continued use of older bioinformatics protocols can limit results and lead to misinterpretation of data. Notably, qPCR identified taxa undetected or underestimated by metagenomic approaches, revealing limitations in current bioinformatics tools for differentiating closely related species and quantifying low abundance taxa. ConclusionsThis study establishes qPCR as a robust tool for large-scale microbiome profiling, offering enhanced accuracy, sensitivity, and quantitative capabilities compared to standard NGS methods. Our findings advocate for the integration of qPCR in standardizing microbiome detection, providing a pathway towards developing human microbiome profiling platforms capable of accurate species quantification. The adoption of qPCR assays could lead to more consistent, reliable, and cost-effective microbiome research and diagnostics.

microbiology↗

bioRxiv2023

Glucose-fed microbiota alters intestinal epithelium and increases susceptibility to bacterial pathogens

Overconsumption of dietary sugar can lead to many negative health effects including the development of Type 2 diabetes, metabolic syndrome, cardiovascular disease, and neurodegenerative disorders. Recently, the human intestinal microbiota strongly associated with our overall health has also been known to be affected by diet. However, mechanistic insight into the importance of the human intestinal microbiota and the effects of chronic sugar ingestion has not been possible largely due to the complexity of the human microbiome which contains hundreds of types of organisms. Here, we use an interspecies C. elegans-E. coli system, where E. coli are subjected to high sugar, then consumed by the bacterivore host C. elegans to become the microbiota. This glucose-fed microbiota results in a significant lifespan reduction accompanied by reduced healthspan including locomotion, stress resistance, and changes in behavior and feeding. Lifespan reduction is also accompanied by two potential major contributors: increased intestinal bacterial density and increased reactive oxygen species. The glucose-fed microbiota accelerated the age-related development of intestinal cell permeability, intestinal distention, and dysregulation of immune effectors. Ultimately, the changes in the intestinal epithelium due to aging with the glucose fed microbiota results in increased susceptibility to multiple bacterial pathogens. Taken together, our data reveal that chronic ingestion of sugar such as a western diet has profound health effects on the host due to changes in the microbiota and may contribute to the current increased incidence of ailments including inflammatory bowel diseases as well as multiple age-related diseases.

physiology↗

bioRxiv2020

Bacterial processing of glucose modulates C. elegans lifespan and healthspan

Intestinal microbiota play an essential role in the health of a host organism. Here, we define how commensal Escherichia coli (E. coli) alters its host after long term exposure to glucose using a C. elegans-E. coli system. Our data reveal that bacterial processing of glucose, rather than direct ingestion by the animal, results in reduced lifespan and healthspan, including reduced locomotion, oxidative stress resistance, and heat stress resistance in C. elegans. Chronic exposure of E. coli to glucose produces growth defects and increased advanced glycation end products within the E. coli. These negative effects are abrogated when the E. coli is not able to process the additional glucose and by the addition of the anti-glycation compound carnosine. Physiological changes of the host C. elegans are accompanied by dysregulation of detoxifying genes including glyoxalase, glutathione-S-transferase, and superoxide dismutase. Loss of gst-4 shortens C. elegans lifespan and blunts the animals response to a glucose-fed bacteria diet. Taken together, we reveal that added dietary sugar may alter intestinal microbial E. coli to decrease lifespan and healthspan of the host and define the critical role of detoxification genes in maintaining health during a chronic high-sugar diet.

genetics↗

bioRxiv2020

Escherichia coli has a Unique Transcriptional Program in Long-Term Stationary Phase

Microbes live in complex and consistently changing environments, but it is difficult to replicate this in the laboratory. Escherichia coli has been used as a model organism in experimental evolution studies for years; specifically, we and others have used it to study evolution in complex environments by incubating the cells into long-term stationary phase (LTSP) in rich media. In LTSP, cells experience a variety of stresses and changing conditions. While we have hypothesized that this experimental system is more similar to natural environments than some other lab conditions, we do not yet know how cells respond to this environment biochemically or physiologically. In this study, we begin to unravel the cells responses to this environment by characterizing the transcriptome of cells during LTSP. We found that cells in LTSP have a unique transcriptional program, and that several genes are uniquely upregulated or downregulated in this phase. Further, we identified two genes, cspB and cspI, which are most highly expressed in LTSP, even though these genes are primarily known to respond to cold-shock. When competed with wild-type cells, these genes are also important for survival during LTSP. These data allow us to compare biochemical responses to multiple environments and identify useful model systems, identify gene products that may play a role in survival in this complex environment, and identify novel functions of proteins. ImportanceExperimental evolution studies have elucidated evolutionary processes, but usually in chemically well-defined and/or constant environments. Using complex environments is important to begin to understand how evolution may occur in natural environments, such as soils or within a host. However, characterizing the stresses cells experience in these complex environments can be challenging. One way to approach this is by determining how cells biochemically acclimate to heterogenous environments. In this study we begin to characterize physiological changes by analyzing the transcriptome of cells in a dynamic complex environment. By characterizing the transcriptional profile of cells in long-term stationary phase, a heterogenous and stressful environment, we can begin to understand how cells physiologically and biochemically react to the laboratory environment, and how this compares to more natural conditions.

microbiology↗