Biology subjects

Crandall, J.

Publications and source records attributed to Crandall, J..

3 recordsLinked to original sources

bioRxiv2024

Comparing Raman and NanoSIMS for heavy water labeling of single cells

Stable isotope probing (SIP) experiments in conjunction with Raman microspectroscopy (Raman) or nano-scale secondary ion mass spectrometry (NanoSIMS) are frequently used to explore single cell metabolic activity in pure cultures as well as complex microbiomes. Despite the increasing popularity of these techniques, the comparability of isotope incorporation measurements using both Raman and NanoSIMS directly on the same cell remains largely unexplored. This knowledge gap creates uncertainty about the consistency of single-cell SIP data obtained independently from each method. Here, we conducted a comparative analysis of 543 Escherichia coli cells grown in M9 minimal medium in the absence or presence of heavy water (2H2O) using correlative Raman and NanoSIMS measurements to quantify the results between the two approaches. We demonstrate that Raman and NanoSIMS yield highly comparable measurements of 2H incorporation, with varying degrees of similarity based on the mass ratios analyzed using NanoSIMS. The 12C2H/12C1H and 12C22H/12C21H mass ratios provide targeted measurements of C-H bonds but may suffer from biases and background interference, while the 2H/1H ratio captures all hydrogen with lower detection limits, making it suitable for applications requiring comprehensive 2H quantification. Importantly, despite its higher mass resolution requirements, the use of C22H/C21H may be a viable alternative to using C2H/C1H due to lower background and higher overall count rates. Furthermore, using an empirical approach to determining Raman wavenumber ranges via the 2nd derivative improved the data equivalency of 2H quantification between Raman and NanoSIMS, highlighting its potential for enhancing cross-technique comparability. These findings provide a robust framework for leveraging both techniques, enabling informed experimental design and data interpretation. By enhancing cross-technique comparability, this work advances SIP methodologies for investigating microbial metabolism and interactions in diverse systems. ImportanceAccurate and reliable measurements of cellular properties are fundamental to understanding the function and activity of microbes. This study addresses to what extent Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS) measurements of single cell anabolic activity can be compared. Here, we study the relationship of the incorporation of a stable isotope (2H through incorporation of 2H2O) as determined by the two techniques and calculate a correlation coefficient to support the use of either technique when analyzing cells incubated with 2H2O. The ability to discern between the comparative strengths and limitations of these techniques is invaluable in refining experimental protocols, enhancing data comparability between studies, data interpretation, and ultimately advancing the quality and reliability of outcomes in microbiome research.

microbiology↗

bioRxiv2023

Assessment of the Effects of Environmental Perturbations on Soil Ecology in A Terrestrial Mesocosm.

Climate change is altering ecosystems in unprecedented ways and necessitates the development of strategies that model ecosystems and allow for the evaluation of environmental impacts of perturbations: including climate events, novel approaches to agronomy or ecosystem management, and impacts of bio-industry and biotechnology innovations. Mesocosms present a platform to model some of the complexity of an ecosystem, while still being controlled and reproducible enough that they can be used to ask targeted questions and systematically assess the impacts of perturbation events. Herein, we established a methodological pipeline to assess the impact of three perturbation events (hydration, nutrification, contamination) upon plant-associated microbial communities using a terrestrial mesocosm. Mesocosms were assessed over a 30-day time-course following environmental perturbations, including modeling contamination with a foreign microbe via the introduction of Saccharomyces cerevisiae. We developed and applied a suite of diagnostic and bioinformatic analyses, including digital droplet PCR, microscopy, and phylogenomic analyses to assess the impacts of a perturbation event in a system that models a terrestrial ecosystem. The resultant data show that our mesocosms are dynamic yet reproducible, and that the analysis pipeline presented here allowed for a longitudinal assessment of microbial population dynamics and abiotic soil characteristics following perturbations, as well as the fate of yeast in the soil. Notably, our data indicate that a single perturbation event can have long-lasting impact upon soil composition and underlying microbial populations. Thus, this approach can be used to ask targeted questions as well as gain insights on broader ecological trends of soil perturbation events. ImportanceSoils are key to a healthy environment, but the impact of human activities and climate change upon soil microbiomes remains unclear. It is challenging to model the complexity of an ecosystem in a laboratory; however, to gain insight on how ecosystems are impacted by outside perturbations it is valuable to develop approaches that mimic an environmental system. Here, we developed a mesocosm that uses readily accessible components that come together to model a terrestrial ecosystem which is coupled with an analysis pipeline to assess how various perturbations impact the soil. We demonstrate the utility of this approach by tracking the effects of three perturbations (water, nutrition, contamination with yeast) on the soil over the course of 30 days. Our results demonstrate that these treatments can have lasting impacts on the soil. These findings and the methods presented here could be useful to other researchers assessing how ecosystems respond to perturbations. HighlightsO_LIWe developed a pipeline using terrestrial mesocosms that allow for the analysis of how perturbations impact soil systems and demonstrate that it is effective for targeted detection of a microbe of interest as well as global phylogenomic observation of ecological changes due to external perturbation events. C_LIO_LIdigital droplet PCR was adapted to track a low abundance, non-native microbe in soil mesocosms. C_LIO_LITemporal sampling allowed for the longitudinal observation of soil response to a one-time perturbance. C_LIO_LIIntroduction of yeast and its associated growth media conferred an expansion of total biomass and increase in alpha-diversity and shifts in the beta-diversity of the soil microbiome. C_LIO_LITreatment with media or yeast resulted in the expansion in the relative contribution of fungal biomass and an increase in the relative abundances of Saccharomycetes and Trellomycetes, with decreases in Sordariomycetes, Leotiomycetes, and Eurotomycetes C_LIO_LIMedia or yeast introduction also resulted in an expansion of the relative abundances of Gammaproteobacteria, Bacilli, and Bacteroidia, and decreases in Actinomycetia and Acidobacteria. C_LI

microbiology↗

bioRxiv2017

Validation and Implementation of CLIA-Compliant Whole Genome Sequencing (WGS) in Public Health Laboratory

BackgroundPublic health microbiology laboratories (PHL) are at the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards.\n\nObjectives1) Establish the performance specifications of WGS applications used in PHL to conform with CLIA (Clinical Laboratory Improvements Act) guidelines for laboratory developed tests (LDT), 2) Develop quality assurance (QA) and quality control (QC) measures, 3) Establish reporting language for end users with or without WGS expertise, 4) Create a validation set of microorganisms to be used for future validations of WGS platforms and multi-laboratory comparisons and, 5) Create modular templates for the validation of different sequencing platforms.\n\nMethodsMiSeq Sequencer and Illumina chemistry (Illumina, Inc.) were used to generate genomes for 34 bacterial isolates with genome sizes from 1.8 to 4.7 Mb and wide range of GC content (32.1%-66.1%). A customized CLCbio Genomics Workbench - shell script bioinformatics pipeline was used for the data analysis.\n\nResultsWe developed a validation panel comprising ten Enterobacteriaceae isolates, five gram-positive cocci, five gram-negative non-fermenting species, nine Mycobacterium tuberculosis, and five miscellaneous bacteria; the set represented typical workflow in the PHL. The accuracy of MiSeq platform for individual base calling was >99.9% with similar results shown for reproducibility/repeatability of genome-wide base calling. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from MLST and genotyping tests were 100%. A test report format was developed for the end users with and without WGS knowledge.\n\nConclusionWGS was validated for routine use in PHL according to CLIA guidelines for LDTs. The validation panel, sequencing analytics, and raw sequences will be available for future multi-laboratory comparisons of WGS in PHL. Additionally, the WGS performance specifications and modular validation template are likely to be adaptable for the validation of other platforms and reagents kits.

microbiology↗