scholarly journals Single-Cell Growth Rates in Photoautotrophic Populations Measured by Stable Isotope Probing and Resonance Raman Microspectrometry

2017 ◽  
Vol 8 ◽  
Author(s):  
Gordon T. Taylor ◽  
Elizabeth A. Suter ◽  
Zhuo Q. Li ◽  
Stephanie Chow ◽  
Dallyce Stinton ◽  
...  
Keyword(s):  
Cell Growth ◽  
Stable Isotope ◽  
Single Cell ◽  
Growth Rates ◽  
Resonance Raman ◽  
Stable Isotope Probing ◽  
Raman Microspectrometry

Using Stable Isotope Probing and Raman Microspectroscopy to measure growth rates of heterotrophic bacteria

2021 ◽  
Author(s):  
Felix Weber ◽  
Tatiana Zaliznyak ◽  
Virginia P. Edgcomb ◽  
Gordon T. Taylor
Keyword(s):  
Stable Isotope ◽  
Single Cell ◽  
Bacterial Growth ◽  
Growth Rates ◽  
Heterotrophic Bacteria ◽  
Raman Microspectroscopy ◽  
Stable Isotope Probing ◽  
Single Cell Level ◽  
Cell Level ◽  
E Coli

The suitability of stable isotope probing (SIP) and Raman microspectroscopy to measure growth rates of heterotrophic bacteria at the single-cell level was evaluated. Label assimilation into E. coli biomass during growth on a complex 13 C-labeled carbon source was monitored in time course experiments. 13 C-incorporation into various biomolecules was measured by spectral “red shifts” of Raman-scattered emissions. The 13 C- and 12 C-isotopologues of the amino acid phenylalanine (Phe) proved to be a quantitatively accurate reporter molecules of cellular isotopic fractional abundances ( f cell ). Values of f cell determined by Raman microspectroscopy and independently by isotope-ratio mass spectrometry (IRMS) over a range of isotopic enrichments were statistically indistinguishable. Progressive labeling of Phe in E. coli cells among a range of 13 C/ 12 C organic substrate admixtures occurred predictably through time. Relative isotopologue abundances of Phe determined by Raman spectral analysis enabled accurate calculation of bacterial growth rates as confirmed independently by optical density (OD) measurements. Results demonstrate that combining stable isotope probing (SIP) and Raman microspectroscopy can be a powerful tool for studying bacterial growth at the single-cell level when grown on defined or complex organic 13 C-carbon sources even in mixed microbial assemblages. Importance: Population growth dynamics and individual cell growth rates are the ultimate expressions of a microorganism’s fitness to its environmental conditions, whether natural or engineered. Natural habitats and many industrial settings harbor complex microbial assemblages. Their heterogeneity in growth responses to existing and changing conditions is often difficult to grasp by standard methodologies. In this proof of concept study, we tested whether Raman microspectroscopy can reliably quantify assimilation of isotopically-labeled nutrients into E. coli cells and enable determination of individual growth rates among heterotrophic bacteria. Raman-derived growth rate estimates were statistically indistinguishable from those derived by standard optical density measurements of the same cultures. Raman microspectroscopy also can be combined with methods for phylogenetic identification. We report development of Raman-based techniques that enable researchers to directly link genetic identity to functional traits and rate measurements of single cells within mixed microbial assemblages, currently a major technical challenge in microbiological research.

scholarly journals Host-compound foraging by intestinal microbiota revealed by single-cell stable isotope probing

2013 ◽  
Vol 110 (12) ◽  
pp. 4720-4725 ◽  
Author(s):  
D. Berry ◽  
B. Stecher ◽  
A. Schintlmeister ◽  
J. Reichert ◽  
S. Brugiroux ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Single Cell ◽  
Intestinal Microbiota ◽  
Stable Isotope Probing ◽  
Host Compound

scholarly journals Refining the Application of Microbial Lipids as Tracers of Staphylococcus aureus Growth Rates in Cystic Fibrosis Sputum

2018 ◽  
Author(s):  
Cajetan Neubauer ◽  
Ajay S. Kasi ◽  
Nora Grahl ◽  
Alex L. Sessions ◽  
Sebastian H. Kopf ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Microbial Community ◽  
Stable Isotope ◽  
Community Composition ◽  
Growth Rates ◽  
Microbial Community Composition ◽  
Stable Isotope Probing ◽  
Lung Infections

ABSTRACTChronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effect of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. Microbial community composition was measured by NanoString DNA analysis and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two different anteiso fatty acids (a-C15:0 and a-C17:0) using gas chromatography–mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae and hence their isotopic enrichment was interpreted as a growth proxy for S. aureus. Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients, thus deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small non-microbial background source detected in some patient samples, a-C15:0 fatty acid still appear to be a relatively robust proxy for CF pathogens, revealing a median generation time of ~1.5 days, similar to prior observations. Contrary to our expectations, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two best practices for application of stable isotope probing in CF sputum: (1) parallel determination of microbial community composition in CF sputum using culture-independent tools, and (2) analysis of samples with a minimum a-C15:0 concentration of 0.1 weight percent of saturated fatty acids.IMPORTANCEIn chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop an new workflow for the application of stable isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

scholarly journals Refining the Application of Microbial Lipids as Tracers of Staphylococcus aureus Growth Rates in Cystic Fibrosis Sputum

2018 ◽  
Vol 200 (24) ◽  
Author(s):  
Cajetan Neubauer ◽  
Ajay S. Kasi ◽  
Nora Grahl ◽  
Alex L. Sessions ◽  
Sebastian H. Kopf ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Fatty Acids ◽  
Microbial Community ◽  
Stable Isotope ◽  
Community Composition ◽  
Growth Rates ◽  
Microbial Community Composition ◽  
Stable Isotope Probing ◽  
Content Type ◽  
Lung Infections

ABSTRACT Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effects of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of seven pediatric CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. The microbial community composition was determined by counting rRNA with NanoString DNA analysis, and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two branched-chain (“anteiso”) fatty acids (a-C15:0 and a-C17:0) using gas chromatography-mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae; hence, their isotopic enrichment was interpreted as a growth proxy for Staphylococcus aureus. Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients; thus, deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small nonmicrobial background source detected in some patient samples, a-C15:0 fatty acid still appears to be a relatively robust proxy for CF pathogens, revealing a median generation time of ∼1.5 days, similar to prior observations. Contrary to our expectation, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two straightforward “best practices” for application of stable-isotope probing to CF sputum metabolites: (i) parallel determination of microbial community composition in CF sputum using culture-independent tools and (ii) assessing background levels of the diagnostic metabolite. IMPORTANCE In chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop a new workflow for the application of stable-isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

scholarly journals High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays

2016 ◽  
Vol 34 (10) ◽  
pp. 1052-1059 ◽  
Author(s):  
Nathan Cermak ◽  
Selim Olcum ◽  
Francisco Feijó Delgado ◽  
Steven C Wasserman ◽  
Kristofor R Payer ◽  
...  
Keyword(s):  
Cell Growth ◽  
Single Cell ◽  
High Throughput ◽  
Growth Rates ◽  
Sensor Arrays ◽  
Mass Sensor

scholarly journals Competition-based screening secures the evolutionary stability of a defensive microbiome

2020 ◽  
Author(s):  
Sarah F. Worsley ◽  
Tabitha M. Innocent ◽  
Neil A. Holmes ◽  
Mahmoud M. Al-Bassam ◽  
Barrie Wilkinson ◽  
...  
Keyword(s):  
Game Theory ◽  
Stable Isotope ◽  
Rna Sequencing ◽  
Growth Rates ◽  
Evolutionary Stability ◽  
Stable Isotope Probing ◽  
Fungus Garden ◽  
Bacterial Strains ◽  
Leaf Cutting ◽  
Microbial Symbionts

AbstractCuticular microbiomes of Acromyrmex leaf-cutting ants are exceptional because they are freely colonizable, and yet the prevalence of Pseudonocardia, a native vertically transmitted symbiont that controls Escovopsis fungus-garden disease, is never compromised. Game theory suggests that competition-based screening can allow the selective recruitment of antibiotic-producing bacteria from the environment, by fomenting and biasing competition for abundant host resources. Mutual symbiont aggression benefits the host and also maintains native symbiont viability. Here we use RNA-stable isotope probing (RNA-SIP) to confirm predictions that Acromyrmex cuticles can maintain a range of microbial symbionts. We then used dual-RNA-sequencing and bioassays to show that vertically transmitted Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of other microbes, ultimately eliminating non-antibiotic-producing strains that might parasitize the symbiosis while still allowing antibiotic-producing Streptomyces strains to survive. Open cuticular microbiomes can thus maintain a specific co-evolved mutualism by restricting access for other bacterial strains.

scholarly journals Reverse and Multiple Stable Isotope Probing to Study Bacterial Metabolism and Interactions at the Single Cell Level

2016 ◽  
Vol 88 (19) ◽  
pp. 9443-9450 ◽  
Author(s):  
Yun Wang ◽  
Yizhi Song ◽  
Yifan Tao ◽  
Howbeer Muhamadali ◽  
Royston Goodacre ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Single Cell ◽  
Stable Isotope Probing ◽  
Bacterial Metabolism ◽  
Single Cell Level ◽  
Cell Level
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