scholarly journals Development of a Fatty Acid and RNA Stable Isotope Probing-Based Method for Tracking Protist Grazing on Bacteria in Wastewater

2010 ◽  
Vol 76 (24) ◽  
pp. 8222-8230 ◽  
Author(s):  
Steffen Kuppardt ◽  
Antonis Chatzinotas ◽  
Matthias Kästner
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Cell Division ◽  
Stable Isotope ◽  
Pathogenic Bacteria ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Estimation Model ◽  
Large Shift ◽  
Actual Contribution

ABSTRACT Removal of potential pathogenic bacteria, for example, during wastewater treatment, is effected by sorption, filtration, natural die-off, lysis by viruses, and grazing by protists, but the actual contribution of grazing has never been assessed quantitatively. A methodical approach for analyzing the grazing of protists on 13C-labeled prey bacteria was developed which enables mass balances of the carbon turnover to be drawn, including yield estimation. Model experiments for validating the approach were performed in closed microcosms with the ciliate Uronema sp. and 13C-labeled Escherichia coli as model prey. The transfer of bacterial 13C into grazing protist biomass was investigated by fatty acid (FA) and RNA stable isotope probing (SIP). Uronema sp. showed ingestion rates of ∼390 bacteria protist−1 h−1, and the temporal patterns of 13C assimilation from the prey bacteria to the protist FA were identified. Nine fatty acids specific for Uronema sp. were found (20:0, i20:0, 22:0, 24:0, 20:1ω9c, 20:1ω9t, 22:1ω9c, 22:1ω9t, and 24:1). Four of these fatty acids (22:0, 20:1ω9t, 22:1ω9c, and 22:1ω9t) were enriched very rapidly after 3 h, indicating grazing on bacteria without concomitant cell division. Other fatty acids (20:0, i20:0, and 20:1ω9c) were found to be indicative of growth with cell division. The fatty acids were found to be labeled with a percentage of labeled carbon (atoms percent [atom%]) up to 50. Eighteen percent of the E. coli-derived 13C was incorporated into Uronema biomass, whereas 11% was mineralized. Around 5 mol bacterial carbon was necessary in order to produce 1 mol protist carbon (yx / s ≈ 0.2), and the temporal pattern of 13C labeling of protist rRNA was also shown. A consumption of around 1,000 prey bacteria (∼98 atom% 13C) per protist cell appears to be sufficient to provide detectable amounts of label in the protist RNA. The large shift in the buoyant density fraction of 13C-labeled protist RNA demonstrated a high incorporation of 13C, and reverse transcription-real-time PCR (RT-qPCR) confirmed that protist rRNA increasingly dominated in the heavy RNA fraction.

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 Fatty Acid and Stable Carbon Isotope Composition of Slovenian Milk: Year, Season, and Regional Variability

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2892 ◽  
Author(s):  
Doris Potočnik ◽  
Lidija Strojnik ◽  
Tome Eftimov ◽  
Alenka Levart ◽  
Nives Ogrinc
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Stable Isotope ◽  
Isotope Composition ◽  
Stable Carbon Isotope ◽  
Cow Milk ◽  
Analysis Model ◽  
Regional Variability ◽  
Geographical Regions ◽  
Metabolic Transformation

This study examined the percentage and stable isotope ratios of fatty acids in milk to study seasonal, year, and regional variability. A total of 231 raw cow milk samples were analyzed. Samples were taken twice per year in 2012, 2013, and 2014, in winter and summer, covering four distinct geographical regions in Slovenia: Mediterranean, Alpine, Dinaric, and Pannonian. A discriminant analysis model based on fatty acid composition was effective in discriminating milk according to the year/season of production (86.9%), while geographical origin discrimination was less successful (64.1%). The stable isotope composition of fatty acids also proved to be a better biomarker of metabolic transformation processes in ruminants than discriminating against the origin of milk. Further, it was observed that milk from Alpine and Mediterranean regions was healthier due to its higher percentage of ω-3 polyunsaturated fatty acid and conjugated linoleic acid.

scholarly journals Stable isotopes of fatty acids: current and future perspectives for advancing trophic ecology

2020 ◽  
Vol 375 (1804) ◽  
pp. 20190641 ◽  
Author(s):  
Cornelia W. Twining ◽  
Sami J. Taipale ◽  
Liliane Ruess ◽  
Alexandre Bec ◽  
Dominik Martin-Creuzburg ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Stable Isotope ◽  
Best Practices ◽  
Resource Use ◽  
Trophic Ecology ◽  
Isotope Analysis ◽  
Dietary Fatty Acids ◽  
Food Web Ecology ◽  
Dietary Requirements

To understand consumer dietary requirements and resource use across ecosystems, researchers have employed a variety of methods, including bulk stable isotope and fatty acid composition analyses. Compound-specific stable isotope analysis (CSIA) of fatty acids combines both of these tools into an even more powerful method with the capacity to broaden our understanding of food web ecology and nutritional dynamics. Here, we provide an overview of the potential that CSIA studies hold and their constraints. We first review the use of fatty acid CSIA in ecology at the natural abundance level as well as enriched physiological tracers, and highlight the unique insights that CSIA of fatty acids can provide. Next, we evaluate methodological best practices when generating and interpreting CSIA data. We then introduce three cutting-edge methods: hydrogen CSIA of fatty acids, and fatty acid isotopomer and isotopologue analyses, which are not yet widely used in ecological studies, but hold the potential to address some of the limitations of current techniques. Finally, we address future priorities in the field of CSIA including: generating more data across a wider range of taxa; lowering costs and increasing laboratory availability; working across disciplinary and methodological boundaries; and combining approaches to answer macroevolutionary questions. This article is part of the theme issue ‘The next horizons for lipids as ‘trophic biomarkers’: evidence and significance of consumer modification of dietary fatty acids’.

scholarly journals Effects of including a ruminally protected lipid supplement in the diet on the fatty acid composition of beef muscle

2003 ◽  
Vol 90 (3) ◽  
pp. 709-716 ◽  
Author(s):  
Nigel D. Scollan ◽  
Mike Enser ◽  
Suresh K. Gulati ◽  
Ian Richardson ◽  
Jeff D. Wood
Keyword(s):  
Fatty Acids ◽  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Neutral Lipid ◽  
Acid Composition ◽  
Saturated Fatty Acids ◽  
Fatty Acid Content ◽  
Control Treatment ◽  
Large Shift ◽  
Lipid Supplement

Enhancing the polyunsaturated fatty acid (PUFA) and decreasing the saturated fatty acid content of beef is an important target in terms of improving the nutritional value of this food for the consumer. The present study examined the effects of feeding a ruminally protected lipid supplement (PLS) rich in PUFA on the fatty acid composition of longissimus thoracis muscle and associated subcutaneous adipose tissue. Animals were fed ad libitum on grass silage plus one of three concentrate treatments in which the lipid source was either Megalac (rich in palmitic acid; 16:0) or PLS (soyabean, linseed and sunflower-seed oils resulting in an 18:2n−6:18:3n−3 value of 2·4:1). Treatment 1 contained 100g Megalac/kg (Mega, control); treatment 2 (PLS1) contained 54g Megalac/kg with 500g PLS/d fed separately; treatment 3 (PLS2) contained no Megalac and 1000g PLS/d fed separately. The PLS was considered as part of the overall concentrate allocation per d in maintaining an overall forage:concentrate value of 60:40 on a DM basis. Total dietary fat was formulated to be 0·07 of DM of which 0·04 was the test oil. Total intramuscular fatty acids (mg/100g muscle) were decreased by 0·31 when feeding PLS2 compared with Mega (P<0·05). In neutral lipid, the PLS increased the proportion of 18:2n−6 and 18:3n−3 by 2·7 and 4·1 on diets PLS1 and PLS2 v. Mega, respectively. Similar responses were noted for these fatty acids in phospholipid. The amounts or proportions of 20:4n−6, 20:5n−3 or 22:6n−3 were not influenced by diet whereas the amounts and proportions of 22:4n−6 and 22:5n−3 in phospholipid were decreased with inclusion of the PLS. The amounts of the saturated fatty acids, 14:0, 16:0 and 18:0, in neutral lipid were on average 0·37 lower on treatment PLS2 compared with Mega. Feeding the PLS also decreased the proportion of 16:0 in neutral lipid. The amount of 18:1n-9 (P=0·1) and the amount and proportion of 18:1 trans (P<0·01) were lower on treatments PLS1 and PLS2 in neutral lipid and phospholipid. Conjugated linoleic acid (cis-9, trans-11) was not influenced by diet in the major storage fraction for this fatty acid, neutral lipid. The PUFA:saturated fatty acids value was increased markedly (×2·5) with inclusion of the PLS (P<0·001) while the σn−6:n−3 value increased slightly (×1·2; P=0·015). The results suggest that the protected lipid used, which was rich in PUFA, had a high degree of protection from the hydrogenating action of rumen micro-organisms. The PLS resulted in meat with a lower content of total fat, decreased saturated fatty acids and much higher 18:2n−6 and 18:3n−3. The net result was a large shift in polyunsaturated: saturated fatty acids, 0·28 v. 0·08, on feeding PLS2 compared with Mega, respectively.

scholarly journals Rv3723/LucA coordinates fatty acid and cholesterol uptake inMycobacterium tuberculosis

2017 ◽  
Author(s):  
Evgeniya V. Nazarova ◽  
Christine R. Montague ◽  
Thuy La ◽  
Kaley M. Wilburn ◽  
Neelima Sukumar ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Pathogenic Bacteria ◽  
Bacterial Virulence ◽  
Fatty Acid Transport ◽  
Acid Transport ◽  
Uncharacterized Protein ◽  
Fatty Acid Transporter ◽  
Important Fatty Acid

AbstractPathogenic bacteria have evolved highly specialized systems to extract essential nutrients from their hosts andMycobacterium tuberculosis(Mtb) scavenges lipids (cholesterol and fatty acids) to maintain infection in mammals. While the uptake of cholesterol by Mtb is mediated by the Mce4 transporter, the route(s) of uptake of fatty acids remain unknown. Here, we demonstrate that an uncharacterized protein LucA, integrates the assimilation of both cholesterol and fatty acids in Mtb. LucA interacts with subunits of the Mce1 and Mce4 complexes to coordinate the activities of these nutrient transporters. We also demonstrate that Mce1 functions as an important fatty acid transporter in Mtb and we determine that the integration of cholesterol and fatty acid transport by LucA is required for full bacterial virulencein vivo. These data establish that fatty acid and cholesterol assimilation are inexorably linked in Mtb and reveals a key role for LucA in coordinating both transport activities.

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 Stable Isotope Probing with 15N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density

2007 ◽  
Vol 73 (10) ◽  
pp. 3189-3195 ◽  
Author(s):  
Daniel H. Buckley ◽  
Varisa Huangyutitham ◽  
Shi-Fang Hsu ◽  
Tyrrell A. Nelson
Keyword(s):  
Stable Isotope ◽  
Natural Variation ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Density Gradients ◽  
Isotope Enrichment ◽  
Labeled Compounds ◽  
Contrast Variation ◽  
Functional Capabilities ◽  
Dna Density

ABSTRACT Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N, nearly all applications of this technique to date have used 13C. Successful application of SIP using 15N-DNA (15N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml−1 for 15N-labeled DNA, relative to 0.036 g ml−1 for 13C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml−1. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of 15N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications.

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