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Grahl, N.

Publications and source records attributed to Grahl, N..

2 recordsLinked to original sources
bioRxiv2018

Refining the application of microbial lipids as tracers of Staphylococcus aureus growth rates in cystic fibrosis sputum

Chronic 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.\n\nIMPORTANCEIn 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.

microbiology↗
bioRxiv2018

Profiling of bacterial and fungal microbial communities in cystic fibrosis sputum using RNA

Here, we report an approach to detect diverse bacterial and fungal taxa in complex samples by direct analysis of community RNA in one step using NanoString probe sets. We designed rRNA-targeting probe sets to detect forty two bacterial and fungal genera or species common in cystic fibrosis (CF) sputum, and demonstrated taxon-specificity of these probes as well as a linear response over more than three logs of input RNA. Culture-based analyses correlated qualitatively with relative abundance data on bacterial and fungal taxa obtained by NanoString and the analysis of serial samples demonstrated the use of this method to simultaneously detect bacteria and fungi and to detect microbes at low abundance without an amplification step. The relative abundances of bacterial taxa detected by analysis of RNA correlated with the relative abundances of the same taxa as measured by sequencing of the V4V5 region of the 16S rRNA gene amplified from community DNA from the same sample. We propose that this method may complement other methods designed to understand dynamic microbial communities, may provide information on bacteria and fungi in the same sample with a single assay, and, with further development, may provide quick and easily-interpreted diagnostic information on diverse bacteria and fungi at the genus or species level.\n\nImportanceHere we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities.

microbiology↗