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Published December 2018 | Supplemental Material + Submitted + Published
Journal Article Open

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

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-C_(15:0) and a-C_(17:0)) 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-C_(17:0) 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-C_(15:0) 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.

Additional Information

© 2018 American Society for Microbiology. Received 14 June 2018; Accepted 19 September 2018; Accepted manuscript posted online 24 September 2018. We thank Elise Cowley, Reto Wijker, and Fenfang Wu for helping with our study. We thank Dominique H. Limoli and George O'Toole for providing CFBE cells and Ram Balasubramanian for providing PMN and HL-60 cells. We thank Jennifer Dien Bard and Thomas G. Keens for their guidance with our study, the CHLA CF center team, and patients of the CHLA CF clinic for participating in this study. We thank the anonymous reviewers for their comments. This work was funded by grants from the National Institutes of Health (R01HL117328).

Attached Files

Published - e00365-18.full.pdf

Submitted - 348037.full.pdf

Supplemental Material - zjb999094934s1.pdf

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Additional details

Created:
August 22, 2023
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October 23, 2023