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Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling

DePas, William H. and Starwalt-Lee, Ruth and Van Sambeek, Lindsey and Kumar, Sripriya Ravindra and Gradinaru, Viviana and Newman, Dianne K. (2016) Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling. mBio, 7 (5). e00796-16. ISSN 2150-7511. PMCID PMC5040109. http://resolver.caltech.edu/CaltechAUTHORS:20160927-144217261

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Abstract

Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF) patients, we demonstrate its ability to survey thousands of bacteria (or bacterial aggregates) over millimeter scales and quantify aggregation of individual species in polymicrobial communities. By analyzing aggregation patterns of four prominent CF pathogens, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus sp., and Achromobacter xylosoxidans, we demonstrate a spectrum of aggregation states: from mostly single cells (A. xylosoxidans), to medium-sized clusters (S. aureus), to a mixture of single cells and large aggregates (P. aeruginosa and Streptococcus sp.). Furthermore, MiPACT-HCR revealed an intimate interaction between Streptococcus sp. and specific host cells. Lastly, by comparing standard rRNA fluorescence in situ hybridization signals to those from HCR, we found that different populations of S. aureus and A. xylosoxidans grow slowly overall yet exhibit growth rate heterogeneity over hundreds of microns. These results demonstrate the utility of MiPACT-HCR to directly capture the spatial organization and metabolic activity of bacteria in complex systems, such as human sputum.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1128/mBio.00796-16DOIArticle
http://mbio.asm.org/content/7/5/e00796-16PublisherArticle
http://mbio.asm.org/content/suppl/2016/09/21/mBio.00796-16.DCSupplementalPublisherSupplementary Data
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040109/PubMed CentralArticle
ORCID:
AuthorORCID
Gradinaru, Viviana0000-0001-5868-348X
Newman, Dianne K.0000-0003-1647-1918
Additional Information:© 2016 DePas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Received 4 May 2016 Accepted 19 August 2016 Published 27 September 2016. A portion of the imaging was performed in the Caltech Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. We thank Elise Cowley, Ajay Kasi, and the pulmonary clinic team and patients from Children’s Hospital of Los Angeles (CHLA) for their assistance with sample collection and support of this study; Harry Choi (and the entire Beckman Institute Molecular Instruments team), Brittany Belin, Antti Lignell, Bin Yang, Alex Persat, and Octavio Mondragón-Palomino for technical advice, and members of the Newman and Gradinaru groups for helpful feedback and discussions. This study was funded by grants from the NIH (grant no. 5R01HL117328-04) to D.K.N., from HHMI to D.K.N., and the Center for Environmental Microbial Interactions (CEMI) at Caltech to D.K.N. and V.G. V.G. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute. This work was also funded by the NIH Director’s New Innovator program (IDP20D017782-01) and PECASE (V.G.) and the Beckman Institute for the Resource Center on CLARITY, Optogenetics and Vector Engineering for technology development and broad dissemination.
Group:Heritage Medical Research Institute, Caltech Center for Environmental Microbial Interactions (CEMI)
Funders:
Funding AgencyGrant Number
NIH5R01HL117328-04
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
Heritage Medical Research InstituteUNSPECIFIED
NIHIDP20D017782-01
Caltech Beckman InstituteUNSPECIFIED
Beckman Institute for the Resource Center on CLARITY, Optogenetics and Vector EngineeringUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
PubMed Central ID:PMC5040109
Record Number:CaltechAUTHORS:20160927-144217261
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160927-144217261
Official Citation:DePas WH, Starwalt-Lee R, Van Sambeek L, Kumar SR, Gradinaru V, Newman DK. 2016. Exposing the three-dimensional biogeography and metabolic states of pathogens in cystic fibrosis sputum via hydrogel embedding, clearing, and rRNA labeling. mBio 7(5):e00796-16. doi:10.1128/mBio.00796-16
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:70618
Collection:CaltechAUTHORS
Deposited By: Joy Painter
Deposited On:27 Sep 2016 22:05
Last Modified:24 Oct 2017 16:45

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