Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published September 27, 2016 | Published + Supplemental Material
Journal Article Open

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


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.

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.

Attached Files

Published - mBio-2016-DePas-.pdf

Supplemental Material - mbo004162984sf1.jpg

Supplemental Material - mbo004162984sf10.jpg

Supplemental Material - mbo004162984sf2.jpg

Supplemental Material - mbo004162984sf3.jpg

Supplemental Material - mbo004162984sf4.jpg

Supplemental Material - mbo004162984sf5.jpg

Supplemental Material - mbo004162984sf6.jpg

Supplemental Material - mbo004162984sf7.jpg

Supplemental Material - mbo004162984sf8.jpg

Supplemental Material - mbo004162984sf9.jpg


Files (24.5 MB)
Name Size Download all
6.3 MB Preview Download
899.1 kB Preview Download
406.0 kB Preview Download
1.1 MB Preview Download
1.3 MB Preview Download
2.1 MB Preview Download
2.8 MB Preview Download
1.8 MB Preview Download
2.5 MB Preview Download
2.8 MB Preview Download
2.4 MB Preview Download

Additional details

August 20, 2023
August 20, 2023