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

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.

Pseudomonas aeruginosa Alginate Overproduction Promotes Coexistence with Staphylococcus aureus in a Model of Cystic Fibrosis Respiratory Infection

“…While we are beginning to advance our knowledge of the distribution of microbial species during CF pulmonary infections, and how and to what extent interspecies interactions occur (50, 51), our understanding remains quite limited. We predict that in certain areas of the lung, P. aeruginosa and S. aureus are spatially segregated, which also likely contributes to S. aureus survival of during coinfection, independent of P. aeruginosa competitive phenotypes.…”

Microenvironmental characteristics and physiology of biofilms in chronic infections of CF patients are strongly affected by the host immune response

“…Several studies have investigated the growth pattern of bacteria in the lungs of patients with CF. It is interesting that these studies have shown that species frequently classified as obligate aerobes, such as Staphylococcus aureus, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans, exhibit virtually zero growth, which is in line with depletion of O 2 in infected parts of the CFs lungs, whereas the facultative anaerobe P. aeruginosa exhibits slow growth under these conditions (6,(55)(56)(57)(58). We conclude that PMNs apparently play a major role in modifying the chemical microenvironment thereby imposing growth restriction upon pathogens in biofilm aggregates associated with chronic lung infections of CF patients.…”

Quantitative visualization of gene expression inPseudomonas aeruginosaaggregates reveals peak expression of alginate in the hypoxic zone

“…HCR is a fluorescent in situ hybridization-like approach that includes a signal amplification step to help visualize low-abundant RNAs (8, 9). We previously used single HCR v2.0 probes to detect bacterial taxa in CF sputum samples (10), and HCR 2.0 was also used by Nikolakakis et al to detect host and bacterial mRNAs in the Hawaiian bobtail squid- Vibrio fischeri symbiosis (11). We chose to test HCR v3.0 as a tool to quantify bacterial gene expression in situ because of its improved specificity over HCR v2.0.…”