Real-time Imaging of Response to Host-Induced Stress in Pseudomonas aeruginosa Aggregates

Summary

Pseudomonas aeruginosa aggregates demonstrate increased tolerance to antibiotics and immune cells. The role of membrane integrity in aggregate survival is currently underexplored. This study demonstrates a method for quantifying changes in cell membrane dynamics within P. aeruginosa aggregates to understand how cellular integrity can impact overall aggregate viability.

Abstract

Chronic infections such as those in cystic fibrosis (CF) are sustained by small, highly tolerant Pseudomonas aeruginosa aggregates that persist despite immune and therapeutic pressures. Unlike classical biofilms, these aggregates represent a distinct pathogenic unit - microscale, spatially organized communities that maintain structural integrity and physiological homeostasis under host-induced stress. However, the mechanisms that enable aggregates to remain intact under these conditions, and whether these homeostatic processes can be selectively disrupted, remain poorly defined. A key barrier to addressing this gap has been the lack of tools capable of capturing the dynamic, spatially resolved processes that link physiological stress to structural stability and collapse in real time. This study presents an integrated imaging and analytical workflow to quantify aggregate responses to host-relevant stressors. Using the voltage-sensitive dye DiBAC₄(5), membrane depolarization was monitored as an early indicator of physiological disruption within aggregates formed in synthetic cystic fibrosis sputum medium (SCFM2). High-resolution time-lapse confocal microscopy enables visualization of aggregates in both stable and stress-induced states, while image segmentation and voxel-based analysis provide quantitative mapping of spatial heterogeneity in membrane integrity and aggregate disassembly at single-cell resolution. This workflow establishes a reproducible and adaptable platform for linking physiological stress to structural outcomes in multicellular bacterial aggregates. By enabling quantitative dissection of the processes that preserve - or compromise - aggregate integrity, this approach provides a critical foundation for identifying and targeting the homeostatic mechanisms that underpin aggregate resilience, advancing new strategies to disrupt this clinically significant mode of bacterial persistence.

Introduction

Chronic bacterial infections are often sustained not by large, surface-attached biofilms, but by small, multicellular aggregates that form within host-associated environments. In cystic fibrosis (CF) and other chronic airway diseases, Pseudomonas aeruginosa commonly exists as suspended aggregates embedded within mucus or sputum rather than as classical biofilms attached to epithelial surfaces. These aggregates represent a distinct mode of growth, characterized by microscale spatial organization, pronounced physiological heterogeneity, and exceptional tolerance to immune and antimicrobial stressors^1,2,3,4. Despite their clinical relevance, the mechanisms governing aggregate stability, which in this protocol is defined as the ability to maintain membrane integrity and spatial organization under such stressors, remain poorly understood, with previous studies focusing more on matrix components that support surface-attached biofilm formation^5,6 .

A major barrier to studying events that contribute to aggregate destabilization, such as changes in membrane depolarization and aggregate architecture, is the lack of experimental workflows that capture dynamic physiological and structural changes in real time^7,8. Traditional bulk assays and endpoint imaging approaches cannot resolve how stress responses emerge within aggregates, how these responses are spatially distributed, or how physiological weakening, such as membrane depolarization and loss of ion homeostasis, translates into early signs of cellular stress and eventual physical collapse. While confocal microscopy has been widely applied to surface-attached biofilms, fewer approaches integrate time-resolved imaging with quantitative analysis that directly links cellular physiology to aggregate architecture and membrane activity.

Changes in bacterial membrane potential provide an early and sensitive indicator of cellular stress and loss of homeostasis. Voltage-sensitive dyes such as bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC₄(5)) accumulate in depolarized cells and offer a non-destructive, fluorescence-based readout of membrane integrity. Although DiBAC dyes have been used to assess membrane depolarization in planktonic cultures and surface-attached biofilms^9,10, their application to multicellular aggregates, particularly under physiologically relevant growth conditions and time-lapse imaging, has been limited.

Here, we present an integrated imaging and analytical workflow that enables real-time visualization and quantification of membrane depolarization and structural failure in P. aeruginosa aggregates. Aggregates are formed in synthetic cystic fibrosis sputum medium (SCFM2), a chemically defined medium that recapitulates key nutritional and ionic features of the CF airway and supports spontaneous aggregate formation without imposed surfaces or flow^11,12. Under these conditions, P. aeruginosa forms suspended aggregates of sizes and structural heterogeneity comparable to those observed in CF sputum and airway samples, providing a physiologically relevant platform for studying aggregate behavior¹³ .

Using time-lapse confocal laser scanning microscopy under controlled temperature and humidity, this workflow captures both stable aggregate states and stress-induced states marked by increased membrane depolarization and loss of cellular homeostasis over extended imaging periods. By combining DiBAC-based membrane potential reporting with high-resolution z-stack imaging, this method enables quantification of DiBAC₄(5) fluorescence at the single-cell level, with values mapped and analyzed within the spatial context of individual aggregates. Downstream image segmentation and voxel-based surface analysis allow changes in aggregate architecture and membrane depolarization to be quantified in parallel.

Importantly, while this protocol is demonstrated using P. aeruginosa aggregates formed in SCFM2, the workflow is broadly generalizable. The imaging strategy, dye-based physiological readout, and analytical framework can be readily adapted to other bacterial species, aggregate-promoting media, and stressors, including antimicrobial agents, immune effectors, or environmental perturbations. As such, this approach provides a transferable and reproducible platform for interrogating the stability of bacterial communities across diverse experimental contexts.

Protocol

All work with P. aeruginosa must be conducted under appropriate BSL-2 containment conditions and institutional biosafety approvals. The reagents and the equipment used in this study are listed in the Table of Materials.

1. Generation of Pseudomonas aeruginosa aggregates in synthetic cystic fibrosis sputum medium (SCFM2)

NOTE: This step describes preparation of P. aeruginosa cultures and formation of suspended multicellular aggregates under physiologically relevant conditions that mimic the CF airway environment.

1. Preparation of bacterial cultures
   1. The day before the experiment, inoculate wild-type Pseudomonas aeruginosa PAO1 carrying plasmid pMRP9-1 (GFP)¹⁴ from frozen glycerol stocks into 5 mL lysogeny broth (LB).
   2. Add carbenicillin (300 µg/mL) to maintain plasmid selection.
   3. Grow cultures overnight at 37 °C with shaking at 200 rpm.
2. Back-dilution and preparation for aggregate formation
   1. On the day of the experiment, back-dilute overnight cultures 1:5 (1 mL overnight: 4 mL LB. Antibiotics are not re-added at this stage; residual carryover is minimal and does not affect growth).
   2. Grow cultures for ~2 h at 37 °C with shaking (200 rpm) to reach logarithmic phase.
      NOTE: Cells are typically in the early exponential phase after ~2 h under these conditions. Additionally, cells are subsequently washed prior to inoculation into SCFM2 to minimize any residual antibiotic carryover.
3. Preparation and equilibration of SCFM2
   1. Approximately 30 min before the end of the back-dilution period, aliquot pre-prepared SCFM2 into sterile tubes and warm to 37 °C.
   2. For imaging in a 4-well glass-bottom optical dish, allocate 500 µL SCFM2 per well. Prepare excess volume to account for pipetting error.
      NOTE: SCFM2 is stored at 4 °C. Pre-warming minimizes temperature shock and improves reproducibility of aggregate formation.
4. Washing and standardization of inoculum
   1. Pellet cultures by centrifugation at 4200 x g for 4 min at room temperature.
      NOTE: If calculations to rpm are required, the rotor radius for the swinging bucket rotor is 172 mm.
   2. Remove supernatant and resuspend pellets in 3 mL of sterile PBS (pH 7.0).
   3. Repeat wash steps twice more (total of three washes).
   4. Measure OD₆₀₀ and calculate the volume required to inoculate SCFM2 to a starting OD₆₀₀ of 0.05.
   5. Vortex briefly to homogenize cells and inoculate each well containing 500 µL SCFM2.
5. Aggregate formation under static conditions
   1. Incubate inoculated SCFM2 statically at 37 °C for 4 h to allow aggregate formation.
      NOTE: A 4-h incubation reproducibly yields suspended aggregates with sizes and heterogeneity comparable to those observed in CF sputum and airway samples, while minimizing later-stage biofilm maturation^1,15.

2. Preparation of immune-associated stressor (human neutrophil elastase)

NOTE: This step describes the preparation of a host-derived stressor used to induce cellular stress and destabilization of bacterial aggregates.

1. Reconstitution and storage of human neutrophil elastase (HNE)
   1. Reconstitute human neutrophil elastase according to the manufacturer’s recommendations using 50 mM sodium acetate, 200 mM NaCl, and sterile water.
   2. Prepare a 50 µg/L stock solution and filter sterilize using a 0.2 µm filter.
   3. Aliquot into 50 mL volumes and store at −20 °C.
   4. The night before use, thaw an aliquot at 4 °C.
      ​NOTE: Aliquoting minimizes freeze–thaw cycles, which can reduce enzymatic activity and introduce variability. Aliquots are stable at 4 °C for approximately 1 month.

3. Preparation and application of DiBAC₄(5) to report membrane depolarization

NOTE: This step describes the preparation and use of a voltage-sensitive dye to visualize membrane depolarization within aggregates.

1. Preparation and storage of DiBAC₄(5)
   1. Begin by preparing a stock solution of DiBAC₄(5). Reconstitute DiBAC₄(5) powder in anhydrous DMSO to 1 mg/mL.
      NOTE: DiBAC₄(5) reconstitution in DMSO follows standard manufacturer recommendations.
   2. Aliquot into 100 µL volumes and store at −20 °C protected from light.
   3. Use each aliquot no more than twice.
      NOTE: DiBAC₄(5) is functionally equivalent to DiBAC₄(3) but selected here to match GFP excitation/emission spectra. In this protocol, DiBAC₄(3) is used only for an additional stain discussed in the results section. The primary dye used throughout this protocol is DiBAC₄(5).

4. Time-lapse confocal imaging of membrane depolarization in P. aeruginosa aggregates

NOTE: This step describes the acquisition of high-resolution, time-resolved confocal images to capture dynamic physiological and structural changes in aggregates.

1. Microscope and environmental chamber preparation
   1. Install a heated microplate insert or environmental chamber on the confocal microscope stage.
   2. Turn on temperature and humidity control at least 1 h before imaging and set to 37 °C.
   3. In the Acquisition tab, locate the Laser tab and approximately 1 h prior to use, power on the required lasers to allow adequate warm-up (argon laser for GFP; DPSS 561-10 for DiBAC₄(5)) (Supplementary Figure 1A).
      NOTE: Stable environmental control reduces focus drift and preserves aggregate integrity during long time-lapse experiments.
2. Application of the immune stressor
   1. After 4 h of aggregate formation, add HNE to experimental wells to a final concentration of 20 µg/L¹⁶.
   2. Leave control wells untreated.
   3. Transfer the 4-well dish to the microscope stage.
      NOTE: HNE was prepared at physiologically relevant concentrations (µg/L), based on reported levels in induced sputum, rather than as a concentrated stock. Concentrations used for this study can be found in McGarvey et al.¹⁶ under IS (induced sputum) NE-AAT (NE/a1-anti-trypsin complex) HV (healthy volunteer).
3. Identification of aggregate regions of interest
   1. Using the Locate tab and a 20× objective, switch on the Transmitted Light to identify aggregate-rich regions using brightfield imaging (Supplementary Figure 1B).
   2. While still using brightfield imaging, navigate to the Acquisition tab and save x-, y-, and z-coordinates using the Positions module (Supplementary Figure 1C).
   3. Repeat for multiple technical replicates per well.
4. High-resolution z-stack and time-series acquisition
   1. Move to a saved position and switch to a 63× oil-immersion objective.
   2. Apply immersion oil before positioning the dish.
   3. In the Acquisition tab, configure channels using Smart Setup (Supplementary Figure 1D): GFP (488/509 nm) – assign green, DiBAC₄(5) (591/615 nm) – assign red.
   4. Navigate to Live view, then using the Z-stack module, identify the bottom of the coverslip by adjusting the fine focus and then set it as the first z-position by clicking Set First (Supplementary Figure 1E).
   5. Move upward until the Range indicates 60 µm and set the final z-position by clicking Set Last (Supplementary Figure 1E).
   6. Turn off Live view and activate Definite Focus. Select Find Surface, and subsequently select Store Focus only once the status indicates Reflex Found (Supplementary Figure 1F).
   7. Repeat focus calibration for each saved position by using the Z-stack and Definite Focus modules.
   8. Configure Time-Series acquisition to image each position every 3 min for 6 h (Supplementary Figure 1G).
      NOTE: The protocol may be paused here to thaw, add, and incubate DiBAC₄(5) dye, and can be resumed after the incubation period.
   9. Select Start Experiment.
      NOTE: Three-minute intervals balance temporal resolution with phototoxicity and photobleaching considerations.
5. DiBAC₄(5) addition and incubation
   1. Add DiBAC₄(5) to each well to a final concentration of 5 µg/mL⁹.
   2. Incubate in the dark for 30 min before initiating time-lapse imaging.
      ​NOTE: This incubation allows dye equilibration while minimizing background fluorescence.

5. Quantitative image analysis of aggregate structure and DiBAC₄(5) co-localization

NOTE: This step describes a quantitative workflow to link membrane depolarization with aggregate architecture and destabilization.

1. Image import and channel separation
   1. Upload image files (.czi or equivalent) into Imaris x64 software version 10.2.0 or higher.
   2. Verify voxel calibration by checking that the x, y, and z pixel dimensions (voxel size) in the image metadata accurately reflect the confocal acquisition parameters (objective, zoom, z-step size). Incorrect calibration will lead to errors in spatial and volumetric measurements.
2. Surface rendering of bacterial aggregates and DiBAC₄(5) signal
   1. Use the Surfaces module to render P. aeruginosa aggregates:
      1. Under Segmentation Setup, select the GFP channel (Supplementary Figure 2A).
      2. Select enable Split Touching Objects (Region Growing) and adjust intensity Threshold to fully capture aggregates (Supplementary Figure 2B).
      3. Under the Filter Seed Points module, change the filter type to Number of Voxels Img = 1 and set to 90% (Supplementary Figure 2C).
      4. After rendering is processed, set surfaces to 100% (Supplementary Figure 2D).
   2. Complete the rendering and assign green coloring to bacterial surfaces (Supplementary Figure 2E).
   3. Repeat rendering for the DiBAC₄(5) channel and assign red coloring.
3. Co-localization and volume-based quantification
   1. Select the DiBAC₄(5) surface rendering (Supplementary Figure 3A) and determine which timepoint(s) co-localization analysis will be performed at.
   2. Navigate to Statistics > Detailed tab > Specific Values (Supplementary Figure 3B).
   3. Define co-localization by selecting objects with a Shortest Distance to Bacterial Surfaces of 0.00 (Supplementary Figure 3B).
   4. Locate Edit, then Duplicate these objects to generate a DiBAC-overlapping-with-cells surface (Supplementary Figure 3C). Ensure to rename this surface to indicate its overlapping qualities.
   5. In the Detailed tab under Average Values, record summed volume values at each selected timepoint(s) for: (1) Total DiBAC₄(5) signal (Supplementary Figure 3D); (2) Co-localized DiBAC₄(5) signal (Supplementary Figure 3E).
   6. Calculate percent overlap as:
      
      NOTE: Percent overlap reflects the proportion of cell-associated DiBAC₄(5) signal within aggregates, reflecting membrane depolarization at a given time point.
      ​NOTE: Although demonstrated using P. aeruginosa aggregates in SCFM2 and stressed with HNE, this workflow is broadly adaptable to other bacterial species, aggregate-promoting media, fluorescent reporters, and stressors, including antimicrobial agents or environmental perturbations.

Results

Provided in the protocol described above is a method to track changes in the membrane dynamics of aggregates of P. aeruginosa modeled in a cystic fibrosis lung environment (Figure 1). Using a wild-type PAO1 strain carrying a pMRP9-1 (GFP) plasmid (Figure 1A), HNE to simulate immune stress, and DiBAC₄(5) membrane dye (Figure 1C), changes in membrane depolarization, reflecting disruption of membran...

Discussion

Membrane potential serves as a sensitive, early readout of cellular stress and disrupted homeostasis. Within multicellular bacterial aggregates, these physiological changes occur prior to, and may drive, subsequent structural destabilization. Here, we present an imaging-based workflow that uses the slow-response, potential-sensitive dye bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC₄(5)) to track depolarization in Pseudomonas aeruginosa aggregates exposed to host-derived stress. Tracking ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(3))	AAT Bioquest	21411	lipophilic, anionic molecule
1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(5))	AAT Bioquest	21410	lipophilic, anionic molecule
Carbenicillin	Research products international	C46000-5.0	used for selection of GFP expressing strains of PAO1
Cellvis 4-chamber 35mm glass bottom dish	Fisher	NC0600518	glass bottom chamber dish allows for maitinance of suspended aggregates in growth media while imaging
Centrifuge	Beckman Coulter Life Sciences	B06320	paired with Beckman Coulter Life Sciences SX4400 swinging bucket rotor (part no. B01425)
Confocal laser scanning microscope (CLSM)	Zeiss	LSM 880	the model of CLSM used for the experiments described in this protocol is no longer available but the LSM 900 is an updated equivalent option
Culture Tubes	Genesee	21-130	sterile; polypropylene
DAPI	Thermo Fisher	62248	used for proposed future applications staining neutrophils with DiBAC4(5)
Disposable cuvettes	Brand Tech	759086D	1.5mL, semi-micro
DMSO	Millipore-Sigma	276855-100ML	used for the reconstitution of DiBAC4(3,5)
DRAQ5	Thermo Fisher	65-0880-92	used for proposed future applications staining neutrophils with DiBAC4(3)
Environmental Chamber for CLSM	PECON	LSM 880	this system was made exclusively for Zeiss products and is generally referred to as the "Incubation System S" and all components can be ordered through Zeiss as an addition to the LSM 880 (or similar) system
Gentamycin sulfate	Fisher	BP918-1	used for selection of RFP expressing strains of PA1633
Glycerol	Genesee	18-205	used to freeze strains for long-term storage at -80 °C
Human Neutrophil Elastase (HNE)	Millipore-Sigma	324681-50UG	once reconstituted it is stable for 1 month at 4 °C or 1 year at 20 °C
Imaris x64 software	Oxford Instruments		Version 10.2.0
Immersion Oil	Electron Microscopy Scienes	16919-12	non-drying; standardized at 37 °C
Incubator	Benchmark Scientific	H1001-M	capable of shaking
Lysogeny Broth (LB)	Genesee	11-118	miller mix
mCherry plasmid			obtained from a collaboorator
PA1633 transposon mutant strain			obtained from a collaboorator
Phosphate Buffered Saline (PBS)	Thermo Fisher	3002	pH 7.0
Sodium Acetate	Thermo Fisher	AM9740	3M; pH 5.5; RNase-free
Sodium Chloride	Fisher	S271-500	used for the reconstiutuion of HNE
Spectrophotometer	VWR	634-0882	used to measure cell density for inoculation of PAO1/PA1633 into SCFM2
Sterile .22µm Filter	Genesee	25-244	used to purify reconstituted HNE
Sterile Microcentrifuge Tubes	Genesee	24-272LR	0.6mL
Sterile Syringe	Fisher	14955459	used to purify reconstituted HNE
SX4400 swinging bucket rotor	Beckman Coulter Life Sciences	B01425	paired with Beckman Coulter Life Sciences centrifuge (part no. B06320)
Synthetic Cystic Fibrosis Sputum Medium (SCFM2)	SyntheBiome	10002-500	Batch #: 001-CF2-0724
Ultra Pure Water	Genesee	18-195	used for the reconstitution of HNE
Wild type Pseudomonas aeruginosa PAO1 with pMRP9-1 Plasmid			This pMRP9-1 plasmid contains GFP and expresses carbenicillin resistance
ZEN Black Edition 2.3 Zeiss Imaging software	Zeiss		compatible with the LSM 880