(M1530-03-17) Unveiling PNAG Dynamics in Bacterial Biofilms through Microfluidics and Fluorescence Microscopy

Purpose: Exopolysaccharides play a vital role in the bacterial biofilm’s ability to endure unfavorable environmental conditions such as the host immune systems and have been identified as important virulence factor in chronic microbial infections. Although significant progress has been made on identifying the structure various exopolysaccharides, there is a lack of understanding of how these polymers are distributed within the biofilms. A common exopolysaccharide found in both Gram- and Gram+ bacteria biofilms is the poly-N-acetylglucosamine (PNAG), which undergoes different modifications including succination and deacetylation depending on bacterial strain. Despite its prevalence, spatial organization of these polymers within biofilms remain a mystery. This raises the question: Do different bacterial strains utilize exopolysaccharides differently? If so, how does this impact biofilm dynamics such as cellular cohesion? Unveiling the distributions of these exopolysaccharides could provide novel insight on biofilm structure, and lead to new strategies for biofilm dispersal.  This abstract highlight the combination of fluorescent microscopy and innovative microfluidic device design for the mapping of PNAG distribution in different bacterial strains and its impact on biofilm architecture.

Methods: Bacteria strains and growth conditions: Gram positive (Staphylococcus epidermidis RP62A) and Gram negative (Escherichia coli MG1655∆crsA) were chosen for comparison. Both strains have been used in previous literature to study PNAG dependent biofilm formation. S. epidermidis and E. coli biofilms were cultured in Tryptic Soy broth and buffered Luria-Bertani broth, respectively. Microfluidic cell device, design and manufacture: Microfluidic design and manufacture were previously described in with small adaptations to the dimensions.¹  Original study described a 6micron height for thin uniform biofilm formation. The height of the device described in this study was increased to 300 microns for a larger biofilm model. Thin scotch tape (0.5-1mm) was used to designate seeding zone prior to plasma treatment. Devices were made using PDMS and adhered to glass slides after oxygen plasma treatment. Device growth, labeling, and dispersal: Overnight starter cultures were harvest and resuspended in 1/10x of final growth media. The cells were introduced to the device and left to grow at room temperature for 20hrs before labeling. Biofilms were labeled using a catalytically inactive PNAG specific hydrolase, Dispersin B (DspB), fused with eGFP (DspB-eGFP) and imaged on through the ECHO revolve microscope.²  The fluorescent probe was flowed in the device via syringe pump until the fluorescent intensity saturates within the biofilm. Dispersal was conducted with the use of active Dispersin B until biofilm fully dissipates or no observable degradation is observed.

Results: Inverted labeling pattern of biofilms, S. epidermidis vs E. coli: Interestingly, the labeling pattern of S. epidermidis was the direct inverse to that of E. coli. As shown in image B, the fluorescent probe accumulates initially at the peripheral of the S. epidermidis biofilm. On the contrary, the fluorescent signal for the E. coli biofilm begins at the center, and its intensity fades at the peripheral. The labelling pattern suggest different spatial concentration of PNAG within different strains of bacterial biofilms. Different dispersal pattern by PNAG glycosidase Dispersin B: After fluorescent labeling of PNAG, hydrolysis of the exopolysaccharide was conducted to observe differences between the two species of biofilm (Figure C). Biofilm dispersal was conducted with the addition of active PNAG specific hydrolase, Dispersin B. Dispersal begins similarly for both biofilm strains with initial dissociation at the peripheral regions. Fluorescent loss can be seen as disposal continues with a more drastic morphology change with the E. coli biofilm, a complete loss of 3-dimensional architecture. Surprisingly, the core of the S. epidermidis biofilm remains intact even after extensive glycosidase treatment suggesting alternative intercellular interactions at the center.

Conclusion: Unveiling how different biofilms organize these exopolysaccharides can provide insight on biofilm structure, leading to tailored strategies for dispersal and therapeutic treatments. To investigate these differences, biofilms are cultivated in a more controlled manner as shown in (Figure A), where cells are seeded in a specific area. As a result, labeling of the different strains of biofilms can be continuously monitored which revealed an inverse in labeling pattern. This suggests differential cohesion factors employed by both biofilms where PNAG is the predominate adhesin in E. coli but not S. epidermidis. This was further supported by the timelapse dispersal of the biofilm, where both strains were treated with PNAG specific glycosidase DspB. Treatment of with DspB led to complete dissociation of the E. coli biofilm, while the S. epidermidis biofilm was left with a center core of bacteria cells (Figure C). By cultivating biofilms in this manner allowed for dimensional resolution of exopolysaccharide distribution in different biofilms. This unveiled differences in PNAG utilization in E. coli and S. epidermidis biofilms and demonstrated that hydrolases of exopolysaccharides may not always result in complete dispersal of all biofilms.

References: 1. Zhang, Y. et al. A Microfluidic Approach for Quantitative Study of Spatial Heterogeneity in Bacterial Biofilms. Small Sci. 2, (2022).
2. Eddenden, A., Kitova, E. N., Klassen, J. S. & Nitz, M. An Inactive Dispersin B Probe for Monitoring PNAG Production in Biofilm Formation. ACS Chem. Biol. 15, 1204–1211 (2020)

Acknowledgements: We are grateful to the NSF for funding. A special thanks to the University of Maryland and the Department of Chemistry & Biochemistry and Mark Lecates (UMD FAB Lab) for assisting in the creation of the microfluidic device.


Figure A. Microscopy imaging with glass slide versus microfluidic device of PNAG dependent biofilms from S. epidermidis (Gram+) and E. coli (Gram-). All samples were labeled by DspB(inactive)-eGFP (inactive PNAG glycosidase fused with eGFP). Seeding zone for microfluidic is shown as green rectangle. Spatial distribution can be observed by samples in microfluidic device. Scale bar 100 microns.


Figure B. Fluorescent images of S. epidermidis and E. coli biofilms after treating with DspB(inactive)-eGFP. Fluorescence signal was measured in fiji by plotting a line segment through center of each biofilm (yellow line) and normalized to max gray value (GV). The fluorescent GVs were plotted versus distance from start point of the biofilms. Scale bar 100 microns.


Figure C. Timelapse of biofilm dispersal by active PNAG specific hydrolase DspB wt. Fluorescent signal reduces as PNAG is hydrolyzed, leading to shedding of cells. E. coli can be seen to completely dissolve while S. epidermidis biofilms retains its center core. Scale bar 100 microns.