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Immunology and Infection

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Published: April 6th, 2021

DOI:

10.3791/62420

1Department of Molecular Medicine, Morsani College of Medicine, University of South Florida

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Pseudomonas aeruginosa (Pa) is an opportunistic pathogen that establishes chronic infections in immune-compromised individuals. For those with the genetic disease cystic fibrosis (CF), these infections can span the course of a lifetime. CF causes the buildup of a viscous, nutrient-rich sputum in the airways, which becomes colonized by a variety of microbial pathogens over time. Pa is one of the most prevalent CF pathogens, colonizing the airways in early childhood and establishing difficult-to-treat infections1. Pa remains a significant clinical problem and is considered a leading cause of mortality in those with....

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1. Prepare synthetic cystic fibrosis medium (SCFM2)

NOTE: Preparation of SCFM2 comprises three main stages outlined below (Figure 2). For full details and references, see9,10,12.

Figure 2
Figure 2.......

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This work details methods to observe Pa aggregates at a high resolution and in an environment similar to that of chronic infection of the CF lung9,10,12. SCFM2 provides an in vitro system that promotes natural aggregation of Pa cells in sizes similar to those observed during actual infection10. The adaptability of SCFM2 as a defined medium can be leveraged to approach many resea.......

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This work has introduced methodologies that can be combined to study bacterial aggregate populations in the presence and absence of antibiotic treatment. High-resolution CLSM allows the visualization of changes in aggregate biomass and the structural orientation of aggregates over real time when exposed to antibiotics. In addition, physical and structural features of the biomass that remain after treatment with antibiotics can be quantified, with the goal to correlate these observations with future gene expression studie.......

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S.E.D is supported by start-up funds provided by the Department of Molecular Medicine, The University of South Florida, as well as a CFF research grant (DARCH19G0) the N.I.H (5R21AI147654 - 02 (PI, Chen)) and the USF Institute on Microbiomes. We thank the Whiteley lab for ongoing collaboration involving data sets related to this manuscript. We thank Dr. Charles Szekeres for facilitating FACS sorting. Figures were created by A.D.G and S.E.D using Biorender.com.

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Name Company Catalog Number Comments
Amino acids
Alanine AcrEquation 1s Organics 56-41-7
Arginine HCl MP 1119-34-2
Asparagine AcrEquation 1s Organics 56-84-8 Prepared in 0.5 M NaOH
Cystine HCl Alfa Aesar L06328
Glutamic acid HCl AcrEquation 1s Organics 138-15-8
Glycine AcrEquation 1s Organics 56-40-6
Histidine HCl H2O Alfa Aesar A17627
Isoleucine AcrEquation 1s Organics 73-32-5
Leucine Alfa Aesar A12311
Lysine HCl Alfa Aesar J62099
Methionine AcrEquation 1s Organics 63-68-3
Ornithine HCl Alfa Aesar A12111
Phenylalanine AcrEquation 1s Organics 63-91-2
Proline Alfa Aesar A10199
Serine Alfa Aesar A11179
Threonine AcrEquation 1s Organics 72-19-5
Tryptophan AcrEquation 1s Organics 73-22-3 Prepared in 0.2 M NaOH
Tyrosine Alfa Aesar A11141 Prepared in 1.0 M NaOH
Valine AcrEquation 1s Organics 72-18-4
Antibiotic
Carbenicillin Alfa Aesar J6194903
Day-of Stocks
CaCl2 * 2H2O Fisher Chemical C79-500
Dextrose (D-glucose) Fisher Chemical 50-99-7
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) Fisher (Avanti Polar Lipids) 4235-95-4 shake 15-20 min at 37 °C to evaporate chloroform
FeSO4 * 7H2O AcrEquation 1s Organics 7782-63-0 this stock equals 1 mg/mL, MUST make fresh
L-lactic acid Alfa Aesar L13242 pH stock to 7 with NaOH
MgCl2 * 6H2O AcrEquation 1s Organics 7791-18-6
N-acetylglucosamine  TCI A0092
Prepared solids
Porcine mucin Sigma M1778-100G UV-sterilize
Salmon sperm DNA Invitrogen 15632-011
Stain
Propidium iodide Alfa Aesar J66764MC
Salts
K2SO4 Alfa Aesar A13975
KCl Alfa Aesar J64189 add solid directly to buffered base
KNO3 AcrEquation 1s Organics 7757-79-1
MOPS Alfa Aesar A12914 add solid directly to buffered base
NaCl Fisher Chemical S271-500 add solid directly to buffered base
Na2HPO4 RPI S23100-500.0
NaH2PO4 RPI S23120-500.0
NH4Cl AcrEquation 1s Organics 12125-02-9 add solid directly to buffered base
Consumables
Conical tubes (15 mL) Olympus plastics 28-101
Conical tubes (50 mL) Olympus plastics 28-106
Culture tubes w/air flow cap Olympus plastics 21-129
35 mm four chamber glass-bottom dish CellVis NC0600518
Luria Bertani (LB) broth Genessee Scientific 11-118
Phosphate-buffered saline (PBS) Fisher Bioreagents BP2944100
Pipet tips (p200) Olympus plastics 23-150RL
Pipet tips (p1000) Olympus plastics 23-165RL
Serological pipets (5 mL) Olympus plastics 12-102
Serological pipets (25 mL) Olympus plastics 12-106
Serological pipets (50 mL) Olympus plastics 12-107
Ultrapure water (RNAse/DNAse free); nanopure water Genessee Scientific 18-194 Nanopure water used for preparation of solutions in Table 1
Syringes (10  mL) BD 794412
Syringes (50 mL) BD 309653
0.22 mm PES syringe filter Olympus plastics 25-244
PS cuvette semi-mico Olympus plastics 91-408
Software
Biorender To prepare the figures
FacsDiva6.1.3 Becton Dickinson, San Jose, CA
Imaris Bitplane version 9.6
Zen Black
Equipment
FacsAriallu Becton Dickinson, San Jose, CA
LSM 880 confocal laser scanning microscope Zeiss

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