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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Prepare synthetic cystic fibrosis medium (SCFM2)

NOTE: Preparation of SCFM2 comprises three main stages outlined below (Figure 2). For full details and references, see^9,10,12.

Figure 2.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Amino acids
Alanine	Acrs Organics	56-41-7
Arginine HCl	MP	1119-34-2
Asparagine	Acrs Organics	56-84-8	Prepared in 0.5 M NaOH
Cystine HCl	Alfa Aesar	L06328
Glutamic acid HCl	Acrs Organics	138-15-8
Glycine	Acrs Organics	56-40-6
Histidine HCl H2O	Alfa Aesar	A17627
Isoleucine	Acrs Organics	73-32-5
Leucine	Alfa Aesar	A12311
Lysine HCl	Alfa Aesar	J62099
Methionine	Acrs Organics	63-68-3
Ornithine HCl	Alfa Aesar	A12111
Phenylalanine	Acrs Organics	63-91-2
Proline	Alfa Aesar	A10199
Serine	Alfa Aesar	A11179
Threonine	Acrs Organics	72-19-5
Tryptophan	Acrs Organics	73-22-3	Prepared in 0.2 M NaOH
Tyrosine	Alfa Aesar	A11141	Prepared in 1.0 M NaOH
Valine	Acrs 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	Acrs 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	Acrs 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	Acrs 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	Acrs 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