Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Summary

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Abstract

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Introduction

Chronic lung infections with Pseudomonas aeruginosa are a major cause of morbidity and mortality in patients with cystic fibrosis (CF). During early childhood, patients are colonized by multiple bacterial pathogens including nonmucoid isolates of P. aeruginosa^1,2. Emergence of the small colony variant (SCV) isolates as well as mucoid isolates is a marker for the onset to chronic infections. SCV isolates are highly drug resistant³ due to their slow growth rates⁴, which renders them a severe deterrent in the treatment regiments and other chronic i....

Protocol

1. SCV Growth Conditions and Physiological Activation of the Salvage Pathway

1. Detection of SCV.
   1. Streak the P. aeruginosa strains PAO1, PAO1ΔpyrD, PAO581, and PAO581ΔpyrD on prewarmed Pseudomonas isolation agar (PIA) plates and grow at 37 °C for 48 h. On the growth plate identify a single colony isolate that has the SCV phenotype (colony size of 1−3 mm as opposed to the normal 3−5 mm colony size).
   2. Repeat step 1.1.1 to obtain a.......

Discussion

Both SCV and alginate are important disease markers implicated in several chronic infections. Therefore, the ability to grow SCV as well as study the regulation and production of alginate by P. aeruginosa is integral to the discovery of novel treatments for these chronic illnesses.

SCV strains are notoriously difficult to grow due to their slow growth rate⁴ as compared to other P. aeruginosa strains, which aids in their antimicrobial resistance

Materials

Name	Company	Catalog Number	Comments
1-Step Ultra TMB-ELISA	Thermo Scientific	34028	via Fisher Scientific
Absolute Ethanol (200 Proof)	Fisher Scientific	BP2818-4	Molecular Bio-grade
Accu Block Digital Dry Bath	Labnet	NC0205808	via Fisher Scientific
Assay Plates 96-well	CoStar	2021-12-20
Bench Top Vortex-Genie 2	Scientific Industries	G560
Boric Acid	Research Products International Corp.	10043-35-3
Cabinet Incubator	VWR	1540
Carbazole	Sigma	C-5132
Carbonate-Bicarbonate Buffer	Sigma	C3041
Centrifuge Tubes (50 ml)	Fisher Scientific	05-539-13	via Fisher Scientific
Culture Test Tubes	Fisher Scientific	14-956-6D	via Fisher Scientific
Cuvette Polystyrene (1.5 ml)	Fisher Scientific	14955127	via Fisher Scientific
Cytosine	Acros Organics	71-30-7
Diposable Inoculation Loops	Fisher Scientific	22-363-597
D-Mannuronic Acid Sodium	Sigma Aldrich	SMB00280
FMC Alginate	FMC	2133
Glycerol	Fisher Scientific	BP906-5	For Molecular Biology
Mouse Anti-Alginate Monoclonal Antibody	QED Biosciences	N/A	Lot # :15725/15726
Phosphate Buffered Saline Powder (PBS)	Sigma	P3813
Pierce Goat Anti-Mouse Poly-HRP Antibody	Thermo Scientific	32230	via Fisher Scientific
Potassium Hydroxide	Fisher Scientific	1310-58-3	via Fisher Scientific
Prism 7	GraphPad
Pseudomonas Isolation Agar (PIA)	Difco	292710	via Fisher Scientific
Pseudomonas Isolation Broth (PIB)	Alpha Biosciences	P16-115	via Fisher Scientific
Round Toothpicks	Diamond		Any brand
Seaweed alginate (Protanal CR 8133)	FMC Corporation
Skim Milk	Difco	232100	via Fisher Scientific
SmartSpec Plus Spectrophotometer	BioRad	170-2525	or preferred vendor
Sodium Chloride (NaCl)	Sigma	S-5886
SpectraMax i3x Multi-mode MicroPlate Reader	Molecular Devices	i3x	or preferred vendor
Sterile Petri Dish 100mm x 15mm	Fisher Scientific	FB0875713	via Fisher Scientific
Sulfuric Acid	Fisher Scientific	A298-212	Technical Grade
Sulfuric Acid (2 Normal -Stop Solution)	R&D Systems	DY994
Tween 20	Sigma	P2287
Uracil	Acros Organics	66-22-8