Analysis and comparison of different methods for the quantification of alginate produced by Pseudomonas aeruginosa

Abstract

Cystic fibrosis is a life-limiting genetic condition that affects multiple organ systems, lung disease usually being the main contributor to disease and mortality. The cause for CF is a defective ion channel called CFTR, which leads to altered ion concentration, dehydrated, sticky mucus in the airways, impaired mucociliary clearance and weakened pulmonary immune defense. This promotes early and chronic airway infection, Pseudomonas aeruginosa being the predominant pathogen in adult CF patients and contributing strongly to lung disease severity. It is highly capable of adapting to the CF lung environment. Due to mutations the bacteria can for example convert to a mucoid phenotype that is caused by the overproduction of the exopolysaccharide alginate. That mucoid conversion allows for the formation of a biofilm which contributes to the difficulty to permanently eradicate P. aeruginosa during the chronic stage of infection. As alginate production is the hallmark of chronic P. aeruginosa infection of CF patients, further research of the polysaccharide is of great importance and a sensitive, user-friendly method of alginate measurement is crucial for CF research. However, P. aeruginosa alginate is a complex polysaccharide that is acetylated, of variable composition and difficult to purify to high purity. Furthermore, anoxic conditions - which are found in the CF lung - have been shown to stimulate alginate production in non-mucoid P. aeruginosa. Several methods to measure and detect alginate exist, however, they all have different advantages and disadvantages, such as the requirement of expensive equipment or hazardous chemicals. The aim of this work was to find a sensitive and user-friendly method for the measurement of alginate produced by P. aeruginosa by testing a recently published crystal violet staining technique and the Alcian blue dye for staining alginate on P. aeruginosa cells. First, it was analyzed if the anoxia-induced increase in alginate production by the non-mucoid PAO1 strain can be macroscopically detected similar to mucoid isolates with a slimy colony appearance. Indeed, after incubation on modified M9 agar plates under anaerobic conditions, PAO1 colonies showed a more mucoid appearance than the aerobically grown bacteria. This assay could be used as crude technique to differentiate between alginate non-producers and producers. However, it does not allow for a quantification of alginate. Second, the spectrophotometric crystal violet assay was tested for suitability and reproducibility using P. aeruginosa strains producing different levels of alginate. A standard curve of crystal violet- stained purified commercially available alginate at concentrations ranging from 0.0 to 2.0 mg/ml was prepared. Statistical analysis showed that there was a linear relation between the concentrations up to 0.8 mg/ml but high variation at higher concentrations. Using P. aeruginosa cultures to measure alginate concentrations with this technique showed that there was a considerable variability between experiments and a lack of reproducibility, which could be due to the experimental procedure. Thus, this technique was considered not suitable for reliable measurements of alginate concentrations. Finally, it was tested whether the Alcian blue dye could be used to stain and microscopically detect alginate on P. aeruginosa bacteria. Blue halo-like structures around some of the P. aeruginosa PDO300 bacteria were microscopically observed. These were absent from PAO1 bacteria suggesting that the structures represent the alginate matrix surrounding the bacteria. With this assay, it was also possible to observe alginate production at the single cell level albeit some variation between and within experiments. The major disadvantage of both the crystal violet and the Alcian blue assay is the inconsistent and highly variable results. Even though both are fast, straightforward and do not require special equipment or hazardous chemicals, they are currently not as reliable as other established methods. Some potential lies in further optimization of both assays but is to be doubted that this will lead to a great improvement in reliability. A novel, sensitive method of alginate measurement is Fourier transform infrared spectroscopy, which should be further explored in the future.