Environmental conditions which influence mucoid conversion Pseudomonas aeruginosa PAO1

Abstract

Growth and conversion to the mucoid phenotype by nonmucoid Pseudomonas aeruginosa PAO1 was studied in a chemostat system under conditions designed to reflect those likely to be present during chronic infection in the lung in cystic fibrosis patients. Mucoid variants were consistently isolated during continuous culture in the presence of 0.3 M NaCl or 5 or 10% glycerol. Mucoid subpopulations were also detected under conditions of carbon, nitrogen, or phosphate limitation. During carbon or nitrogen limitation, mucoid conversion was dependent upon the choice of substrate. Phosphate-limited cultures exhibited an inverse relationship between culture growth rate and number of mucoid organisms detected. Mucoid variants were not detected when dilution rates (D) exceeded 0.173 h-'. Conversely, at a D of 0.044 h-1, 40% of the population expressed the mucoid phenotype. Phosphorylcholine, a product of phosphojipase C activity on the major lung surfactant phosphatidylcholine, was also used as a growth substrate in nutrient limitation studies. Under all conditions, growth of PAO1 supplied with phosphorylcholine resulted in isolation of mucoid variants, indicating that the lung may provide at least one nutrient source conducive to mucoid conversion. Continuous culture also resulted in detection of a phage associated with strain PAO1. High titers of phage were present under all conditions, including those which yielded no mucoid organisms, suggesting that environmental conditions rather than the phage regulated the appearance of mucoid variants.Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with chronic pulmonary infections in cystic fibrosis (CF) patients (10,15,24). With prolonged chronic infection, P. aeruginosa undergoes alteration from the classical nonmucoid form to an atypical mucoid form, which correlates with the production of the exopolysaccharide alginic acid (6). In the context of chronic lung infection, alginate is an important virulence determinant. The appearance of alginate-producing P. aeruginosa strains is usually associated with a poor patient prognosis (10). Alginate has been implicated in adherence to tracheal epithelium (21, 28) and has been associated with antiphagocytic function (29). While the relationship between mucoid P. aeruginosa and CF is well documented, the environmental factors of the CF lung responsible for conversion of nonmucoid P. aeruginosa to the alginate-producing mucoid form have yet to be defined.A chemostat model for the study of this organism under chronic diseaselike growth conditions (phosphate and nitrogen limitation) which would reflect the likely physiological status of organisms in the lungs of CF patients has recently been reported (18). In this study, phosphate-limited cultures of mucoid P. aeruginosa were capable of utilizing phosphorylcholine (PC), a product of phospholipase C activity on phosphatidylcholine (a major component of lung surfactant), as a sole source of phosphate for growth (18).In an effort to understand the environmen...

Influence of nutrient media on the chemical composition of the exopolysaccharide from mucoid and non-mucoid Pseudomonas aeruginosa

“…Other experiments also demonstrated that growth in glycerol-containing media resulted in maximal alginate production [3,33]. Gluconate and glutamate used as nutrient factors failed to yield mucoid colonies [33]. We obtained the same result with the nM-BR strain.…”

“…Several in vitro conditions have been investigated and resulted in isolation of mucoid variants or in enhancement of the EPS production. Such environmental factors are nutrient media enriched with magnesium [30], aeration of growing cultures [31], restriction of nutrients such as iron [32] and osmotic stress [33].…”

“…Another study has shown that a strain produced alginate only when grown in a special medium [34]. Other experiments also demonstrated that growth in glycerol-containing media resulted in maximal alginate production [3,33]. Gluconate and glutamate used as nutrient factors failed to yield mucoid colonies [33].…”

“…Although the medium-dependent expression of the mucoid phenotype is known [12], no previous reports compare the chemical composition of the exopolysaccharide produced by P. aeruginosa when the bacteria are grown on different media. However, the effect of different conditions such as aeration, osmolarity, magnesium and iron content on the EPS amount has been shown [30][31][32][33]. Moreover, several genetic studies [13][14][15][16][17] demonstrated that mucoidy in P. aeruginosa is at least partially under environmental control.…”

Investigation of critical inter-related factors affecting the efficacy of pulsed light for inactivating clinically relevant bacterial pathogens

“…aeruginosa chromogenic agar (bioMérieux, La Balme-les-Grottes, France) at 37°C and visually checked thereafter for mucoid colony development after 24 and 48 h incubation as recommended by Laine et al (2009) (Table 2). Isolated colonies of mucoid strains (and nonmucoid control strain ATCC 27853) were then separately grown with shaking at 150 rev min )1 for 20 h at 37°C in 50 ml of chemically defined alginate promoting medium of Terry et al (1991) that was adjusted to pH 7 before media sterilization. To confirm alginate production in culture suspensions for PL studies, cells were pelleted after incubation by centrifugation at 10 000 rev min )1 for 10 min at 4°C.…”