Anaerobic culture conditions favor biofilm-like phenotypes in Pseudomonas aeruginosa isolates from patients with cystic fibrosis

Pseudomonas aeruginosa causes chronic infections in the lungs of cystic fibrosis (CF) individuals and remains the leading cause of morbidity and mortality associated with the disease. Biofilm growth and phenotypic diversification are factors thought to contribute to this organism's persistence. Most studies have focused on laboratory isolates such as strain PAO1, and there are relatively few reports characterizing the properties of CF strains, especially under decreased oxygen conditions such as occur in the CF lung. This study compared the phenotypic and functional properties of P. aeruginosa from chronically infected CF adults with those of strain PAO1 and other clinical non-CF isolates under aerobic and anaerobic culture conditions. The CF isolates overall displayed a reduced ability to form biofilms in standard in vitro short-term models. They also grew more slowly in culture, and exhibited decreased adherence to glass and decreased motilities (swimming, swarming and twitching). All of these characteristics were markedly accentuated by anaerobic growth conditions. Moreover, the CF strain phenotypes were not readily reversed by culture manipulations designed to encourage planktonic growth. The CF strains were thus inherently different from strain PAO1 and most of the other non-CF clinical P. aeruginosa isolates tested. In vitro models used to research CF isolate biofilm growth need to take the above properties of these strains into account

Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions

The success of Pseudomonas aeruginosa in cystic fibrosis (CF) and other chronic infections is largely attributed to its ability to grow in antibiotic-resistant biofilm communities. This study investigated the effects of limiting iron levels as a strategy for preventing/disrupting P. aeruginosa biofilms. A range of synthetic and naturally occurring iron-chelating agents were examined. Biofilm development by P. aeruginosa strain PAO1 and CF sputum isolates from chronically infected individuals was significantly decreased by iron removal under aerobic atmospheres. CF strains formed poor biofilms under anaerobic conditions. Strain PAO1 was also tested under anaerobic conditions. Biofilm formation by this model strain was almost totally prevented by several of the chelators tested. The ability of synthetic chelators to impair biofilm formation could be reversed by iron addition to cultures, providing evidence that these effective chelating compounds functioned by directly reducing availability of iron to P. aeruginosa. In contrast, the biological chelator lactoferrin demonstrated enhanced anti-biofilm effects as iron supplementation increased. Hence biofilm inhibition by lactoferrin appeared to occur through more complex mechanisms to those of the synthetic chelators. Overall, our results demonstrate the importance of iron availability to biofilms and that iron chelators have potential as adjunct therapies for preventing biofilm development, especially under low oxygen conditions such as encountered in the chronically infected CF lung

Biofilm dispersal cells of a cystic fibrosis Pseudomonas aeruginosa isolate exhibit variability in functional traits likely to contribute to persistent infection

Persistent lung infection by Pseudomonas aeruginosa is typically associated with the development of biofilms, the appearance of morphotypic variants and reduction in the expression of acute virulence factors. We have characterised and compared functional traits [carbon substrate utilisation, attachment and biofilm formation, protease and elastase activity, quorum-sensing (QS)] of the biofilm dispersal populations of a representative P. aeruginosa isolate from a chronically infected cystic fibrosis individual and P. aeruginosa strain PAO1. The dispersal variants of the clinical strain exhibited significantly greater heterogeneity in all of the phenotypes tested. All morphotypic variants from the dispersal population of the clinical strain showed a significant increase in QS signal and elastase production compared to the parental strain. In contrast, isolates from planktonic cultures were phenotypically identical to the inoculum strain, suggesting that the appearance of these variants was biofilm specific. The clinical strain was shown to have a 3.4-fold higher mutation frequency than PAO1 which corroborated with the increased diversity of dispersal isolates. These data suggest that the development of a chronic infection phenotype can be reversed to recover acute infection isolates and that growth within a biofilm facilitates diversification of P. aeruginosa which is important for ecological adaptation

Lateral Flagella and Swarming Motility in Aeromonas Species

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility. Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for ≈8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30°C) for Aeromonas spp. The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined