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Pseudomonas aeruginosa strain 18A is a clinical, nonclonal isolate retrieved from the sputum of a chronically infected cystic fibrosis patient. The genome of 18A was sequenced for comparison with environmental and clinical isolates to identify genes that might facilitate its persistence during infection.

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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.

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Little is known about the colonization mechanisms of Aeromonas spp. Previous work has suggested that the type IV bundle-forming pilus (Bfp) is an aeromonad intestinal colonization factor. This study provides the first genetic characterization of this structure. To define the role of Bfp in Aeromonas veronii bv. Sobria adherence, a 22-kb locus encoding the bundle-forming pilus was isolated; this contained 17 pilus-related genes similar to the mannose-sensitive hemagglutinin (MSHA) of Vibrio cholerae. Reverse transcriptase PCR (RT-PCR) demonstrated that the locus had two major transcriptional units, mshI to mshF and mshB to mshQ. Transcriptional fusion experiments demonstrated the presence of two strong promoters upstream of mshI and mshB. The locus encoded four putative prepilin proteins, one of which (MshA) corresponded to the N-terminal sequence of the previously isolated major pilin protein. All the pilin genes were inactivated, mutation of each minor or major pilin gene greatly reduced the bacterium's ability to adhere and form biofilms, and complementation of each mutant in trans rescued this phenotype. Mutation of the major pilin MshA and MshB, a minor pilin, resulted in their loss. The position of the mshH gene is conserved within a number of bacteria, and we have shown it is not transcriptionally linked to the other msh genes; moreover, its mutation did not have a dramatic effect on either adhesion or biofilm formation. We conclude that the bundle-forming pilus is required for A. veronii bv. Sobria adherence and biofilm formation; furthermore, both the major and minor pilin proteins are essential for this process.

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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.

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Intractable biofilm infections with Pseudomonas aeruginosa are the major cause of premature death associated with cystic fibrosis (CF). Few studies have explored the biofilm developmental cycle of P. aeruginosa isolates from chronically infected individuals. This study shows that such clinical isolates exhibit biofilm differentiation and dispersal processes similar to those of the better-studied laboratory P. aeruginosa strain PAO1 in the glass flow-cell (continuous-culture) biofilm model, albeit they are initially less adherent and their microcolonies are slower to develop and show heterogeneous, strain-specific variations in architecture. Confocal scanning laser microscopy combined with LIVE/DEAD viability staining revealed that in all CF biofilms bacterial cell death occurred in maturing biofilms, extending from the substratum to the central regions of mature microcolonies to varying degrees, depending on the strain. Bacteriophage activity was detected in the maturing biofilms of all CF strains examined and the amount of phage produced paralleled the degree of cell death seen in the biofilm. Some CF strains exhibited 'seeding dispersal' associated with the above phenomena, producing 'hollowing' as motile cells evacuated from the microcolony interiors as has been described for strain PAO1. Moreover, morphotypic cell variants were seen in the biofilm effluents of all CF strains. For those CF strains where marked cell death and seeding dispersal occurred in the microcolonies, variants were more diverse (up to five morphotypes) compared to those of strain PAO1 (two morphotypes). Given that variants of strain PAO1 have enhanced colonization traits, it seems likely that the similar biofilm dispersal events described here for CF strains contribute to the variability seen in clinical isolates and the overall persistence of the P. aeruginosa in the CF airway.

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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.

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Aeromonas spp. (gram-negative, aquatic bacteria which include enteropathogenic strains) have two distinct flagellar systems, namely a polar flagellum for swimming in liquid and multiple lateral flagella for swarming over surfaces. Only approximately 60% of mesophilic strains can produce lateral flagella. To evaluate flagellar contributions to Aeromonas intestinal colonization, we compared polar and lateral flagellar mutant strains of a diarrheal isolate of Aeromonas caviae for the ability to adhere to the intestinal cell lines Henle 407 and Caco-2, which have the characteristic features of human intestinal enterocytes. Strains lacking polar flagella were virtually nonadherent to these cell lines, while loss of the lateral flagellum decreased adherence by approximately 60% in comparison to the wild-type level. Motility mutants (unable to swim or swarm in agar assays) had adhesion levels of approximately 50% of wild-type values, irrespective of their flagellar expression. Flagellar mutant strains were also evaluated for the ability to form biofilms in a borosilicate glass tube model which was optimized for Aeromonas spp. (broth inoculum, with a 16- to 20-h incubation at 37 degrees C). All flagellar mutants showed a decreased ability to form biofilms (at least 30% lower than the wild type). For the chemotactic motility mutant cheA, biofilm formation decreased >80% from the wild-type level. The complementation of flagellar phenotypes (polar flagellar mutants) restored biofilms to wild-type levels. We concluded that both flagellar types are enterocyte adhesins and need to be fully functional for optimal biofilm formation.

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Flagella are much more than organelles of locomotion and have multiple roles that contribute to pathogenesis. Bacteria, such as Vibrio parahaemolyticus and Aeromonas spp., that possess two distinct flagellar systems (a polar flagellum for swimming in liquid and lateral flagella for swarming over surfaces) are relatively uncommon and provide ideal models for the independent investigation of the contributions of these different types of motility and other flagellar functions to virulence and how they are controlled. Studies with the above organisms have already increased our understanding of how bacteria sense and colonize surfaces forming biofilms that enable them to survive and persist in hostile environments. These insights are helping to identify possible new targets for novel antimicrobials that will both prevent or disrupt these processes and enhance the effectiveness of existing antibiotics. Aeromonas lateral flagella, in addition to mediating swarming motility, appear to be adhesins in their own right, contribute to microcolony formation and efficient biofilm formation on surfaces, and possibly facilitate host cell invasion. It is, therefore, likely that the ability to express lateral flagella is a significant virulence determinant for the Aeromonas strains able to cause persistent and dysenteric infections in the gastrointestinal tract, but further work is needed to establish this.

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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 approximately 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 degrees 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.

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The incidence and properties of Aeromonas species found in milk were examined to evaluate the potential of milk as a vehicle for the transmission of Aeromonas gastroenteritis. Aeromonads are common in raw milk (60%, 43 of 72 samples, positive). Pasteurization is effective at removing this contamination. Nevertheless, around 4% (seven of 183) pasteurized milk samples contained potentially significant strains, apparently introduced by subsequent handling of the milk. Some of these strains were indistinguishable from diarrhea-associated strains and were able to produce exotoxins at 37°C and adhere to epithelial cells. Adhesive ability and piliation increased when these strains were grown at low temperature. Such strains, although mesophilic, could reach high numbers in refrigerated milk without detectable spoilage of the milk. They pose the risk of colonization and in vivo toxin production. Further studies are required, but ingestion of preformed toxins produced in stored pasteurized milk may be of less concern, as psychrotrophic aeromonads, with the ability to produce large amounts of exotoxins in milk, appear to be uncommon and exotoxin production in milk was significantly lower than in bacteriological medium.