The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.

The emergence of virulent Pseudomonas aeruginosa clones is a threat to cystic fibrosis (CF) patients globally. Characterization of clonal P. aeruginosa strains is critical for an understanding of its clinical impact and developing strategies to meet this problem. Two clonal strains (AES-1 and AES-2) are circulating within CF centers in eastern Australia. In this study, phenotypic characteristics of 43 (14 AES-1, 5 AES-2, and 24 nonclonal) P. aeruginosa isolates were compared to gain insight into the properties of clonal strains. All 43 isolates produced bands of the predicted size in PCRs for vfr, rhlI, rhlR, lasA, lasB, aprA, rhlAB, and exoS genes; 42 were positive for lasI and lasR, and none had exoU. Thirty-seven (86%) isolates were positive in total protease assays; on zymography, 24 (56%) produced elastase/staphylolysin and 22 (51%) produced alkaline protease. Clonal isolates were more likely than nonclonal isolates to be positive for total proteases (P ‫؍‬ 0.02), to show elastase and alkaline protease activity by zymography (P ‫؍‬ 0.04 and P ‫؍‬ 0.01, respectively), and to show elastase activity by the elastin-Congo red assay (P ‫؍‬ 0.04). There were no other associations with genotype. Overall, increasing patient age was associated with decreasing elastase activity (P ‫؍‬ 0.03). Thirty-two (74%) isolates had at least one N-acylhomoserine lactone (AHL) by thin-layer chromatography. rhl-associated AHL detection was associated with the production and level of total protease and elastase activity (all P < 0.01). Thirty-three (77%) isolates were positive for ExoS by Western blot analysis, 35 (81%) produced rhamnolipids, and 34 (79%) showed chitinase activity. Findings suggest that protease activity during chronic infection may contribute to the transmissibility or virulence of these clonal strains.Pseudomonas aeruginosa lung infection is a major determinant of morbidity and mortality in cystic fibrosis (CF) patients (16). The pathogenesis of P. aeruginosa infection depends on multiple cell-associated and extracellular virulence factors including proteases (elastase [LasB], alkaline protease [AprA], and staphylolysin [LasA]), hemolysins (rhamnolipids), and toxins such as exoenzyme S (ExoS) and exotoxin A (13). Proteases contribute to pathogenesis in the lung through the induction of tissue necrosis and inflammation, destruction of surface receptors on neutrophils resulting in the inhibition of chemotaxis, phagocytosis, and the oxidative burst and degradation of surfactant proteins (27, 32).Many P. aeruginosa virulence factors, including the proteases, are regulated by cell-to-cell communication systems that rely on diffusible N-acylhomoserine lactones (AHLs) to monitor population size in a process known as "quorum sensing" (QS) (7, 31). P. aeruginosa has two AHL-regulated circuits: las, consisting of the transcriptional activator LasR and the AHL synthase LasI, which directs the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and rhl, consisting of RhlR and RhlI, which directs the synthesis of N-...

These results indicate that quorum-sensing deficiency may occur naturally in clinical isolates, and the possession of lasI and hence a functional Las quorum-sensing system may be important in development of corneal infection.

Pseudomonas aeruginosa is a ubiquitous bacterium that causes opportunistic infections in a range of host tissues and organs. Infections by P. aeruginosa are difficult to treat and hence there is interest in the development of effective therapeutics. One of the key mechanisms that P. aeruginosa uses to control the expression of many virulence factors is the N-acylated homoserine lactone (AHL) regulatory system. Hence, there is considerable interest in targeting this regulatory pathway to develop novel therapeutics for infection control. P. aeruginosa is the principal cause of microbial keratitis and of infections in cystic fibrosis (CF) sufferers, and AHL-dependent cell-to-cell signalling has been shown to be important for both infection types. However, keratitis tends to be an acute infection whereas infection of CF patients develops into a chronic, lifelong infection. Thus, it is unclear whether AHL-regulated virulence plays the same role during these infections. This review presents a comparison of the role of AHL signalling in P. aeruginosamediated microbial keratitis and chronic lung infections of CF patients. IntroductionAll evidence to date indicates that we are losing the race to combat what have been for the last 40 years simple bacterial infections. This is primarily due to the emergence of, and strong selection for, antibiotic-resistant bacteria, which has led to the current situation where many bacteria are so antibiotic resistant that patients can only be treated with what are considered to be drugs of last defence. A contributing factor in this steady failure of current drugs has been the strategy of making simple modifications of existing classes of antimicrobials to maintain a drug's activity over the short term. This process is no longer working, and what needs to be done is to identify and develop new drug targets. This will take significant effort and may require a paradigm shift in thinking about how we deal with pathogenic bacteria, and the development of therapeutics that are highly specific for bacterial groups or even individual organisms. However, such outcomes should not be seen as limitations, but as benefits, reducing the likelihood of general resistance developing. Furthermore, bacterial control does not necessarily require bactericidal activity; it may be sufficient to target genetic pathways that are essential for virulence or the infection process, but that are otherwise non-essential systems. One such system is the acylated homoserine lactone (AHL) quorum sensing (QS) system found in a range of pathogenic Gram-negative bacteria, e.g. Pseudomonas aeruginosa, Burkholderia cepacia and Serratia marcescens. P. aeruginosa is of particular interest because it forms biofilms that contribute to the infection process and many of its virulence factors, including biofilm development, are QS regulated. Furthermore, it causes infections in wounds, eyes and lungs (both chronic and acute). In fact, P. aeruginosa infection is the most frequent cause of mortality in cystic fibrosis sufferers and is...