1,741 research outputs found
Optimization of nitric oxide donors for investigating biofilm dispersal response in Pseudomonas aeruginosa clinical isolates
No full textPseudomonas aeruginosa biofilms contribute heavily to chronic lung infection in cystic fibrosis patients, leading to morbidity and mortality. Nitric oxide (NO) has been shown to disperse P. aeruginosa biofilms in vitro, ex vivo and in clinical trials as a promising anti-biofilm agent. Traditional NO donors such as sodium nitroprusside (SNP) have been extensively employed in different studies. However, the dosage of SNP in different studies was not consistent, ranging from 500 nM to 500 μM. SNP is light sensitive and produces cyanide, which may lead to data misinterpretation and inaccurate predictions of dispersal responses in clinical settings. New NO donors and NO delivery methods have therefore been explored. Here we assessed 7 NO donors using P. aeruginosa PAO1 and determined that SNP and Spermine NONOate (S150) successfully reduced > 60% biomass within 24 and 2 h, respectively. While neither dosage posed toxicity towards bacterial cells, chemiluminescence assays showed that SNP only released NO upon light exposure in M9 media and S150 delivered much higher performance spontaneously. S150 was then tested on 13 different cystic fibrosis P. aeruginosa (CF-PA) isolates; most CF-PA biofilms were significantly dispersed by 250 μM S150. Our work therefore discovered a commercially available NO donor S150, which disperses CF-PA biofilms efficiently within a short period of time and without releasing cyanide, as an alternative of SNP in clinical trials in the future
Microbial colonization and competition on the marine alga ulva australis
No full textPseudalteromonas tunicata and Roseobacter gallaeciensis are biofilm-forming marine bacteria that are often found in association with the surface of the green alga Ulva australis. They are thought to benefit the plant host by producing inhibitory compounds that are active against common fouling organisms. We investigated factors that influence the ability of P. tunicata and R. gallaeciensis to attach to and colonize the plant surface and also the competitive interactions that occur between these organisms and other isolates from U. australis during biofilm formation on the plant surface. A surprisingly high number of P. tunicata cells, at least 108 cells ml–1, were required for colonization and establishment of a population of cells that persists on axenic surfaces of U. australis. Factors that enhanced colonization of P. tunicata included inoculation in the dark and pregrowth of inocula in medium containing cellobiose as the sole carbon source (cellulose is a major surface polymer of U. australis). It was also found that P. tunicata requires the presence of a mixed microbial community to colonize effectively. In contrast, R. gallaeciensis effectively colonized the plant surface under all conditions tested. Studies of competitive interactions on the plant surface revealed that P. tunicata was numerically dominant compared with all other bacterial isolates tested (except R. gallaeciensis), and this dominance was linked to production of the antibacterial protein AlpP. Generally, P. tunicata was able to coexist with competing strains, and each strain existed as microcolonies in spatially segregated regions of the plant. R. gallaeciensis was numerically dominant compared with all strains tested and was able to invade and disperse preestablished biofilms. This study highlighted the fact that microbial colonization of U. australis surfaces is a dynamic process and demonstrated the differences in colonization strategies exhibited by the epiphytic bacteria P. tunicata and R. gallaeciensi
Competitive interactions in mixed-species biofilms containing the marine bacterium pseudoalteromonas tunicata
No full textRole of mutation in pseudomonas aeruginosa biofilm development.
No full textThe 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
Bacterial biofilms: prokaryotic adventures in multicellularity
No full textThe development of bacterial biofilms includes both the initial social behavior of undifferentiated cells, as well as cell death and differentiation in the mature biofilm, and displays several striking similarities with higher organisms. Recent advances in the field provide new insight into differentiation and cell death events in bacterial biofilm development and propose that biofilms have an unexpected level of multicellularity
Down-regulation of DNA mismatch repair enhances initiation and growth of neuroblastoma and brain tumour multicellular spheroids
No full textMulticellular tumour spheroid (MCTS) cultures are excellent model systems for simulating the development and microenvironmental conditions of in vivo tumour growth. Many documented cell lines can generate differentiated MCTS when cultured in suspension or in a non-adhesive environment. While physiological and biochemical properties of MCTS have been extensively characterized, insight into the events and conditions responsible for initiation of these structures is lacking. MCTS are formed by only a small subpopulation of cells during surface-associated growth but the processes responsible for this differentiation are poorly understood and have not been previously studied experimentally. Analysis of gene expression within spheroids has provided clues but to date it is not known if the observed differences are a cause or consequence of MCTS growth. One mechanism linked to tumourigenesis in a number of cancers is genetic instability arising from impaired DNA mismatch repair (MMR). This study aimed to determine the role of MMR in MCTS initiation and development. Using surface-associated N2a and CHLA-02-ATRT culture systems we have investigated the impact of impaired MMR on MCTS growth. Analysis of the DNA MMR genes MLH1 and PMS2 revealed both to be significantly down-regulated at the mRNA level compared with non-spheroid-forming cells. By using small interfering RNA (siRNA) against these genes we show that silencing of MLH1 and PMS2 enhances both MCTS initiation and subsequent expansion. This effect was prolonged over several passages following siRNA transfection. Down-regulation of DNA MMR can contribute to tumour initiation and progression in N2a and CHLA-02-ATRT MCTS models. Studies of surface-associated MCTS differentiation may have broader applications in studying events in the initiation of cancer foci
Nitric oxide signaling in pseudomonas aeruginosa biofilms mediates phosphodiesterase activity, decreased cyclic Di-GMP levels, and enhanced dispersal
No full textBacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa. Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO
Enhanced benzaldehyde tolerance in zymomonas mobilis biofilms and the potential of biofilm applications in fine-chemical production
No full textBiotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 ?m, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)?1 day?1 with a 90% molar yield over a 45-h production perio
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