Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel.

Recent studies have indicated that biosurfactants play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. A combination of caprylic acid (0.01 % v/v) together with rhamnolipids (0.04 % v/v) was applied to biofilms of Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 9144 and a mixed culture under BioFlux flowthrough conditions and caused disruption of the biofilms. The biofilms were also treated with a combination of rhamnolipids (0.04 % v/v) and sophorolipids (0.01 %). Control treatments with PBS 1× had no apparent effect on biofilm disruption. The Gram-positive bacterium (S. aureus ATCC 9144) was more sensitive than P. aeruginosa ATCC 15442 in terms of disruption and viability as shown by Live/Dead staining. Disruption of biofilms of P. aeruginosa ATCC 15442 was minimal. Oxygen consumption by biofilms, after different treatments with biosurfactants, confirms that sophorolipid on its own is unable to kill/inhibit cells of P. aeruginosa ATCC 15442, and even when used in combination with rhamnolipids, under static conditions, no decrease in the cell viability was observed. Cells in biofilms exposed to mono-rhamnolipids (0.04 % v/v) showed behaviour typical of exposure to bacteriostatic compounds, but when exposed to di-rhamnolipids (0.04 % v/v), they displayed a pattern characteristic of bactericidal compounds

Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations

Contribution of Cell Elongation to the Biofilm Formation of Pseudomonas aeruginosa during Anaerobic Respiration

Pseudomonas aeruginosa, a gram-negative bacterium of clinical importance, forms more robust biofilm during anaerobic respiration, a mode of growth presumed to occur in abnormally thickened mucus layer lining the cystic fibrosis (CF) patient airway. However, molecular basis behind this anaerobiosis-triggered robust biofilm formation is not clearly defined yet. Here, we identified a morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its effect on the biofilm formation in vitro. A standard laboratory strain, PAO1 was highly elongated during anaerobic respiration compared with bacteria grown aerobically. Microscopic analysis demonstrated that cell elongation likely occurred as a consequence of defective cell division. Cell elongation was dependent on the presence of nitrite reductase (NIR) that reduces nitrite (NO2−) to nitric oxide (NO) and was repressed in PAO1 in the presence of carboxy-PTIO, a NO antagonist, demonstrating that cell elongation involves a process to respond to NO, a spontaneous byproduct of the anaerobic respiration. Importantly, the non-elongated NIR-deficient mutant failed to form biofilm, while a mutant of nitrate reductase (NAR) and wild type PAO1, both of which were highly elongated, formed robust biofilm. Taken together, our data reveal a role of previously undescribed cell biological event in P. aeruginosa biofilm formation and suggest NIR as a key player involved in such process

Hacking into bacterial biofilms: a new therapeutic challenge

Microbiologists have extensively worked during the past decade on a particular phase of the bacterial cell cycle known as biofilm, in which single-celled individuals gather together to form a sedentary but dynamic community within a complex structure, displaying spatial and functional heterogeneity. In response to the perception of environmental signals by sensing systems, appropriate responses are triggered, leading to biofilm formation. This process involves various molecular systems that enable bacteria to identify appropriate surfaces on which to anchor themselves, to stick to those surfaces and to each other, to construct multicellular communities several hundreds of micrometers thick, and to detach from the community. The biofilm microbial community is a unique, highly competitive, and crowded environment facilitating microevolutionary processes and horizontal gene transfer between distantly related microorganisms. It is governed by social rules, based on the production and use of "public" goods, with actors and recipients. Biofilms constitute a unique shield against external aggressions, including drug treatment and immune reactions. Biofilm-associated infections in humans have therefore generated major problems for the diagnosis and treatment of diseases. Improvements in our understanding of biofilms have led to innovative research designed to interfere with this process

Reactive oxygen species in phagocytic leukocytes

Phagocytic leukocytes consume oxygen and generate reactive oxygen species in response to appropriate stimuli. The phagocyte NADPH oxidase, a multiprotein complex, existing in the dissociated state in resting cells becomes assembled into the functional oxidase complex upon stimulation and then generates superoxide anions. Biochemical aspects of the NADPH oxidase are briefly discussed in this review; however, the major focus relates to the contributions of various modes of microscopy to our understanding of the NADPH oxidase and the cell biology of phagocytic leukocytes

The mechanism for activation of the neutrophil NADPH-oxidase by the peptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs from that for interleukin-8

Neutrophil chemotaxis has been shown to be regulated by two different signalling pathways that allow strong chemoattractants, such as bacterial-derived formylated peptides, to dominate over endogenous attractants, such as interleukin-8 (IL-8). Here we show that triggering of the formyl peptide receptor (FPR) with f-Met-Leu-Phe (fMLF) substantially reduced the neutrophil superoxide production induced by activation of the CXC receptors with IL-8. When the order of agonists was reversed, the cells were primed in their response to fMLF, suggesting that the signalling hierarchy between strong, so-called end-type (i.e. fMLF) and weak or intermediate-type (i.e. IL-8) chemoattractants, is also operating during activation of the NADPH-oxidase. The same result was obtained when fMLF was replaced with the hexapeptide, WKYMVM, specific for the formyl peptide-like receptor 1 (FPRL1). There were additional differences between the agonist receptor pairs fMLF/FPR, WKYMVM/FPRL1 and IL-8/CXCR. In contrast to FPR and FPRL1, no reserve pool of CXCR was present in subcellular granules and it was impossible to prime the oxidative response transduced through CXCR by the addition of priming agents such as tumour necrosis factor-α and platelet-activating factor. Moreover, the cytoskeleton-disrupting substance, cytochalasin B, had no effect either on IL-8-triggered oxidase activation or on CXCR reactivation. A pertussis toxin-sensitive G-protein is involved in signalling mediated through both FPR and CXCR, and the signalling cascades include a transient intracellular calcium increase, as well as downstream p38 MAPK and phosphoinositide 3-kinase activation. The data presented in this study provide support for two different signalling pathways to the neutrophil NADPH-oxidase, used by ligand binding to FPR/FPRL1 or CXCR, respectively

Anti-a actinin antibodies as new predictors of response to treatment in autoimmune hepatitis type 1

Background We reported that combined presence of autoantibodies (Abs) against filamentous-actin (AFA) and a-actinin are specific for autoimmune hepatitis type 1 (AIH-1) diagnosis. Aim To explore our data and assess whether anti-a-actinin and AFA Abs could be used as indicators of response to treatment and predictors of AIH-1 flares in a large cohort of AIH-1 patients. Methods Seven hundred and sixty-four serial serum samples of 86 consecutive AIH-1 patients, 509 pathological and 110 normal controls were tested for the presence of anti-a-actinin and AFA Abs by an in-house IgG-specific ELISA and a standardised commercially available ELISA respectively. Patients sera were divided into baseline group (active disease before treatment initiation, n = 86) and then according to treatment response into group A-responders (n = 40 patients), group B-relapsers/incomplete responders (n = 37 patients) and group C-not-treated (n = 9 patients). Results Anti-a-actinin and AFA levels were significantly higher at baseline. Double reactivity against a-actinin and AFA was associated with disease activity (OR 4.9; 95% CI: 2.79). Anti-a-actinin optical densities (ODs) before treatment decreased significantly at first remission (P < 0.05). Treatment response was associated with anti-a-actinin Abs negativity before treatment (OR 3.4; 95% CI: 1.38.9) and absence of double positivity for anti-a-actinin and AFA Abs before treatment (OR 3.8; 95% CI: 1.410.4). Responders had lower baseline levels of anti-a-actinin than relapsers and/or incomplete responders (P = 0.002). Binary logistic regression revealed lower levels of anti-a-actinin as the only independent predictors of response (P = 0.05). Conclusions Anti-a-actinin Abs at baseline appear to predict treatment response and therefore they might be used for monitoring treatment outcome in AIH-1