Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Hospital for Sick Children

Pseudomonas aeruginosa is an opportunistic pathogen that can form biofilms in the lungs and airways of cystic fibrosis (CF) patients, resulting in chronic endobronchial infection. Two clonal strains of P. aeruginosa, named type A and type B, have recently been identified and have been found to infect more than 20% of CF patients in Ontario, Canada. In this study, 4 type A and 4 type B isolates retrieved from 8 CF patients in Ontario, Canada, were characterized. All 8 isolates grew well in rich medium and formed biofilms in vitro. Antibiotic resistance profiles of bacteria grown in biofilms and planktonic culture were studied via minimal bactericidal concentration assays for tobramycin, gentamicin, and ciprofloxacin. Compared to laboratory strains of P. aeruginosa, all 8 isolates showed increased resistance to all antibiotics studied in both biofilm and planktonic assays. Gene expression analysis of mexX, representing the MexXY-OprM efflux pump, and mexA, representing MexAB-OprM, revealed that these genes were up-regulated in the 8 clinical isolates. These results suggest clonal type A and type B isolates of P. aeruginosa isolated from CF patients in Ontario, Canada, show a multidrug resistance pattern that can be partially explained as being due to the increased expression of common antibiotic efflux systems.

Characterization of clonal strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Ontario, Canada.

Bacteria growing in biofilms are responsible for a large number of persistent infections and are often more resistant to antibiotics than are free-floating bacteria. In a previous study, we identified a Pseudomonas aeruginosa gene, ndvB, which is important for the formation of periplasmic glucans. We established that these glucans function in biofilm-specific antibiotic resistance by sequestering antibiotic molecules away from their cellular targets. In this study, we investigate another function of ndvB in biofilm-specific antibiotic resistance. DNA microarray analysis identified 24 genes that were responsive to the presence of ndvB. A subset of 20 genes, including 8 ethanol oxidation genes (ercS', erbR, exaA, exaB, eraR, pqqB, pqqC, and pqqE), was highly expressed in wild-type biofilm cells but not in ΔndvB biofilms, while 4 genes displayed the reciprocal expression pattern. Using quantitative real-time PCR, we confirmed the ndvB-dependent expression of the ethanol oxidation genes and additionally demonstrated that these genes were more highly expressed in biofilms than in planktonic cultures. Expression of erbR in ΔndvB biofilms was restored after the treatment of the biofilm with periplasmic extracts derived from wild-type biofilm cells. Inactivation of ethanol oxidation genes increased the sensitivity of biofilms to tobramycin. Together, these results reveal that ndvB affects the expression of multiple genes in biofilms and that ethanol oxidation genes are linked to biofilm-specific antibiotic resistance.

The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.

The presence of the mucoid phenotype of Pseudomonas aeruginosa is a marker of poor survival in cystic fibrosis. As CF lung disease results from chronic infection leading to airway inflammation, we determined whether the switch to a mucoid phenotype by P. aeruginosa has an impact on the inflammatory response of airway epithelial cells. Exposure of airway epithelial cells to non-mucoid and mucoid P. aeruginosa-derived material leads to p38α MAPK activation, a key protein kinase involved in transmitting inflammatory signals. However, while the non-mucoid strain PAO1 activates p38α MAPK pathway solely via TLR5, the mucoid strain PACF508 activates p38α MAPK via both TLR5 and TLR2. Inactivation of mucA (the gene responsible for the mucoid phenotype) in PAO1 leads to p38α MAPK activation by both TLR2 and TLR5, as observed in the clinical mucoid isolate PACF508. Therefore, the switch to mucoid phenotype may contribute to more inflammation via TLR2 activation in addition to TLR5. Our findings highlight an important and under recognized role for TLR2 in the response of airway epithelial cells to infection.

Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells.

Biofilm microcolonies of Pseudomonas aeruginosa chronically infect the airways of patients with cystic fibrosis and fuel ongoing destructive inflammation, yet the impact of the switch from planktonic to biofilm growth on host responses is poorly understood. We report that in airway epithelial cells a threshold of p38α mitogen-activated protein kinase (MAPK) activation was required to trigger neutrophil recruitment, which is influenced by extrinsic and intrinsic factors. Planktonic P. aeruginosa diffusible material (PsaDM) induced stronger p38α MAPK activation as compared to biofilm PsaDM. Biofilm PsaDM activated p38α MAPK in a Toll-like receptor-independent fashion via the lasI/lasR quorum-sensing system, but this activation was insufficient to recruit neutrophils. However, in airway epithelial cells from patients with cystic fibrosis with hypersensitivity to injurious stimuli, biofilm PsaDM activated p38α MAPK strongly enough to recruit neutrophils, which can contribute to lung injury.

The level of p38α mitogen-activated protein kinase activation in airway epithelial cells determines the onset of innate immune responses to planktonic and biofilm Pseudomonas aeruginosa.

Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease- dependent cytokine degradation. In subacute pulmonary infections, lasR mutant-infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients.

Cystic fibrosis-adapted Pseudomonas aeruginosa quorum sensing lasR mutants cause hyperinflammatory responses.

Pulmonary infection with Burkholderia cepacia complex in cystic fibrosis (CF) patients is associated with more-rapid lung function decline and earlier death than in CF patients without this infection. In this study, we used confocal microscopy to visualize the effects of various concentrations of tobramycin, achievable with systemic and aerosolized drug administration, on mature B. cepacia complex biofilms, both in the presence and absence of CF sputum. After 24 h of growth, biofilm thickness was significantly reduced by exposure to 2,000 μg/ml of tobramycin for Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis; 200 μg/ml of tobramycin was sufficient to reduce the thickness of Burkholderia dolosa biofilm. With a more mature 48-h biofilm, significant reductions in thickness were seen with tobramycin at concentrations of ≥100 μg/ml for all Burkholderia species. In addition, an increased ratio of dead to live cells was observed in comparison to control with tobramycin concentrations of ≥200 μg/ml for B. cepacia and B. dolosa (24 h) and ≥100 μg/ml for Burkholderia cenocepacia and B. dolosa (48 h). Although sputum significantly increased biofilm thickness, tobramycin concentrations of 1,000 μg/ml were still able to significantly reduce biofilm thickness of all B. cepacia complex species with the exception of B. vietnamiensis. In the presence of sputum, 1,000 μg/ml of tobramycin significantly increased the dead-to-live ratio only for B. multivorans compared to control. In summary, although killing is attenuated, high-dose tobramycin can effectively decrease the thickness of B. cepacia complex biofilms, even in the presence of sputum, suggesting a possible role as a suppressive therapy in CF.

Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms.

MICs and biofilm inhibitory concentrations (BICs) were measured for 68 cystic fibrosis (CF) Achromobacter isolates for amikacin, aztreonam, colistin, levofloxacin, and tobramycin. With the exception of colistin and levofloxacin, the remaining antibiotics had MIC90s, BICs at which 50% of the isolates were susceptible (BIC50s), and BICs at which 90% of the isolates were susceptible (BIC90s) equal to or above the highest concentrations tested. In a biofilm model, tobramycin was able to significantly increase killing of bacterial cells compared to controls, for intermediate-resistant strains only, at concentrations of 1,000 and 2,000 μg/ml.

Effect of High-Dose Antimicrobials on Biofilm Growth of Achromobacter Species Isolated from Cystic Fibrosis Patients.

Lung disease in cystic fibrosis (CF) is often exacerbated following acute upper respiratory tract infections caused by the human rhinovirus (HRV). Pathophysiology of these exacerbations is presently unclear and may involve deficient innate antiviral or exaggerated inflammatory responses in CF airway epithelial cells. Furthermore, responses of CF cells to HRV may be adversely affected by pre-exposure to virulence factors of Pseudomonas (P.) aeruginosa, the microorganism that frequently colonizes CF airways. Here we examined production of antiviral cytokine interferon-β and inflammatory chemokine interleukin-8, expression of the interferon-responsive antiviral gene 2'-5'-oligoadenylate synthetase 1 (OAS1), and intracellular virus RNA load in primary CF (delF508 CFTR) and healthy airway epithelial cells following inoculation with HRV16. Parallel cells were exposed to virulence factors of P. aeruginosa prior to and during HRV16 inoculation. CF cells exhibited production of interferon-β and interleukin-8, and expression of OAS1 at levels comparable to those in healthy cells, yet significantly higher HRV16 RNA load during early hours post-inoculation with HRV16. In line with this, HRV16 RNA load was higher in the CFBE41o- dF cell line overexpessing delF508 CFTR, compared with the isogenic control CFBE41o- WT (wild-type CFTR). Pre-exposure to virulence factors of P. aeruginosa did not affect OAS1 expression or HRV16 RNA load, but potentiated interleukin-8 production. In conclusion, CF cells demonstrate elevated HRV RNA load despite preserved interferon-β and OAS1 responses. High HRV load in CF airway epithelial cells appears to be due to deficiencies manifesting early during HRV infection, and may not be related to interferon-β.

Rhinovirus Load Is High despite Preserved Interferon-β Response in Cystic Fibrosis Bronchial Epithelial Cells.

Infections With Biofilm Formation: Selection of Antimicrobials and Role of Prolonged Antibiotic Therapy.

Chronic Pseudomonas aeruginosa lung infections are associated with progressive epithelial damage and lung function decline. In addition to its role in tissue injury, the persistent presence of P. aeruginosa-secreted products may also affect epithelial repair ability, raising the need for new antivirulence therapies. The purpose of our study was to better understand the outcomes of P. aeruginosa exoproducts exposure on airway epithelial repair processes to identify a strategy to counteract their deleterious effect. We found that P. aeruginosa exoproducts significantly decreased wound healing, migration, and proliferation rates, and impaired the ability of directional migration of primary non-cystic fibrosis (CF) human airway epithelial cells. Impact of exoproducts was inhibited after mutations in P. aeruginosa genes that encoded for the quorum-sensing (QS) transcriptional regulator, LasR, and the elastase, LasB, whereas impact was restored by LasB induction in ΔlasR mutants. P. aeruginosa purified elastase also induced a significant decrease in non-CF epithelial repair, whereas protease inhibition with phosphoramidon prevented the effect of P. aeruginosa exoproducts. Furthermore, treatment of P. aeruginosa cultures with 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a QS inhibitor, abrogated the negative impact of P. aeruginosa exoproducts on airway epithelial repair. Finally, we confirmed our findings in human airway epithelial cells from patients with CF, a disease featuring P. aeruginosa chronic respiratory infection. These data demonstrate that secreted proteases under the control of the LasR QS system impair airway epithelial repair and that QS inhibitors could be of benefit to counteract the deleterious effect of P. aeruginosa in infected patients.-Ruffin, M., Bilodeau, C., Maillé, É., LaFayette, S. L., McKay, G. A., Trinh, N. T. N., Beaudoin, T., Desrosiers, M.-Y., Rousseau, S., Nguyen, D., Brochiero, E. Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

Trevor Beaudoin

Physiology and Experimental Medicine

Hospital for Sick Children

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Trevor Beaudoin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Physiology and Experimental Medicine

Hospital for Sick Children

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Physiology and Experimental Medicine