Mucoid Biofilms Research Articles

Co-spray dried inhalable composite powders of ciprofloxacin and alginate oligosaccharide as anti-biofilm therapy

The treatment of chronic respiratory infections caused by biofilm formation are extremely challenging owing to poor drug penetration into the complex biofilm structure and high drug resistance. Local delivery of an antibiotic together with a non-antibiotic adjuvant to the lungs could often enhance the therapeutic responses by targeting different bacterial growth pathways and minimizing drug resistance. In this study, we designed new inhalable dry powders containing ciprofloxacin (CIP) and OligoG (Oli, a low-molecular-weight alginate oligosaccharide impairing the mucoid biofilms by interacting with their cationic ions) to combat respiratory bacterial biofilm infections. The resulting powders were characterized with respect to their morphology, solid-state property, surface chemistry, moisture sorption behavior, and dissolution rate. The aerosol performance and storage stability of the dry powders were also evaluated. The results showed that inhalable dry powders composed of CIP and Oli could be readily accomplished via the wet milling and spray drying process. Upon the storage under 20 ± 2 °C/20 ± 2 % relative humidity (RH) for one month, there was no significant change in the in vitro aerosol performances of the dry powders. In contrast, the dry powders became non-inhalable following the storage at 20 ± 2 °C/53 ± 2 % RH for one month due to the hygroscopic nature of Oli, which could be largely prevented by incorporation of leucine. Collectively, this study suggests that the newly developed co-spray-dried powders composed of CIP and Oli might represent a promising and alternative treatment strategy against respiratory bacterial biofilm infections.

Extracellular DNA enhances biofilm integrity and mechanical properties of mucoid Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most common biofilm-forming pathogens responsible for lung infections of individuals with cystic fibrosis (CF). P. aeruginosa becomes tolerant to antimicrobials in the biofilm state and is difficult to treat. Production of extracellular polymeric substances (EPS), such as alginate and extracellular DNA (eDNA), can allow adherence to abiotic and biotic surfaces, antimicrobial evasion, and resilience to environmental pressures. Alginate-producing mucoid variants of P. aeruginosa are frequently isolated from CF airway samples and are associated with worsening patient outcomes. While eDNA is a major structural component of nonmucoid P. aeruginosa biofilms, the potential role of eDNA in mucoid biofilms is unclear. Here, we investigate how eDNA contributes to clinical mucoid biofilm physiology and integrity. We predicted that eDNA plays a structural and mechanical role in mucoid biofilms. To test this, we quantified biofilm eDNA in mucoid biofilms and used microscopy and rheology to visualize eDNA and detect changes in biofilm structure and mechanics upon DNaseI treatment. We showed that biofilm eDNA abundance is diverse across clinical mucoid strains and observed a temporal increase in foci of eDNA within intact mucoid biofilms. Increased cell dispersal and reduced biomass were also observed following DNaseI treatment of mucoid biofilms. Degradation of eDNA also impacted the mechanical integrity of mucoid biofilms by increasing the stiffness and decreasing the cohesion of the biofilm. These findings advance our understanding of clinical mucoid P. aeruginosa biofilms and facilitate the development of new approaches to target biofilms by exploiting the functions of EPS components. IMPORTANCE Understanding the role of eDNA in mucoid Pseudomonas aeruginosa biofilms will lead to therapeutic strategies that combat the biophysical and structural function of EPS for the eradication of bacteria in mucoid biofilms during chronic infections. This knowledge can be used to further identify unknown matrix component interactions within pathogenic biofilm-forming clinical isolates.

Bacterial Infection in Head and Neck Space Regions: A Narrative Review

Abstract: Head and neck infection (HNI) is more complicated, as most of the sites of infection in this regions are very complex. Bacterial head and neck infections can usually originate through the upper airway, sinusitis, and dental or oral cavity and then extend deeper into other head and neck compartment sites. Both aerobic and anaerobic bacteria induce bacterial head and neck infections. This narrative review discusses the bacterial association, sites of infection, host-pathogen interaction, and secondary complications of head and neck bacterial infection. Staphylococcus aureus, Klebsiella spp, Escherichia coli, Peptostreptococcus spp., Pseudomonas putida, Pseudomonas aeruginosa, Fusobacterium spp, Citrobacter freundii, Streptococcus gordonii, Enterobacter spp, Gemella haemolysans, Haemophilus influenzae, and Enterococcus spp., Fusobacterium Spp are commonly responsible bacteria behind the bacterial head and neck infection (BHNI). Immunosuppression, alcohol consumption, and smoking risk factors are associated with it. The immune cell maintains a defense mechanism in host-pathogen interaction. Antibiotic-resistant genes in mucoid biofilm raise multidrug resistance against pathogenic bacteria. Inflammatory condition of the complete head and neck region can be demonstrated by computed tomography (CT) scan. The secondary complication may lead to induce cancer. Microbial invasions can be bacterial, fungal, or viral.

Association between phenotypic characteristics of Escherichia coli and UTI recurrence in immunocompromised patients: A case-control study

Urinary tract infection (UTI) recurrence is important in immunocompromised patients. There is a trend to study genotypically and phenotypically the role of certain virulence factors of Escherichia coli in the diagnosis of recurrent UTI. The main objective of this study was to determine if there is an association between phenotypic characteristics of E coli and UTI recurrence in immunocompromised patients. A case-control study was performed on immunocompromised patients from Hospital Regional de Alta Especialidad del Bajío, Mexico. E coli strains isolated from these patients were identificated and antimicrobial susceptibility test were performed. Strains with filamented cell morphology, mucoid colonial phenotype, or biofilm production were considered cases. Strains without the characteristics were considered controls. UTI recurrence was identified based on clinical records. The odds ratio (OR) was calculated to quantify the magnitude of the association. An association between filamented cell morphology and UTI recurrence was found (OR=2.19 95% CI 1.06-4.51; P=.031). No association was found between mucoid colony morphology (P>.05) or biofilm production (P>.05) and UTI recurrence. An association between mucoid colony morphology and extended-spectrum β-lactamase production was found (OR=3.09 95% 1.59-5.99; P<.001). Filamented cell morphology and mucoid colonial phenotype may have a possible diagnostic value for the detection of UTI recurrence and antimicrobial resistance. Further diagnostic test studies are needed to fully assess their clinical utility.

Polyguluronate simulations shed light onto the therapeutic action of OligoG CF-5/20

Chronic mucoid P. aeruginosa cystic fibrosis (CF) lung infections are associated with the development of a biofilm composed of anionic acetylated exopolysaccharide (EPS) alginate, electrostatically stabilised by extracellular Ca2+ ions. OligoG CF-5/20, a low molecular weight guluronate rich oligomer, is emerging as a novel therapeutic capable of disrupting mature P. aeruginosa biofilms. However, its method of therapeutic action on the mucoid biofilm EPS is not definitively known at a molecular level. This work, utilising molecular dynamics (MD) and Density-Functional Theory (DFT), has revealed that OligoG CF-5/20 interaction with the EPS is facilitated solely through bridging Ca2+ ions, which are not liberated from their native EPS binding sites upon OligoG CF-5/20 dispersal, suggesting that OligoG CF-5/20 does not cause disruptions to mature P. aeruginosa biofilms through breaking EPS-Ca2+-EPS ionic cross-links. Rather it is likely that the therapeutic activity arises from sequestering free Ca2+ ions and preventing further Ca2+ induced EPS aggregation.

Electrochemical Detection of Tobramycin Resistance in Pseudomonas Aeruginosa

Pseudomonas aeruginosa are Gram-negative anaerobic bacteria and is listedby the World Health Organization as one of the critical antibiotic-resistant pathogens1. The treatment for P. aeruginosa infection can be challenging, since this pathogen often exhibits an enhanced resistance to antibiotics, including aminoglycosides, such as Tobramycin (TOB). These are called mucoid strains and are mainly found in the lungs of cystic fibrosis patients, where it is a common cause of death for these individuals2. The overall goal of the presented work is the development of an electrochemical method capable of detecting TOB uptake in P. aeruginosa. Drug susceptible and drug resistant strains of P. aeruginosa were exposed to a TOB solution for different time intervals (Figure 1). By removing organisms from the solution after incubation, the remaining TOB in solution can be determined by electrochemistry. Resistant bacteria strains showed significantly lower TOB uptake than susceptible organisms, because cellular drug resistant mechanisms enable them to inhibit TOB absorption. The presented drug uptake studies demonstrate the sensitivity of electrochemistry to detect drug resistance in living bacteria. References Singh, M., Yau, Y. C. W., Wang, S., Waters, V. & Kumar, A. MexXY efflux pump overexpression and aminoglycoside resistance in cystic fibrosis isolates of pseudomonas aeruginosa from chronic infections. Canadian Journal of Microbiology 63, 929–938 (2017).Germoni, L. A. P., Bremer, P. J. & Lamont, I. L. The effect of alginate lyase on the gentamicin resistance of Pseudomonas aeruginosa in mucoid biofilms. Journal of Applied Microbiology 121, 126–135 (2016). Figure 1

Does ibuprofen affect the expression of alginate genes in pathogenic Pseudomonas aeruginosa strains?

Conversion to mucoid form is a crucial step in the pathogenesis of P. aeruginosa in burns and cystic fibrosis (CF) patients. Alginate is considered the major component of biofilm and is highly associated with the formation of mucoid biofilm in this species. Nonsteroid anti-inflammatory drugs (NSAIDs), including ibuprofen, have shown promising antibacterial and antibiofilm potential for bacterial pathogens. In this study, we aimed to evaluate the effect of ibuprofen on the expression of alginate synthetase (alg8), GDP-mannose dehydrogenase (algD), and alginate lyase (algL) genes in multiple drug-resistant (MDR) P. aeruginosa strains. The biofilm formation potential and the expression of alg8, algD, and algL among the bacteria treated with ibuprofen (at sub-inhibitory concentration) were investigated using the crystal violet staining and real-time PCR assays, respectively. The minimum inhibitory concentration of ibuprofen for the studied strains was determined 1024-2048µg/mL. We observed that ibuprofen was able to reduce bacterial biofilm by 51-77%. Also, the expression of alg8, algD, and algL decreased by 32, 52, and 48%, respectively. The reduction of the genes responsible for alginate synthesis indicates promising antivirulece potential of ibuprofen to combat P. aeruginosa infection, especially in burns and CF patients. Our findings suggest that ibuprofen could be used to reduce the pathogenicity of P. aeruginosa that could be used in combination with antibiotics to treat drug-resistant infections.

Quorum quenchers affect the virulence regulation of non-mucoid, mucoid and heavily mucoid biofilms co-cultured on cell lines

Biofilm formation conferring pathogenicity is a survival strategy for Pseudomonas aeruginosa. P. aeruginosa’s virulence may differ due to differences in host-microbe interactions and the growth environment. The epithelial cell line within the respiratory system and the keratinocytes on the skin form the first physical barrier of defence. P. aeruginosa spp. biofilm formation and virulence factor secretion with and without quorum quenching (QQ) treatment was studied in co-culture using A549 and HaCaT cell lines; pyocyanin and rhamnolipid productions and elastolytic activity as virulence factors were quantified by independent assays. Biofilm formation was evaluated under dynamic conditions by quantifying total carbohydrates, alginate, proteins and eDNA. A sandwich ELISA was performed to study IL-8 secretion by the epithelial cells. The difference in gene expression of the quorum sensing (QS) and virulence factors between strains during individual and combination treatments was analysed by qPCR. Combination treatment by farnesol and tyrosol was more effective against P. aeruginosa biofilms when grown in co-cultures. The strain RBHi was found to be 3 to 4 times more virulent compared to PAO1 and NCTC 10,662, respectively, and combination treatment was more effective against RBHi strain when grown in co-culture with A549 cell line. The addition of quorum quenchers (QQs) individually and in combination reduced IL-8 secretion by A549 cells. Relative mRNA expression showed upregulation of the QS genes and virulence factors. Co-culture of P. aeruginosa and HaCaT cell line showed a general decrease in gene expression, especially in the case of P. aeruginosa RBHi when treated with farnesol and tyrosol combination.Key points• Differentiating the interactions of biofilm formed by different phenotypes of P. aeruginosa, NCTC 10,662 (non-mucoid), PAO1 (semi mucoid) and RBHi (heavily mucoid).• Biofilm formed by these P. aeruginosa strains on two commonly afflicted tissues represented by A549 (lung) and HaCaT (skin) cell lines.• Anti-biofilm/anti-virulence roles of quorum quenchers, tyrosol and farnesol in co-cultures.

Susceptibility Testing of Exophiala dermatitidis to Inform Peri-Operative Prophylaxis at Time of Lung Transplantation

Purpose Exophiala dermatitidis is a black fungus that frequently colonises the lungs of people with cystic fibrosis (CF). Mucoid and non-mucoid variants can be detected from the same sputum for some patients, but their role in chronic CF lung disease is not fully understood and attempts at early eradication are not routinely performed. Invasive infections with this species are rare; however in 2014 we reported our first case of fatal, invasive infection with a highly mucoid strain of E. dermatitidis in a 34 year old post lung transplant patient with CF. This prompted us to include peri-operative antifungal prophylaxis for all patients colonised with this species. To inform the choice of optimal antifungal, we performed susceptibility testing of 38 isolates from 34 patients including lung transplant patients. The ability of these isolates to produce biofilm was also investigated. Methods The minimum inhibitory concentration (MIC) of 8 antifungal agents was measured using a commercial broth microdilution assay. Biofilm formation was assessed using a standard in vitro assay using staining with crystal violet. Results Using breakpoints for Candida albicans, all isolates were susceptible to voriconazole and amphotericin B with most isolates also susceptible to itraconazole (89% of isolates) and posaconazole (71% of isolates). In contrast, all isolates were non-susceptible to caspofungin and most isolates (92%) non-susceptible to fluconazole. All strains were inhibited by ketoconazole at ≤0.25 mg/ L whereas MICs of 5-fluorocytosine ranged from 1->64 mg/ L. 31 of 38 isolates were biofilm producers including 4 isolates that were classified as strong biofilm producers. We did not find a correlation between mucoid phenotype and biofilm production. Conclusion E. dermatitidis is commonly found to colonise the lungs of patients with CF, typically produces biofilm, and may rarely produce serious infection post transplantation. We demonstrate in vitro susceptibility to voriconazole and amphotericin B. For patients colonised with this species, we now elect to include voriconazole as part of the peri-operative prophylactic regimen.

P. aeruginosa flow-cell biofilms are enhanced by repeated phage treatments but can be eradicated by phage-ciprofloxacin combination.

Phage therapy has shown promising results in the treatment of Pseudomonas aeruginosa biofilm infections in animal studies and case reports. The aim of this study was to quantify effects of phage treatments on P. aeruginosa biofilm production and structure. Confocal scanning microscopy was used to follow the interaction between a cocktail of three virulent phages and P. aeruginosa flow-cell biofilms. The role of (i) biofilm age, (ii) repeated phage treatments, (iii) alginate production and (iv) the combination with sub-MIC levels of ciprofloxacin was investigated. Single phage treatment in the early biofilm stages significantly reduced P. aeruginosa PAO1 biovolume (85%-98% reduction). Repeated phage treatments increased the biovolume from 18.25 (untreated biofilm) to 22.24 and 31.07 µm3/µm2 for biofilms treated with phages twice and thrice, respectively. Alginate protected against the phage treatment as the live biovolume remained unaffected by the phage treatment in the mucoid biofilm (20.11 µm3/µm2 in untreated and 21.74 µm3/µm2 in phage-treated biofilm) but decreased in the PAO1 biofilm from 27.35 to 0.89 µm3/µm2. We show that the combination of phages with antibiotics at sub-MIC levels caused a ∼6 log units reduction in the abundance of P. aeruginosa cells in biofilms and that phage treatment increased the size of microcolonies in flow-cell system.

Optimization of colistin dosing regimen for cystic fibrosis patients with chronic Pseudomonas aeruginosa biofilm lung infections.

The present study was performed to explore dosing regimens of colistin in patients of cystic fibrosis (CF) with Pseudomonas aeruginosa chronic biofilm lung infection. Ten CF patients were involved. One dose colistimethate sodium (CMS) of 6 MIU (million international units) and 9 MIU were administered by intravenous infusion over 45 and 90 min. Venous blood was collected at different time points after the infusion of CMS. Pharmacokinetic parameters of colistin were calculated. Minimum inhibitory concentration for planktonic P. aeruginosa, minimum biofilm inhibitory concentration and minimum biofilm eradication concentration of P. aeruginosa were determined. Monte Carlo simulation was performed to determine the clinical probability of target attainment of different dosing regimens of colistin in CF patients. For 90 min (6 MIU), 45 min (6 MIU), and 45 min (9 MIU) intravenous infusion of colistin, Cmax was 8.9 ± 1.8, 15 ± 5.5, and 31.7 ± 5.3 μg/mL, respectively; Tmax was 1.2 ± 0.4, 0.7 ± 0.2, and 0.8 ± 0.2 h, respectively; AUCtot were 31 ± 3.8, 34 ± 10, and 135 ± 31mg · h/L, respectively; t1/2 was 2.1 ± 0.4, 2 ± 0.3, and 3.3 ± 0.4 h, respectively. MBIC and MBEC of colistin on biofilms at 24 h period treatment were 16-128 μg/mL for non-mucoid and mucoid biofilms of P. aeruginosa. For 90 min (6 MIU), 45 min (6 MIU) and 45 min iv infusion (9 MIU) with one dose colistin, PTA was 49.8%, 53.8%, 99.4% for planktonic infection, and 11.3%, 14.6%, 65.3%, respectively for biofilm infection. colistin treatment using 45 min iv infusion is better than 90 min iv infusion in this study. Colistin dosage of 9 MIU is better than 6 MIU on both planktonic and biofilm infections of P. aeruginosa in this study.

Viscoelastic properties of Pseudomonas aeruginosa variant biofilms

Pseudomonas aeruginosa evolves during chronic pulmonary infections of cystic fibrosis (CF) patients, forming pathoadapted variants that are persistent. Mucoid and rugose small-colony variants (RSCVs) are typically isolated from sputum of CF patients. These variants overproduce exopolysaccharides in the biofilm extracellular polymeric substance (EPS). Currently, changes to the biophysical properties of RSCV and mucoid biofilms due to variations in EPS are not well understood. This knowledge may reveal how lung infections resist host clearance mechanisms. Here, we used mechanical indentation and shear rheometry to analyse the viscoelasticity of RSCV and mucoid colony-biofilms compared to their isogenic parent at 2-, 4-, and 6-d. While the viscoelasticity of parental colony-biofilms underwent fluctuating temporal changes, in contrast, RSCV and mucoid colony-biofilms showed a gradual progression to more elastic-solid behaviour. Theoretical indices of mucociliary and cough clearance predict that mature 6-d parental and RSCV biofilms may show reduced cough clearance from the lung, while early mucoid biofilms may show reduced clearance by both mechanisms. We propose that viscoelasticity be considered a virulence property of biofilms.

Sputum Active Polymyxin Lipopeptides: Activity against Cystic Fibrosis Pseudomonas aeruginosa Isolates and Their Interactions with Sputum Biomolecules.

The mucoid biofilm mode of growth of Pseudomonas aeruginosa (P. aeruginosa) in the lungs of cystic fibrosis patients makes eradication of infections with antibiotic therapy very difficult. The lipopeptide antibiotics polymyxin B and colistin are currently the last-resort therapies for infections caused by multidrug-resistant P. aeruginosa. In the present study, we investigated the antibacterial activity of a series of polymyxin lipopeptides (polymyxin B, colistin, FADDI-003, octapeptin A3, and polymyxin A2) against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa cystic fibrosis isolates grown under planktonic or biofilm conditions in artificial sputum and their interactions with sputum component biomolecules. In sputum media under planktonic conditions, the lipopeptides FADDI-003 and octapeptin A3 displayed very promising activity against the polymyxin-resistant isolate FADDI-PA066 (polymyxin B minimum inhibitory concentration (MIC) = 32 mg/L), while retaining their activity against the polymyxin-sensitive strains FADDI-PA021 (polymyxin B MIC = 1 mg/L) and FADDI-PA020 (polymyxin B MIC = 2 mg/L). Polymyxin A2 was only effective against the polymyxin-sensitive isolates. However, under biofilm growth conditions, the hydrophobic lipopeptide FADDI-003 was inactive compared to the more hydrophilic lipopeptides, octapeptin A3, polymyxin A2, polymyxin B, and colistin. Transmission electron micrographs revealed octapeptin A3 caused reduction in the cell numbers in biofilm as well as biofilm disruption/“antibiofilm” activity. We therefore assessed the interactions of the lipopeptides with the component sputum biomolecules, mucin, deoxyribonucleic acid (DNA), surfactant, F-actin, lipopolysaccharide, and phospholipids. We observed the general trend that sputum biomolecules reduce lipopeptide antibacterial activity. Collectively, our data suggests that, in the airways, lipopeptide binding to component sputum biomolecules may reduce antibacterial efficacy and is dependent on the physicochemical properties of the lipopeptide.

Regulation of ribonucleotide synthesis by the Pseudomonas aeruginosa two-component system AlgR in response to oxidative stress

Ribonucleotide reductases (RNR) catalyze the last step of deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. Three forms of RNR exist: classes I, II, and III. While eukaryotic cells use only class Ia RNR, bacteria can harbor any combination of classes, granting them adaptability. The opportunistic pathogen Pseudomonas aeruginosa surprisingly encodes all three classes, allowing it to thrive in different environments. Here we study an aspect of the complex RNR regulation whose molecular mechanism has never been elucidated, the well-described induction through oxidative stress, and link it to the AlgZR two-component system, the primary regulator of the mucoid phenotype. Through bioinformatics, we identify AlgR binding locations in RNR promoters, which we characterize functionally through EMSA and physically through AFM imaging. Gene reporter assays in different growth models are used to study the AlgZR-mediated control on the RNR network under various environmental conditions and physiological states. Thereby, we show that the two-component system AlgZR, which is crucial for bacterial conversion to the mucoid phenotype associated with chronic disease, controls the RNR network and directs how the DNA synthesis pathway is modulated in mucoid and non-mucoid biofilms, allowing it to respond to oxidative stress.

Insights from the analysis of alginate lyase protein model from Pseudomonas fluorescens towards the understanding of mucoid biofilm disruption.

Bacterial biofilm is a protective, slippery and slimy coat secreted by bacterial cells. It helps in attaching to moisturized surfaces during colonization. Alginate is an important component as it is essential for retention of water and nutrients in biofilms. It is a polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) monomers with 1-4 linkage. The alginate lyase (AlgL) secreted by certain bacteria is capable of degrading alginate into oligo-uronides by β-elimination of the glycosidic bond. Therefore, it is of interest to analyze the simulated (GROMACS force filed) structure protein model (homology based on template 4OZV) of AlgL from Pseudomonas fluorescens to gain functional insight mucoid biofilm disruption. We report root mean square deviation (RMSD) and radius of gyration (Rg) profiles of the simulated (molecular dynamics) AlgL protein homology model in this context towards biofilm discruption.

Spatial Mapping of Pyocyanin in Pseudomonas Aeruginosa Bacterial Communities Using Surface Enhanced Raman Scattering.

Surface enhanced Raman spectroscopy (SERS) imaging was used in conjunction with principal component analysis (PCA) for the in situ spatiotemporal mapping of the virulence factor pyocyanin in communities of the pathogenic bacterium Pseudomonas aeruginosa. The combination of SERS imaging and PCA analysis provides a robust method for the characterization of heterogeneous biological systems while circumventing issues associated with interference from sample autofluorescence and low reproducibility of SERS signals. The production of pyocyanin is found to depend both on the growth carbon source and on the specific strain of P. aeruginosa studied. A cystic fibrosis lung isolate strain of P. aeruginosa synthesizes and secretes pyocyanin when grown with glucose and glutamate, while the laboratory strain exhibits detectable production of pyocyanin only when grown with glutamate as the source of carbon. Pyocyanin production in the laboratory strain grown with glucose was below the limit of detection of SERS. In addition, the combination of SERS imaging and PCA can elucidate subtle differences in the molecular composition of biofilms. PCA loading plots from the clinical isolate exhibit features corresponding to vibrational bands of carbohydrates, which represent the mucoid biofilm matrix specific to that isolate, features that are not seen in the PCA loading plots of the laboratory strain.

Polydopamine-Mediated Immobilization of Alginate Lyase to Prevent P. aeruginosa Adhesion.

Given alginate's contribution to Pseudomonas aeruginosa virulence, it has long been considered a promising target for interventional therapies, which have been performed by using the enzyme alginate lyase. In this work, instead of treating pre-established mucoid biofilms, alginate lyase is immobilized onto a surface as a preventive measure against P. aeruginosa adhesion. A polydopamine dip-coating strategy is employed for functionalization of polycarbonate surfaces. Enzyme immobilization is confirmed by surface characterization. Surfaces functionalized with alginate lyase exhibit anti-adhesive properties, inhibiting the attachment of the mucoid strain. Moreover, surfaces modified with this enzyme also inhibit the adhesion of the tested non-mucoid strain. Unexpectedly, treatment with heat-inactivated enzyme also inhibits the attachment of mucoid and non-mucoid P. aeruginosa strains. These findings suggest that the antibacterial performance of alginate lyase functional coatings is catalysis-independent, highlighting the importance of further studies to better understand its mechanism of action against P. aeruginosa strains.

Antibacterial Action of Nitric Oxide-Releasing Chitosan Oligosaccharides against Pseudomonas aeruginosa under Aerobic and Anaerobic Conditions.

Chitosan oligosaccharides were modified with N-diazeniumdiolates to yield biocompatible nitric oxide (NO) donor scaffolds. The minimum bactericidal concentrations and MICs of the NO donors against Pseudomonas aeruginosa were compared under aerobic and anaerobic conditions. Differential antibacterial activities were primarily the result of NO scavenging by oxygen under aerobic environments and not changes in bacterial physiology. Bacterial killing was also tested against nonmucoid and mucoid biofilms and compared to that of tobramycin. Smaller NO payloads were required to eradicate P. aeruginosa biofilms under anaerobic versus aerobic conditions. Under oxygen-free environments, the NO treatment was 10-fold more effective at killing biofilms than tobramycin. These results demonstrate the potential utility of NO-releasing chitosan oligosaccharides under both aerobic and anaerobic environments.

Characterization of a mucoid-like Pseudomonas aeruginosa biofilm

Pseudomonas aeruginosa biofilms are implicated in chronic infections. A key element of P. aeruginosapathogenicity is the formation of a biofilm, a community of bacteria encased in an exopolymeric substance (EPS) that shields the bacteria from the host immune response and antibiotic treatment. A crucial step in biofilm production is a switch in motility from freely swimming, planktonic bacteria to twitching movement and then to attached and sedentary bacteria that develop into a mature pillar-shaped biofilm. A mucoid biofilm produces an excess of alginate and is clinically the most pathogenic and the most resistant to antibiotics. Biofilms from patients exhibit a wide variety of structure, motility, and levels of attachment. In vitrobiofilms do not exhibit such a wide variety of structure and physiology. The difference between in vivo and in vitro biofilms has made the translation of in vitro studies into in vivo treatments difficult. Under different growth conditions in our lab, the P. aeruginosa strain PAO1 demonstrates two phenotypes: a non-mucoid and a mucoid-like phenotype. Confocal laser scanning microscopy (CLSM) indicates the mucoid-like phenotype is intermediate in height to the non-mucoid phenotype and biofilms formed in a once-flow-through chamber. Both mucoid-like and non-mucoid phenotypes exhibit a significant increase in twitching between 24 and 72 hours of development. The mucoid-like phenotype had greater attachment at 72 hours compared to non-mucoid phenotype. Therefore, the two phenotypes observed in our lab may represent the effect of environment to stimulate development of two types of biofilms by PAO1.

Nitric oxide production by polymorphonuclear leucocytes in infected cystic fibrosis sputum consumes oxygen

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O(2)) due to production of superoxide (O(2)(-)). In this study, we show that the PMNs also consume O(2) for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with N(G) -monomethyl-L-arginine (L-NMMA) resulted in reduced O(2) consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO(3)(-) (P < 0·04, r = 0·66, n = 10) and NO(2)(-) (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O(2) for production of reactive oxygen species, the PMNs in CF sputum also consume O(2) for production of NO.