Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa

Fluorescently labelled lectins were used in combination with epifluorescence microscopy and confocal laser scanning microscopy to allow the visualization and characterization of carbohydrate-containing extracellular polymeric substances (EPS) in biofilms of Pseudomonas aeruginosa. A mucoid strain characterized by an overproduction of the exopolysaccharide alginate, and an isogenic, non-mucoid strain were used. Model biofilms grown on polycarbonate filters were treated with lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) that were fluorescently labelled with fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate. Fluorescently labelled ConA yielded cloud-like regions that were heterogeneously distributed within mucoid biofilms, whereas these structures were only rarely present in biofilms of the non-mucoid strain. The bacteria visualized with the fluorochrome SYTO 9 were localized both within and between the ConA-stained regions. In WGA-treated biofilms, the lectin was predominantly associated with bacterial cells. Alginate seemed to be involved in the interaction of ConA with the EPS matrix, since (i) pre-treatment of biofilms with an alginate lyase resulted in a loss of ConA biofilm staining, and (ii) using an enzyme-linked lectinsorbent assay (ELLA), ConA was shown to bind to purified alginate, but not to alginate that was degraded by alginate lyase. The application of fluorescently labelled lectins in combination with ELLA was found to be useful for the visualization and characterization of extracellular polysaccharide structures in P. aeruginosa biofilms.     

Expression of extracellular polysaccharides and proteins by clinical isolates of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in response to environmental conditions

The opportunistic pathogen Pseudomonas aeruginosa causes chronic respiratory infections in patients with cystic fibrosis (CF). Persistence of this bacterium is attributed to its ability to form biofilms which rely on an extracellular polymeric substance matrix. Extracellular polysaccharides (EPS) and secreted proteins are key matrix components of P. aeruginosa biofilms. Recently, nebulized magnesium sulfate has been reported as a significant bronchodilator for asthmatic patients including CF. However, the impact of magnesium sulfate on the virulence effect of P. aeruginosa is lacking. In this report, we investigated the influence of magnesium sulfate and other environmental factors on the synthesis of alginate and secretion of proteins by a mucoid and a non-mucoid strain of P. aeruginosa, respectively. By applying the Plackett-Burman and Box-Behnken experimental designs, we found that phosphates (6.0 g/l), ammonium sulfate (4.0 g/l), and trace elements (0.6 mg/l) markedly supported alginate production by the mucoid strain. However, ferrous sulfate (0.3 mg/l), magnesium sulfate (0.02 g/l), and phosphates (6.0 g/l) reinforced the secretion of proteins by the non-mucoid strain.     

The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment

Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.     

Biofilm inhibitory effect of alginate lyases on mucoid P. aeruginosa from a cystic fibrosis patient

Chronic mucoid Pseudomonas aeruginosa infections are a major scourge in cystic fibrosis patients. Mucoid P. aeruginosa displays structured alginate-rich biofilms that are resistant to antibiotics. Here, we have assessed the efficacy of a panel of alginate lyases in combating mucoid P. aeruginosa biofilms in cystic fibrosis. Albeit we could not demonstrate alginate degradation by alginate lyases in sputum, we demonstrate that the endotypic alginate lyases, CaAly (from Cellulophaga algicola) and VspAlyVI (from Vibrio sp. QY101) and the exotypic alginate lyases, FspAlyFRB (from Falsirhodobacterium sp. alg1), and SA1-IV (from Sphingomonas sp. A1), indeed inhibit biofilm formation by a mucoid P. aeruginosa strain isolated from the sputum of a cystic fibrosis patient with comparative effect to that of the glycoside hydrolase PslG, a promising candidate for biofilm treatment. We believe that these enzymes should be explored for in vivo efficacy in future studies.     

Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation

Background

Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.

Methods

Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.

Results

Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.

Conclusion

We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.     

Factors Affecting Catalase Expression in Pseudomonas aeruginosa Biofilms and Planktonic Cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 μM as FeCl₃) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

MucA-mediated coordination of type III secretion and alginate synthesis in Pseudomonas aeruginosa

The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence factor. The T3SS of P. aeruginosa can be induced by a low calcium signal or upon direct contact with the host cells. The exact pathway of signal sensing and T3SS activation is not clear. By screening a transposon insertion mutant library of the PAK strain, mutation in the mucA gene was found to cause repression of T3SS expression under both type III-inducing and -noninducing conditions. Mutation in the mucA gene is known to cause alginate overproduction, resulting in a mucoid phenotype. Alginate production responds to various environmental stresses and plays a protective role for P. aeruginosa. Comparison of global gene expression of mucA mutant and wild-type PAK under T3SS-inducing conditions confirmed the down regulation of T3SS genes and up regulation of genes involved in alginate biosynthesis. Further analysis indicated that the repression of T3SS in the mucA mutant was AlgU and AlgR dependent, as double mutants mucA/algU and mucA/algR showed normal type III expression. An algR::Gm mutant showed a higher level of type III expression, while overexpression of the algR gene inhibited type III gene expression; thus, it seems that the AlgR-regulated product inhibits the expression of the T3SS genes. It is likely that P. aeruginosa has evolved tight regulatory networks to turn off the energy-expensive T3SS when striving for survival under environmental stresses.     

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ²²). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.     

The Effects of CFTR and Mucoid Phenotype on Susceptibility and Innate Immune Responses in a Mouse Model of Pneumococcal Lung Disease

Recent studies have reported the isolation of highly mucoid serotype 3 Streptococcus pneumoniae (Sp) from the respiratory tracts of children with cystic fibrosis (CF). Whether these highly mucoid Sp contribute to, or are associated with, respiratory failure among patients with CF remains unknown. Other mucoid bacteria, predominately Pseudomonas aeruginosa, are associated with CF respiratory decline. We used a mouse model of CF to study pneumococcal pneumonia with highly mucoid serotype 3 and non-mucoid serotype 19A Sp isolates. We investigated susceptibility to infection, survival, and bacterial counts from bronchoaviolar lavage samples and lung homogenates, as well as associated inflammatory cytokines at the site of infection, and lung pathology. Congenic CFTR^–/– mice and wild-type (WT)-mice were infected intranasally with CHB756, CHB1126, and WU2 (highly mucoid capsular serotype 3, intermediately mucoid serotype 3, and less mucoid serotype 3, respectively), or CHB1058 (non-mucoid serotype 19A). BAL, lung homogenates, and blood were collected from mice 5 days post-infection. Higher CFU recovery and shorter survival were observed following infection of CFTR^–/– mice with CHB756 compared to infection with CHB1126, WU2, or CHB1058 (P≤0.001). Additionally, CFTR^–/– mice infected with CHB756 and CHB1126 were more susceptible to infection than WT-mice (P≤0.05). Between CFTR^–/– mice and WT-mice, no significant differences in TNF-α, CXCL1/KC concentrations, or lung histopathology were observed. Our results indicate that highly mucoid type 3 Sp causes more severe lung disease than non-mucoid Sp, and does so more readily in the lungs of CFTR^–/– than WT-mice.     

Enzymes involved in the biosynthesis of alginate byPseudomonas aeruginosa

Summary Analysis of the enzymes involved in the biosynthesis of alginic acid by mucoidPseudomonas aeruginosa PAO strain's determined the presence of enzymes required to synthesise GDP-mannuronic acid. Addition of polymannuronic acid to an ammonium sulphate precipitate of a cell free alginate suspension indicated the presence of an enzyme which catalysed the epimerisation of mannuronic acid to guluronic acidafter the polymer had been synthesised. The epimerase was shown to be calcium dependant.Various non-mucoid mutants were also studied. The non-mucoid parental strain PAO 381 also contained the enzymes required for alginate synthesis but they were not expressed. Synthesis of alginic acid led to an increase in the level of these enzymes. In the non-mucoid mutants derived from mucoid parents GDP-mannose dehydrogenase was absent in all strains studied. In some of these strains GDP-mannose pyrophosphorylase was also absent, while in other strains, phosphomannase isomerase was absent or greatly reduced.     

The carbon monoxide releasing molecule CORM-2 attenuates Pseudomonas aeruginosa biofilm formation

Chronic infections resulting from biofilm formation are difficult to eradicate with current antimicrobial agents and consequently new therapies are needed. This work demonstrates that the carbon monoxide-releasing molecule CORM-2, previously shown to kill planktonic bacteria, also attenuates surface-associated growth of the gram-negative pathogen Pseudomonas aeruginosa by both preventing biofilm maturation and killing bacteria within the established biofilm. CORM-2 treatment has an additive effect when combined with tobramycin, a drug commonly used to treat P. aeruginosa lung infections. CORM-2 inhibited biofilm formation and planktonic growth of the majority of clinical P. aeruginosa isolates tested, for both mucoid and non-mucoid strains. While CORM-2 treatment increased the production of reactive oxygen species by P. aeruginosa biofilms, this increase did not correlate with bacterial death. These data demonstrate that CO-RMs possess potential novel therapeutic properties against a subset of P. aeruginosa biofilm related infections.     

A gacS Deletion in Pseudomonas aeruginosa Cystic Fibrosis Isolate CHA Shapes Its Virulence

Pseudomonas aeruginosa, a human opportunistic pathogen, is capable of provoking acute and chronic infections that are associated with defined sets of virulence factors. During chronic infections, the bacterium accumulates mutations that silence some and activate other genes. Here we show that the cystic fibrosis isolate CHA exhibits a unique virulence phenotype featuring a mucoid morphology, an active Type III Secretion System (T3SS, hallmark of acute infections), and no Type VI Secretion System (H1-T6SS). This virulence profile is due to a 426 bp deletion in the 3′ end of the gacS gene encoding an essential regulatory protein. The absence of GacS disturbs the Gac/Rsm pathway leading to depletion of the small regulatory RNAs RsmY/RsmZ and, in consequence, to expression of T3SS, while switching off the expression of H1-T6SS and Pel polysaccharides. The CHA isolate also exhibits full ability to swim and twitch, due to active flagellum and Type IVa pili. Thus, unlike the classical scheme of balance between virulence factors, clinical strains may adapt to a local niche by expressing both alginate exopolysaccharide, a hallmark of membrane stress that protects from antibiotic action, host defences and phagocytosis, and efficient T3S machinery that is considered as an aggressive virulence factor.     

Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients

Pseudomonas aeruginosa is the predominant microorganism in chronic lung infection of cystic fibrosis patients. The chronic lung infection is preceded by intermittent colonization. When the chronic infection becomes established, it is well accepted that the isolated strains differ phenotypically from the intermittent strains. Dominating changes are the switch to mucoidity (alginate overproduction) and loss of epigenetic regulation of virulence such as the Quorum Sensing (QS). To elucidate the dynamics of P. aeruginosa QS systems during long term infection of the CF lung, we have investigated 238 isolates obtained from 152 CF patients at different stages of infection ranging from intermittent to late chronic. Isolates were characterized with regard to QS signal molecules, alginate, rhamnolipid and elastase production and mutant frequency. The genetic basis for change in QS regulation were investigated and identified by sequence analysis of lasR, rhlR, lasI and rhlI. The first QS system to be lost was the one encoded by las system 12 years (median value) after the onset of the lung infection with subsequent loss of the rhl encoded system after 17 years (median value) shown as deficiencies in production of the 3-oxo-C12-HSL and C4-HSL QS signal molecules respectively. The concomitant development of QS malfunction significantly correlated with the reduced production of rhamnolipids and elastase and with the occurrence of mutations in the regulatory genes lasR and rhlR. Accumulation of mutations in both lasR and rhlR correlated with development of hypermutability. Interestingly, a higher number of mucoid isolates were found to produce C4-HSL signal molecules and rhamnolipids compared to the non-mucoid isolates. As seen from the present data, we can conclude that P. aeruginosa and particularly the mucoid strains do not lose the QS regulation or the ability to produce rhamnolipids until the late stage of the chronic infection.