Adherence of mucoid and nonmucoid Pseudomonas aeruginosa to acid-injured tracheal epithelium.

A model of the adherence of Pseudomonas aeruginosa to injured lower respiratory tract cells is described. Mouse tracheas were injured by exposure to 0.1 N HCl for 15 min, cut into pieces of 2 to 4 rings, and placed in petri dishes for adherence studies. Adherence was quantitated by direct count of the number of bacteria attached to a fixed surface area as viewed by scanning electron microscopy. Optimal conditions for study were 1 h of incubation with an inoculum of 10(7) to 10(8) CFU/ml. Both mucoid and nonmucoid P. aeruginosa adhered to the injured cells in this system, and no major differences in adherence between these strains was apparent. Adherence to injured tracheal cells was a phenomenon limited to P. aeruginosa and did not occur with Escherichia coli and Klebsiella pneumoniae. Besides adherence to injured cells, this model allowed the demonstration of adherence of mucoid P. aeruginosa and K. pneumoniae to mucin strands of uninjured control tracheas. This model is an alternative to the buccal cell model and has the advantage of allowing the study of the adherence of both mucoid and nonmucoid strains of P. aeruginosa.     

Adherence of Pseudomonas aeruginosa to cilia of human tracheal epithelial cells.

Pseudomonas aeruginosa was found to adhere selectively to cilia of human ciliated tracheal epithelial cells (TECs). P. aeruginosa bound in equal numbers to TECs of smokers and nonsmokers, with the mean adhesion index for binding of P. aeruginosa 492c to TECs of healthy individuals (+/- standard deviation) being 6.83 +/- 6.00 bacteria per TEC.     

Quantitation of adherence of mucoid and nonmucoid Pseudomonas aeruginosa to hamster tracheal epithelium.

Adherence of mucoid and nonmucoid isolates of Pseudomonas aeruginosa to tracheal epithelium was quantitated by using hamster tracheas mounted in a perfusion chamber. The strains of P. aeruginosa used were clinical isolates from cystic fibrosis patients and a series of laboratory strains. Aseptically excised hamster tracheas were mounted in perfusion chambers and embedded in minimal essential medium containing 1.5% agarose. The tracheas were infected with various numbers of bacteria for various periods, rinsed, homogenized, and plated on Trypticase soy agar. A 4-mm segment from each trachea was prepared for quantitation, and the other segment was prepared for examination by scanning electron microscopy. Adherence increased with time and with increasing concentrations of inoculum. Standard conditions of inoculation were set at an inoculum of 10(7) CFU/ml and a 2-h incubation. Under these conditions, the mucoid organisms adhered to the ciliated epithelium 10- to 100-fold better than did the nonmucoid organisms. Adherence of the mucoid isolates did not appear to be pilus mediated and did not involve hydrophobic interactions. The mucoid P. aeruginosa isolates could be seen adhering to the epithelium in the form of microcolonies embedded in an extracellular matrix which attaches the organisms to the cilia and to each other. The adherence may be involved in the establishment of infection of the lungs of these patients and in the inability to clear the organisms from the lungs. The model will be useful in determining the mechanism of adherence of the bacteria to the ciliated epithelium of the respiratory tract.     

Role of pili in the adherence of Pseudomonas aeruginosa to injured tracheal epithelium.

Pili have been demonstrated to be the adhesins of nonmucoid Pseudomonas aeruginosa for buccal cells. In this study, we examined their role in the adherence of both mucoid and nonmucoid strains to injured tracheal cells. Pili incubated with tracheal cells inhibited the adherence of a nonmucoid strain in a dose-dependent manner. Both homologous and heterologous pili inhibited this nonmucoid strain. Antibody against pili from the nonmucoid strain inhibited adherence of the homologous but not a heterologous strain. Pili failed to inhibit two mucoid strains, but inhibited nonmucoid variants derived from mucoid strains. These studies suggest that pili mediate the adherence of nonmucoid strains to injured tracheal cells but that they are not the final mediators of adherence of mucoid strains. It is also inferred that there are differences in the receptor for mucoid and nonmucoid strains.     

Adherence of Pseudomonas aeruginosa to respiratory epithelium and the effect of leucocyte elastase

The tracheobronchial secretions from patients with cystic fibrosis often contain high amounts of free proteases. To evaluate whether human leucocyte elastase (HLE) can favour the persistence of bacterial airways infection, we exposed the frog palate mucosa to HLE and then to radiolabelled Pseudomonas aeruginosa and followed the sequence of events by scanning electronmicroscopy. In response to HLE there was a marked outpouring of mucus and a desquamation of the epithelium. P. aeruginosa was shown to adhere to recently secreted granules of mucus and to the exposed submucosal underlying connective tissues. For the eight different bacterial strains studied, a significative adherence to HLE-injured mucosa was observed only in strains that possessed internal haemagglutinating activity. Neither the presence of fimbriae, nor of the mucoid exopolysaccharide, nor of the bacterial surface haemagglutinating activity could be related to adherence of P. aeruginosa to the injured mucosa. These results support the hypothesis that HLE enhances bacterial infection of the respiratory mucosa both by inducing mucus hypersecretion and by exposing receptors to the microbial adhesins. It is also suggested that P. aeruginosa internal lectins may be implicated in adherence to host tissues.     

Adherence of Pseudomonas aeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation.

Adherence of Pseudomonas aeruginosa to normal, injured, and regenerating tracheal mucosa was examined by scanning electron microscopy. Uninfected and influenza-infected murine tracheas were exposed to six strains of P. aeruginosa isolated from human sources and one strain of platn origin. All of the strains tested adhered to desquamating cells of the infected tracheas, but not to normal mucosa, the basal cell layer, or the regenerating epithelium. Adherence increased when the incubation time of the bacteria with the trachea was prolonged. Strains isolated from human tracheas appeared to adhere better than strains derived from the urinary tract. After endotracheal intubation of ferrets, P. aeruginosa adhered only to the injured cells and to areas of exposed basement membrane. We call this phenomenon "opportunistic adherence" and propose that alteration of the cell surfaces or cell injury facilitates the adherence of this bacterium and that adherence to injured cells may be a key to the pathogenesis of opportunistic Pseudomonas infections.     

Adherence of Pseudomonas aeruginosa to human tracheobronchial mucin.

A microtiter plate assay was developed to study the adherence of Pseudomonas aeruginosa to purified human tracheobronchial mucin. The wells of the plates were treated with silicon to minimize nonspecific binding of bacteria and then coated with a solution of purified human tracheobronchial mucin. Bacteria were added to the wells, and the plates were incubated at 37 degrees C. The wells were washed 15 times in an automated microtiter plate washer, and the bacteria bound to wells were desorbed with Triton X-100 and plated for enumeration. Scanning electron microscopy verified bacterial adherence to the mucin-coated wells and desorption of bacteria by Triton X-100. Adherence of P. aeruginosa increased as the concentration of mucin used to coat the wells was increased, with saturation occurring at 0.5 microgram of mucin protein per ml. Other parameters that affected adherence included the time of incubation and concentration of bacteria. Similar studies with strains of Escherichia coli and Klebsiella pneumoniae indicated a relative lack of binding of these bacteria to mucin. In comparing different strains of P. aeruginosa, there were small differences in binding between strains. It is inferred that there may be specific sites on human tracheobronchial mucin which facilitate this preferential binding.     

Adherence of Pseudomonas aeruginosa to hydrophilic contact lenses and other substrata.

Nonmucoid Pseudomonas aeruginosa isolated from corneal ulcers and contact lens cases and solutions were examined for their ability to adhere to polystyrene, glass, and hydrophilic contact lenses of varying water content and polymer composition. Adherence to the various substrates was strain specific. Adherence also was influenced by hydrophobicity and chemical composition of the substratum, as well as pH and electrolyte concentration. An extracellular polymeric adhesive appeared to be involved in the firm attachment of cells to soft contact lenses.     

Role of Pseudomonas aeruginosa mucoid exopolysaccharide in adherence to tracheal cells.

The mucoid exopolysaccharide of Pseudomonas aeruginosa is thought to confer antiphagocytic properties on mucoid strains of P. aeruginosa, thus allowing them to persist in the respiratory tract. It has also been speculated that the mucoid exopolysaccharide may be the adhesin for mucoid strains, but proof is lacking. We studied the role of the mucoid exopolysaccharide in adherence of mucoid strains in competitive experiments with purified mucoid exopolysaccharide, by measuring the binding of 14C-labeled mucoid exopolysaccharide to injured tracheas and testing whether an antibody against the major epitope of the mucoid exopolysaccharide inhibits adherence of these organisms. Our data show that the purified mucoid exopolysaccharide increased the adherence of four of the mucoid strains tested (by 50 to 300%; P less than 0.001) instead of inhibiting adherence. Radiolabeled mucoid exopolysaccharide bound much better to injured tracheal cells than to normal tracheal cells (P less than 0.001), and antibody against the antigen of strain 2192, the strain from which mucoid exopolysaccharide was prepared, inhibited the adherence of four of five mucoid strains but not the strain lacking this antigen. This antibody also failed to inhibit a nonmucoid revertant from strain 2192, which was previously shown to be inhibited by pili. These data strongly support the thesis that the mucoid exopolysaccharide is the adhesion for mucoid strains of P. aeruginosa.     

Binding of Pseudomonas aeruginosa to neutral glycosphingolipids of rabbit corneal epithelium.

35S-labeled Pseudomonas aeruginosa isolates were shown to bind to neutral glycosphingolipids (NGSLs) of rabbit corneal epithelia in culture by a thin-layer chromatogram overlay procedure. The lipids of the corneal epithelial cells grown in culture were extracted and partitioned into a chloroform-rich lower phase containing NGSLs and an aqueous upper phase containing gangliosides. By using a dot-blot assay, at least six times more radiolabeled P. aeruginosa isolates were shown to bind to the lipids in the lower phase compared with those in the upper phase. Thin-layer chromatography of the lower-phase lipids followed by staining with an orcinol spray revealed at least 10 NGSL components and several fast-migrating, nonglycosylated neutral lipid components (including cholesterol). 35S-labeled P. aeruginosa was shown to bind to NGSL components 1, 2, 5, 6, and 9. P. aeruginosa-reactive NGSL components 6 and 9 migrated with chromatographic mobilities similar to those of the standards ceramide trihexoside (CT) and ceramide monohexoside, respectively. Components 1 and 2 migrated slightly ahead of asialo GM1, and component 5 migrated faster than globoside but slower than CT. Among the various standards tested, P. aeruginosa bound to asialo GM1 and, to a lesser extent, to ceramide dihexoside and CT but not to GM1, GD1A, GM3, or ceramide monohexoside. It remains to be determined whether any of the five P. aeruginosa-reactive NGSL components of corneal epithelium identified in this study plays a role in the development of corneal infection. However, we have previously shown that component 9, one of the five P. aeruginosa-reactive NGSL components identified in this study, is present in significantly greater amounts in migrating epithelia than it is in nonmigrating epithelia (N. Panjwani, G. Michalopoulos, J. Song, G. Yogeeswaran, and J. Baum, Invest. Ophthalmol. Vis. Sci., in press). This may prove to be of biological significance because it is generally believed that traumatized (migrating) epithelia are more susceptible to infection than normal (nonmigrating) epithelia are.     

Characterization of Pseudomonas aeruginosa adherence to cultured hamster tracheal epithelial cells

This study reports an in vitro system that allows the convenient study of both microenvironmental and bacterial factors affecting adherence of Pseudomonas aeruginosa to tracheal epithelium. Primary cultures of mixed ciliated and nonciliated epithelial cells isolated from hamster tracheas were grown on collagen-coated multiwell plates containing 10(5) epithelial cells/well at confluence. When 10(7) 14C-labeled P. aeruginosa (nonmucoid, strain Y-4) suspensions were added to each well, 8.13 +/- 2.6% (mean +/- SD) of the initial inoculum bound to the cultured cells, an amount comparable to that measured using suspensions of human tracheal epithelial cells and the same bacteria. The bacteria adhered preferentially to the cultured cells rather than to an acellular collagen matrix. Five additional nonmucoid strains of P. aeruginosa also bound well to the cultured cells, while two mucoid strains were less adherent. Strains of two other gram-negative bacteria, Pseudomonas maltophilia and Klebsiella pneumoniae, did not bind significantly, emphasizing the bacterial species specificity of the adherence interaction being measured. The binding interaction with P. aeruginosa was both pH-sensitive and altered by the presence of the divalent cation calcium. Thus, the in vitro assay system described provides a consistent surface of tracheal epithelial cells that binds P. aeruginosa in a specific manner and can be used to examine the effects of bacterial variables and microenvironmental conditions that may be present in the human airway.     

Protection of human respiratory epithelium from Pseudomonas aeruginosa adherence by phosphatidylglycerol liposomes.

The ability of phosphatidylglycerol (DSPG) liposomes to prevent adherence of Pseudomonas aeruginosa to primary cultures of non-cystic fibrosis (CF) and delta F508 homozygous CF human respiratory epithelium was studied. The culture model was characterized by the simultaneous presence of various cellular phenotypes: well-differentiated respiratory epithelial cells, ciliated and nonciliated cells, and migrating cells which can be assimilated into a regenerating epithelium after injury. DSPG liposomes significantly decreased the binding of P. aeruginosa to migrating cells of both non-CF and delta F508 homozygous CF cultures compared with control cultures (35.5 x 10(-3) +/- 8.1 x 10(-3) bacteria per micron 2 versus 23.9 x 10(-3) +/- 2.5 x 10(-3); P < 0.01 for non-CF cultures and 88.8 x 10(-3) +/- 17.2 x 10(-3) bacteria per micron 2 versus 29.1 x 10(-3) +/- 0.6 x 10(-3), P < 0.001 for CF cultures). After treatment with DSPG liposomes, the size of P. aeruginosa aggregates bound to migrating cells in both non-CF cultures and delta F508 homozygous CF cultures was significantly decreased (14.4 +/- 3 bacteria per aggregate versus 11.9 +/- 2.5 bacteria per aggregate [P < 0.05] and 29.9 +/- 8.4 bacteria per aggregate versus 17.3 +/- 2.3 bacteria per aggregate [P < 0.01], respectively). Moreover, the control cultures were characterized by a differential P. aeruginosa adherence according to both the cellular phenotype and the mutation. The migrating cells bound more bacteria than the stationary cells of both non-CF and delta F508 homozygous CF cultures. The CF migrating cells bound significantly more bacteria than the non-CF migrating cells (88.8 x 10(-3) +/- 17.2 x 10(-3) bacteria per microns 2 versus 35.5 x 10(-3) +/- 8.1 x 10(-3) bacteria per micron 2, P < 0.001). These results suggest that DSPG liposomes are able to decrease P. aeruginosa adherence to CF and non-CF respiratory epithelium, particularly to migrating cells, which mimic a regenerating epithelium after injury. DSPG liposomes could also represent a hydrophobic barrier limiting the deleterious action of P. aeruginosa exoproducts.     

Adherence of streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa to fibronectin-coated and uncoated epithelial cells.

The relationship between the variability in the fibronectin (Fn) content on human buccal epithelial cells and the capacity of the cells to bind gram-positive (Streptococcus pyogenes) or gram-negative (Escherichia coli or Pseudomonas aeruginosa) bacteria was investigated. Adhesion experiments performed with mixtures of epithelial cells and mixed suspensions of either S. pyogenes and E. coli or S. pyogenes and P. aeruginosa exhibited three major populations of buccal cells: one of these was able to bind S. pyogenes (gram positive) but neither of the gram-negative bacteria; a second population was able to bind the gram-negative but not the gram-positive bacteria; and a third was able to bind various numbers of both types of organisms. Further adhesion experiments performed with a mixture of epithelial cells and a mixed suspension of S. pyrogens, E. coli, and fluoresceinconjugated methacrylate beads coated with immune immunoglobulin G directed against Fn revealed that the epithelial cells recognizing the gram-positive bacteria were rich in Fn, whereas those recognizing the gram-negative organisms were poor in Fn. Immunoelectron microscopy confirmed that cells of S. pyogenes bound to epithelial cells coated with Fn, whereas cells of E. coli bound to epithelial cells lacking Fn. These results suggest that Fn on the surfaces of epithelial cells may modulate the ecology of the human oropharyngeal cavity, especially with respect to the colonization of these surfaces by pathogenic gram-negative or gram-positive bacteria.     

Adherence to and Penetration of Human Intestinal Caco-2 Epithelial Cell Monolayers by Pseudomonas aeruginosa

Clinical isolates of Pseudomonas aeruginosa from blood adhered to and penetrated intestinal Caco-2 cell monolayers to a greater degree than did isolates from sputum, with a concomitant drastic decrease in transepithelial electrical resistance. PAO-PR1, an avirulent exotoxin A mutant of PAO1, did not cause a decrease in the resistance. The Caco-2 monolayer system may be useful for the evaluation of certain P. aeruginosa virulence factor activities.     

Monoclonal antibodies to Pseudomonas aeruginosa ferripyochelin-binding protein.

Hybridomas secreting specific monoclonal antibodies against the Pseudomonas aeruginosa ferripyochelin-binding protein (FBP) were isolated. These monoclonal antibodies reacted with FBP in immunoblots of outer membrane preparations from all serotypes of P. aeruginosa. Two of the monoclonal antibodies also reacted with FBP in strains of P. putida, P. fluorescens, and P. stutzeri. These antibodies did not react with outer membranes of P. cepacia, "P. multivorans," P. maltophilia, or other gram-negative organisms. The monoclonal antibodies were opsonophagocytic and blocked the binding of [59Fe]ferripyochelin to isolated outer membranes of strain PAO. By indirect immunofluorescence techniques, the monoclonal antibodies were used to demonstrate that FBP is present on the cell surface of P. aeruginosa cells grown in low-iron but not high-iron medium. These observations were confirmed by using 125I in surface-labeling techniques.     

Adherence of enteropathogenic Escherichia coli to intestinal epithelium in vivo.

Two porcine strains of enteropathogenic Escherichia coli, one possessing K88 antigen and one lacking K88, were orally inoculated into conventional neonatal piglets. Athough both strains caused severe diarrhea, only the K88-possessing strain was able to proliferate in the anterior small intestine. Both K88-possessing and K88-lacking strains were found in large numbers in the posterior small intestine and, using fluorescent antibodies and scanning and transmission electron microscopy, were found adhering to the epitheial surface in these regions. The presence of an unusual surface structure on the bacterial cell of the K88-lacking strain was described.     

Polysaccharide antigens of Pseudomonas aeruginosa.

The major polysaccharide antigens of P. aeruginosa are the cell-wall lipopolysaccharides many of which have an acidic polysaccharide chain (O-antigen) rich in unusual amino sugars. The D-rhamnose-rich polysaccharide antigen common to many serologically distinct strains is also associated with the lipopolysaccharide. The high-molecular-weight polysaccharides with O-specificity are present in extracellular slime produced by strains isolated from the environmental and from the immunocompromised hosts. The extracellular antigenic polysaccharide of another type (bacterial alginate) is expressed by mucoid strains isolated from patients with cystic fibrosis. Serotype-specific immune responses after infection are directed at the lipopolysaccharides and these heat-stable antigens serve as the basis for differentiation of P. aeruginosa strains. Both the cell-wall antigens including conjugates of the O-polysaccharides with different proteins and the extracellular antigens have been used to prepare specific antibodies tested for protection against infections due to P. aeruginosa.