Extended contact lens wear enhances Pseudomonas aeruginosa adherence to human corneal epithelium.

Extended wear of soft contact lenses is associated with an increased risk of Pseudomonas aeruginosa infection of the cornea. To assess the role of bacterial adherence in the pathogenesis of these infections, superficial corneal epithelial cells and leukocytes from ten patients who use extended-wear soft lenses and ten control eyes were compared for their propensity to attach P. aeruginosa in vitro. Cells were washed from the cornea by saline irrigation, incubated with a 10-ml solution containing 10(7) colony-forming units/ml of bacteria at 35 degrees C for 30 min, collected on a filter, and prepared using a modified acridine orange staining method. Fluorescence microscopy showed bacterial adherence to corneal epithelial cells, leukocytes, and ocular mucus. The mean number of bacteria adhering to epithelial cells was 2.6 for control eyes and 6.6 for the lens-wearing eyes (P = 0.002). The percentage of epithelial cells attaching greater than or equal to four bacteria was higher for lens-wearing eyes than control eyes (57.4% versus 26.0%, P = 0.0005). There was no significant difference between contact lens-wearing eyes and control eyes in the number of leukocytes collected or in the number of bacteria attached to these cells. These results show that P. aeruginosa adherence to epithelial cells is enhanced in those who use extended-wear soft contact lenses, and this may contribute to the increased incidence of P. aeruginosa keratitis for this population.

Lipopolysaccharide in adherence of Pseudomonas aeruginosa to the cornea and contact lenses.

PURPOSE: To determine the role of smooth or rough lipopolysaccharide on adherence of Pseudomonas aeruginosa bacteria to the rat cornea in vitro and on contact lenses of differing types. METHODS: Adherence of a smooth (AK957) and isogenic rough strain (AK1012) of P. aeruginosa bacteria to rat corneas that were either normal, traumatized using a 20-gauge needle or treated for 15 min with 0.1N sodium hydrochloric acid was assessed by homogenization and viable counting. Adherence of these organisms to 43 unworn contact lenses representing the four Food and Drug Administration lens groups was also assessed using viable counts. RESULTS: Attachment to contact lenses was greater for the smooth strain for all four lens types (P < 0.001). No variation in adherence to the different lens types was observed. Smooth bacteria also adhered to the cornea to a greater extent than the rough strain, regardless of trauma type (P < 0.001). Adherence to traumatized corneas was greater than to nontraumatized corneas for both strains of P. aeruginosa bacteria (P < 0.01). Measurement of surface hydrophobicity of the two bacterial strains revealed that the smooth strain was more hydrophobic than the rough strain (P < 0.001), perhaps accounting for the adherence pattern. CONCLUSIONS: These results indicate that bacterial surface characteristics may be important determinants of adherence and could explain the propensity of certain bacterial strains to infect the cornea.

Lipopolysaccharide outer core is a ligand for corneal cell binding and ingestion of Pseudomonas aeruginosa.

PURPOSE: Pseudomonas aeruginosa has been observed to be adherent to and inside epithelial cells during experimental corneal infection. The authors identified bacterial ligands involved in adherence and entry of P. aeruginosa into corneal epithelial cells. METHODS: In vitro gentamicin survival assays were used to determine the intracellular survival of a panel of P. aeruginosa mutants. Strains (10(6) to 10(7) colony-forming units) were added to primary cultures of rabbit corneal epithelial cells (approximately 10(5)/well) for 3 hours, nonadherent bacteria were washed away, and extracellular bacteria were killed with gentamicin. The antibiotic was then washed away, and epithelial cells were lysed with 0.5% Triton X-100 to release internalized bacteria. Bacterial association (sum of bound and internalized bacteria) was measured by the omission of gentamicin. Similar assays were carried out with whole mouse eyes in situ. RESULTS: A lipopolysaccharide core with an exposed terminal glucose residue was found to be necessary for maximal association and entry of P. aeruginosa into corneal cells. Bacterial pili and flagella were not involved. Mutants of P. aeruginosa strains that do not produce an LPS core with a terminal glucose residue had a significantly lower level of association with (approximately 50%) and ingestion by ( > 90%, P < 0.01) corneal cells than did strains with this characteristic. Complementation of the LPS productions defect by plasmid-borne DNA returned association and ingestion to near parental levels. Lipopolysaccharides and delipidated oligosaccharides with a terminal glucose residue in the core inhibited bacterial association and entry into corneal cells. Experiments using P. aeruginosa LPS mutants and corneal cells on whole mouse eyes confirmed the role of the LPS core in cellular entry. CONCLUSIONS: Corneal epithelial cells bind and internalized P. aeruginosa by the exposed LPS core.

Different strains of Pseudomonas aeruginosa isolated from ocular infections or inflammation display distinct corneal pathologies in an animal model.

PURPOSE: The present investigation sought to define the responses of mouse eyes to challenge with three different strains of P. aeruginosa isolated from human corneas or contact lenses: two different strains produced an ulcerative keratitis, and one strain produced contact lens-induced acute red eye (CLARE). METHODS: The corneas of BALB/c mice were inoculated with three different strains of P aeruginosa. The strains were allowed to interact with the corneas for up to 24 h. In addition, strain Paerl, isolated from CLARE, was subjected to in vitro assays to measure its ability to invade corneal epithelial cells, or to produce cytotoxicity in these cells. Both these assays used cultured rabbit corneal epithelial cells. RESULTS: Both MK isolates were able to infect the corneas of mice, but the CLARE isolate was non-infective. The predominant response to infection with the cytotoxic strain was severe corneal edema and infiltration of the corneal stroma with polymorphonuclear leukocytes (PMNs). The predominant response with the invasive MK isolate was corneal ulceration and infiltration with PMNs. The CLARE strain produced only low levels of PMN infiltration. In in vitro assays the CLARE strain was non-invasive and non-cytotoxic. CONCLUSIONS: This study has identified that P. aeruginosa produces at least three different types of corneal pathology and that not all strains are able to infect mouse corneas.

The role of pili in the attachment of Pseudomonas aeruginosa to unworn hydrogel contact lenses.

Contamination of contact lenses is thought to increase the risk of infectious keratitis, yet factors promoting attachment of bacteria to contact lenses are not fully understood. It has been suggested that strains of Pseudomonas aeruginosa attach to mucosal surfaces via pili which are appendages found on some strains. This study investigated the role of pili and the effect of incubation time on the attachment of P. aeruginosa to 20 unworn hydrogel lenses representative of each of the four FDA categories. Ten lenses were incubated for 15 minutes and another ten for 180 minutes. Lenses were incubated with either PAK + P. aeruginosa which possessed pili or its isogenic mutant pair, PAK-, which was genetically similar except for the absence of pili. Bacteria were quantified, following homogenization of the contact lens, by viable counts. Non-piliated bacteria were significantly more likely to adhere to the lenses (p < 0.001). A significant interaction between lens type and incubation time was observed (p < 0.05); thus it is difficult to generalize about either of these effects in isolation. These results show that surface characteristics may confer an attachment advantage to bacteria.

Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity.

We have previously shown that ExoU, a type III secreted cytotoxin of Pseudomonas aeruginosa, causes acute cytotoxicity towards corneal epithelial cells in vitro, and contributes to corneal disease pathology and ocular colonization in vivo. Subsequently, we reported that ExoU represses phagocyte infiltration of infected corneas in vivo. ExoU has patatin-like phospholipase activity that is required for cytotoxic activity in vitro (mammalian cell injury and death) and for disease in a murine model of pneumonia. We hypothesized that the phospholipase activity was required for ExoU-mediated corneal disease and ocular colonization. Using the murine scarification model, corneal disease pathology was examined after inoculation with approximately 10(6)cfu of a P. aeruginosa effector mutant (PA103DeltaexoUexoT::Tc) complemented with either exoU (pUCPexoU), phospholipase-inactive exoU (pUCPexoUD344A) or a plasmid control (pUCP18). Eyes were photographed and disease severity scored at 24 and 48h post-infection. Viable bacteria colonizing infected eyes were quantified at 6 and 48h. Complementation with exoU caused significantly more pathology (increased disease severity scores) and enabled bacteria to better colonize (by approximately 1000-fold) at 48h as compared to phospholipase-inactive exoU which did not differ from plasmid control. Surprisingly, exoU did not contribute to early (6h) colonization. In-vitro assays confirmed that the phospholipase domain of exoU was required for cytotoxicity towards human corneal epithelial cells. Taken together these data show that the phospholipase activity of the P. aeruginosa cytotoxin, ExoU, plays a role in the pathogenesis of corneal infection via mechanism(s) occurring after initial colonization of a susceptible cornea.

Exposure of human corneal epithelial cells to contact lenses in vitro suppresses the upregulation of human beta-defensin-2 in response to antigens of Pseudomonas aeruginosa.

Bacterial keratitis is a sight-threatening complication of contact lens wear, and Pseudomonas aeruginosa is a commonly isolated pathogen. The mechanisms by which lenses predispose the cornea to P. aeruginosa infection are unknown. Corneal epithelial cells express numerous innate defenses, some of which have bactericidal effects against P. aeruginosa. One of these is human beta-defensin-2 (hBD-2), which is upregulated in response to lipopolysaccharide or flagellin antigens. We hypothesized that prior exposure of corneal epithelia to a contact lens would interfere with upregulation of hBD-2 in response to P. aeruginosa. A novel in vitro model was used in which cultured human corneal epithelial cells were exposed to a hydrophilic contact lens for up to 3.5 days prior to challenge with a culture supernatant of P. aeruginosa antigens for 6h. Without prior lens exposure, the supernatant caused >2-fold upregulation of hBD-2 mRNA message and expression of hBD-2 peptide. Prior contact lens exposure blocked this upregulation without obvious effects on cell health. Western immunoblot and luciferase reporter studies showed that Pseudomonas-induced hBD-2 upregulation involved MyD88, c-Jun N-terminal kinase and both AP-1 and NF-kappaB transcription factors. Contact lenses did not affect surface expression of Toll-like receptor-2, -4 or -5, but did block antigen activation of AP-1, but not NF-kappaB, transcription factors. These data show that contact lenses can interfere with epithelial defense responses to bacterial antigens in vitro, and if translated in vivo, could help predispose the cornea to infection.

Resistance of Pseudomonas aeruginosa isolates to hydrogel contact lens disinfection correlates with cytotoxic activity.

One of the most common pathogens in infection of hydrogel contact lens wearers is Pseudomonas aeruginosa, which can gain access to the eye via contamination of the lens, lens case, and lens care solutions. Only one strain per species is used in current regulatory testing for the marketing of chemical contact lens disinfectants. The aim of this study was to determine whether P. aeruginosa strains vary in their susceptibility to hydrogel contact lens disinfectants. A method for rapidly screening bacterial susceptibility to contact lens disinfectants was developed, based on measurement of the MIC. The susceptibility of 35 P. aeruginosa isolates to two chemical disinfectants was found to vary among strains. MICs ranged from 6.25 to 100% for both disinfectants at 37 degrees C, and a number of strains were not inhibited by a 100% disinfectant concentration in the lens case environment at room temperature (22 degrees C). Resistance to disinfection appeared to be an inherent rather than acquired trait, since some resistant strains had been isolated prior to the introduction of the disinfectants and some susceptible P. aeruginosa strains could not be made more resistant by repeated disinfectant exposure. A number of P. aeruginosa strains which were comparatively more resistant to short-term disinfectant exposure also demonstrated the ability to grow to levels above the initial inoculum in one chemical disinfectant after long-term (24 to 48 h) disinfectant exposure. Resistance was correlated with acute cytotoxic activity toward corneal epithelial cells and with exsA, which encodes a protein that regulates cytotoxicity via a complex type III secretion system. These results suggest that chemical disinfection solutions may select for contamination with cytotoxic strains. Further investigation of the mechanisms and factors responsible for resistance may also lead to strategies for reducing adverse responses to contact lens wear.

Microbial flora in eyes of current and former contact lens wearers.

Microbial flora from the right eye conjunctival sac of 84 consecutively presenting contact lens patients were compared with cultures from both surfaces of their lens after aseptic removal and with the flora of their storage cases. Similar results were obtained from contact lens and conjunctival cultures of each individual; however, there was no correlation between storage case isolates and lens or conjunctival flora, suggesting that in uncomplicated lens wear, the eye is highly efficient in eradicating microorganisms introduced via handling. Conjunctival flora during daily contact lens wear was similar to the conjunctival flora of a matched control group of non-lens wearers. However, bacteria that are considered to be part of the normal ocular flora were isolated significantly more often from former contact lens wearers. The data also indicated that the use of nonperoxide chemical lens disinfection was associated with a higher proportion of positive cultures for pathogenic microorganisms than the use of other forms of disinfection, for both current and former contact lens wearers. The isolation of potential pathogens was particularly common among elderly subjects using thick contact lenses for extended wear. These changes to conjunctival flora may contribute to the increased risk of ocular infection associated with contact lens wear.

Conjunctival flora in extended wear of rigid gas permeable contact lenses.

A longitudinal study was performed to examine the effect of rigid gas permeable (RGP) contact lenses (Boston Equalens II and Quantum II) on the conjunctival flora of 45 young healthy subjects. Microbial flora were determined before delivery of lenses. Subjects wore lenses on an extended wear basis, removing them every 7 days for cleaning and disinfection. Cultures were repeated after 2 months of lens wear and the microbial flora were found to be significantly altered compared to the prelens wear results (0.05 greater than p greater than 0.02). Changes to conjunctival flora included an increase in the number of eyes from which potentially pathogenic microorganisms were isolated, an increase in the number of eyes that were culture-negative, and a decrease in the number of eyes harboring only normal conjunctival flora. The increase in potentially pathogenic flora was not specific for Gram-negative bacteria, which are most often associated with infectious keratitis during contact lens wear.

Pseudomonas aeruginosa invasion of and multiplication within corneal epithelial cells in vitro.

Pseudomonas aeruginosa is usually considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have previously demonstrated that P. aeruginosa is able to invade corneal cells during infectious keratitis in mice. In vitro, P. aeruginosa was found to enter the following cells: human corneal cells removed by irrigation; epithelial cells in the cornea of rats, mice, and rabbits; and primary corneal epithelial cells cultured from rat and rabbit eyes. The level of invasion was related to the level of adherent or associated bacteria. In general, invasion was more efficient with cultured epithelial cells than with cells tested in situ. Invasion did not occur when assays were performed at 4 degrees C. Cytochalasin D but not colchicine inhibited bacterial invasion, suggesting that bacterial entry was an endocytic process dependent on actin microfilaments but not microtubules. Bacteria that invaded cultured corneal epithelial cells were found to multiply within cells. The ability of P. aeruginosa to invade and multiply within corneal epithelial cells may contribute to the virulence of this organism during infectious keratitis, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.

Modulation of Pseudomonas aeruginosa adherence to the corneal surface by mucus.

To gain access to the corneal epithelium and cause infections keratitis, bacterial pathogens must first interact with ocular surface factors that could affect bacterial adherence. In this study, we demonstrated that the mucus layer, and, in particular, the mucin fraction of mucus, modulated adherence to intact corneal epithelium of Pseudomonas aeruginosa but not that of Staphylococcus aureus or Streptococcus pyogenes. Removal of endogenous mucus from rat or rabbit eyes increased the adherence of P. aeruginosa by 3- to 10-fold. Ocular mucus obtained from rat eyes, porcine stomach mucin, or bovine submaxillary gland mucin inhibited adherence of P. aeruginosa to uninjured corneal epithelium. The mucin fraction of ocular mucus, purified by ultracentrifugation, was found to contain the inhibitory activity, and inhibition was demonstrated at concentrations of mucin as low as 35 micrograms/ml. Ocular mucin was the only material tested that inhibited adherence of P. aeruginosa to an injured cornea. However, the binding of P. aeruginosa to immobilized substrates in vitro did not predict which fraction would possess antiadherence activity: bacteria bound well to whole ocular mucus, mucin, the nonmucin fraction of ocular mucus, and dilute human tears as well as to porcine stomach mucin and bovine submaxillary gland mucin. The effectiveness of the mucin fraction of ocular mucus at inhibiting the binding of P. aeruginosa to the cornea implies that this material is a barrier that protects the surface of the eye from P. aeruginosa adherence.

FlhA, a component of the flagellum assembly apparatus of Pseudomonas aeruginosa, plays a role in internalization by corneal epithelial cells.

Pseudomonas aeruginosa invades various epithelial cell types in vitro and in vivo. The P. aeruginosa genome possesses a gene (flhA) which encodes a protein that is believed to be part of the export apparatus for flagellum assembly and which is homologous to invA of Salmonella spp. Because invA is required for invasion of Salmonella spp., a role for flhA in P. aeruginosa invasion was explored using cultured rabbit corneal epithelial cells. An flhA mutant of P. aeruginosa strain PAO1 was constructed and was shown to be nonmotile. Complementation with flhA in trans restored motility. Corneal cells were infected for 3 h with the wild type (PAO1), the flhA mutant, the flhA mutant complemented with flhA in trans, an flhA mutant containing the plasmid vector control, or an fliC mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the flhA and the fliC mutants invaded at a lower level than the wild-type strain did, suggesting that both fliC and flhA played roles in invasion. However, loss of motility was not sufficient to explain the reduced invasion by flhA mutants, since centrifugation of bacteria onto cells did not restore invasion to wild-type levels. Unexpectedly, the flhA mutant adhered significantly better to corneal epithelial cells than wild-type bacteria or the fliC mutant did. The percentage of adherent bacteria that invaded was reduced by approximately 80% for the flhA mutant and approximately 50% for the fliC mutant, showing that only part of the role of flhA in invasion involves fliC. Invasion was restored by complementing the flhA mutant with flhA in trans but not by the plasmid vector control. Intracellular survival assays, in which intracellular bacteria were enumerated after continued incubation in the presence of antibiotics, showed that although flhA and fliC mutants had a reduced capacity for epithelial cell entry, they were not defective in their ability to survive within those cells after entry. These results suggest that the flagellum assembly type III secretion system plays a role in P. aeruginosa invasion of epithelial cells. Since the flhA mutants were not defective in their ability to adhere to corneal epithelial cells, to retain viability at the cell surface, or to survive inside epithelial cells after entry, the role of flhA in invasion of epithelial cells is likely to occur during the process of bacterial internalization.

Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity.

Pseudomonas aeruginosa clinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene, exsA, that controls the expression of several extracellular proteins, is inactivated. exsA mutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specific exsA-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the exsA-regulated extracellular factor exoU was neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by an exsA-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.

Ocular surface epithelia express mRNA for human beta defensin-2.

Human skin, lung and trachea produce human beta defensin-2 (hBD-2), an inducible, transcriptionally regulated antibiotic peptide with activity against gram negative bacteria, which may explain the unusual resistance of these tissues to infection. Since an intact corneal epithelium is also highly resistant to infection, we examined whether human ocular surface epithelia might produce hBD-2. Conjunctival epithelial cells were obtained from a human cadaver eye, while corneal epithelial cells were obtained from both a cadaver eye and the eye of a living human patient. Using reverse transcription-polymerase chain reaction and custom primers for hBD-2, a 257 bp sequence was amplified from both human corneal and conjunctival epithelial cell cDNA, and the amino acid sequence of this DNA band was computer-matched with the known gene sequence of hBD-2 available through GenBank (Z71389). To determine whether bacterial by-products upregulate hBD-2 mRNA expression, we stimulated confluent SV 40-immortalized human corneal epithelial cells with bacterial culture supernatant prepared from either wild-type P. aeruginosa strain PAO1 or two different lipopolysaccharide (LPS) mutants of PAO1. Both of these mutants, strains AK1012 and PAO1 algC::tet, are deficient in phosphomannomutase activity which is required for the synthesis of both a complete polysaccharide core and the O side chain structures of the LPS molecule. Neither of these mutations affects the lipid A portion of LPS. Cells treated with P. aeruginosa wild-type PAO1 bacterial culture supernatant demonstrated strong upregulation of hBD-2 mRNA expression, whereas cells stimulated with culture supernatant produced by either of the LPS mutants showed little or no change in hBD-2 gene expression. LPS extracted from the bacterial culture supernatant was used to demonstrate that upregulation of hBD-2 is caused by LPS. Genistein blocked this upregulation suggesting that protein tyrosine kinase activity is involved. Thus, both human corneal and conjunctival epithelium express mRNA for hBD-2, and this expression is upregulated by bacterial LPS. Data obtained from LPS mutants suggest that lipid A, which is responsible for initiating a number of the pathophysiological manifestations induced by endotoxin in mammals, is not required. Stimulation of endogenous hBD-2 production via the active portion of LPS might have therapeutic potential.

Pseudomonas aeruginosa invades corneal epithelial cells during experimental infection.

Pseudomonas aeruginosa is considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have demonstrated that some strains of P. aeruginosa are able to invade corneal cells during experimental bacterial keratitis in mice. Although intracellular bacteria were detectable 15 min after inoculation, the number of intracellular bacteria increased in a time-dependent manner over a 24-h period. Levels of invasion were similar when bacteria were grown as a biofilm on solid medium and when they were grown in suspension. Intracellular bacteria survived in vitro for at least 24 h, although only minimal bacterial multiplication within cells was observed. P. aeruginosa PAK and Escherichia coli HB101 did not cause disease in this model and were not isolated from corneas after 24 h even when an inoculum of 10(8) CFU was applied. Transmission electron microscopy of corneal epithelium from eyes infected for 8 h revealed that intracellular bacteria were present within membrane-bound vacuoles, which suggests that bacterial entry was an endocytic process. At 24 h, the observation of many bacteria free in the cytoplasm indicated that P. aeruginosa was able to escape the endocytic vacuole. The ability of some P. aeruginosa strains to invade corneal epithelial cells may contribute to the pathogenesis or to the progression of disease, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.

Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity.

Intact tissues are relatively resistant to Pseudomonas aeruginosa-induced disease, and injury predisposes tissue to infection. Intact epithelia contain polarized cells that have distinct apical and basolateral membranes with unique lipids and proteins. In this study, the role of cell polarity in epithelial cell susceptibility to P. aeruginosa virulence mechanisms was tested. Madin-Darby canine kidney (MDCK) cells, human corneal epithelial cells, and primary cultures of two different types of airway epithelial cells were grown on Transwell filters or in plastic tissue culture wells. P. aeruginosa invasion of cells was quantified by gentamicin survival assays with two isolates that invade epithelial cells (6294 and PAO1). Cytotoxic activity was assessed by trypan blue exclusion assays with two cytotoxic strains (6206 and PA103). Basolateral surfaces of cells were exposed by one of two methods: EGTA pretreatment of epithelial cells or growth of cells in low-calcium medium. Both methods of exposing basolateral membranes increased epithelial cell susceptibility to P. aeruginosa invasion and cytotoxicity. Migrating cells were also found to be more susceptible to P. aeruginosa invasion than confluent monolayers that had established membrane polarity. Monolayers of MDCK cells that had been selected for resistance to killing by concanavalin A were resistant to both cytotoxicity and invasion by P. aeruginosa because they were more efficiently polarized for their susceptibility to P. aeruginosa virulence factors than regular MDCK cells and not because they were defective in glycosylation. These results suggest that there are factors on the basolateral surfaces of epithelial cells that promote interaction with P. aeruginosa or that there are inhibitory factors on the apical cell surface. Thus, cell polarity of intact epithelia is likely to contribute to defense against P. aeruginosa infection.