Pseudomonas aeruginosa acquires biofilm-like properties within airway epithelial cells - PubMed (original) (raw)

Pseudomonas aeruginosa acquires biofilm-like properties within airway epithelial cells

Raquel Garcia-Medina et al. Infect Immun. 2005 Dec.

Abstract

Pseudomonas aeruginosa can notably cause both acute and chronic infection. While several virulence factors are implicated in the acute phase of infection, advances in understanding bacterial pathogenesis suggest that chronic P. aeruginosa infection is related to biofilm formation. However, the relationship between these two forms of disease is not well understood. Accumulating evidence indicates that, during acute infection, P. aeruginosa enters epithelial cells, a process viewed as either a host-mediated defense response or a pathogenic mechanism to avoid host-mediated killing. We investigated the possibility that epithelial cell entry during early P. aeruginosa-epithelial cell contact favors bacterial survival and is linked to chronic infection. Using electron microscopy and confocal microscopy to analyze primary culture airway epithelial cells infected with P. aeruginosa, we found that epithelial cells developed pod-like clusters of intracellular bacteria with regional variation in protein expression. Extracellular gentamicin added to the medium after acute infection led to the persistence of intracellular P. aeruginosa for at least 3 days. Importantly, compared to bacterial culture under planktonic conditions, the intracellular bacteria were insensitive to growth inhibition or killing by antibiotics that were capable of intraepithelial cell penetration. These findings suggest that P. aeruginosa can use airway epithelial cells as a sanctuary for persistence and develop a reversible antibiotic resistance phenotype characteristic of biofilm physiology that can contribute to development of chronic infection.

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Figures

FIG. 1.

FIG. 1.

Effect of P. aeruginosa on integrity of primary cultured mouse tracheal epithelial cell layers. A. Transepithelial resistance (Rt) of MTEC incubated with P. aeruginosa. PAO1 (106 to 107 CFU) was applied to the apical surface of MTEC (2 × 105 cells) for up to 10 h. Values are the means ± standard deviations (n = 3 to 9 samples/time). The dashed line represents the lower limit of Rt corresponding to maintenance of tight junctions (46). Mechanical injury (mech inj) induced by scraping an uninfected cell layer is shown as a reference. A significant difference at 10 h (P < 0.05) compared to other times is indicated (*). B. Scanning EM of cells obtained 10 h after inoculation with control medium or P. aeruginosa (Pa) as in panel A. The boxed region (center panel) showing bacteria either entering or emerging from the apical membrane of cells is enlarged in the right panel. Bars = 5 μm (left and center panels); 1 μm (right panel).

FIG. 2.

FIG. 2.

Time-dependent changes in intracellular abundance of P. aeruginosa. A. Representative laser scanning confocal photomicrograph (xy) and _z_-axis reconstruction of MTEC stained with rhodamine-labeled phalloidin (red) to identify filamentous actin, obtained 4 h after incubation with PAO1-GFP and then gentamicin to kill extracellular bacteria. Bar = 10 μm. B. Gentamicin survival assay of MTEC incubated for 4 h with indicated strains of P. aeruginosa (CS1, clinical strain). PAO1-GFP treated with paraformaldehyde prior to assay is indicated (“fixed”). MTEC were incubated with bacteria and then treated with gentamicin, lysed, and cultured on solid medium to determine surviving intracellular bacteria (CFU/well). Shown is the mean ± standard deviation of triplicate samples from a representative experiment. C. Gentamicin survival assay of P. aeruginosa PAO1-GFP strain following 0.5 to 8 h of incubation with MTEC performed as for panel B. Shown are means ± standard deviations of replicate samples from at least three experiments. A significant difference (P < 0.05) from 0.5 h and 2 h is at 4 h (*) and from 0.5 and 4 h is at 8 h (**).

FIG. 3.

FIG. 3.

P. aeruginosa forms intracellular pods. Scanning electron photomicrographs of MTEC obtained 8 and 10 h after incubation with strain PAO1. Cells were incubated as in Fig. 1. Images are from two independent preparations (top and bottom). Bars = 5 μm.

FIG. 4.

FIG. 4.

Persistence and heterogeneity of gene expression of intracellular P. aeruginosa. A. Transepithelial cell resistance (Rt) of MTEC incubated with PAO1-GFP for 4 h, treated with fresh medium containing gentamicin daily for up to 72 h. The dashed line represents the level of Rt associated with maintenance of epithelial cell tight junctions (46). Values are the means ± standard deviations of replicate samples from three independent preparations. B. Gentamicin survival assay of recovered intracellular bacteria (CFU/well) from MTEC incubated with PAO1-GFP as in panel A. Values are the means ± standard deviations of triplicate samples from three independent experiments. C. Laser scanning confocal photomicrograph (x, y) and _z_-axis reconstruction of MTEC stained with rhodamine-labeled phalloidin (red), obtained after incubation with PAO1-GFP for 4 h followed by gentamicin for 24 h. Bar = 10 μm. D. Immunofluorescence photomicrograph of P. aeruginosa strain CS1 cultured using planktonic conditions, applied to a glass slide, and then immunostained for FlgA (red) and/or OprF (green). Accompanying DAPI (4′,6′-diamidino-2-phenylindole)-stained images are below (background was inverted to white for contrast). Bar = 1 μm. E. Confocal photomicrograph (x, y) and _z_-axis reconstruction of MTEC obtained after incubation with CS1 immunostained for FlgA (red) and OprF (green). White lines indicate Transwell membrane and apical cell border. Bar = 10 μm.

FIG. 5.

FIG. 5.

Efficacy of intracellular P. aeruginosa killing by antibiotics. A. Survival of P. aeruginosa PAO1-GFP (104 CFU) cultured using planktonic conditions in the presence of control medium or ceftazidime for an additional 2 h. B. Recovery of intracellular PAO1-GFP from MTEC, 2 and 24 h after incubation with ceftazidime. Intracellular bacteria were established by incubation of MTEC with P. aeruginosa for 4 h, washed, treated with gentamicin for 30 min as in Fig. 2, and then incubated with control medium or ceftazidime for 2 h or 24 h, followed by cell lysis and culture on solid medium. C. Survival of planktonic PAO1-GFP treated with ciprofloxacin as in panel A. D. Recovery of intracellular PAO1-GFP from MTEC infected as in panel B, after incubation with ciprofloxacin. E. Survival of strain CS1 cultured planktonically and treated with ciprofloxacin as in panel C. F. Recovery of intracellular CS1 from MTEC infected as in panel C and treated with ciprofloxacin. Values represent the means ± standard deviations of three to six samples from a representative experiment of at least three different preparations. A significant difference (P < 0.05) from untreated bacteria is indicated (*), as is a significant difference at 2 h compared to 24 h (**).

FIG. 6.

FIG. 6.

Fate of intraepithelial P. aeruginosa and model. A. Scanning electron photomicrograph of sloughed pod of airway epithelial cell containing bacteria on the MTEC layer obtained 10 h after incubation with PAO1. Arrows indicate other bacteria adherent to cilia. Bar = 5 μm. B. Confocal scanning photomicrograph (_z_-axis reconstruction) of MTEC incubated with P. aeruginosa clinical sample (CS1) for 4 h and then with gentamicin for 24 h, demonstrating sloughed cells filled with bacteria (arrows) on top of the MTEC layer. Cells were immunostained with antibody to OprF detected with fluorescein isothiocyanate (green) to identify P. aeruginosa (Pa) and rhodamine-phalloidin (red). C. Model of bacterial internalization and release into the airway lumen. Following entry into airway epithelial cells, P. aeruginosa bacteria proliferate to form pods. Bacteria within cells form biofilm-like communities. Subsequent cell death results in ejection of epithelia filled with bacteria into the airway, where bacteria may contribute to airway biofilm formation and persistent P. aeruginosa infection in the lung.

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