Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa.

We have reported that some strains of Pseudomonas aeruginosa can enter corneal epithelial cells during experimental murine eye infection and when the cells are cultured in vitro. Following invasion, both the host cell and the intracellular bacteria can remain viable for up to 24 h. Others have reported that toxin-mediated damage of epithelial cells contributes to the pathogenesis of P. aeruginosa keratitis. To clarify the relationship between cell invasion and cytotoxicity, fourteen P. aeruginosa isolates were compared for their capacity to enter epithelial cells and for their ability to induce cytotoxicity. Bacterial invasion was quantified by gentamicin survival assays both in vivo and in vitro. Cytotoxicity was examined qualitatively by trypan blue exclusion assays and quantitatively by chromium release assays in vitro. A significant inverse correlation was found between the ability to induce cytotoxicity and epithelial cell invasion as measured by gentamicin survival assays. Both cytotoxic and noncytotoxic strains were identified among corneal and noncorneal isolates; all isolates that were not cytotoxic were capable of epithelial cell invasion. Efficient host cell invasion could not be demonstrated for cytotoxic strains; however, the gentamicin survival assay relies upon host cells retaining viability in order to yield useful results, and this may limit the effectiveness of this assay for testing epithelial cell invasion by cytotoxic strains. Since all of the corneal isolates that were tested were virulent in vivo, the results show that there are at least two different types of P. aeruginosa-induced disease, one caused by strains that are cytotoxic and the other involving bacteria that can enter epithelial cells and survive intracellularly without killing the host cell.     

CFTR-dependent susceptibility of the cystic fibrosis-host to Pseudomonas aeruginosa

Cystic fibrosis is the most common autosomal recessive disorder in western countries. The disease is characterized by recurrent and chronic infections of the lung in particular with Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia, and Haemophilus influenzae. Albeit intensive research in the last years, the molecular mechanisms causing the high susceptibility of cystic fibrosis patients to bacterial infections are still unknown. Animal models provided important novel information on the pathophysiology of cystic fibrosis and mimicked many of the pathological findings in humans, for instance chronic inflammation and increased infection susceptibility. These animal models were recently employed to identify several proteins and lipids that are critically involved in the pathophysiology of cystic fibrosis. Thus, several studies identified death receptors, caveolin proteins, membrane rafts, and alterations of the ceramide metabolism with an accumulation of ceramide in cystic fibrosis lungs to be critically involved in the infection susceptibility, the chronic inflammation, and the reduced mucociliary clearance in cystic fibrosis.     

Age-related susceptibility to Pseudomonas aeruginosa ocular infections in mice

The susceptibility of newborn and infant mice to eye infection by Pseudomonas aeruginosa was studied in 5-, 10-, 15- to 16-, and 21-day-old mice. In the first of three age-related susceptibility experiments, inoculation of P. aeruginosa under the unopened eyelids of infant (5- and 10-day-old) mice in the absence of prior corneal wounding resulted in acute infection and rapid death of many of the animals. However, endophthalmitis was observed in about 30% of bacteremic animals that survived to age 14 to 15 days. In the second experiment, 15- to 16-day-old mice whose eyes were open received P. aeruginosa topically onto either wounded or unwounded corneas. At least 50% of the mice that received both corneal wounding and the bacteria exhibited keratitis, endophthalmitis, and subsequent phthisis bulbi. None of the infected mice died of bacteremia. In addition, mice infected in the absence of corneal wounding did not exhibit any eye damage. In the third experiment, the wounded-cornea responses of 21-day-old mice to P. aeruginosa were more variable. Thirty seven percent of the mice exhibited an intermediate response of decreased eye size and cataracts which was not observed in 15- to 16-day-old mice, 32% recovered spontaneously, and 29% exhibited complete shrinkage of the infected eyes. The variability of the latter responses may reflect a transitional maturation period of natural immunity to the organism in some of the animals, since all 4- to 6-week-old adult mice respond routinely to ocular wounding and similar infections with the organism by undergoing a spontaneous resolvable keratitis (3 to 4 weeks).     

Differential Sensitivity of Human Epithelial Cells to Pseudomonas aeruginosa Exoenzyme S

Exoenzyme S (ExoS) is an ADP-ribosyltransferase produced and directly translocated into eukaryotic cells by the opportunistic pathogen Pseudomonas aeruginosa. Model systems that allow bacterial translocation of ExoS have found ExoS to have multiple effects on eukaryotic cell function, affecting DNA synthesis, actin cytoskeletal structure, and cell matrix adherence. To understand mechanisms underlying differences observed in cell sensitivities to ExoS, we examined the effects of bacterially translocated ExoS on multiple human epithelial cell lines. Of the cell lines examined, confluent normal kidney (NK) epithelial cells were most resistant to ExoS, while tumor-derived cell lines were highly sensitive to ExoS. Analysis of the mechanisms of resistance indicated that cell association as well as an intrinsic resistance to morphological alterations were associated with increased resistance to ExoS. Conversely, increased sensitivity to ExoS appeared to be linked to epithelial cell growth, with tumor cells capable of undergoing non-contact-inhibited, anchorage-independent growth all being sensitive to ExoS, and NK cells becoming sensitive to ExoS when subconfluent and growing. Consistent with the possibility that growth-related, actin-based structures are involved in sensitivity to ExoS, scanning electron microscopy revealed cellular extensions from sensitive, growing cells to bacteria, which were not readily evident in resistant cells. In all studies, the severity of effects of ExoS on cell function directly correlated with the degree of Ras modification, indicating that sensitivity to ExoS in some manner related to the efficiency of ExoS translocation and its ADP-ribosylation of Ras. Our results suggest that factors expressed by growing epithelial cells are required for the bacterial contact-dependent translocation of ExoS; as normal epithelial cells differentiate into polarized confluent monolayers, expression of these factors is altered, and cells in turn become more resistant to the effects of ExoS.     

Epithelial cell invasion and survival of Bordetella bronchiseptica.

Wild-type Bordetella bronchiseptica and a bvg mutant strain were used for invasion and survival experiments in human Caco-2 and A549 epithelial cells. Both bacterial strains were able to enter and persist within the host cells for at least a week. A significant proportion of the bacteria from both B. bronchiseptica strains but not from Bordetella pertussis were found free in the cytoplasm, suggesting different invasion and survival strategies of the two species in epithelial cells.     

Age-related susceptibility of mice to ocular challenge with Pseudomonas aeruginosa exotoxin A.

We studied the responses of mice to ocular challenge with purified exotoxin A from Pseudomonas aeruginosa in 5-, 10-, 16-, 21-, and 30-day-old animals. In the absence of trauma, injection of 3 to 6 microgram of exotoxin per mouse beneath the fused eyelids of 5-day-old Swiss-Webster mice resulted in death of all animals within 24 h. Administration of 1.5, 0.75, and 0.375 microgram of exotoxin per mouse resulted in 24-h mortality rates of 50, 22, and 20%, respectively. Additional deaths were recorded throughout the next 4 days. Similar lethality results were obtained with 10-day-old animals that received equivalent amounts of exotoxin beneath the fused eyelid and in these experiments, the 72-h 50% lethal dose was 0.49 microgram of exotoxin per mouse. Mice that were 16 and 21 days old, whose eyelids were open, each received from 0.375 to 15 microgram of exotoxin topically applied to the surface of a wounded cornea. Cataracts were observed within 1 week in both groups, and none of the animals that received the higher concentrations of toxin died. Young adult (30-day-old) animals also received from 1.8 to 15 microgram of exotoxin topically on the surfaces of wounded corneas. Corneal swelling and slight opacity were observed at 24 h and within 1 month; 80% of these mice had cataracts of the ocular lens.     

Serotypes of Pseudomonas aeruginosa in clinical specimens in relation to antibiotic susceptibility.

Ninety-eight hospital strains of Pseudomonas aeruginosa isolated from six different hospitals in Athens were serotyped by a slide agglutination test with unabsorbed commercial antisera. Serotypes O6, O11, O12, and "pool E" strains (strains that agglutinated only in pool E, which contained antisera against O2, O5, O15, and O16 antigens, but did not agglutinate in the individual antisera) predominated, accounting for more than 62% of all isolates tested. In respect to serotypes, (i) there was no apparent correlation with hospital of origin, (ii) most strains of serotypes O6 and O11 were sensitive to gentamicin and carbenicillin (iii) most strains of pool E were from urine and were resistant to these drugs, (iv) all 9 strains of serotype O12 tested were resistant to carbenicillin and all 5 strains tested hydrolyzed this drug, and (v) 24 of 25 strains of pool E were resistant to carbenicillin but only 2 of 17 strains hydrolyzed it.     

Pseudomonas aeruginosa Induces Type-III-Secretion-Mediated Apoptosis of Macrophages and Epithelial Cells

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that is cytotoxic towards a variety of eukaryotic cells. To investigate the effect of this bacterium on macrophages, we infected J774A.1 cells and primary bone-marrow-derived murine macrophages with the P. aeruginosa strain PA103 in vitro. PA103 caused type-III-secretion-dependent killing of macrophages within 2 h of infection. Only a portion of the killing required the putative cytotoxin ExoU. By three criteria, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assays, cytoplasmic nucleosome assays, and Hoechst staining, the ExoU-independent but type-III-secretion-dependent killing exhibited features of apoptosis. Extracellular bacteria were capable of inducing apoptosis, and some laboratory and clinical isolates of P. aeruginosa induced significantly higher levels of this form of cell death than others. Interestingly, HeLa cells but not Madin-Darby canine kidney cells were susceptible to type-III-secretion-mediated apoptosis under the conditions of these assays. These findings are consistent with a model in which the P. aeruginosa type III secretion system transports at least two factors that kill macrophages: ExoU, which causes necrosis, and a second, as yet unidentified, effector protein, which induces apoptosis. Such killing may contribute to the ability of this organism to persist and disseminate within infected patients.     

Epithelial cell invasion by bovine septicemic Escherichia coli.

Little is known regarding the pathogenesis of Escherichia coli-induced septicemic colibacillosis of calves. To understand the mechanism by which these strains penetrate the intestinal epithelium and gain access to the bloodstream, we examined the potential of bovine septicemic E. coli to invade cultured epithelial cells. By using a gentamicin survival assay, we demonstrated bacterial invasion of Madin-Darby canine kidney (MDCK) cells. Transcytosis of polarized MDCK cell monolayers was also observed, but only when bacteria were added to the basolateral surface. Electron microscopy confirmed the presence of intracellular organisms which appeared to be within membrane-bound vacuoles. The bovine septicemic isolate used in this study expressed the fimbrial adhesion CS31A. To examine the role of CS31A-mediated adherence in invasion and transcytosis of MDCK cell monolayers, a CS31A-deficient mutant was constructed by suicide vector-mediated insertional mutagenesis. Although nonadherent, the mutant showed a level of invasion similar to that of the wild-type parent. E. coli DH5 alpha carrying the cloned CS31A determinant was noninvasive. These findings suggest that expression of CS31A is neither required nor sufficient to mediate invasion.     

Pseudomonas aeruginosa ATCC 49189, a new quality control strain for testing P. aeruginosa susceptibility to the aminoglycosides.

Quality control for in vitro antimicrobial susceptibility testing of Pseudomonas aeruginosa versus aminoglycosides is improved by P. aeruginosa ATCC 49189, which was developed in the Clinical Microbiology Laboratory at St. Paul-Ramsey Medical Center. This strain, used by us for daily testing for the past 6 years, requires MICs that approximate therapeutic concentrations, are midrange in most dilution schemes, and are stable and reproducible.     

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.     

Macrophages and Epithelial Cells Respond Differently to the Pseudomonas aeruginosa Type III Secretion System

The multiple effects of Pseudomonas aeruginosa type III secretion have largely been attributed to variations in cytotoxin expression between strains. Here we show that the target cell type is also important. While lung epithelial cells showed significant changes in morphology but not viability when infected with P. aeruginosa, macrophages were efficiently killed by P. aeruginosa. Both responses were dependent on the type III secretion system.     

Exercise affects platelet-promoted tumor cell adhesion and invasion to endothelium

This investigation explored how exercise intensity impacts platelet-mediated interactions of nasopharyngeal carcinoma cells (NPCs) and vascular endothelial cells (ECs) under shear flow in 33 males. Our results showed that (a) platelet–NPC aggregates (PNA) were associated with higher shear-induced P-selectin expression and glycoprotein α_IIβ₃ activation than platelet–platelet aggregates (PPA); (b) strenuous exercise (SE, up to ), but not moderate exercise (ME, 60% for 30 min), increased both PPA and PNA in mimicked venous and arterial circuits and enhanced PNA in mimicked flow of stenotic vessels; (c) the percentages of PNA that remained bound to ECs in mimicked flow of post-capillary venules increased, while platelet-induced CD44 cleavage on NPC and trans-endothelial migration of NPC were enhanced following SE, but were unchanged in response to ME. We conclude that SE, but not ME, enhances the capacity of PNA to adhere to ECs, withstand flowing blood, and facilitate the invasion of NPCs toward ECs.     

Pseudomonas aeruginosa in French hospitals between 2001 and 2011: back to susceptibility

The European Antimicrobial Resistance Surveillance Network (EARS-Net) reported an increase in the rates of resistance of Pseudomonas aeruginosa to antimicrobials between 2008 and 2011 in France. This alarming report was based on data collected during the harmonisation of breakpoints by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) committee. However, these data were not supported by the findings of other national surveillance networks. In this study, we assessed the trends in P. aeruginosa antimicrobial drug resistance at six French hospitals over a longer period of time (2001–2011) and with a constant definition of resistance. After the exclusion of incomplete data and duplicates, we sorted 34,065 isolates into the antimicrobial resistance patterns defined by the European Centre for Disease Prevention and Control (ECDC). The proportion of isolates with a resistant pattern (non-susceptible to one or two antimicrobial categories), a multidrug-resistant pattern (non-susceptible to three or four antimicrobial categories) or an extensively drug-resistant pattern (non-susceptible to five or six antimicrobial categories) decreased significantly over time. Logically, the proportion of isolates with a wild-type resistance pattern has increased significantly over the same period. No significant changes in the rates of resistance to cephalosporins and penicillins were observed, whereas carbapenem resistance rates increased. By contrast, the proportion of isolates resistant to fluoroquinolones, aminoglycosides and monobactams decreased significantly over time. In conclusion, our data do not confirm the EARS-net data, suggesting instead that antimicrobial drug resistance in P. aeruginosa might not have increased in French hospitals over the last decade.     

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.     

Accuracy and cost of antibiotic susceptibility testing of mixed morphotypes of Pseudomonas aeruginosa.

Chronic Pseudomonas aeruginosa colonization of the lower respiratory tract of patients with cystic fibrosis frequently results in pulmonary exacerbations requiring treatment with antimicrobial agents. Multiple morphotypes with different antibiotic susceptibilities are often isolated from a single sputum sample. Determination of MICs of antibiotics for each sputum morphotype is used to guide therapy but is time-consuming and expensive. We explored an alternative assay for determining MICs for all P. aeruginosa morphotypes cultured from a homogenized sputum sample. We sought correlations of those MICs with the MIC for the most resistant morphotypes tested separately. The MICs determined for a mixture of morphotypes correctly predicted the highest MICs (+/- one dilution) determined for isolated morphotypes 73.5% of the time. The MIC for the mixed morphotypes correctly predicted susceptibility in 90.4% of samples. In contrast, determination of the MIC for the mixture of morphotypes correctly predicted resistance in only 57.0%. For sputa containing susceptible isolates, testing the mixed culture may provide adequate susceptibility data with significant laboratory time and cost savings. However, for sputa with resistant strains, the traditional method of testing isolated morphotypes should still be used.     

Pseudomonas aeruginosa internalization by human epithelial respiratory cells depends on cell differentiation, polarity, and junctional complex integrity

Internalization of Pseudomonas aeruginosa by epithelial respiratory cell lines has been suggested to be dependent on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Because we have observed intracellular (IC) P. aeruginosa only in cells that do not express apical CFTR, we addressed the question of whether bacterial internalization by epithelial cells depends on the degree of cell differentiation and polarity. Internalization of piliated P. aeruginosa PAO-1 and PAK by human epithelial respiratory cells in primary culture and by the 16 human bronchial epithelial 14o- cell line cultured either on thick collagen gels or on thin collagen films was evaluated by the gentamicin exclusion assay. Cells cultured on thick gels were differentiated, polarized, and tight. They exhibited CFTR at their apical membranes, expressed beta1 integrins at their basal membranes, excluded lanthanum nitrate, and uniformly expressed ZO-1 protein. In contrast, in cells cultured on thin films, CFTR was present mainly in the cytoplasm, whereas beta1 integrins were detected at apical membranes. Most cells cultured on thin films did not exclude lanthanum nitrate and rarely expressed ZO-1 protein. Cells grown on thick and thin collagen substrates differed markedly in bacterial internalization: no IC bacteria could be detected in cells cultured on gels, whereas high IC bacterial concentrations were isolated from cells cultured on thin films. Treatment of cells cultured on thin films with ethylenediaminetetraacetic acid, to disrupt intercellular junctions further, significantly enhanced P. aeruginosa internalization. Our results suggest that P. aeruginosa internalization by epithelial respiratory cells does not depend on CFTR protein expression at the epithelial cell surface but rather on cell polarity and junctional complex integrity.     

Antibodies to cell envelope proteins of Pseudomonas aeruginosa in cystic fibrosis patients.

Many vaccines containing somatic and secreted antigens of Pseudomonas aeruginosa have been reported. The vaccines containing lipopolysaccharide have been found to provide type-specific protection, but the endotoxin content of these vaccines does not make it feasible to use them in patients who are already debilitated. Outer membrane proteins could be effective as vaccines, as they can be purified free of lipopolysaccharide, and also because they are common to all serotypes of P. aeruginosa. To be effective as a vaccine, such proteins must be immunogenic and accessible from the outside of the intact bacterial cell. In this study, we showed that systemic antibodies were produced frequently to two cell envelope proteins with masses of 58,500 and 37,500 daltons and occasionally to 34,000-dalton protein of P. aeruginosa in cystic fibrosis patients with chronic lung infections. In rabbits immunized with whole, fixed cells of P. aeruginosa, antibodies were also produced against the 58,500-dalton proteins. Thus, the 58,500-dalton cell envelope protein of P. aeruginosa was the only immunogenic protein that was accessible to the immune system when whole, fixed cells were used for immunization. These serum antibodies did not protect the cystic fibrosis patients against further lung infection with P. aeruginosa.