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.     

Chinese hamster ovarian cell glycoproteins that mediate type 1 piliated gram-negative bacterial adherence.

We used Chinese hamster ovary (CHO) cell lines to define the structures of glycoproteins responsible for Type 1 piliated bacterial adherence. CSH 50 Escherichia coli, a Type 1 piliated bacteria, adhered significantly better than an isogenic nonpiliated E. coli to all CHO lines tested. CSH 50 E. coli adhered least well to CHO cells expressing intact complex type oligosaccharides on cell surface glycoproteins. CSH 50 adherence increased when shorter oligosaccharides were present and was maximal when mannose groups were present in terminal, nonreducing positions. Five high mannose type glycoproteins, with molecular weights of 79, 75, 55, 50, and 37 kD, were identified as high affinity ligands for Type 1 piliated bacteria. Our results suggest that alterations in cell surface carbohydrates may increase adherence of Type 1 piliated gram-negative bacteria to cells.     

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.     

Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes.

Strains of enteroaggregative Escherichia coli (EAggEC) have been implicated in several studies as important agents of persistent diarrhea among infants in the developing world. We have previously shown that the aggregative adherence (AA) property of EAggEC is associated with the presence of a 60-MDa plasmid which confers AA when introduced into E. coli HB101. Here, we report the cloning of the AA determinant from EAggEC strain 17-2 into the 21.5-kb cosmid vector pCVD301. TnphoA mutagenesis of the AA cosmid clone pJPN31 implicated an AA region of approximately 12 kb. Transmission electron microscopy of HB101 (pJPN31) revealed the presence of bundle-forming fimbriae, which were absent in AA- TnphoA insertion mutants. The presence of these fimbriae, AA, and hemagglutination (HA) of human erythrocytes were all concurrently lost by single-insertion mutations. A 14-kDa protein was seen on polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of surface shear preparations from fimbriated clones. Twelve of nineteen volunteers fed EAggEC 17-2 developed rises in antibodies to the 14-kDa protein as determined by Western blot. We have termed the cloned bundle-forming fimbriae aggregative adherence fimbriae I (AAF/I); positivity with a previously described EAggEC probe and human erythrocyte HA appear to correlate with the presence of AAF/I.     

Adherence of Pseudomonas aeruginosa to tracheal epithelium.

Adherence of mucoid and nonmucoid strains of Pseudomonas aeruginosa to tracheal epithelium was studied with a perfused-trachea model. The species specificity of adherence was studied by infecting tracheas from hamsters, guinea pigs, or mice. Perfused tracheas from hamsters were infected with strains of P. aeruginosa in the presence of various sugars, lectins, cations, or charged polymers. Adherence of mucoid strains of P. aeruginosa was greatest for guinea pigs; that for hamsters and mice was approximately the same. Nonmucoid strains did not adhere well to epithelium from any of the species tested. N-Acetylglucosamine, galactose, and N-acetylneuraminic acid were the best inhibitors of adherence of mucoid strains of P. aeruginosa. Phaseolus vulgaris agglutinin and Arachis hypogaea agglutinin enhanced adherence of mucoid strains. Adherence of mucoid strains was also enhanced by the presence of Ca2+ in the incubation medium. Poly-L-lysine, poly-L-aspartic acid, and polyglycine inhibited adherence of a mucoid strain by 96, 86, and 52%, respectively. In general, the adherence of nonmucoid strains was not affected. The results indicate that carbohydrates are involved in the interaction of mucoid strains of P. aeruginosa with tracheal cells and that divalent cations may enhance this interaction. The lectin data show that lectins can interact with the mucoid organisms and the host and suggest that lectins may play a role in the adhesion process.     

Type I interferon response to extracellular bacteria in the airway epithelium

The airway epithelium possesses many mechanisms to prevent bacterial infection. Not only does it provide a physical barrier, but it also acts as an extension of the immune system through the expression of innate immune receptors and corresponding effectors. One outcome of innate signaling by the epithelium is the production of type I interferons (IFNs), which have traditionally been associated with activation via viral and intracellular organisms. We discuss how three extracellular bacterial pathogens of the airway activate this intracellular signaling cascade through both surface components as well as via secretion systems, and the differing effects of type I IFN signaling on host defense of the respiratory tract.     

Factors influencing bacterial adherence to biomaterials.

The adherence of bacteria to implanted medical devices is believed to be important in the development of implant associated infections. Measures which reduce bacterial adherence should reduce the incidence of these infections. However, in order to assess the importance of adherence, the effectiveness of methods to reduce adherence, and compare data from different laboratories, the conditions of the in vitro studies on adherence need to be specified. There are currently no correct and incorrect methods, however, methods used need to be carefully described. The studies reported here indicate that the definition of adherence needs to be established, with the use of polystyrene as the reference material recommended. Since the adherent organisms lose adherence traits with culture, cultures must be selected for adherence regularly. It is important to control the number of organisms/ml but the volume used is not important. The medium used to grow the organisms and the use of stationary, rocking or flow conditions will alter adherence and need to be specified and be consistent within a set of experiments. Culture conditions, methods of rinsing the material, methods of elution and counting, or direct counting of organisms on the material need to be specified. Finally, as much information as possible on the bulk and surface properties of the material should be provided. The handling of the material for the experiments should be careful and defined. Fingerprints, contact with protein, wet surfaces vs dry surfaces, etc., will all affect the subsequent adherence. The materials should not be re-used since the removal of the adherent proteins or the biofilm is very difficult. Progress can be made in this important area if the details of procedures are specified.     

Gingipain adhesin domains mediate Porphyromonas gingivalis adherence to epithelial cells

Porphyromonas gingivalis, a Gram-negative oral anaerobe, interacts with epithelium lining the gingival sulcus. Continuing our studies on the role of gingipain cysteine proteinases in P. gingivalis adherence to epithelial cells, we showed that antibody raised to the recombinant adhesin domain of arg-gingipain A blocked bacterial attachment, providing new additional evidence that P. gingivalis adherence to epithelial cells is mediated by gingipain adhesin peptides.     

Effect of lacto-N-neotetraose, asialoganglioside-GM1 and neuraminidase on adherence of otitis media-associated serotypes of Streptococcus pneumoniae to chinchilla tracheal epithelium

The adherence of Streptococcus pneumoniae (Spn) otitis media-associated serotypes 3, 6A and 14 to ciliated chinchilla respiratory epithelium was investigated using a whole organ perfusion technique. We demonstrated that Spn adhere to chinchilla tracheal epithelium within 30 min and exhibit saturation kinetics indicating that the effect being observed is receptor mediated. Inhibition of adherence was achieved by prior incubation of Spn with lacto-N-neotetraose (LNnT) or asialoIganglioside GM1 (aGM1), recognized by glycoconjugate analogs of known Spn receptors. NeurIaminidase treatment of the tracheae increased Spn adherence in vitro and reversed the inhibition effect of LNnT suggesting that neuraminidase treatment resulted in an increase in the number of available receptors for Spn. The chinchilla trachea organ perfusion culture system used in this study imitates eustachian tube conditions more closely than isolated cell culture systems and is a useful model for investigating the role of Spn adherence in vitro in the pathogenesis of OM.     

Role of pili in the adherence of Pseudomonas aeruginosa to mouse epidermal cells.

Pili have been demonstrated to be the adhesins of Pseudomonas aeruginosa for mouse epidermal cells. The mechanisms of adhesion of P. aeruginosa to mouse epidermal cells was studied by using four mutants derived from a single strain: flagellated and piliated (F+P+), flagellated and nonpiliated (F+P-), nonflagellated and piliated (F-P+), and nonflagellated and nonpiliated (F-P-) mutants. F+P+ and F-P+ bacteria efficiently adhered to mouse epidermal cells, while F+P- and F-P- bacteria hardly adhered to mouse epidermal cells. The number of F+P+ bacteria that adhered to mouse epidermal cells was almost the same as that of F-P+ bacteria. The number of F+P- bacteria that adhered to mouse epidermal cells was almost the same as that of F-P- bacteria. The adhesion of P+ (F+P+ and F-P+) bacteria was inhibited by antipilus serum, while that of P- (F+P- and F-P-) bacteria was not inhibited by antipilus serum. There were no significant differences between the number of bacteria adhering to mouse epidermal cells isolated from normal skin and those adhering to cells isolated from burned skin. Heating of the mouse epidermal cell suspension had no effect on the adhesion of P. aeruginosa. These results suggest that pili mediate the adhesion of P. aeruginosa to mouse epidermal cells and that P. aeruginosa adheres efficiently to mouse epidermal cells despite the loss of cell viability caused by burning.     

Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells.

Adherence of Pseudomonas aeruginosa organisms to the upper respiratory epithelium of seriously ill patients in vitro is correlated with subsequent colonization of the respiratory tract by this opportunistic pathogen. The role of pili in the attachment to epithelial cells of P. aeruginosa was studied in an in vitro system employing human buccal epithelial cells and P. aeruginosa pretreated by various means. Pretreatment of the bacteria with proteases, heat, or Formalin caused a significant decrease in adherence. A decrease when compared with controls was also noted in the adherence of P. aeruginosa organisms to buccal epithelial cells preincubated with purified pili prepared from the strain used for adherence testing; however, pili prepared from a heterologous strain failed to block adherence. Similar results were obtained in serological studies when antisera to purified pili prepared from the strain used for adherence testing decreased adherence, whereas heterologous antiserum to pili did not decrease adherence. From these results it appears that pili mediate the adherence of P. aeruginosa organisms to human buccal epithelial cells.     

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.     

Development of hamster tracheal epithelium: I. A quantitative morphologic study in the fetus

The development of the tracheal epithelium was studied in hamsters, beginning on fetal day 10 and ending on fetal day 16, shortly after birth. The epithelial morphology was characterized and as soon as the cells could be recognized by type (day 14) their proportions were quantified along dorsal and ventral surfaces from larynx to carina. At all times, columnar cells of the dorsal surface were taller than those of the ventral surface. On days 10 and 11 the simple epithelium was composed of poorly differentiated columnar cells, but on day 12 organoid clusters, consistent with the morphology of neuroepithelial bodies, were observed; four clusters were seen along the dorsal epithelium in one section. The epithelium was pseudostratified on day 13, composed of short and columnar cells. Most columnar cells were poorly differentiated but a few preciliated and ciliated cells were recognizable dorsally, especially at the tracheal poles. Hemidesmosomes were seen at the base of some short cells on day 14, and rough endoplasmic reticulum was moderately developed in some columnar cells, suggesting that these cells were prebasal and presecretory cells, respectively. Preciliated and ciliated cells, which were most prevalent caudally, accounted for about 14% of all dorsal epithelial cells on day 14, but they were rare in the ventral surface, about 0.1%. The epithelial cells were sufficiently specialized by days 15 and 16 to allow quantification by type. Proportions of basal, presecretory, and preciliated-ciliated cells were similar on both days but the cellular makeup of dorsal and ventral surfaces was significantly different. There were more basal cells ventrally (36-40%) than dorsally (22-23%), and more preciliated-ciliated cells dorsally (18-21%) than ventrally (about 1%). On days 15 and 16 differences also existed along both surfaces between cranial and caudal parts of the trachea. Basal cells were more prevalent cranially and preciliated-ciliated cells were more prevalent caudally.     

Effect of antibiotics on bacterial adherence to foreign material

Microbial adherence is the first step of foreign body infection. In vitro and ex vivo experiments have studied the mechanisms of the anti-adherent effect of antimicrobial agents. Adherence of bacteria onto foreign bodies is a complex phenomenon where several factors from the bacteria, the foreign body and the host, are involved. Bacterial factors are physicochemical factors such as cell surface charges, hydrophobicity, and adhesins such as exopolysaccharides or "slime". Foreign body factors are irregularities of the surface and hydrophobicity. Human factors are serum proteins as fibronectin, collagen, laminin and vitronectin, which are deposited onto the material and act as receptors for bacteria. In vitro models can study the interactions between the bacteria, the foreign body and the antimicrobial agent. They can be useful to analyse the molecular mechanisms of the effects of the antimicrobial agents on microbial adherence. Different studies have shown: 1) no relation between the effect of antimicrobial agents on microbial adherence and their bacteriostatic or bactericidal effects, 2) antimicrobial agents of the same class may have different effects on the same strain, 3) no correlation between effect of antimicrobial agents on microbial hydrophobicity, slime production or fibronectin receptors, and effect on adherence.     

Model for studying bacterial adherence to skin wounds.

The adherence of bacterial pathogens to wounded skin is probably the first step in wound infection. This report describes the development of a bioassay to simulate the adherence of bacteria to wounds. The adherence of bacteria was examined by exposing wounds to known quantities of pathogens, washing the wounds with distilled water, and quantitating the number of adherent bacteria per cm2 of tissue. Our studies focused on the effects of naturally occurring mediators of bacterial adherence, such as wound fluid, serum, and fibronectin. Bacterial adherence was shown to be challenge dependent. Addition of wound fluid, serum, and heat-inactivated serum was shown to reduce the adherence of Pseudomonas aeruginosa to the wound surface compared with that of a saline control. Additional fibronectin treatment of the wound had no effect on the adherence of Staphylococcus aureus. The ability to identify what affects the binding of bacteria to wounded skin can lead to a better understanding of wound infection.     

SspA对生物材料细菌黏附及生物膜形成的影响

金黄色葡萄球菌（staphylococcus aureus,SA）是以生物材料为中心感染的主要致病菌之一,主要通过细菌黏附并形成生物膜造成感染,给患者带来灾难性的后果.SA的表面蛋白纤维连接蛋白结合蛋白（fibronectin-binding protein,FnBP）是其关键的黏附因子,而金黄色葡萄球菌丝氨酸蛋白酶（staphylococcal serine protease,SspA）可降低细菌的FnBP,抑制自溶素（autolysin,AtlE）分泌,降解表面A蛋白（surfaceproteinA,Spa）从而抑制细菌黏附和生物膜（bacterial biofilm,BF）的形成,为SspA在控制SA感染方面提供了一个重要的治疗途径.     

SspA对生物材料细菌黏附及生物膜形成的影响

金黄色葡萄球菌(staphylococcusaureus,SA)是以生物材料为中心感染的主要致病菌之一,主要通过细菌黏附并形成生物膜造成感染,给患者带来灾难性的后果。SA的表面蛋白纤维连接蛋白结合蛋白(fibronectin-bindingprotein,FnBP)是其关键的黏附因子,而金黄色葡萄球菌丝氨酸蛋白酶(staphy-lococcalserineprotease,SspA)可降低细菌的FnBP,抑制自溶素(autolysin,AtlE)分泌,降解表面A蛋白(surfaceproteinA,Spa)从而抑制细菌黏附和生物膜(bacterialbiofilm,BF)的形成,为SspA在控制SA感染方面提供了一个重要的治疗途径。     

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.     

Functional heterogeneity of intestinal Escherichia coli strains expressing type 1 somatic pili (fimbriae): assessment of bacterial adherence to intestinal membranes and surface hydrophobicity.

Although the role of host-specific, nonmannose-sensitive pilus adhesins in the intestinal adherence of pathogenic Escherichia coli is well established, a similar role for mannose-sensitive type 1 or common pili is less clear, since these structures can be expressed by most E. coli, even nonpathogens. We first examined whether type 1 pili, expressed by the rabbit-effacing, adherent, enteropathogenic E. coli strain RDEC-1, mediated interactions with intestinal membranes of several species and compared these interactions with those mediated by the nonmannose-sensitive adhesin of RDEC-1. We next grew a series of E. coli intestinal strains in static broth to promote type 1 pilus expression and determined whether E. coli expressing type 1 pili differed in their affinity for intestinal membranes (as measured by phase-contrast microscopy and aggregometry), hydrophobic surface properties, net negative surface charge (as measured by hydrophobic interaction chromatography and salt aggregation), and hemagglutination patterns. In contrast to the species-specific attachment to rabbit brush borders of RDEC-1 expressing its nonmannose-sensitive adhesin, type 1 pili on RDEC-1 mediated mannose-sensitive attachment to intestinal membranes of all four species tested. Expression of type 1 pili on other E. coli strains resulted in varying degrees of nonspecies-specific, mannose-sensitive attachment to intestinal membranes. This attachment correlated with increasing surface hydrophobicity rather than with hemagglutination patterns. These results indicate that various E. coli strains expressing type 1 pili are functionally heterogeneous and suggest that some type 1 pili might contribute to in vivo enteroadherence.