RrgA is a pilus-associated adhesin in Streptococcus pneumoniae

Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production.     

Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial ceils

Hemophilus influenzae type b is a Gram-negative bacillus that initiates infection by colonizing the upper respiratory tract. Previous studies have established that H. influenzae haemagglutinating pili possess adhesive properties and influence the process of colonization. Additional studies suggest the presence of a second H. influenzae adhesin distinct from haemagglulinating pili. In the present study, we examined a non-piliated H. influenzae type b strain by transmission electron microscopy and visualized occasional short, thin, surface fibrils. Subsequently, we isolated a spontaneous mutant that lacked surface fibrils and was deficient in adherence to cultured human epithelial cells. Using a cloning strategy that exploited this mutant, we isolated a fragment of DNA that promotes in vitro adherence to human epithelial cells when expressed in laboratory strains of Escherichia coli. Mutagenesis of this fragment in a series of H. influenzae type b strains resulted in loss of expression of surface fibrils and a marked decrease in attachment. Furthermore, restoration of surface fibril expression was associated with reacquisition of wild-type levels of adherence. Our results are consistent with the conclusion that H. influenzae type b surface fibrils have adhesive capacity. We speculate that these organelles facilitate colonization of the human respiratory tract.     

Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.     

In vitro adherence of Candida albicans strains to murine gastrointestinal mucosal cells and explants and the role of environmental pH

Two in vitro adherence assays involving isolated mucosal cells or mucosal explants were used to study the adherence of five Candida albicans strains to murine gastrointestinal mucosal surfaces. Adherence was found to be dependent on the strain used, and on the cellular arrangement, as well as the site of origin of the mucosal surface. Adherence of strains NCPF 3436 and 3310 to stomach and jejunal surfaces was affected by the pH of the medium. Binding between the C. albicans strains and stomach mucosal cells fluctuated as the pH was raised from pH 1.2 to pH 3.4. However, adherence increased with a rise in pH when the strains were incubated with stomach mucosal explants. Optimal adherence by both strains to jejunal mucosal surfaces occurred at neutral pH.     

Kingella kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells

Kingella kingae is a gram-negative bacterium that colonizes the respiratory tract and is a common cause of septic arthritis and osteomyelitis. Despite the increasing frequency of K. kingae disease, little is known about the mechanism by which this organism adheres to respiratory epithelium and seeds joints and bones. Previous work showed that K. kingae expresses long surface fibers that vary in surface density. In the current study, we found that these fibers are type IV pili and are necessary for efficient adherence to respiratory epithelial and synovial cells and that the number of pili expressed by the bacterium correlates with the level of adherence to synovial cells but not with the level of adherence to respiratory cells. In addition, we established that the major pilin subunit is encoded by a pilA homolog in a conserved region of the chromosome that also contains a second pilin gene and a type IV pilus accessory gene, both of which are dispensable for pilus assembly and pilus-mediated adherence. Upon examination of the K. kingae genome, we identified two genes in physically separate locations on the chromosome that encode homologs of the Neisseria PilC proteins and that have only a low level homology to each other. Examination of mutant strains revealed that both of the K. kingae PilC homologs are essential for a wild-type level of adherence to both respiratory epithelial and synovial cells. Taken together, these results demonstrate that type IV pili and the two PilC homologs play important roles in mediating K. kingae adherence.     

The Haemophilus Cryptic Genospecies Cha Adhesin Has at Least Two Variants That Differ in Host Cell Binding,Bacterial Aggregation,and Biofilm Formation Properties

The Haemophilus cryptic genospecies (HCG) causes genital tract infections in pregnant and postpartum women and respiratory infections in neonates. The major surface adhesin in HCG is called Cha, which mediates bacterial adherence to cultured human epithelial cells. In this study, we report that there are two antigenically distinct variants of Cha, dubbed Cha1 and Cha2. These variants are encoded by the same genetic locus in diverse strains and have nearly identical N-terminal export and C-terminal surface anchoring domains but significantly different internal adhesive domains. Based on the comparison of derivatives of a laboratory strain of Haemophilus influenzae expressing either surface-associated Cha1 or surface-associated Cha2, Cha1 mediates a higher level of adherence to cultured human epithelial cells and Cha2 mediates a higher level of adherence to abiotic surfaces. We hypothesize that variation in the Cha1 and Cha2 internal region results in changes in binding specificity or binding affinity and may be associated with adaptation to different host environments during colonization and disease.     

Stenotrophomonas maltophilia interaction with human epithelial respiratory cells in vitro

Bacteria of Stenotrophomonas maltophilia have been isolated with increasing frequency from the airways of cystic fibrosis (CF) patients, usually following P. aeruginosa infections, but their adherence to human epithelial respiratory cells has never been investigated. In this study, various S. maltophilia strains were seen to adhere to epithelial respiratory cells in vitro, mainly along intercellular junctions. Bacteria could also enter into host cells, as determined by the gentamicin exclusion assay and transmission electron microscopy. Cells co-incubated with P. aeruginosa and S. maltophilia exhibited a significantly decreased adherence of these latter bacteria. No decrease in S. maltophilia adherence was observed when co-infection was carried out with heat-killed P. aeruginosa or when respiratory cells were first incubated with P. aeruginosa, before incubation with S. maltophilia. Our data suggest that P. aeruginosa infections do not account for the increased prevalence of S. maltophilia in CF patient airways, that thermolabile products from P. aeruginosa can control the adherence of S. maltophilia to respiratory cells and also that these two bacteria do not compete for cell receptors.     

Comparative adherence to human A549 cells, plant fibronectin-like protein, and polystyrene surfaces of four Pseudomonas fluorescens strains from different ecological origin

The main objective of this study was to compare the adherence properties of four Pseudomonas fluorescens isolates from different ecological niches (human tissue, rhizosphere, drinking water, and cow milk). The substrates used to test P. fluorescens adherence were as follows: cultured human respiratory epithelial cells A549, immobilized plant fibronectin-like protein, and polystyrene. For all the experiments, bacteria were grown at 27 degrees C. The adherence assay to human cells was performed at 37 degrees C, whereas adherence to fibronectin and polystyrene was done at 27 degrees C. The four strains tested adhered to A549 cells but showed different adherence patterns. At 3 h, the milk isolate showed an aggregative adherence phenotype, whereas the three other isolates showed a diffuse adherence pattern. With a longer incubation time of 24 h, the aggregative pattern of the milk isolate disappeared, the adherence of the clinical strain increased, the adherence of the water isolate decreased, and morphological changes in A549 cells were observed with the clinical, water, and soil isolates. The four strains tested formed biofilms on polystyrene dishes. The clinical and milk isolates were the more efficient colonizers of polystyrene surfaces and also the more adherent to immobilized plant fibronectin-like protein. There was no relation between bacterial surface hydrophobicity and P. fluorescens adherence to the substrates tested. The main conclusions of these results are that P. fluorescens is an adherent bacterium, that no clear correlation exists between adherence and ecological habitat, and that P. fluorescens can adhere well to substrates not present in its natural environment.     

Identification of a novel trimeric autotransporter adhesin in the cryptic genospecies of Haemophilus

Haemophilus biotype IV strains belonging to the recently recognized Haemophilus cryptic genospecies are an important cause of maternal genital tract and neonatal systemic infections and initiate infection by colonizing the genital or respiratory epithelium. To gain insight into the mechanism of Haemophilus cryptic genospecies colonization, we began by examining prototype strain 1595 and three other strains for adherence to genital and respiratory epithelial cell lines. Strain 1595 and two of the three other strains demonstrated efficient adherence to all of the cell lines tested. With a stably adherent variant of strain 1595, we generated a Mariner transposon library and identified 16 nonadherent mutants. All of these mutants lacked surface fibers and contained an insertion in the same open reading frame, which encodes a 157-kDa protein designated Cha for cryptic haemophilus adhesin. Analysis of the predicted amino acid sequence of Cha revealed the presence of an N-terminal signal peptide and a C-terminal domain bearing homology to YadA-like and Hia-like trimeric autotransporters. Examination of the C-terminal 120 amino acids of Cha demonstrated mobility as a trimer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the capacity to present the passenger domain of the Hia trimeric autotransporter on the bacterial surface. Southern analysis revealed that the gene that encodes Cha is conserved among clinical isolates of the Haemophilus cryptic genospecies and is absent from the closely related species Haemophilus influenzae. We speculate that Cha is important in the pathogenesis of disease due to the Haemophilus cryptic genospecies and is in part responsible for the apparent tissue tropism of this organism.     

Surface hydrophobicity of "rheumatogenic" and "nephritogenic" strains of group A streptococci and the ultrastructural surface feature of pharyngeal cells exposed to group A streptococci.

The present study was carried out to determine the surface hydrophobicity of group A streptococcal strains responsible for rheumatic fever (RF), "rheumatogenic" strains (RG strains) and strains causing glomerulonephritis, "nephritogenic" strains (NG strains) in relation to their adhesion to human pharyngeal cells. Scanning electronmicroscopic (SEM) studies were carried out to the difference, if any, in the adherence of group A streptococci (M type 5) to pharyngeal and buccal cells (PEC and BEC). By employing two techniques for hydrophobicity determination, salt aggregation titre (SAT) and n-hexadecane binding technique, it was observed that RG strains (M5, M1 and M6) were more hydrophobic than NG strain, M49. However, NG strain M12 was almost equally as hydrophobic as RG strains. The adherence of RG strains, except M1 and M24, to PEC was greater in number than that of NG strains. Although M1 strain was hydrophobic, its adherence to PEC was less. Pepsin and trypsin treatment with streptococci reduced the hydrophobicity and adherence of RG and NG strains to PEC. SEM studies revealed firmly adhered indigenous bacteria on PEC and BEC. Streptococci (M5) adhered more to PEC than to BEC. SEM studies also showed that PEC had a peculiar ultrastructural surface feature to which streptococci adhered. These findings suggest that streptococcal hydrophobicity alone does not determine their adhesion to PEC. The surface nature of PEC might be a characteristic feature of the epithelial cells that allows streptococci to adhere and colonize or it might be a consequence of streptococcal adhesion.     

Occurrence of the Fimbria Gene hifA in clinical isolates of nonencapsulated Haemophilus influenzae

The adherence of Haemophilus influenzae to epithelial cells plays a crucial role in infections. However, little is known about the occurrence of fimbriae. In this study, we examined the distribution of the fimbria gene (hifA) by PCR among 167 H. influenzae strains isolated from patients with respiratory infections. Almost all (163; 98%) of the isolates were nonencapsulated strains. The carriage rate of hifA by the nonencapsulated strains was 18.4%. Electron microscopy showed that fimbriae were abundantly present on the cell surface of hifA-positive strains tested. Only four (2.4%) isolates were encapsulated, all of which were type b and did not possess hifA. The present work suggests that fimbriae may play a considerable role as adhesins in nonencapsulated H. influenzae strains.     

Moraxella (Branhamella) catarrhalis Adherence to Human Bronchial and Oropharyngeal Cells: The Role of Adherence in Lower Respiratory Tract Infections

To study the role of Moraxella (subgenus Branhamella) catarrhalis (B. catarrhalis) adherence to airway cells in lower respiratory tract infections, the in vitro attachments of B. catarrhalis to upper airway (oropharyngeal) and lower airway (bronchial) epithelial cells were compared. The adherence of 4 strains (1 nonfimbriated and 3 fimbriated) of B. catarrhalis to respiratory tract epithelial cells collected from 11 patients with chronic pulmonary disease (CPD) and 11 healthy individuals was evaluated. Both the fimbriated and nonfimbriated strains showed increased attachment to oropharyngeal cells in the CPD patients (mean ± SEM; 25.0 ± 3.2/cell; P < 0.01) when compared to the control subjects (12.1 ± 1.1/cell). On the average, the attachment to bronchial cells was 6.1 to 13.6 times greater per surface area (bacteria/μ²) than the attachment to oropharyngeal cells. The fimbriated strains tended to adhere in higher numbers to bronchial cells (19.0 ± 1.8/cell) than the nonfimbriated strain (8.7 ± 1.2/cell), although there was no difference between the CPD and control groups. In conclusion, the attachment of B. catarrhalis to oropharyngeal cells may be an enhancing factor for colonization in the upper respiratory tract in patients with CPD, and elevated adherence of the bacteria to bronchial cells may suggest pathogenic importance when mucociliary function is impaired.     

Adherence of ovine and human Bordetella parapertussis to continuous cell lines and ovine tracheal organ culture

The adherence of ovine and human isolates of Bordetella parapertussis to ovine and human continuous culture cell lines and to ovine tracheal organ culture was compared. Adherence to non-ciliated respiratory continuous culture cells did not reveal any host-specificity of the isolates. In contrast, adherence of ovine B. parapertussis strains to ciliated ovine tracheal organ culture was significantly greater than that of human strains. These results indicate that tracheal organ culture is a useful tool for studying host-specific adherence of B. parapertussis and suggest that adherence of B. parapertussis to ciliated epithelia is species-specific making it unlikely that the transfer of B. parapertussis between humans and sheep will result in an infection.     

Cell Surface Hydrophobicity and Slime Production of <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> Brazilian Isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M − <2M, SAT 2M − <4M, and SAT ≥4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT ≥ 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0–0.4 O.D. group, including non-glass adherent isolates; 0.5–0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8–1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT. Received: 13 May 2002 / Accepted: 5 July 2002     

Adherence ofPasteurella multocida to porcine upper respiratory tract cells

A model to study the adherence ofPasteurella multocida to porcine upper respiratory tract cells is described. The ability of 27 differentP. multocida isolates to adhere to isolated tracheal epithelial cells was examined. The mean number of adherent bacterial cells was significantly greater (p<0.005) for capsular type A cells than for capsular type D cells. No significant differences were observed between toxigenic and nontoxigenic isolates, or between isolates exhibiting different somatic antigens. However, isolates from pigs without atrophic rhinitis showed only 65% of the adherence of isolates from pigs with atrophic rhinitis. Adherence ofP. multocida to porcine tracheal cells decreased with animal age; adherence to cells from adults was only half of the adherence to cells from newborn animals. The data indicate that, in the present experimental conditions, theP. multocida strains tested possess different abilities to attach to porcine upper respiratory tract cells.     

Adherence of Vibrio parahaemolyticus to rabbit intestinal epithelial cells in vitro

Kanagawa positive strains of Vibrio parahaemolyticus showed adherence whereas most of the Kanagawa negative strains were non-adhering to rabbit intestinal epithelial cells. Anti-hemolysin antisera did not inhibit the adherence of V. parahaemolyticus strains. Moreover, the adhesive capacity of non-adhering strains was not enhanced by purified hemolysin. Cell surface hydrophobicity remained the same in both Kanagawa positive and negative strains of V. parahaemolyticus. Fetuin strongly inhibited the adherence to rabbit intestinal epithelial cells followed by -mannose and D-glucose.     

Adherence to epithelial cells and ultrastructure of fosfomycin-resistant mutants of group A streptococci

Adherence of three strains of group A streptococci and their fosfomycin-resistant mutants to HEp-2 tissue culture cells was compared with some cell-surface characteristics, i.e. ultrastructure and hydrophobicity. Among Fosr mutants, both well-adhering and weakly adhering mutants were found. Clonal analysis of the mutants proved their greater stability in the adherence. Well-adhering parent strains of streptococci and Fosr mutants exhibited surface fibrillae in contrast to weakly adhering Fosr mutants which were devoid of fibrillae or contined fibrillae of lower electron density. Decrease of adherence of Fosr mutants of two strains was accompanied by a decrease of their hydrophobicity.     

A rapid bioluminescence method for quantifying bacterial adhesion to polystyrene

Bioluminescence ATP analysis has been used to assess bacterial adhesion with hydrophobic polystyrene tubes as the attachment surface. The assay was performed at 37 degrees C and pH 6.8 with a 10 min incubation period. A variation of more than 200-fold was observed in the adherence capacity of 34 urinary isolates of Escherichia coli, and organisms could be classified as strongly or weakly adherent. All strains capable of strong adhesion possessed both type 1 fimbriae and flagella, and maximum adhesion was expressed during the exponential growth phase. Attachment was in all cases virtually eliminated by addition of 2.5% (w/v) D-mannose to the incubation buffer. Conversely, strains which were deficient in type 1 fimbriae or flagella, or both, were weakly adherent during all phases of growth. There was no correlation between adherence of E. coli to polystyrene and adherence to buccal or uroepithelial cells, but there was a significant association with adherence to uromucoid (P less than 0.002).     

Bacteroides fragilis adherence to Caco-2 cells

The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76-100%) to non- or weakly adherent (0-25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.     

The fibronectin-binding capacity and host cell adherence of Streptococcus pyogenes strains are discordant with each other

Surface exposed fibronectin-binding proteins (FBPs) play an important role in the adherence of Streptococcus pyogenes (group A streptococcus, GAS) to host cells. This pathogen expresses numerous FBPs, of which SfbI, SfbII and PrtF2 are major surface exposed FBPs. However, GAS strains differ in the genetic potential to express these proteins. To test whether this difference reflects in differences in fibronectin (Fn) binding, a set of circulating strains previously examined for adherence to host cells was used. The 68 distinct strains were isolated from throat, skin and blood. They were analyzed for (a) the presence of genes for SfbI, SfbII and PrtF2 and (b) the extent of Fn binding. The results suggest that strains possessing two or more of the genes for these FBPs bound Fn significantly more than strains possessing none or one of the genes. No correlation between the extent of Fn binding and the tissue site of isolation was found. Furthermore, together with our previous studies on adherence capacity of these GAS strains, we found no correlation between Fn binding ability and the avidity of the strains to adhere to epithelial cells. We suggest that while Fn binding is important for adhesion, for many GAS strains the extent of Fn binding is not the critical determinant of adherence.