Characteristics of capsules in enterotoxemic Escherichia coli O139:K12 strains causing swine edema disease

The characteristics of the capsule of the enterotoxemic Escherichia coli (ETEEC) O139:K12 strains that strongly adhere to Hep-2 cells were examined. Electron microscopic studies using the freeze-substitution technique revealed that ETEEC strains had a capsule of approximately 25 nm. These strains show hydrophobic surface properties and strong adherence to human polymorphonuclear leukocytes (PMNs). In contrast, ETEEC strains RK-O139 and ED-1 show weak adherence to HEp-2 cells and fail to express the capsule layer on the cell surface. These ETEEC strains possess hydrophilic surface properties and also adhere to PMNs. The lipopolysaccharide (LPS) analysis by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that ETEEC strains had the same LPS profile and long O-side chains of LPS. Furthermore, all strains were resistant to serum killing activity. These results suggest that the capsule of ETEEC strains does not contribute as an antiphagocytic factor, but as an adherence factor to host cells.     

Morphological and physical characterization of the capsular layer of Vibrio cholerae O139

The morphological and physical characteristics of the capsule of Vibrio cholerae O139 were examined. An electron microscopic study using the freeze-substitution technique showed that all of the V. cholerae strains of the O139 serogroup examined have a very thin fibrous layer on the outside of the outer membrane. In contrast, the mutants of strain O139, strain MO10T4 (which lacks capsule synthesis), and strain Bengal-2R1 (which fails to synthesize both the capsule and the O-antigen of lipopolysaccharide) were all found to have lost the surface layer. In addition, the capsule layer could also not be observed on the surface of V. cholerae strain O1. To determine the biological characteristics of the capsule of strains of the O139 serogroup, we investigated the serum killing activity and bacterial phagocytosis by polymorphonuclear leukocytes. The O139 strains were more resistant to the serum killing activity than were the V. cholerae O1 strain and the O139 mutant strains, thus suggesting that the existence of the capsule gave a serum-resistant character to the O139 strains. The surface character of the O139 strains had the same hydrophobic character as did that of the O139 mutant strains and the O1 strain. In addition, all the V. cholerae O1 and O139 strains examined, including the mutant strains, were effectively ingested by the human polymorphonuclear leukocytes. The number of ingested bacteria was not significantly different among the strains, and the ingestion of the acapsular O139 mutants thus showed that the capsule does not play an antiphagocytic role. These data suggest that the capsule of V. cholerae O139 has a physiological function different from that of the ordinal hydrophilic capsule that is found in invasive bacteria such as Klebsiella pneumoniae. Received: 23 March 1998 / Accepted: 28 July 1998     

Identification of entero-aggregative Escherichia coli based on surface properties

Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a'stacked brick' pattern (EAggEC), and four nonadherent control strains, wereexamined for the ability to hybridize with gene probes for aggregative (AA) and diffuse (DA)HEp-2 cell adhesion phenotypes. These strains were also tested for the ability to express an 18 kDa membrane-associated outer- membrane protein (MAP), to agglutinate erythrocytes, and toproduce a pellicle during broth culture. Thirteen of the 29 HEp-2 adherent strains of E. coli hybridized with the gene probes for both AA and DA, and expressed an 18 kDa outermembrane protein (OMP) which was antigenically related to the MAP expressed by strains of E. coli O126:H27. The strains that did not carry the additional DA genes did notexpress an 18 kDa OMP. Although strains of EAggEC share the ability to adhere to HEp-2 cellswith a stacked brick pattern, these strains exhibit a diverse range of physical and biochemicalproperties. From the results of this study, it was concluded that currently, the possession ofEAggEC genes or the ability to adhere to HEp-2 cells in a stacked brick formation, remain theonly reliable means of identifying EAggEC.     

Non-Invasive Providencia alcalifaciens Strains Fail to Attach to HEp-2 Cells

We investigated the adherence properties of six P. alcalifaciens strains with previously characterized differential invasive capabilities in HEp-2 cells. Highly invasive strains were found to attach to HEp-2 cell monolayers within 2 h post-infection and in large numbers on the eukaryotic cell surfaces within 3 h post-infection. In contrast, weakly or non-invasive P. alcalifaciens strains were non-adherent to HEp-2 cells even at 3 h post-infection. Highly invasive isolates were found to weakly bind F-actin using the fluorescent actin staining assay although these strains were negative for Escherichia coli attachment and effacing gene (eaeA) of enteropathogenic E. coli (EPEC). These results suggest that the strain variation in the ability of P. alcalifaciens to invade HEp-2 cells previously noted by several investigators may be linked to expression of key adhesin(s) on the cell surface of invasive isolates. Received: 10 April 2001 / Accepted: 17 April 2001     

Comparison of adherence to and penetration of a human laryngeal epithelial cell line by group A streptococci of various M protein types

Clinically isolated group A streptococci (GAS) of different M protein types were studied using aminoglycoside exclusion and [2,8-3H]adenine radiolabeled GAS assays to compare the abilities of different strains to adhere to and internalize within human laryngeal epithelial (HEp-2) cells. GAS isolated from patients with pharyngitis and GAS isolated from patients with more severe disease, such as necrotizing fasciitis, adhered to and penetrated HEp-2 cells equally well. M3, M4, M6, and M12 strains adhered to and were internalized within HEp-2 cells more than M1 strains. M18 GAS producing hyaluronic acid capsules were less adherent and less invasive than the M3, M4, M6, and M12 strains. An M3-producing GAS strain and its M protein-deficient isogenic strain adhered similarly to HEp-2 cells, but the M protein-deficient strain exhibited greater penetration. Preincubation of HEp-2 cells with an N-terminal synthetic M3 peptide did not alter the adherence or penetration by an M3 strain. In summary, this study demonstrates that GAS from invasive and non-invasive disease adhere to and penetrate HEp-2 cells equally well and that multiple strains of GAS with various M protein types have the ability to adhere to and penetrate HEp-2 cells.     

HEp-2 cell adherence and Vero cell cytotoxin production by EPEC strains isolated from children with diarrhoea in New Zealand

Abstract A total of 112 EPEC strains isolated from children with diarrhoea in New Zealand were examined for mannose-resistant HEp-2 cell adherence and production of exotoxins. Enterotoxin production was not detected in any of the strains examined. Verotoxin production was detected in 13 (11.6%) strains and of these 4 were also found to adhere to HEp-2 cells. HEp-2 cell adherence was displayed by a total of 29 (25.8%) strains of which 22 were diffusely adherent. Only 3 (2.7%) strains were shown to belong to the new virulence phenotype, entero-aggregative adherence, when examined in the adherence assay. We identified one strain with the novel characteristics of causing detachment of HEp-2 cells from glass coverslips and are further investigating this possible virulence mechanism. These results suggest that if EPEC strains are to be considered as a cause of diarrhoea, the search for new virulence factors must be extended.     

Enzymic detection of adhesion of enteropathogenic Escherichia coli to HEp-2 cells

We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.     

Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7.

Intimin-gamma is an outer membrane protein of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that is required for the organism to adhere tightly to HEp-2 cells and to colonize experimental animals. Another EHEC O157:H7 protein, the Transferred intimin receptor (Tir), is considered the primary receptor for intimin-gamma. Nevertheless, Tir-independent binding of intimin-gamma to HEp-2 cells has been reported. This observation suggests the existence of a eukaryotic receptor(s) for intimin-gamma. In this study, we sought to identify that receptor(s). First, we determined by equilibrium binding titration that the association of purified intimin-gamma with HEp-2 cells was specific and consistent with a single host cell receptor. Second, we isolated a protein from lysates of HEp-2 cells that bound intimin-gamma and subsequently identified this molecule as nucleolin, a protein involved in cell growth regulation that can be cell surface-expressed. Third, we established that purified intimin-gamma and nucleolin were co-localized on the surface of HEp-2 cells and that the site of EHEC O157:H7 attachment was associated with regions of nucleolin expression. Finally, we demonstrated that mouse anti-nucleolin sera significantly decreased the adherence of EHEC O157:H7 to HEp-2 cells. From this, we conclude that nucleolin is the HEp-2 cell receptor for intimin-gamma expressed by EHEC O157:H7.     

Sulfatides Inhibit Binding of Helicobacter pylori to the Gastric Cancer Kato III Cell Line

Helicobacter pylori adhere to Kato III and Hela S3 cells in monolayer cultures. To explore whether cell surface glycoconjugates on these two cell lines mediate binding of H. pylori, various carbohydrates, glycoproteins, and glycolipids were tested to inhibit H. pylori cell adhesion. The adhesion was measured (i) with a urease-based assay and (ii) by cells stained with fluorescein. Sodium periodate and sialidase treatment (but not α- or β-galactosidase, heparitinase, lysozyme, or trypsin) inhibited H. pylori binding to both cell lines. Sulfatides and sulfated glycoconjugates (50 μg/ml) but not heparin or a number of simple carbohydrates inhibited binding (1 mg/ml). The two H. pylori strains studied (CCUG 17874 and strain 25) showed high binding of soluble ¹²⁵I-labeled heparin and other sulfated carbohydrate compounds. Received: 5 July 1996 / Accepted: 17 October 1996     

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

Invasion of HEp-2 and Other Eukaryotic Cell Lines by Providenciae: Further Evidence Supporting the Role of Providencia alcalifaciens in Bacterial Gastroenteritis

Wild-type strains of Providencia species were evaluated for their ability to invade HEp-2 monolayers based upon microscopic and semi-quantitative assays. Of 14 P. alcalifaciens strains tested, 3 (17%) were found to be highly invasive, 4 (22%) moderately invasive, and the remaining 61% weakly or noninvasive. HEp-2 invasion results were confirmed by thin-section electron microscopy. Invasive capabilities of P. alcalifaciens were greater at higher MOIs (100 to 1000) than at lower inocula (<10 MOI). No strain of P. stuartii or P. rettgeri tested invaded HEp-2 cells. Quantitative assays of Triton X-100-lysed, HEp-2-invaded cells indicated that between 0.001% and 0.013% of the initial bacterial inoculum was gentamicin resistant. Further testing of select strains on various cell lines indicated the efficiency of invasion was Vero > Y1 > INT-407 > HEp-2. Two isolates recovered from a father and son with prolonged diarrhea after returning from Mexico were found to be identical on the basis of biotype, serotype, and genotype. These results provide additional evidence that some P. alcalifaciens strains cause gastroenteritis. Received: 1 December 1997 / Accepted: 25 March 1998     

Collagen Binding of Bifidobacterium adolescentis

In this study, 13 bifidobacterial strains were tested for their ability to adhere to immobilized extracellular matrix (ECM) proteins. Only two Bifidobacterium adolescentis strains adhered to immobilized type I and type V collagens, but not to laminin, fibronectin, and type III and IV collagens. The adhesion of B. adolescentis BB-119 to type V collagen was inhibited by type I and V collagens and gelatin, and was diminished after protease treatment of the cells. Periodate treatment of immobilized collagen and the presence of galactose inhibited the adhesion of strain BB-119 to type V collagen. Two cell surface proteins with molecular masses of 36 kDa and 52 kDa from strain BB-119 were found to bind to horseradish peroxidase-conjugated type V collagen by ligand blotting. We concluded that B. adolescentis BB-119 binds to type V collagen at galactose chains as target via these two cell surface proteins by their lectin-like activity. Received: 15 October 1996 / Accepted: 20 November 1996     

HEp-2 adhesion and the expression of a 94 kDa outer-membrane protein by strains of Escherichia coli belonging to enteropathogenic serogroups

Sixty strains of Escherichia coli belonging to enteropathogenic serogroups (EPEC) were examined for the ability to adhere to HEp-2 cells, the possession of the genes encoding EPEC adherence factor (EAF) and the ability to express an outer-membrane protein (OMP) of 94 kDa thought to be involved in bacterial adhesion to eukaryotic cells. An absolute correlation was found between HEp-2 adhesion and the possession of the genes encoding EAF. An OMP of 94 kDa was observed in the SDS-PAGE profile of most adhesive strains. In some strains this protein was prone to proteolytic degradation. An antiserum raised to a HEp-2 adhesive strain of EPEC did not react with the 94 kDa OMP of all EPEC which were EAF-positive and HEp-2 adhesive, indicating some interstrain antigenic variation of this protein. Although this 94 kDa protein was surface-exposed, specific antibodies binding to the 94 kDa protein in situ in the outer membrane did not interfere with adhesion of EPEC to HEp-2 cells. Therefore, these studies question the value of this protein as a potential vaccine component.     

Iron-limited condition modulates biofilm formation and interaction with human epithelial cells of enteroaggregative Escherichia coli (EAEC)

Aims:  The aim of this study was to investigate the influence of low iron availability on biofilm formation and adherence to HEp-2 cells of enteroaggregative Escherichia coli (EAEC) strains isolated from diarrhoea cases.
Methods and Results:  The ability of EAEC to form biofilm on a plastic surface was evaluated quantitatively and qualitatively after 3 and 18 h of incubation of strains with or without the iron chelator 2,2-dipyridyl. When submitted to low iron conditions, prototype EAEC 042 strain showed a decrease in biofilm formation. Conversely, an increase in biofilm formation was observed for the clinical EAEC strains cultured in restricted iron condition. Moreover, the reduction of iron concentration inhibited the aggregative adherence to HEp-2 cells of all EAEC strains tested. However, all effects promoted by iron chelation were suppressed by thiourea.
Conclusions:  Low iron availability may modulate biofilm formation and adhesive properties of EAEC strains to HEp-2 cells.
Significance and Impact of the Study:  The data obtained in this study provide useful insights on the influence of low iron conditions possibly associated with redox stress on the pathogenesis of EAEC strains.     

Analysis of the surface proteins of <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strain SP5/1 and the new,pyrite-oxidizing <Emphasis Type="Italic">Acidithiobacillus</Emphasis> isolate HV2/2, and their possible involvement in pyrite oxidation

Two strains of rod-shaped, pyrite-oxidizing acidithiobacilli, their cell envelope structure and their interaction with pyrite were investigated in this study. Cells of both strains, Acidithiobacillus ferrooxidans strain SP5/1 and the moderately thermophilic Acidithiobacillus sp. strain HV2/2, were similar in size, with slight variations in length and diameter. Two kinds of cell appendages were observed: flagella and pili. Besides a typical Gram-negative cell architecture with inner and outer membrane, enclosing a periplasm, both strains were covered by a hitherto undescribed, regularly arranged 2-D protein crystal with p2-symmetry. In A. ferrooxidans, this protein forms a stripe-like structure on the surface. A similar surface pattern with almost identical lattice vectors was also seen on the cells of strain HV2/2. For the surface layer of both bacteria, a direct contact to pyrite crystals was observed in ultrathin sections, indicating that the S-layer is involved in maintaining this contact site. Observations on an S-layer-deficient strain show, however, that cell adhesion does not strictly depend on the presence of the S-layer and that this surface protein has an influence on cell shape. Furthermore, the presented data suggest the ability of the S-layer protein to complex Fe³⁺ ions, suggesting a role in the physiology of the microorganisms.     

The role of Tir, EspA, and NleB in the colonization of cattle by Shiga toxin producing Escherichia coli O26:H11

Shiga toxin producing Escherichia coli (STEC) O26:H11 is an enteric pathogen capable of causing severe hemorrhagic colitis that can lead to hemolytic uremic syndrome. This organism is able to colonize cattle and human intestinal epithelial cells by secreting effectors via a type III secretion system (T3SS). In this investigation, we examined the role of 2 effectors, Tir and NleB, and the structural translocator component EspA in the adherence of STEC to epithelial cells and in the colonization of cattle. Isogenic deletion mutants were constructed and using microscopy and flow cytometry compared to the wild-type strain in their ability to adhere to HEp-2 cells. A competitive assay was also used to measure the capacity of the mutants to colonize the intestinal tract of cattle, where both the mutant and the parental strains were introduced orally at the same time. Genomic DNA was extracted from enriched fecal samples collected at various time points, and quantitative real-time PCR was used to quantify bacteria. A significant reduction in fecal shedding was observed, and adherence to HEp-2 cells was decreased for the tir and espA mutants. Deletion of the nleB gene did not have a significant effect on the adherence of HEp-2 cells; however, in an in vivo model, it strongly reduced the ability of STEC O26:H11 to colonize the bovine intestinal tract.     

Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells

AIMS: To investigate the prevalence of Aeromonas in a major waterway in South East Queensland, Australia, and their interactions with a gut epithelial model using Caco-2 cells. METHODS AND RESULTS: A total of 81 Aeromonas isolates, collected from a major waterway in South East Queensland, Australia, were typed using a metabolic fingerprinting method, and tested for their adhesion to HEp-2 and Caco-2 cells and for cytotoxin production on Vero cells and Caco-2 cells. Aeromonas hydrophila had the highest (43%) and Aeromonas veronii biovar sobria had the lowest (25%) prevalence. Four patterns of adhesion were observed on both HEp-2 and Caco-2 cell lines. Representative isolates having different phenopathotypes (nine strains) together with two clinical isolates were tested for their translocation ability and for the presence of virulence genes associated with pathogenic Escherichia coli. The rate and degree of translocation across Caco-2 monolayers varied among strains and was more pronounced with LogA pattern. Translocation was associated with the adherence of strains to Caco-2 cells microvilli, followed by internalization into Caco-2 cells. Two Aer. veronii biovar sobria strains were positive for the presence of heat-labile toxin genes, with one strain also positive for Shiga-like toxin gene. CONCLUSIONS: Pathogenic strains of Aeromonas carrying one or more virulence characteristics are highly prevalent in the waterways studied and are capable of translocating across a human enterocyte cell model. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that Aeromonas strains carrying one or more virulence properties are prevalent in local waterways and are capable of translocating in a human enterocyte cell culture model. However, their importance in human gastrointestinal disease has yet to be verified under competitive conditions of the gut.     

Effect of viral preinfection upon cell adherence in some staphylococcus strains from specific infections or carriers

The adherence of bacteria to eukaryote cells has been largely investigated as an essential step in the occurrence of bacterial infection. Some clinical and epidemiological studies have revealed the frequent association of certain viral infections with bacterial infections originating in the same ecological niche. Therefore, we investigated the effect of the viral preinfection (ADV4) of some cultivated cells (HEp-2 and IC.SK-27) upon the adherence of staphylococcus to these cells. The analysis of cell adherence within the mentioned conditions, estimated by flow cytometry, allowed of the following conclusions: 1. bacterial adherence to cultivated and virally preinfected cells is augmented by the viral preinfection, and its value on a given cell substrate may characterize a bacterial strain; 2. bacterial adherence to the investigated cell substrates does not correlate with the origin of the tested staphylococcus strains (infections or carriers) and some cell lines can differentiate bacterial strains depending upon the ecological niche or inside it.     

Properties of adherence factor plasmids of enteropathogenic Escherichia coli and the effect of host strain on expression of adherence to HEp-2 cells

EPEC adherence factor (EAF) plasmids from three strains of enteropathogenic Escherichia coli (EPEC) - E2347/69 (O127:H6), E20517 (O111:H2) and E24582 (O142:H6) - were examined. The EAF plasmids were all marked with ampicillin resistance by transposition of Tn801 to give pDEP1, pDEP2 and pDEP11, respectively. All three plasmids showed incompatibility with an FIme and an FIV plasmid and had some similarity in restriction enzyme digest patterns. Plasmid pDEP1 differed from pDEP2 and pDEP11 in being autotransferring and fertility-inhibition positive. An EAF probe consisting of a 1 kb BamHI-SalI restriction endonuclease fragment of the prototype EAF-associated plasmid pMAR2 hybridized to similar-sized SalI-BamHI fragments of pDEP1 and pDEP11 but to a different-sized fragment of plasmid pDEP2. Loss of the EAF plasmids from EPEC strains resulted in a marked reduction in the ability of these strains to adhere to HEp-2 cells. The EAF-plasmid-negative variants did not express a 94 kDa outer-membrane protein (OMP). When these EAF plasmids were reintroduced into EAF-plasmid-negative EPEC strains a high level of adherence equivalent to that of the parent EPEC strains was restored and a 94 kDa OMP was usually expressed. However, when EAF plasmids were transferred into E. coli K12 or non-EPEC E. coli the host strains either did not adhere or adhered poorly to the HEp-2 cells. These transconjugants did not express a 94 kDa OMP.     

Surface properties of lactobacilli isolated from the small intestine of pigs

One hundred wild-type strains of the genus Lactobacillus were isolated from the small intestine of newly-slaughtered pigs up to 6 months of age. Cell surface hydrophobicity and capsule formation were studied on a number of strains. Strains showing high surface hydrophobicity as measured by the salt-aggregation test and hydrophobic interaction chromatography on Octyl Sepharose were commonly found to adhere in high numbers to isolated pig intestinal epithelial cells. Heat and protease treatment of bacteria of high surface hydrophobicity, including autoaggregating strains in phosphate-buffered saline, showed a drastic decline in this surface property. Three hydrophilic strains (LBp 1044, 1068 and 1073) also showed binding to intestinal cells but at a lower level (approx. 5 bacteria/cell) as compared with the best binding hydrophobic strain (LBp 1063, approx. 11 bacteria/cell). These findings suggest that different or multiple adhesion mechanisms may be involved in the colonization of the small intestinal mucosa of pigs. Cultures of selected strains grown in liquid media rich in carbohydrates did not affect their hydrophobic cell surface character. Therefore it seems less likely that carbohydrate capsule polymers are the major determinants of intestinal colonization of lactobacilli in pigs.