Chemiluminescence response of human phagocytes against IgG-opsonized staphylococci with and without protein A activities

The effects of immunological IgG binding to Staphylococcus aureus and IgG binding via protein A on the chemiluminescence (CL) response of human phagocytes were examined. The results obtained by enzyme immunoassay showed a clear correlation between the magnitude of the CL response and amount of IgG on protein A-deficient HL-87 strain. Despite no difference in protein A activity between 209P and Cowan I strains, the CL response to IgG-opsonized 209P cells was lower than that to Cowan I cells similarly opsonized. Moreover, the CL response to opsonized HL-87 cells was identical with that of opsonized Cowan I cells, which was a protein A-rich parent strain of the HL-87. The protein A activity of Cowan I cells was significantly decreased when the cells were treated with the Fc fraction of IgG before opsonization, but such a treatment did not change the phagocytic CL response. These results strongly suggest that IgG bound to protein A via its Fc portion has no effect on the phagocytic CL response and that IgG immunologically bound to S. aureus is responsible for the opsonization of the bacteria.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37°C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37 degrees C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity.

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.     

Evaluation of interactions between strains of S. aureus isolated from different clinical specimens with peripheral blood phagocytic cells

The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S. aureus isolated from different clinical specimens (blood, respiratory tract, pus). To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal elutriation were incubated with suspensions of opsonized bacteria. In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement. To investigate efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocyanate (FITC) and the percentage of cells containing FITC-labelled bacteria was analysed by flow cytometry. The data obtained show that strains of S. aureus originated from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from respiratory tract show the lowest sensitivity for killing. These strains differ too in their ability to trigger monocyte CL response. Contrary, there was no difference in toxicity of bacteria against phagocytes. Strains isolated from peripheral blood showed significant negative correlation between the ability to trigger CL response and toxicity against phagocytes.     

Evaluation of the interactions between strains of S. aureus and peripheral blood phagocytic cells

The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S. aureus isolated from different clinical specimens (blood, respiratory tract, pus). To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal evaluation these cells were incubated with suspensions of opsonized bacteria. In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement. To investigate the efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocynate (FITC) and the percentage of cells containing FITC-labelled bacteria were analysed by flow cytometry. The data obtained show the strains of S. aureus derived from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from the respiratory tract have the lowest sensitivity to killing. These strains differ too in their ability to trigger monocyte CL response. On the contrary, there was no difference in toxicity of bacteria against phagocytes. Strains isolated from peripheral blood showed a significant negative correlation between the ability to trigger CL response and toxicity against phagocytes.     

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.     

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.     

Cell surface hydrophobicity ofStaphylococcus aureus measured by the salt aggregation test (SAT)

Well-defined laboratory strains as well as 72 clinical strains ofStaphylococcus aureus isolated from bovine mastitis were investigated for surface hydrophobicity by the salt aggregation test (SAT).Staphylococcus aureus strain Cowan 1, rich in protein A and fibronectin-binding surface proteins, was found to show high surface hydrophobicity, whereas strain Wood 46, deficient in these surface proteins, showed low surface hydrophobicity. SAT showed a significant difference in surface hydrophobicity (P<0.001) between protein A-positive and A-negative strains measured by ²-test analysis. Comparison of SAT values with results obtained from hydrophobic interaction chromatography (HIC) showed a good correlation (P<0.025). A high-level protein-A-producing mutant (SA 113prA-3) showed increased surface hydrophobicity as compared with the parent strain (SA 113), whereas ten protein-A-negative mutants showed low surface hydrophobicity in SAT. Of the 72 clinical isolates tested by SAT, 47 (65%) showed autoaggregating properties, i.e., the strains aggregated even in isotonic buffers. Tween 80 (1% vol/vol) and ethylene glycol (50% vol/vol) prevented autoaggregation of some hydrophobic strains aggregating in phosphate-buffered saline. However, 2M of a chaotropic agent (NaSCN) was more efficient in preventing autoaggregation of the strains tested. Heating of cell suspensions to 80°C or 100°C as well as trypsin andStreptomyces griseus protease treatment generally caused a decrease in the cell surface hydrophobicity. This indicates that protein A, fibronectin-binding proteins, and probably other as yet unidentified proteins contribute to the high surface hydrophobicity of most strains isolated from bovine mastitis.     

Role and behaviour of the hydrophobic conditions in bacterial adhesion to incurrent siphon in a bivalve mollusc

Surface hydrophobicity is a widely distributed characteristic among human bacterial pathogens playing an important role in microbes retention by the incurrent siphon of a marine bivalve. Feeding retention experiments with the bivalve Mesodesma donacium was done with hydrophobic strains alone (Staphylococcus aureus, Salmonella paratyphi, Vibrio cholerae) or with mixed cultures using a hydrophilic strain (Aeromonas hydrophila). Results showed that hydrophobic bacteria are retained in greater numbers than hydrophilic bacteria. This difference is statistically significant. Hydrophobic strains also survive longer than hydrophilic ones in sea water. Surface hydrophobicity is to be considered as a factor influencing concentration of hydrophobic pathogens by filter feeding molluscs.     

Compositional factors influencing cell surface hydrophobicity ofPasteurella multocida variants

The influence of macromolecules other than lipopolysaccharide on the hydrophobic properties ofPasteurella multocida was investigated by assessing cell surface hydrophobicity (CSH) after experimentally modifying surfaces of various strains. CSH of hydrophobic variants was enhanced by growth on blood-supplemented medium and mechanical shearing, whereas chloramphenicol, oxytetracycline, trypsin, and pronase E treatments decreased CSH. No such modifications were observed for hydrophilic strains. Microscopic observations revealed hydrophilic strains to be heavily encapsulated in contrast to hydrophobic strains. Repeated subculturing reduced encapsulation with a concomitant increase in CSH for one hydrophilic strain while exerting no changes in the other hydrophilic strain examined. Hyaluronidase removal of capsular material from a serotype A strain resulted in increased CSH; subsequent exposure to pronase E resulted in partial restoration of hydrophilicity. These data suggest the encapsulation of hydrophilicP. multocida strains masks a relatively hydrophobic surface that is conferred, at least in part, by the presence of one or more surface-exposed proteins common to both hydrophilic and hydrophobic variants.     

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

The influence of saliva on the hydrophobic surface properties of bacteria isolated from oral sites of macaque monkeys

Abstract The surface hydrophobicity of 64 bacterial strains isolated from discrete, intra-oral sites of monkeys ( Macaca fascicularis ) was determined by measuring their affinity for hexadecane. Bacteria were also exposed to monkey saliva which either increased or reduced the surface hydrophobicity of the cells. After exposure to saliva those bacteria isolated solely from the mucosal surfaces were significantly more hydrophobic than bacteria ( Streptococcus mutans and Actinomyces spp.) whose major habitat was the dentition. Streptococcus sanguis strains isolated from all intra-oral sites and among the early plaque formers were as hydrophobic as the organisms isolated only from the mucosal surfaces.     

Autoaggregation,hydrophobic, and hydrophylic properties of Moraxella catarrhalis strains

The hydrophobicity of the bacterial cell surface was evaluated via the salt aggregation test (SAT) in 58 strains (19 from the lower and 39 from the upper respiratory tracts) of Moraxella catarrhalis in hospitalized patients aged 25 to 65. Based on the SAT results, the strains were divided into three groups: autoaggregating (highly hydrophobic), hydrophobic, and hydrophilic. At a temperature of 37 degrees C, the autoaggregating, hydrophobic or hydrophilic properties did not depend on the choice of a medium, whereas at 22 degrees C the investigated properties did (p<0.0001). Taking into account the origin of the strains (lower vs. upper respiratory tract), it was found that: in the strains cultivated in liquid medium, both highly hydrophobic, hydrophobic and hydrophilic surfaces were present with a comparative frequency, independent of the strain isolation site and cultivation conditions; strains with highly hydrophobic and hydrophobic surfaces, but only those cultivated on solid media at 22 degrees C, were much more often isolated from sputum rather than from nose and throat swabs, whereas a statistically significant incidence of hydrophilic strains was found in samples from the upper rather than lower respiratory tract.     

Characterization of a cell-surface protein antigen of hydrophilic Streptococcus mutans strain GS-5

Fourteen strains of Streptococcus mutans serotype c were examined for their cell-surface protein antigens in terms of hydrophobicity, Mr and immunochemical specificities. Thirteen strains were hydrophobic, while strain GS-5 was markedly hydrophilic as compared to the other strains tested. Cell-surface protein antigens were then analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting. A protein antigen of Mr 190,000 (PAc) was found in cell extracts and culture supernatants of all the hydrophobic strains. Neither culture supernatant nor cell extract of strain GS-5 contained PAc. Strain GS-5, however, produced extracellularly a large amount of a protein of Mr 155,000 (PAGS-5) which reacted with rabbit anti-PAc serum. Immunodiffusion analysis showed that PAGS-5 lacked a part of the antigenic moieties in the PAc molecule. SDS-PAGE and radioimmunoassay showed a small amount of PAGS-5 on the cell surface of strain GS-5. These findings suggest that PAGS-5 may correspond to PAc which lacks a region participating in binding of PAc to the streptococcal cell.     

The assessment of relative surface hydrophobicity as a factor involved in the activation of human polymorphonuclear leukocytes by uropathogenic strains of Escherichia coli

The initiation of the respiratory burst and the degranulation of human polymorphonuclear leukocytes (PMN) in response to stimulation by uropathogenic strains of Escherichia coli is dependent on the expression of Type 1 fimbriae by those strains. These PMN responses correlate with an increasing tendency of the interacting E. coli strain to be retained on hydrophobic columns. The present work assessed the measurement of relative surface hydrophobicity in relation to PMN activation. Type 1 fimbriate organisms bound most readily to Octyl-Sepharose columns and were strongly agglutinated in the salt aggregation test. In contrast, the same organisms partitioned to the dextran-rich (hydrophilic) phase of aqueous two-polymer phase systems. Electron microscopic observation of the organisms eluted from the Octyl-Sepharose columns and of the organisms recovered from both phases of the aqueous two-phase systems demonstrated, however, that both Type 1 and P-fimbriate organisms were retained on the columns and partitioned into the dextran-rich phase as a consequence of their being fimbriate and failed to identify this as a major factor in the activation of PMN. In addition electron microscopy demonstrated that each P-fimbriate population had fewer organisms expressing fimbriae than did Type 1 fimbriate populations, confirming the importance of phase variation as a factor affecting the physicochemical characteristics of a bacterial population.     

Debaryomyces hansenii strains with different cell sizes and surface physicochemical properties adhere differently to a solid agarose surface

The initial adhesion of four Debaryomyces hansenii strains to a solid agarose surface was investigated and correlated with their cell size and some cell surface physicochemical properties, i.e. (i) hydrophobicity and (ii) electron donor/acceptor ability. One strain adhered very poorly, whereas the three other strains were more adhesive. The former strain had a very hydrophilic cell surface, whereas the latter strains had more hydrophobic cell surfaces. In addition, the strain with the lowest adhesion among the adhesive strains had a more hydrophobic cell surface than the two most adhesive strains. Finally, the more adhesive the strain was, the larger it was, and the better it was to donate electrons from its cell surface. These results show a clear relationship between the cell size, the cell surface physicochemical properties, and the initial adhesion of D. hansenii. A possible explanation of this relationship is discussed.     

Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones.

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The 2.7-kb DNA fragment containing the norA gene had a long open reading frame coding for 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was similar in the two strains. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.     

Escherichia coli strains of serogroup O139 isolated from oedema disease of swine bind fibronectin

Abstract 15 Escherichia coli strains of the serogroup O139, isolated from oedema disease of swine, were examined for their ability to interact with ¹²⁵I-labelled fibronectin. All strains were positive, and all except one showed higher fibronectin binding than Staphylococcus aureus strain Cowan 1 cells (to which fibronectin bound in the order of 15% of total protein added). 7 E. coli strains isolated from diarrhoea in young piglets were also tested, and 3 were positive. 2 of these strains showed higher binding than S. aureus Cowan 1 cells. E. coli strains expressing either K99 or K88 antigen were poor binders, comparable to cells of S. aureus strain Wood 46. There was no correlation between cell surface hydrophobicity, as determined by chromatography on Octyl-Sepharose, and the fibronectin-binding property.