Biomedical subjects

B A Sanford

Publications and source records attributed to B A Sanford.

At least 19 recordsLinked to original sources

PubMed1997

Enzyme-linked lectinsorbent assay measures N-acetyl-D-glucosamine in matrix of biofilm produced by Staphylococcus epidermidis.

An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification of in situ biofilm produced by Staphylococcus epidermidis in polystyrene 96-well tissue culture plates with phosphatase-labeled wheat germ agglutinin (WGA) as a specific probe for the GlcNAcbeta-1,4n component of exocellular matrix material (EMM) that is responsible for intercellular adhesion and accumulation. The ELLA and the modified Christensen dye assay were used to test 13 laboratory strains of coagulase-negative staphylococci and 10 clinical isolates of S. epidermidis. Four biofilm-positive laboratory strains of S. epidermidis were positive by both tests, and six biofilm-negative strains were negative by both. One strain of S. hemolyticus was positive by the ELLA only. Two of the 10 clinical isolates of S. epidermidis were positive by both assays, two were negative by both, and the remaining were positive by the ELLA only. The ELLA was objective, reproducible, specific, sensitive, and useful for screening strains for their capacity to adhere to plastic, produce EMM, and form biofilm.

Acetylglucosamine↗

PubMed1996

A dual fluorescence technique for visualization of Staphylococcus epidermidis biofilm using scanning confocal laser microscopy.

A new dual fluorescence technique is described which, when combined with scanning confocal laser microscopy (SCLM), can be used to visualize the components of biofilm produced by Staphylococcus epidermidis. Chemostat cultures of RP62A (a well-characterized slime-producing strain of S. epidermidis) were used to produce mature biofilm on polyvinylchloride (PVC) disks immobilized in a modified Robbins device using a 'seed' and 'feed' model system. Serial horizontal and vertical optical thin sections, as well as three-dimensional computer reconstructions, were obtained on in situ biofilm using the dual fluorescence procedure. Bacteria were visualized by green autofluorescence excited at 488 nm with an Argon laser. Cell-associated and exocellular matrix material (slime) was visualized by red fluorescence excited at 568 nm with a Krypton laser after interaction of the biofilm with Texas Red-labeled wheat germ agglutinin which is a slime-specific lectin marker. Structural analysis revealed that the cocci grew in slime-embedded cell clusters forming distinct conical-shaped microcolonies. Interspersed open channels served to connect the bulk liquid with the deepest layers of the mature, hydrated biofilm which increased overall surface area and likely facilitated the exchange of nutrients and waste products throughout the biofilm. The combined dual fluorescence technique and SCLM is potentially useful as a specific noninvasive tool for studying the effect of antimicrobial agents on the process of biofilm formation and for the characterization of the architecture of S. epidermidis biofilm formed in in vivo and in vitro on medical grade virgin or modified inert polymer surfaces.

Biofilms↗

PubMed1995

Lectin-biotin assay for slime present in in situ biofilm produced by Staphylococcus epidermidis using transmission electron microscopy (TEM).

A lectin-biotin assay was developed for use in the specific detection of slime produced by Staphylococcus epidermidis RP62A and M187sp11 grown in a chemically defined medium. Mature biofilm was formed on polyvinylchloride (PVC) disks using a combined chemostat-modified Robbins device (MRD) model system. Specimens fixed in situ were: 1) stained with ruthenium red; 2) reacted overnight with biotin-labeled lectins (WGA, succinyl-WGA, Con A, or APA) followed by treatment with gold-labeled extravidin; or 3) reacted with antibodies against S. epidermidis RP62A capsular polysaccharide/adhesin (PS/A) using an immunogold procedure. WGA and succinyl-WGA (S-WGA), which specifically bind N-acetylglucosamine, were shown by TEM to react only with slime, both cell-associated and exocellular. In contrast, Con A, APA and anti-PS/A reacted with the bacterial cell surface but did not react with slime. These results indicate the usefulness of WGA lectin as a specific marker for detection of the presence and distribution of slime matrix material in S. epidermidis biofilm.

Biocompatible Materials↗

PubMed1993

Calcium- and mucin-binding proteins of staphylococci.

The association of staphylococci with the mucus gel that overlays the mucosa of the respiratory tract may lead to clearance of cocci or, in certain conditions such as cystic fibrosis (CF), to colonization. In the present study, a quantitative radioassay was used to study the effect of Ca2+, which is elevated in CF sputa, on the adhesion of 3H-labelled Staphylococcus aureus to submaxillary gland mucin immobilized in MaxiSorp 96-well, break-apart modules. Ca2+ significantly enhanced the adhesion of S. aureus (five strains) and Staphylococcus epidermidis (four strains). The reaction was specific because adhesion was not enhanced in the presence of Mg2+, Ca(2+) + EGTA (a Ca2+ chelator) or protamine and was not attributable to hydrophobicity of the test strains. Staphylococcal adhesion was significantly (P < or = 0.005) blocked in the presence of highly sialated and sulphated reagents, which suggests that Ca2+ binds to the sialic acid and sulphate residues of immobilized mucin. The Ca(2+)-binding sites on the surface of S. aureus were trypsin-sensitive; in addition, 125I-labelled solubilized S. aureus surface proteins reacted with immobilized mucin in a direct binding assay, and the reaction was significantly enhanced by Ca2+. Autoradiography demonstrated that 45Ca bound directly to two polypeptides (M(r) 170,000 and 150,000) of solubilized staphylococcal surface proteins separated by SDS-PAGE, and that 125I-labelled mucin bound directly to three staphylococcal polypeptides (M(r) 40,000, 35,000, and 29,000). These results suggest that S. aureus adhesion to mucin is mediated by at least two mechanisms: via Ca(2+)-binding surface proteins in the presence of Ca2+ and via mucin-binding surface proteins unrelated to Ca2+.

Bacterial Adhesion↗

PubMed1989

Binding of staphylococci to mucus in vivo and in vitro.

The association of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus with tissues of the upper respiratory tract were compared by using an in vivo ferret model. Ferrets were challenged intranasally with a 1-ml volume of radiolabeled staphylococci (3 mg [dry weight]), were allowed to clear the bacteria in vivo for 90 min, and were sacrificed. Tissues from the right nasal fossa were harvested and processed for radioassay or histology. Of the recoverable staphylococci, greater than or equal to 96% was associated with mucus gel overlaying mucosa of the turbinates. A quantitative radioassay was developed to study the binding of labeled staphylococci to immobilized crude ferret nasal mucin (FM) and bovine submaxillary gland mucin (BM). Binding showed saturation kinetics and was blocked specifically by BM but not by human Tamm-Horsfall glycoprotein nor orosomucoid. Binding to both FM and BM was significantly inhibited (P less than or equal to 0.01) when cocci were pretreated with trypsin but not when treated with beta-galactosidase or sodium metaperiodate (except for binding of S. saprophyticus to FM). These results suggest that mucin-binding receptors of the cocci may have protein components. The staphylococcus-binding receptors of both FM and BM appear to contain protein components, based on sensitivity to pretreatment with trypsin.

Amino Sugars↗

PubMed1989

Antibody response to Staphylococcus aureus surface proteins in rabbits with persistent osteomyelitis after treatment with demineralized bone implants.

A rabbit model was used to study the effect of allogeneic demineralized bone powder (DBP) implants on the persistence of Staphylococcus aureus osteomyelitis. Thirty-one rabbits with chronic osteomyelitis of the tibia established by day 21, were started on systemic antibiotics followed by either no additional treatment or debridement plus either DBP (with or without supplemental antibiotics) or supplemental antibiotics only. On day 21, cultures showed a mean of 2 x 10(4) CFU/mg of debrided osseous material. By day 70, the treatment most effective in clearing infection was found in animals treated with supplemental antibiotics only (mean of 142 +/- 116 CFU/mg). In contrast, infection persisted at a 7- to 10-fold-higher level in animals receiving DBP with and without supplemental antibiotics; these results suggest that DBP contributed to persistence of infection. Longitudinal sera were tested again staphylococcal sonic extracts by immunoblot. Detection of numerous probe-positive bands indicated complex but remarkably similar antibody responses among infected animals. Antibodies attached directly to the cell surfaces of staphylococci as shown by immunogold and blocked the binding of organisms to HEp-2 and human fetal lung cells in a radioadherence assay. Antibodies could be absorbed out by intact organisms and were unreactive by immunoblot against antigens derived from cells pretreated with pronase, proteinase K, or lysostaphin. These results indicate that the major response was directed against staphylococcal cell surface proteins. Surprisingly, only one major band (molecular weight, approximately 12,000) was detected when a homologous in vivo antigen preparation was studied by immunoblot. Antibody reactive against this peptide did not appear to react with staphylococci grown in vitro.

Animals↗

PubMed1989

In vivo localization of Staphylococcus aureus in nasal tissues of healthy and influenza A virus-infected ferrets.

An in vivo ferret model was used to study the association of Staphylococcus aureus with specific tissues of the nasal cavity in both control and influenza A virus-infected animals. Ferrets were inoculated intranasally with various doses of influenza A3/Hong Kong/1/68 virus. On Days 2, 5, 9 and 14, four or five virus-inoculated and two uninoculated controls were challenged intranasally with a 1-ml volume of radiolabeled S. aureus (3 mg dry wt), a clinical isolate of low passage history. Ferrets were allowed to clear the staphylococci in vivo for 60 to 90 min before sacrifice. The animals were anesthetized, exsanguinated, and decapitated, and the lower jaw was removed. The nasal fossae were exposed by dissection and turbinates from the left nasal fossa were used for virus isolation. The median septum and tissues from the right nasal fossa, which included vestibule and anterior and posterior turbinates, were harvested and processed for radioassay. The percentage of recoverable staphylococci from virus-infected ferrets (Days 2 and 5) was greater than or equal to 10-fold higher compared with controls and animals infected with suboptimal doses of virus; greater than or equal to 76% of the recoverable staphylococci, whether from controls or virus-infected animals, was associated with the anterior turbinates. Histologic examination of the anterior turbinates from virus-infected ferrets, particularly on Days 2 and 5 postexposure to virus, showed that the staphylococci were adhering to desquamating respiratory epithelial cells. In contrast, the anterior turbinates from control ferrets uninoculated with virus and posterior turbinates from both control and virus-infected animals showed no evidence of bacteria adhering to host cells; instead, the staphylococci were found in association with the mucus gel layer of respiratory mucosa. Examination of vestibular tissue showed staphylococci in association with cells of the stratum granulosum in both virus-infected and control animals. Results of this study suggest that the early events of S. aureus interaction with different sites of ferret nasal tissues are effected by different mechanisms, and that the interaction is significantly enhanced by virus-infection.

Animals↗

PubMed1988

Luminol-dependent chemiluminescence microassay for phagocytic function.

A luminol-dependent chemiluminescence (CL) microassay was developed to measure phagocytic function of peripheral blood leukocytes. Buffy coats, obtained by centrifugation of only 100 microliter of whole blood, provided an enriched population of polymorphonuclear leukocytes (PMNs). The total reaction mixture, consisting of leukocytes-luminol-inducer (opsonized zymosan), was 450 microliter. Peak CL activity was seen 5 min after addition of inducer at 37 degrees C with cells tested within 60 min after collection. Tests to determine precision and reproducibility of the microassay gave a coefficient of variation of 8.5% and 11%, respectively. There was no significant difference between the mean peak CL values for 20 healthy adult donors compared to 14 premature neonates, however, the newborns' CL activity declined more rapidly; CL activity was severely depressed in cells obtained from a patient with chronic granulomatous disease. Results suggest that this microassay provides a simple, rapid, and reliable test of phagocytic function in cases where the amount of blood available for testing is limited.

Humans↗

PubMed1987

Effect of isomers of swainsonine on glycosidase activity and glycoprotein processing.

The chemical synthesis of swainsonine [(1S,2R,8R,8 alpha R)-trihydroxyindolizidine] from trans-1,4-dichloro-2-butene was previously described [Adams, C. E., Walker, F. J., & Sharpless, K. B. (1985) J. Org. Chem. 50, 420-424]. A modification of that synthesis provided two other isomers, referred to here as "Glc-swainsonine" [(1S,2S,8R,8 alpha R)-trihydroxyindolizidine] and "Ido-swainsonine" [(1S,2S,8S,8 alpha R)-trihydroxyindolizidine]. To determine whether these new compounds had biological activity, they were compared to swainsonine as inhibitors of a number of commercially available glycosidases. While swainsonine is a potent inhibitor of jack bean alpha-mannosidase but does not inhibit other glycosidases, its two isomers were inactive on alpha-mannosidase but did inhibit other enzymes. Thus, Glc-swainsonine was an inhibitor of the fungal alpha-glucosidase amyloglucosidase, and this inhibition was of a competitive nature (Ki = 5 X 10(-5) M) with respect to the substrate p-nitrophenyl alpha-D-glucopyranoside. This alkaloid also inhibited beta-glucosidase, but much less effectively than alpha-glucosidase. On the other hand, Ido-swainsonine was more effective toward beta-glucosidase than toward alpha-glucosidase, and this inhibition was also of a competitive nature. None of these inhibitors were effective against beta-mannosidase or alpha- or beta-galactosidase. Glc-swainsonine was also tested against the glycoprotein processing glycosidases. Surprisingly, in this respect, the alkaloid was like swainsonine in that it inhibited mannosidase II but had no effect or only slight effect on glucosidase I, glucosidase II, and mannosidase I. Glc-swainsonine also inhibited glycoprotein processing in cell culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMed1987

Bacterial adherence to the upper respiratory tract of ferrets infected with influenza A virus.

A ferret model was used to study bacterial adherence in animals with influenza. Ferrets were inoculated intranasally with influenza A3/Hong Kong/1/68 virus. Antiviral serum antibodies were apparent by Day 5. On Days 3, 5, 7, 9, and 11, three virus-inoculated and two uninoculated controls were anesthetized, exsanguinated, and decapitated, and the lower jaw was removed. Each animal was inoculated intranasally with a 1-ml suspension containing 20 mg (dry wt) of either 3H-labeled Staphylococcus aureus or 3H-labeled group B Streptococcus type Ia and incubated for 45 min at ambient temperature. In animals challenged with staphylococci, 80% of the original inoculum remained free in suspension; of the remaining 20%, the distribution in the upper respiratory tracts of virus-infected and control animals was significantly different. Of the staphylococci remaining in the nasopharynx of control animals, 74% was present in mucinous plugs, 11% was bound to host cells present in washes of the nasal cavity, and 15% was released by protease treatment of the nasopharynx. Of the staphylococci remaining in the upper respiratory tract of virus-infected ferrets, 36% was recovered in plugs, 24% was bound to cells in nasal washes, and 40% was released by enzyme treatment. Overall, adherence-positive staphylococci represented 64% of recoverable bacteria in virus-infected ferrets versus 26% in controls. Adherence was increased twofold (Days 5 and 7) to threefold (Days 3, 9, and 11) in virus-infected ferrets compared to uninfected controls. In contrast, only 7% of the original streptococcal inoculum was recovered from virus-infected and uninfected control animals and virus infection did not enhance streptococcal adherence except for an approximately threefold increase that was seen on Day 11.

Animals↗

PubMed1986

Detection of staphylococcal membrane receptors on virus-infected cells by direct adhesin overlay.

We partially characterized the interaction between 125I-labeled surface proteins of Staphylococcus aureus, obtained by thermal extraction, and purified plasma membranes from uninfected and influenza virus A/FM/1/47-infected Madin-Darby canine kidney cells. The radioactivity profile of surface-labeled proteins, derived from anion-exchange high-pressure liquid chromatography, showed a mixture of acidic polypeptides in a peak which contained approximately 5% of the total protein injected; adsorption with purified plasma membranes reduced radioactivity by 5 to 7% and altered elution profiles. Using a direct adhesin overlay procedure, we found that these surface-labeled proteins reacted with polypeptides located on the external surface of plasma membranes which were shared by both virus-infected and uninfected cells or were unique to virus-infected cells. Our data may help explain the enhanced adherence of S. aureus to influenza virus A-infected cells in vitro.

Adhesiveness↗

PubMed1986

Characterization of clinical strains of Staphylococcus aureus associated with pneumonia.

A total of 5 Staphylococcus aureus strains from patients with postinfluenzal staphylococcal pneumonia, 7 from burn patients with staphylococcal pneumonia, and 21 from the nasopharynx of carriers were phenotypically characterized. All or most strains produced coagulase, clumping factor, DNase, thermostable DNase, protease, gelatinase, lipase, and pigment; the strains were low to moderate producers of extracellular protein A, fibrinolysin, and alpha-hemolysin. All strains were sensitive to mercury, half were sensitive to arsenate and cadmium, and 67 to 92% were resistant to penicillin. Differences between strains were not statistically significant. Cell surface hydrophobicity was determined by measuring percent adsorption to hexadecane. Hydrophobicity of postinfluenzal staphylococcal pneumonia strains was significantly lower than that of pneumonia strains from burn patients and carriers (P less than 0.005). Immunoblot experiments with sera immune to one clinical test strain allowed the separation of all strains into three groups based on probe-positive reactions with primarily four staphylococcal polypeptides (154,200, 130,000, 77,100, and 64,400 molecular weight). The difference in distribution of clinical and carrier strains was highly significant (P = 0.007).

Adolescent↗

PubMed1986

Staphylococcus aureus adherence to influenza A virus-infected and control cell cultures: evidence for multiple adhesins.

During major epidemics with influenza, there is an increased number of pneumonias due to Staphylococcus aureus with a subsequent high mortality rate. We have postulated that influenza A virus infection of host cells promotes the adherence of S. aureus ultimately resulting in bacterial superinfection. In the present study we compared the adherence of seven strains of 3H-labeled S. aureus to Madin-Darby canine kidney (MDCK) cell monolayers, uninfected and infected with influenza A/FM/1/47 virus. Test strains included: Cowan I; a Cowan I protein A-deficient mutant (PA-); EMS, a protein A and clumping factor-deficient mutant; HSmR; 52A5, a teichoic acid-deficient mutant of HSmR; M, an encapsulated strain; and, No. 1071, a clinical isolate. By radioassay, six of the seven strains demonstrated significantly enhanced adherence to virus-infected cell monolayers compared to uninfected controls; only the M strain was adherence negative. Surface hydrophobicity of the staphylococci did not correlate with their ability to adhere. Four strains of labeled staphylococci (Cowan I, PA-, EMS, and No. 1071), untreated or treated with 2.5% trypsin, 1.25% protease, or by autoclaving, were tested in the radioassay. Protease treatment, which was more effective than trypsin treatment, reduced adherence of all four test strains by 74-96%. Results of heat treatment suggested the presence of both thermolabile and thermostable adhesins. Staphylococcal thermal extracts, profiled by anion-exchange HPLC, were used to pretreat monolayers in a blocking radioassay. Adherence was decreased to control cells (9-78%) and to virus-infected cells (56-90%). The data suggest that multiple distinct surface proteins mediate the binding of S. aureus to uninfected and influenza A virus-infected cells.

Adhesiveness↗

PubMed1984

The pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, inhibits glycoprotein processing.

2,5-Dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) is a pyrrolidine alkaloid that was isolated from the plant, Lonchocarpus sericeus. In the present study, DMDP was tested as an inhibitor of glycoprotein processing. MDCK cells were infected with influenza virus and the virus was raised in the presence of various amounts of DMDP. The glycoproteins were labeled by the addition of [2-3H]mannose or [1-3H]galactose to the medium. The virus was isolated by differential centrifugation and treated with Pronase to obtain glycopeptides. These glycopeptides were isolated by chromatography on Bio-Gel P-4, then digested with endoglucosaminidase H (Endo H) and rechromatographed on the Bio-Gel P-4 column. In the control virus, more than 70% of the glycopeptides were resistant to Endo H and were previously characterized as complex types of oligosaccharides. The remaining 20-25% are sensitive to Endo H and are of the high-mannose type. However, in the presence of DMDP (250 micrograms/ml), more than 80% of the glycopeptides are susceptible to digestion by Endo H. The oligosaccharide released by this treatment sized like a hexose11-12GlcNAc on a calibrated column of Bio-Gel P-4, and was only slightly susceptible to alpha-mannosidase treatment. This oligosaccharide was also labeled in the glucose moieties by growing the virus in [1-3H]galactose in the presence of DMDP. Following isolation, the oligosaccharide was subjected to complete methylation. Acid hydrolysis of the methylated oligosaccharide gave three methylated glucose derivatives, corresponding to 2,3,4,6-tetramethylglucose, 3,4,6-trimethylglucose, and 2,4,6-trimethylglucose in almost equal amounts. These data indicate that the oligosaccharide is a Glc3Man8-9-GlcNAc and that DMDP inhibits glucosidase I. Similar results were obtained with the cellular glycoproteins. DMDP did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it inhibit virus production as measured by plaque counts or hemagglutination assays. DMDP did cause some inhibition of mannose incorporation into the lipid-linked monosaccharides, but incorporation into lipid-linked oligosaccharides was not greatly affected, and incorporation into protein was stimulated. These results suggest that the pyrrolidine alkaloids are a new class of processing inhibitors.

Alkaloids↗

PubMed1983

Castanospermine inhibits the processing of the oligosaccharide portion of the influenza viral hemagglutinin.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that inhibits alpha- and beta-glucosidase in fibroblast extracts [Saul, R., Chambers, J. P., Molyneux, R. J., & Elbein, A. D. (1983) Arch. Biochem. Biophys. 221, 593-597]. In the present study, castanospermine also proved to be a potent inhibitor of glycoprotein processing by virtue of the fact that it inhibits glucosidase I. Thus, when influenza virus was raised in the presence of castanospermine, at 10 micrograms/mL or higher, 80-90% of the viral glycopeptides were susceptible to the action of endoglucosaminidase H, whereas in the normal virus 70% of the glycopeptides are resistant to this enzyme. The major oligosaccharide released by endoglucosaminidase H from castanospermine-grown virus migrated like a hexose10GlcNac on a calibrated Bio-Gel P-4 column. This oligosaccharide was characterized as a Glc 3 Man 7 GlcNAc on the basis of various enzymatic treatments, as well as by methylation analysis of the [2-3H]-mannose-labeled or [6-3H]galactose-labeled oligosaccharide. The presence of three glucose residues in the oligosaccharide was also confirmed by periodate oxidation studies of the [6-3H]galactose-labeled hexose10GlcNAc. Castanospermine did not inhibit the incorporation of [3H]leucine or [14C]alanine into protein in MDCK cells at levels as high as 50 micrograms/mL. In addition, influenza virus produced in the presence of this alkaloid were fully infective and apparently produced in similar amounts to that of control cells, as determined by plaque counts. Castanospermine did, however, cause considerable changes in cell surface properties, since MDCK cells grown in 10 micrograms/mL castanospermine were able to bind twice as much [3H]concanavalin A as were control cells.

Alkaloids↗

PubMed1983

Antibody to kidney antigen in the urine of patients with urinary tract infections.

One hundred six urine specimens from 26 patients with acute and chronic pyelonephritis and cystitis were tested by radioimmunoassay to determine (1) whether antibody to normal human kidney antigen was present and (2) whether the presence or absence of this antibody correlated quantitatively with antibody to the patient's own infecting organism. Of the 106 urine specimens tested, 55 (52%) contained elevated antibody to human kidney antigen. For 80 (75%) of 106 urine specimens there was a correlation between the results of quantitative assays for antibody levels to kidney antigen and to the bacterial antigen. Indirect fluorescent antibody studies of thin sections of normal human kidneys and a patient's urine containing elevated levels of antibody to kidney antigen and to bacterial antigen demonstrated diffuse renal localization. Results indicate the occurrence of antibody to kidney antigen, particularly in urine specimens from patients with chronic pyelonephritis and from urine specimens containing elevated levels of antibody to bacterial antigen.

Antibodies↗

PubMed1982

Factors influencing adherence of Staphylococcus aureus to influenza A virus-infected cell cultures.

A quantitative radioassay was used to study the factors affecting the adherence of (3)H-labeled Staphylococcus aureus 1071 to Madin-Darby canine kidney cells, either uninfected or infected with the human FM1 strain of influenza A virus. Enhanced adherence to virus-infected cell cultures was independent of nonspecific factors-hydrophobicity, surface charge, and monolayer cell density. Viral hemagglutinin and neuraminidase did not act as the cell receptors for S. aureus because the growth of virus-inoculated monolayers in tunicamycin (an inhibitor of glycosylation) and the pretreatment of virus-infected cells with trypsin or virus-specific antiserum, which inhibit hemadsorption, had no effect on staphylococcal adherence. In contrast, adherence to uninfected and virus-infected cells was significantly reduced by protease treatment of either monolayers or staphylococci and by heat treatment of staphylococci. UV irradiation and treatment of bacteria with 0.1 M EDTA enhanced adherence. Pretreatment of monolayers with a thermal extract of S. aureus decreased adherence by 89 to 97%. The staphylococcal adhesin, which blocks adherence to virus-infected cells, appears to be a remarkably heat-stable, protease- and trypsin-sensitive macromolecule which is distinct from protein A, clumping factor, and teichoic acid. Lastly, pretreatment of S. aureus with human fibrinogen significantly enhanced adherence to virus-infected cells (P < 0.005) compared with binding with untreated S. aureus. The treated bacteria also adsorbed virus out of suspension. These results suggest that fibrinogen forms a bridge between S. aureus and receptors present on virus-infected cells and free virus.

Adhesiveness↗

PubMed1982

Fibrinogen-mediated adherence of group A Streptococcus to influenza A virus-infected cell cultures.

A quantitative radioassay was used to study the adherence of group A Streptococcus to Madin-Darby canine kidney cells infected with influenza A virus (strains FM1, Jap 305, and NWS) and reacted with fibrinogen. Treatment of virus-infected cell cultures with human fibrinogen significantly enhanced streptococcal adherence (P less than 0.0005) compared with adherence to untreated, virus-infected cells and uninfected control cells. Enhanced adherence was not seen with NWS virus-infected cell cultures or with virus-infected cells treated with human fibronectin, canine fibrinogen, or porcine fibrinogen. Human fibrinogen was shown to bind directly to surface membranes of virus-infected cells. Virus-infected cell cultures were incubated in the presence of tunicamycin, an antibiotic that inhibits glycosylation of virus-specific surface membrane glycoproteins. We found that with increasing antibiotic concentration there was a progressive decrease in fibrinogen-mediated streptococcal adherence. Adherence of 3H-labeled streptococci to fibrinogen-treated, virus-infected cell cultures showed saturation kinetics and could be blocked with monospecific antibodies against fibrinogen. These results suggest that human fibrinogen binds to a glycoprotein moiety on the surface of influenza A virus-infected cells, and that once bound the fibrinogen molecule acts as an "acquired" receptor for the attachment of group A Streptococcus. We postulate that this mechanism, it if occurs in vivo, might help explain the observed association between influenza A virus infection and subsequent bacterial superinfection with group A Streptococcus.

Adhesiveness↗