Barbara A. Sanford

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 byStaphylococcus epidermidis. Chemostat cultures of RP62A (a well-characterized slime-producing strain ofS. epidermidis) were used to produce mature biofilm on polyvinylcholoride (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 onin 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 ofS. epidermidis biofilm formedin vivo andin vitro on medical grade virgin or modified inert polymer surfaces.

Bacterial Adherence to the Upper Respiratory Tract of Ferrets Infected with Influenza A Virus

Abstract 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 la 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.

Enzyme-Linked Lectinsorbent Assay Measures N-Acetyl-D-Glucosamine in Matrix of Biofilm Produced by Staphylococcus epidermidis

Abstract. 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 GlcNAcβ-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.

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 byStaphylococcus 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 fixedin 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 againstS. epidermidis RP62A capsular polysaccharide/adhesin (PS/A) using an immunogold procedure. WGA and succinyl-WGA (S-WGA), which specifically bindN-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 inS. epidermidis biofilm.

Staphylococcus aureus Adherence to Influenza A Virus-Infected and Control Cell Cultures: Evidence for Multiple Adhesins

Abstract 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.

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+.

In Vivo Localization of Staphylococcus aureus in Nasal Tissues of Healthy and Influenza A Virus-Infected Ferrets

Abstract 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 ≥ 10-fold higher compared with controls and animals infected with suboptimal doses of virus; ≥ 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.

Adherence of Group B Streptococci and Human Erythrocytes to Influenza A Virus-Infected MDCK Cells

Summary Scanning electron microscopy was used to compare the adherence of group B streptococci (types Ia, Ic, and II) and human erythrocytes to canine kidney cell monolayers inoculated 24-48 hr earlier with influenza A/NWS/33 virus. Streptococci and erythrocytes adhered to the kidney cell surface membranes and filamentous projections of some but not all the cells in virus-inoculated monolayers, while they did not adhere to control monolayers. Streptococcal adherence was blocked when the virus-infected cells were first exposed to erythrocytes. Likewise, hemadsorption was blocked when the virus-infected cells were first exposed to streptococci. In mixed bacterial adherence-hemadsorption tests bacteria adhered only to hemadsorption-positive virus-infected cells. Adherence of streptococci types Ia and Ic and erythrocytes was inhibited when the streptococci and erythrocytes were pretreated with receptor destroying enzyme. However, enzyme treatment of type II streptococci did not affect their ability to adhere to virus-infected cells.

Evidence for chronic viral infections in human arteries.

Summary Evidence is presented that measlesvirus antibody and herpesvirus antibody are sometimes present in human aortic tissues in a state not readily dissociable by extensive washing with buffered physiologic saline. This antibody is readily released from the tissues by high molarity potassium iodide solution, a treatment known to dissociate antigen-antibody complexes. We postulate: (1) that viral antigen-antibody complexes are sometimes present in aortic tissues; and (2) that a significant cause of vascular tissue injury in humans may be viral infection of the blood vessels themselves and/or deposit of viral antigen-antibody complexes in the vascular tissues.

Radioimmunoassay procedure for quantitating bacterial antibody in human sera

A RIA system was developed to detect antibodies in human sera against bacteria. Sonicates of Escherichia coli and Fusobacterium polymorphum were used as antigens to sensitize plastic-coated beads; antibodies to these antigens were detected with 125I-labeled antihuman globulin. Serum antibody titers against E. coli were determined by the serial dilution method; from the results the standard curve principle was applied in determining the relative amounts of antibodies against E. coli in serum samples tested at a single dilution. The coefficient of variation of the RIA procedure was less than 10%. Serum titers obtained by the RIA and indirect immunofluorescence test were compared; RIA was more sensitive, quantitative and objective. Absorption studies, using E. coli and F. polymorphum absorbentes against E. coli and F. polymorphum. This RIA procedure offers a combination of desirable advantages; it is sensitive, specific, objective, quantitative, and easy to perform.