A novel cationic-peptide coating for the prevention of microbial colonization on contact lenses.

AIMS: To develop an antimicrobial peptide with broad spectrum activity against bacteria implicated in biomaterial infection of low toxicity to mammalian cells and retaining its antimicrobial activity when covalently bound to a biomaterial surface. METHODS AND RESULTS: A synthetic peptide (melimine) was produced by combining portions of the antimicrobial cationic peptides mellitin and protamine. In contrast to the parent peptide melittin which lysed sheep red blood cells at >10 microg ml(-1), melimine lysed sheep red blood cells only at concentrations >2500 microg ml(-1), well above bactericidal concentrations. Additionally, melimine was found to be stable to heat sterilization. Evaluation by electron microscopy showed that exposure of both Pseudomonas aeruginosa and Staphylococcus aureus to melimine at the minimal inhibitory concentration (MIC) produced changes in the structure of the bacterial membranes. Further, repeated passage of these bacteria in sub-MIC concentrations of melimine did not result in an increase in the MIC. Melimine was tested for its ability to reduce bacterial adhesion to contact lenses when adsorbed or covalently attached. Approximately 80% reduction in viable bacteria was seen against both P. aeruginosa and S. aureus for 500 microg per lens adsorbed melimine. Covalently linked melimine (18 +/- 4 microg per lens) showed >70% reduction of these bacteria to the lens. CONCLUSIONS: We have designed and tested a synthetic peptide melimine incorporating active regions of protamine and mellitin which may represent a good candidate for development as an antimicrobial coating for biomaterials. SIGNIFICANCE AND IMPACT OF THE STUDY: Infection associated with the use of biomaterials remains a major barrier to the long-term use of medical devices. The antimicrobial peptide melimine is an excellent candidate for development as an antimicrobial coating for such devices.

Lysostaphin treatment of methicillin-resistant Staphylococcus aureus keratitis in the rabbit.

PURPOSE: To determine the efficacy of lysostaphin treatment of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a rabbit model. METHODS: The sensitivity to lysostaphin and vancomycin were compared for 34 MRSA and 12 methicillin-sensitive strains. Methicillin-resistant S. aureus strain 301 (MRSA 301) or a methicillin-sensitive strain of low virulence, ISP546, was intrastromally injected into rabbit corneas. Rabbit eyes were treated topically every 30 minutes from 4 to 9 or 10 to 15 hours postinfection with 0.28% lysostaphin or 5.0% vancomycin. Rabbits were killed and corneas were excised and cultured to determine the number of colony forming units (CFU) per cornea. RESULTS: Ninety percent minimal inhibitory concentrations were at least 19-fold lower for lysostaphin than for vancomycin. With early therapy (4 -9 hours postinfection) lysostaphin sterilized all MRSA 301-infected corneas, whereas untreated corneas contained 6.52 log CFU/cornea (P < or = 0.0001). Corneas infected with MRSA 301 and treated similarly with vancomycin retained 2.3 +/-0.85 log CFU/cornea, and none were sterile. When therapy was begun later (10-15 hours postinfection) the residual bacteria in lysostaphin-treated eyes were significantly less numerous than in vancomycin-treated eyes (0.58 +/- 0.34 vs. 5.83 +/- 0.16 log CFU/cornea, respectively; P < or = 0.0001). Three experiments were performed to demonstrate that lysostaphin penetrated the cornea to kill bacteria in vivo; lysostaphin-treated eyes were found to recover from infection, bacteria that did not cause epithelial defects (ISP546) were susceptible to lysostaphin, and inhibition of lysostaphin when harvesting corneas did not alter the observed therapeutic values of lysostaphin. CONCLUSIONS: Lysostaphin is very effective in treating keratitis mediated by methicillin-sensitive or methicillin-resistant S. aureus.

Staphylococcus corneal virulence in a new topical model of infection.

PURPOSE: To develop a topical inoculation model of Staphylococcus aureus keratitis in which scarification, contact lenses, and spermidine are used to inhibit the host defenses and to investigate the role of alpha-toxin in this infection. METHODS: An alpha-toxin-positive parent strain (8325-4), its isogenic alpha-toxin-negative mutant (DU1090), and a genetically rescued form of the mutant (DU1090/pDU1212) were bound to rabbit-specific contact lenses, treated with spermidine (50 mM), and applied to scarified rabbit corneas. Eyes were treated topically with spermidine before and after lens application. Eyes were graded for disease by slit lamp examination (SLE) every 6 hours until 24 hours PI (PI), and erosion diameters were measured. Histopathologic changes and colony forming units (CFUs) of bacteria were determined. RESULTS: Spermidine treatment and inoculation of eyes with Staphylococcus on contact lenses resulted in significant increases in both CFUs per cornea (P = 0.0041) and SLE score (P or= 0.1959) multilog increase in CFUs over the inoculum at 24 hours PI. The alpha-toxin-producing strains, 8325-4 and DU1090/pDU1212, caused significantly more disease than the alpha-toxin-deficient mutant DU1090 at 24 hours PI (P

Phospholipase A(2) in rabbit tears: a host defense against Staphylococcus aureus.

PURPOSE: This study analyzed rabbit tears for anti-staphylococcal activity, the role of phospholipase A(2) (PLA2) in this reaction, and the ability of enzyme inhibitors to promote bacterial survival. METHODS: Contact lenses with Staphylococcus aureus were applied to scarified rabbit eyes. The colony-forming units (CFU) per cornea or lens were determined and pathology was scored by slit-lamp examination (SLE). The bactericidal activity was measured by incubating bacteria with rabbit tears or PLA2 at 33 degrees or 37 degrees C. Radiolabeled S. aureus was incubated with PLA2 or tears to quantify the release of a membrane component that was identified by thin-layer chromatography. Inhibitors of these reactions were also analyzed. RESULTS: Application of Staphylococcus, on contact lenses, to rabbit corneas resulted in bacterial killing and limited inflammation. Incubation of tears and bacteria (1:1; v/v) in tryptic soy broth at 33 degrees C decreased CFU approximately 4 logs. Tears (> or =30 microl) or PLA2 (> or =30 U) incubated with bacteria in phosphate-buffered saline were bactericidal. PLA2 (> or =0.2 U) or tears (> or =2 microl) cleaved bacterial membranes, liberating arachidonic acid. Spermidine or tetracaine inhibited cleavage of bacterial membranes by tears or PLA2 and spermidine promoted bacterial survival and growth in tears. Tears (60 microl) killed >99% of the bacterial inoculum, whereas bacteria incubated in tears plus spermidine approximately doubled in number. CONCLUSIONS: PLA2 in rabbit tears kills Staphylococcus by hydrolyzing bacterial membranes to release arachidonic acid. Spermidine and tetracaine inhibited PLA2 activity and spermidine protected Staphylococcus from PLA2 in rabbit tears.

Furanones as potential anti-bacterial coatings on biomaterials.

A major barrier to the long-term use of medical devices is development of infection. Staphylococcus epidermidis is one of the most common bacterial isolates from these infections with biofilm formation being their main virulence factor. Currently, antibiotics are used as the main form of therapy. However with the emergence of staphylococcal resistance, this form of therapy is fast becoming ineffective. In this study, the ability of a novel furanone antimicrobial compound to inhibit S. epidermidis adhesion and slime production on biomaterials was assessed. Furanones were physically adsorbed to various biomaterials and bacterial load determined using radioactivity. Slime production was assessed using a colorimetric method. Additionally, the effect of the furanone coating on material surface characteristics such as hydrophobicity and surface roughness was also investigated. The results of this study indicated that there was no significant change in the material characteristics after furanone coating. Bacterial load on all furanone-coated materials was significantly reduced (p<0.001) as was slime production (p<0.001). There is a potential for furanone-coated biomaterials to be used to reduce medical device-associated infections.

Biological performance of a novel synthetic furanone-based antimicrobial.

Infection of medical devices causes significant morbidity and mortality and considerable research effort has been directed at solving this problem. The aim of this study was to assess the biological performance of a novel furanone compound that has potential as an anti-infective coating for medical devices. This study examined in vitro leukocyte response following exposure to the antibacterial 3-(1'-bromohexyl)-5-dibromomethylene-2(5H)-furanone and assessed the tissue response following subcutaneous implantation of the furanone compound covalently bound to polystyrene (PS). Peripheral human blood was exposed to furanones in solution for 1h and flow cytometry used to analyse viability and changes in expression of surface receptors CD11b/CD18 and CD44. Flow cytometry results from propidium iodide stained cell suspensions suggested that the leukocytes were viable after exposure to furanones in whole blood. No significant difference was found in the expression of CD11b/CD18 and CD44 between the furanone exposed samples and the negative control for neutrophils suggesting that the furanones themselves do not activate these leukocytes. The positive control lipopolysaccharide significantly up-regulated CD11b/CD18 and slightly down-regulated CD44 on both PMNs and monocytes. In vivo studies of the tissue response to furanone covalently bound to PS showed that there was no significant difference in cellularity of capsules surrounding the disk and no significant increase in myeloperoxidase expression. These results demonstrate negligible acute inflammatory response to synthetic brominated antibacterial furanones. Future studies will focus on chronic responses and examination of in vivo efficacy.

The control of Staphylococcus epidermidis biofilm formation and in vivo infection rates by covalently bound furanones.

In order to overcome the continuing infection rate associated with biomaterials, the use of covalently bound furanones as an antibiofilm coating for biomaterials has been investigated. Furanones have previously been shown to inhibit growth of Gram-positive and Gram-negative bacteria. The aim of these studies were to covalently bind furanones to polymers and to test their efficacy for inhibiting biofilm formation of Staphylococcus epidermidis and in vivo infection rate. Two methods of covalent attachment of furanones were used. The first, a co-polymerisation with a styrene polymer, and second, a plasma-1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) reaction to produce furanone-coated catheters. Biofilm formation by S. epidermidis in vitro was inhibited by 89% for polystryene-furanone disks and by 78% by furanone-coated catheters (p<0.01). In an in vivo sheep model we found furanones were effective at controlling infection for up to 65 days. Furanones have potential to be used as a coating for biomaterials to control infection caused by S. epidermidis.

Prevention of diseases caused by Staphylococcus aureus using the peptide RIP.

Staphylococcus aureus causes many diseases including cellulitis, keratitis, osteomyelitis, septic arthritis and mastitis. The heptapeptide RIP has been shown to prevent cellulitis in mice, which was induced by S. aureus strain Smith diffuse. Here we show that RIP can also significantly reduce the overall pathology and delay the onset of disease symptoms in several other models of S. aureus infections, including: keratitis (tested in rabbits against S. aureus 8325-4), osteomyelitis (tested in rabbits against S. aureus MS), mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections) and septic arthritis (tested in mice against S. aureus LS-1). These findings substantiate that RIP is not strain specific in its inhibitory activity and that RIP is an effective inhibitor of bacterial pathology at multiple body sites following diverse routes and doses of administration. These findings strongly evidence the potential value of RIP as a chemotherapeutic agent.

An appraisal of the virulence factors associated with streptococcal endocarditis.

Platelet aggregation is believed to be a virulence factor in infective endocarditis. Other factors may be adhesion to components of thrombotic vegetations, particularly platelets, fibronectin and fibrinogen. Two strains from the Streptococcus sanguis group (SSG) were chosen for comparative study on the basis that one aggregated both human and rat platelets and the other lacked this capacity. Both strains caused endocarditis in the rat model but the aggregating strain was found in higher numbers in the excised vegetations. The nonaggregating strain was unable to bind to human or rat platelets but could bind insoluble fibronectin, insoluble fibrinogen and platelet-fibrin clots from both sources, albeit to a lesser extent than the aggregating strain. These results suggest that whereas adhesion to, and aggregation of, platelets are not essential events in the initiation of the pathogenesis of experimental endocarditis, they may be factors contributing to virulence.

An examination of the clonal variants of Serratia marcescens that infect the eye during contact lens wear.

Serratia marcescens colonises contact lenses during wear, although the frequency of isolation is generally low (0.6% contamination rate). A method for typing the S. marcescens colonising the eye or contact lens was developed, based upon ribotyping, serotyping and biotyping. Twelve different types of S. marcescens were isolated from the eyes, contact lenses, contact lens cases and fingers of contact lens wearers in the Sydney area over a 2-year period. There was no evidence of a specific type being more readily able to colonise the contact lenses than other types. Indeed, eight S. marcescens strains were isolated from the lenses and these belonged to seven types. The diversity of types isolated from the eye indicates that there is probably not a subset of S. marcescens that can colonise the eye, although the results suggest that the types of strains isolated from contact lenses are different from those isolated from nosocomial infections.

Pathogenic potential of lactobacilli.

Lactobacilli are often considered to be commensal or beneficial participants in human microbial ecology and considerable research is being carried out into the effects of the use of lactobacilli as additives in both human and animal diets. However, lactobacilli also cause some human diseases (e.g. dental caries, rheumatic vascular disease, septicaemia and infective endocarditis (IE)), and have recently been identified as potential emerging pathogens in elderly and immunocompromised patients, particularly those receiving broad spectrum antibiotic therapy. The identification of potential pathogenic traits amongst lactobacilli will therefore facilitate the use of the organisms for probiotic purposes. The ability to aggregate human platelets is considered to be a possible pathogenic trait in the progression of IE. A comparison of bacterial cell surface properties amongst L. rhamnosus strains showed that platelets were aggregated by 5/5 IE strains and 8/16 laboratory strains. For the L. paracasei subsp. paracasei strains the respective numbers were 2/5 and 2/9. However two strains, morphological mutants of a non-aggregating strain, which had been re-isolated after passaging through rats were found to aggregate platelets. No loss of aggregating function occurred on extensive subculturing of IE strains. Aggregation also occurred with 11/14 strains for five other species, namely, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salvivarius, with each species being represented indicating that the property is not uncommon in the genus. A comparison of IE and oral isolates of L. rhamnosus and L. paracasei subsp. paracasei and seven other Lactobacillus species, has shown that the binding of both fibronectin and fibrinogen by lactobacilli is greatly increased, up to 50 fold, when the pH is reduced from 7.0 to 5.0. Re-exposing the lactobacilli to a neutral pH environment releases most of the bound proteins, but the amount still remaining bound to the cell is several times more than is bound at neutral pH. Lactobacilli will also bind to the proteins that make up the extracellular matrix of endothelial cells. Lactobacilli bound significantly better to collagen types I and V than to types III and IV (p < 0.01). Further, strains isolated from IE cases, particularly L. rhamnosus strains, bound significantly better to types I and V than did 'normal' strains (p < 0.02). Type V collagen has been demonstrated at the sites of endothelial damage. Thus the binding of lactobacilli, particularly L. rhamnosus to these collagen types may be of importance in the early stages of colonization of the damaged heart valve.(ABSTRACT TRUNCATED AT 400 WORDS)

Lysostaphin is effective in treating methicillin-resistant Staphylococcus aureus endophthalmitis in the rabbit.

PURPOSE: To determine the effectiveness of lysostaphin treatment of experimental endophthalmitis caused by methicillin-resistant Staphylococcus aureus (MRSA). METHODS: In one experiment, rabbits were injected in the mid-vitreous with 50 or 200 CFU of S. aureus; untreated groups and groups injected intra-vitreally at 8 or 24 hours postinfection with vehicle or lysostaphin (0.1 mg/ml) were compared in terms of CFU/ml vitreous at 24 or 48 hours postinfection. Histopathology of untreated and treated eyes was also compared. To quantify the potency of lysostaphin, additional rabbits were injected with 50 CFU of S. aureus and untreated eyes and eyes treated at 8 hours with 0.001, 0.01 or 0.05 mg/ml were compared in terms of CFU/ml vitreous at 24 hours postinfection. RESULTS: Vitreous of untreated eyes or vehicle-treated eyes injected with 50 or 200 CFU of S. aureus contained 5-10 million CFU/ml at 24 or 48 hours postinfection. All eyes treated with lysostaphin at 8 hours postinfection had less than 1 log CFU/ml in the vitreous (P >or= 0.0001). Similarly, eyes treated with lysostaphin at 24 hours postinfection had approximately 1 log of CFU/ml at 48 hours postinfection. None of the untreated eyes were sterile and 88% or 50% of the eyes treated at 8 or 24 hours postinfection, respectively, were sterile. Eyes treated with lysostaphin at 8, but not 24, hours postinfection had less pronounced pathologic changes than the untreated eyes (P = 0.002). A significant reduction in the CFU/ml vitreous at 24 hours postinfection was obtained by treating infected eyes at 8 hours postinfection with lysostaphin at concentrations of >or=0.001 mg/ml (P

Clarithromycin for experimental Staphylococcus aureus keratitis.

PURPOSE: Clarithromycin, a macrolide antibiotic not previously tested against the common causes of bacterial keratitis, was analyzed for its effectiveness in reducing the number of viable bacteria in a Staphylococcus keratitis model. An in vivo comparison of the effectiveness of clarithromycin to erythromycin, minocycline, and tetracycline for three strains of Staphylococcus aureus was done. METHODS: Rabbit eyes were intrastromally injected with 100 colony forming units of one of three strains of S. aureus. Two strains were methicillin-sensitive (ATCC 25923 and MSSA 309) and one strain methicillin-resistant (COL). Eyes were treated every 30 minutes with 0.3% clarithromycin, erythromycin, tetracycline, or minocycline from 4 to 9 hours postinfection. The number of colony forming units (CFU) per cornea in all eyes was determined at 10 hours postinfection. RESULTS: Vehicle-treated and untreated eyes (controls) contained over 6 logs of CFU per cornea, a value significantly higher than any of the antibiotic-treated eyes (P < or = 0.0001). Clarithromycin or erythromycin therapy significantly decreased the number of CFU per cornea by approximately 5 logs in the eyes infected with the methicillin-sensitive strains and by approximately 4 logs in the eyes infected with the methicillin-resistant strain. Tetracycline and minocycline were also successful in treating these strains, but overall showed less effectiveness than clarithromycin and erythromycin. CONCLUSIONS: Clarithromycin proved to be an effective ocular medication for the therapy of experimental S. aureus keratitis. The effectiveness of clarithromycin in this model and its known effectiveness for a variety of bacterial pathogens suggests a role for this drug as a useful ocular antibiotic.

Serratia marcescens keratitis: strain-specific corneal pathogenesis in rabbits.

PURPOSE: The purpose of this study was to develop an animal model of Serratia keratitis that is suitable to demonstrate the pathology of specific strains. METHODS: Serratia marcescens ocular strains 93-1399-1 and 94-EI-185-2, and an environmental strain (ATCC 14041) were characterized in vitro in terms of their motility, metabolic profiles, ribotypes, and protease production. The strains were then analyzed in the rabbit intrastromal injection model. Slit lamp examination (SLE) and enumeration of bacteria in the cornea was conducted every 6 hours for 30 hours post-infection. In vivo motilities were analyzed by quantification of bacteria in the peripheral and central areas of infected rabbit corneas. RESULTS: All strains were similar in their metabolic activity and production of extracellular proteases. The ocular isolates were distinct from the environmental strain in their ribotyping patterns and in their motility. Each strain grew logarithmically in the cornea up to 6 hours post-infection. SLE scores increased from 0 to 30 hours post-infection for strains ATCC 14041 and 93-1399-1, while the SLE score of strain 94-EI-185-2 reached its maximum at 18 hours post-infection. Strain-specific differences in pathology were noted from 18 to 30 hours post-infection. Strain 94-EI-185-2 produced iritis but only mild corneal changes. Strain 93-1399-1 produced a severe corneal infiltrate encompassing the entire corneal surface as well as severe conjunctival inflammation and iritis. Strain ATCC 14041 produced a localized, severe, exudative corneal abscess that contained infecting bacteria. CONCLUSIONS: A rabbit model of Serratia keratitis was developed in which bacterial growth kinetics and strain-specific ocular pathologic changes were reproducible.

Role of quorum sensing by Pseudomonas aeruginosa in microbial keratitis and cystic fibrosis.

Pseudomonas aeruginosa is a ubiquitous bacterium that causes opportunistic infections in a range of host tissues and organs. Infections by P. aeruginosa are difficult to treat and hence there is interest in the development of effective therapeutics. One of the key mechanisms that P. aeruginosa uses to control the expression of many virulence factors is the N-acylated homoserine lactone (AHL) regulatory system. Hence, there is considerable interest in targeting this regulatory pathway to develop novel therapeutics for infection control. P. aeruginosa is the principal cause of microbial keratitis and of infections in cystic fibrosis (CF) sufferers, and AHL-dependent cell-to-cell signalling has been shown to be important for both infection types. However, keratitis tends to be an acute infection whereas infection of CF patients develops into a chronic, life-long infection. Thus, it is unclear whether AHL-regulated virulence plays the same role during these infections. This review presents a comparison of the role of AHL signalling in P. aeruginosa-mediated microbial keratitis and chronic lung infections of CF patients.

Differences in the pathogenesis of bacteria isolated from contact-lens-induced infiltrative conditions.

PURPOSE: The purpose of this study was to examine the ability of gram-negative bacteria isolated from the non-infectious condition contact-lens-induced acute red eye (CLARE) and infectious microbial keratitis (MK) to infect the eyes of mice. METHODS: One cornea of BALB/c mice was scratched with a needle and 5 x 10(6) bacteria applied. Bacterial types used were Pseudomonas aeruginosa, Haemophilus influenzae, Serratia marcescens, Stenotrophomonas moltophilia and Aeromonas hydrophilia. The mice were killed after 1, 4 and 24 h and examined by slit lamp, histology and microbiology. RESULTS: CLARE strains were unable to infect the mouse corneas. The eyes inoculated with the CLARE strains had low levels of polymorphonuclear leucocytes (PMN) in the stroma. All eyes that had MK contained bacteria and large numbers of PMN after 24 h. CONCLUSIONS: The development of CLARE or MK is dependent on the type of bacteria with specific bacterial types being responsible for CLARE or MK.

Adhesion and growth of Serratia marcescens on artificial closed eye tears soaked hydrogel contact lenses.

The adhesion to hydrogel contact lenses and growth of Serratia marcescens on artificial tear fluid (ATF) soaked lenses was investigated. Results indicated that a corneal ulcer isolate adhered more avidly to lenses; ATF increased adhesion for all strains tested. The contact lens induced acute red eye (CLARE) isolate adhered poorly, however, it grew to a larger extent on ATF-coated lenses. The ability of the corneal ulcer isolate to adhere to lenses may be an important factor in its pathogenicity whereas the ability of the CLARE isolate to grow on the lens in the presence of antimicrobial tear proteins may be important in the development of inflammation.

Antimicrobial peptides: a potential role in ocular therapy.

Bacterial pathogens are often involved in contact lens-related adverse responses. This study aimed to find antimicrobial peptides and proteins that effectively eradicate or inhibit ocular bacteria. The antimicrobials were screened against gram-negative and gram-positive bacteria originating from ocular sources. The viability of these ocular bacteria was measured after exposure to the peptides and proteins. Two conditions were used to grow bacteria, low nutrient phosphate-buffered saline and high nutrient tryptone soya broth. Samples were taken at different times up to 48 h. In low nutrient conditions, protamine was found to be the most effective against all strains. Melittin was very effectve against all strains except Serratia and one Pseudomonas isolate which were partially affected. In high nutrient condition, only melittin was effective in killing Staphylococcus aureus. Protamine and the combination of protamine and melittin had the greatest effect in eradicating the bacteria tested in low nutrient condition. Protamine alone and its combination with melittin may have potential therapeutic agents for ocular infections in an era of emerging antibiotic resistance.

The aggregation of human platelets by Lactobacillus species.

The ability to aggregate human platelets was examined for five Lactobacillus rhamnosus strains and five Lactobacillus paracasei subsp. paracasei strains isolated from patients with infective endocarditis (IE), 25 laboratory isolates from the same two species, and 14 strains from five other oral species, namely Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salivarius. Amongst the L. rhamnosus strains, platelets were aggregated by all five IE strains and 8/16 laboratory strains. For the L. paracasei subsp. paracasei strains, the respective numbers were 2/5 and 2/9. Aggregation also occurred with 11/14 strains of the other five species; each species was represented. The optimal ratio of bacteria to platelets for aggregation was approximately 1:1, and there was considerable variation in the lag phase that preceded aggregation, depending on the source of the platelets. Overall, the lag phase varied between 0.25 +/- 0.1 and 20.4 +/- 3.2 min and the percentage aggregation ranged between 70 +/- 2.6 and 104 +/- 13.5%. Confirmation that aggregation was being observed came from studies with five strains on the inhibitory effects of EDTA, dipyridamole, apyrase, imipramine, acetylsalicylic acid and quinacrine. Inhibition of aggregation by L. rhamnosus strains by the peptide arginine-glycine-aspartic acid-serine (RGDS) further indicated a role for fibronectin and/or fibrinogen. Pronase treatment of cells for 1 h and extraction of bacterial surface components with 0.1 M-Tris/HCl (pH 8.5) at 37 degrees C for 1 h stopped aggregation in 8/9 IE strains. Extracted surface proteins (200 micrograms) completely inhibited platelet aggregation by 8/9 of the homologous strains.(ABSTRACT TRUNCATED AT 250 WORDS)