Effects of Molecular Weight of Dextran on the Adherence of Streptococcus sanguis to Damaged Heart Valves

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1 INFECTION AND IMMUNITY, July 1980, p /80/ /07$02.00/0 Vol. 29, No. 1 Effects of Molecular Weight of Dextran on the Adherence of Streptococcus sanguis to Damaged Heart Valves CARLOS H. RAMIREZ-RONDA General Medical Research and Medical Services, VA. Medical and Regional Office Center,* and Department ofmedicine, University ofpuerto Rico School ofmedicine, San Juan, Puerto Rico Dextran-producing streptococci such as Streptococcus sanguis are the organisms most frequently associated with infective endocarditis in humans. A series of experiments was designed to study how the molecular weight of dextrans affects the adherence of an endocarditis strain of S. sanguis to canine heart valves covered with platelets and fibrin. The data indicated that this adherence was dependent on dextrans of high molecular weight, such as dextran T-2000 or glucans isolated from S. sanguis or S. mutans. The adherence properties of the strain studied were not modified by prior exposure of the bacterial cells or valve leaflets to high-molecular-weight dextrans. Preexposure of bacterial cells or valve leaflets to low-molecular-weight dextrans decreased their adherence. Low-molecular-weight dextrans interfered with adherence of dextran-positive strains to damaged heart valves. Streptococci are the organisms most frequently associated with endocarditis in humans (24). Most cases of endocarditis are caused by dextran-producing streptococci (16). Streptococcus sanguis, the classic endocarditis streptococcus, is a dextran-producing strain (11, 16, 17, 23). Bacterial dextran and glucans have been shown to be important in the adherence of S. sanguis to rabbit heart valves with preexisting nonbacterial thrombotic endocarditis (19) and in the adherence of glucan-positive streptococci, including S. sanguis, to damaged canine heart valves covered with fibrin and platelets (18). The source of dextran has not been found to be important in the adherence of S. mutans to Actinomyces viscosus, but the molecular weight of dextrans has been found to be important (2). Linear leuconostoc dextran (dextran T-2000) was found to work as well as dextrans isolated from S. sanguis and S. mutans (2). The role of the molecular weight of dextran in adherence was also demonstrated when it was shown that the adherence to smooth glass surfaces by S. mutans requires the synthesis of high-molecular-weight water-insoluble polysaccharide (13). The effects of the molecular weight of dextran on the adherence of S. sanguis to damaged heart valves has not been systematically studied. To define the role of variations in molecular weight on adherence to damaged heart valves, a series of experiments was designed. First, we compared the effects of a given concentration of exogenous dextrans of various molecular weights on adherence. Second, we compared the effects of different concentrations of dextran of a given molecular weight on adherence. Third, we compared the effects of exogenous dextran from commercial sources and glucans isolated from the studied strain and other clinical strains. Finally, we compared the effects of pretreatment of the bacterial cells or the valve sections with dextran on bacterial adherence. The data demonstrate that adherence of S. sanguis to damaged canine heart valves is dependent on dextrans of high molecular weight, including commercial dextran T-2000 and glucans from S. sanguis or S. mutans. MATERIALS AND METHODS Bacterial strains and conditions of cultivation. The bacterial strains used were obtained from the Microbiology Laboratory of the Puerto Rico Medical Center and from R. J. Gibbons, Forsyth Dental Laboratory, Boston, Mass. S. sanguis CR-100 and S. mutanm CR-1 are endocarditis strains isolated from clinical specimens for our previous study (18); S. mutants 6715 is a laboratory strain (4). All strains were lyophilized when originally isolated and used fresh after reconstitution. All strains were grown first in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) and then, for experiments, in the chemically defined medium of Terleckyj et al. (FMC) (22) with or without sucrose supplementation. Preparation of standardized bacterial suspensions. The standardized bacterial suspensions were prepared as previously described (7, 18). Examination of the wet mounts of the bacterial suspensions by light microscopy showed well-dispersed bacterial cells without aggregates except for suspensions of S. mutans 6715, which contained clusters with an average size of three to four bacteria per cluster. Viable counts of the standardized bacterial suspension absorbencyy at 590

2 2 RAMIREZ-RONDA nm = 0.3) were performed in every experiment; for adherence studies, the standardized suspensions were used undiluted and at serial 10-fold dilutions as noted for individual experiments. Radioisotopic labeling of bacteria. The procedure for labeling bacteria with 5"Cr was previously published (7). Briefly, 250 LCi of Na5"CrO4 (363 mci/ mg; New England Nuclear Corp., Boston, Mass.) was added to 10-ml samples of standardized bacterial suspensions with an absorbance of 0.3. The suspensions were incubated at 370C for 3 h, centrifuged at 12,000 x g for 10 min, and then washed three times with cold phosphate-buffered saline (PBS). The 51Cr-labeled bacteria were resuspended in 10 ml of cold PBS. Bacterial clumps were eliminated by the same dispersion and filtration procedures used in standardization, and suspensions of the 51Cr-labeled bacteria with an absorbance of 0.3 were prepared. Preparation of standardized sections of traumatized aortic valves with nonbacterial thrombotic endocarditis. Mongrel dogs of either sex weighing 14 to 16 kg were anesthetized by intravenous injection of pentobarbital (50 mg/kg). The neck region was shaved, and an incision was made to expose the left carotid artery. The vessel was ligated in its distal portion, and a polyethylene-160 catheter (inside diameter, inch [about 0.1 cm]; outside diameter, inch [about 0.16 cm]; Clay-Adams, Div. of Becton, Dickinson & Co., Parsippany, N.J.) containing sterile saline was introduced into the heart. The catheter was left in place for 18 to 20 h. The aortic valve leaflets were removed by sterile techniques and placed in sterile PBS. Sections were cut only from areas of the valves showing macroscopically visible damage. Microscope examination of representative sections revealed deposition of fibrin and platelets on the traumatized endothelial surface. The method for preparing standardized sections of canine valves with a 2-mmdiameter skin biopsy punch under sterile conditions has been described previously (7, 18). Quantitative measurement of bacterial adherence to valve sections. The bacterial suspensions were standardized as previously described (7). The following standardized conditions were used for all experiments unless otherwise noted. Four valve sections were placed in 3-ml samples of 5"Cr-labeled or unlabeled suspensions of bacteria in box-type plastic petri dishes (50 by 12 mm; Falcon Plastics, Oxnard, Calif.) and were agitated at 16 cycles/min on a reciprocating shaker at 250C for 1 h. Two pairs of valve sections were washed three times with ph 7.2 PBS solution (NaCl, g; Na2HPO4, g; KH2PO4, 7.75 g; distilled water, 19 liters); then each pair was placed in a mortar with 3 ml of PBS and 0.5 g of sterile sand and homogenized with a pestle until discrete pieces of tissue were no longer visible. Viable bacteria were counted by using samples of the standardized bacterial suspensions, the homogenates of the valve sections, and the residual bacterial suspensions after removal of the heart valve sections as previously described (7, 18). 5'Cr-labeled bacteria were counted by comparing the radioactivity of 1-ml samples of the original bacterial suspensions and of the homogenized valve sections. Counting was done in a Beckman model 7000 gamma scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.). INFECT. IMMUN. The adherence ratio was defined as the proportion of bacteria in the initial suspension that was recovered from the washed, homogenized aortic valve sections. Experimental determinations of adherence ratios were based either on viable counts or on measurements of radioactivity in 5'Cr-labeled bacteria and were calculated by the following formulas: adherence ratio = (viable bacteria recovered from heart valves [CFU/ ml])/(viable bacteria in incubation media [CFU/ml]) and adherence ratio = (radioactivity adsorbed to heart valves [cpm/ml])/(radioactivity in incubation medium [cpm/ml]), where CFU represents colony-forming units, as described previously (7, 18). Statistical data were compared by using the Student t test (12). Glucan production determination and preparation of bacterial glucans. Glucan production in broth was determined by the method of Gibbons and Banghart (5), with modifications. Organisms were grown in brain heart infusion broth (phosphate buffered to ph 6) supplemented with 5% sucrose for 48 h, and 10 ml was blended in a Sorvall Omnimixer (DuPont Instrument Products Div., Sorvall Operations, Wilmington, Del.) for 30 s at 4 C to remove cellassociated dextran. The organisms were removed by centrifugation at 1,200 rpm for 20 min. The polysaccharide in the supernatant was precipitated with the addition of 1.5 vol of 95% ethanol and centrifuged at 3,500 rpm for 20 min. The pellet was washed twice in 65% ethanol, recentrifuged as above, rinsed in distilled water, and again centrifuged at 3,500 rpm for 20 min. Residual protein (less than 2%) was removed by the addition of trichloroacetic acid to a 10% concentration and subsequently dialyzed overnight. The polysaccharide was then precipitated in 2 vol of 95% ethanol, centrifuged at 3,500 rpm for 20 min, lyophilized, and weighed. S. sanguis CR-100 produced 2,019 ± 118,g of glucan in broth and S. mutans 6715 produced 5,770 ± 211 gtg of glucan in broth per ml. The glucans were then used in other experiments. Determination of the effects of variations in molecular weight and concentration of dextran on adherence. Dextran (Pharmacia Fine Chemicals, Uppsala, Sweden) T-2000 (lot 8122), T-500 (lot 3447), T-70 (lot 4765), T-40 (lot 2771), and T-10 (lot 6930) were suspended in PBS, ph 7.2, in concentrations of 1, 2, 5, 10, 20, and 40 mg/ml. In experiments where the effect of exogenous dextran was studied, the overnight cultures grown in FMC were centrifuged at 12,000 X g for 10 min, washed with PBS, and then suspended in the PBS-dextran suspension; then the bacterial suspension was standardized as previously described (7). In experiments where the glucans used were bacterial glucans, they were suspended in PBS, ph 7.2, in a concentration of 5 mg/ml and used as described above. Determination of the effects of contact with dextran before adherence studies. Dextran T-2000 (lot 8122) was suspended in PBS, ph 7.2, at 5 mg/ml. The standardized bacterial suspension prepared from bacterial cells grown in a sucrose-free medium was centrifuged at 12,000 x g for 10 min and suspended into an equal volume of PBS-dextran T-2000 for 30 min. The dextran-treated cells were centrifuged at 12,000 x g for 10 min, washed three times with PBS, and resuspended in PBS; then adherence studies were

3 VOL. 29, 1980 carried out. In other experiments, the circular valve leaflet sections were exposed to the PBS-dextran T for 30 min and washed three times in PBS before adherence studies were carried out. A third variation in the experiments was to expose the bacterial cells or the valve leaflets to dextrans of lower molecular weight dextrann T-70 in concentrations of 5 mg/nil) as previously described. After the bacterial cells grown in a sucrose-free medium or valves were exposed to dextran T-70, adherence studies were carried out with the following modifications: (i) adherence in PBS; (ii) adherence in PBS-dextran T-70; and (iii) adherence in PBS-dextran T RESULTS Dextran-induced aggregation before adherence: possible effects. The possible effect of dextran-induced aggregation of bacterial cells before adherence studies was minimized by performing the following procedures. First, all bacterial suspensions were restandardized after the addition of exogenous dextran or glucan. Second, the following ratio was calculated in all experiments: ([number of bacteria in the standardized bacterial suspension or initial inoculum] - [number of bacteria adherent to the surface])/ (number of bacteria eluted and present in the supernatant after exposure). The predicted ratio is 1.0; the actual ratio for 431 observations was Third, we performed most experiments with radioactively labeled bacteria also and demonstrated the same adherence ratios by bacterial counts and radiolabeled bacteria (see Tables 1, 3, and 5). Effects of molecular weight and concentration of dextran on adherence of S. sangui8 Comparison of the effects of variations in concentration of dextran T-2000 on the adherence of S. sanguis grown in a sucrose-containing medium and in a sucrose-free medium to damaged heart valves is shown in Table 1. The adherence ratio in a sucrose-containing medium in the absence of dextran was almost six times the adherence ratio under sucrose-free conditions. The addition of exogenous dextran T-2000 in concentrations ranging from 1 to 40 mg/ml to cells grown under sucrose-free conditions restored adherence ratios to those found in cells grown in sucrose conditions in the absence of dextran, but had no effect on the adherence when endogenous glucans were present. The effects of variations in the molecular weight of dextran on adherence can be seen in Table 2. The adherence ratio of S. sanguis grown under sucrose-free conditions and absence of exogenous dextran ranged from 801 to 891. The addition of exogenous dextrans of molecular weights below 70,000 had a minor effect on restoring adherence ratios to those found under endogenous dextran-generating condi- EFFECTS OF DEXTRAN ON ADHERENCE OF S. SANGUIS TABLE 1. Effect of dextran T-2000 concentration on adherence of unlabeled and "'Cr-labeled S. sanguis CR-100 to damaged aortic valve leaflets" Adherence ratio (x105; mean ± standard error) Dextran FMC + 5% SucroseFM T-2000 Control FMC concn (mg/ml) Unlabeled Labeled Unlabeled Labeled cells' cela cells cells 0 4,950 ± 149 4,900 ± ± ± ,900 ± 209 4,910 ± 129 2,600 ± 302 2,540 ± ,808 ± 301 4,950 ± 142 4,790 ± 269 4,801 ± ,102 ± 203 5,200 ± 200 5,108 ± 302 5,200 ± ,100 ± 204 5,098 ± 201 5,202 ± 290 5,194 ± ,100 ± 203 5,201 ± 198 4,980 ± 60 5,000 ± ,909 ± 300 4,992 ± 269 4,842 ± 296 4,903 ± 294 a Bacterial suspensions in all experiments were standardized at absorbency at 590 nm Determinations of viable counts of all standardized suspensions of unlabeled cells were performed and varied from 3.9 X 10' to 4.7 X 10' CU/ml. Determinations of radioactivity of all standardized suspensions of labeled cells were performed and varied from 483,000 to 501,000 cpm/ml. 'Calculated from viable counts (four observations per experiment). c Calculated from "'Cr-labeled bacteria (four observations per experiment). tions. Concentrations of up to 40 mg of dextran T-70 per ml increased the adherence ratio from a base line of 891 ± 30 to 1,690 ± 99, but was never able to restore control values of 4,890 ± 211. Dextran T-500 increased adherence ratios under sucrose-free conditions in the ranges of concentrations studied 3.7 to 4.7 times, restoring adherence close to control values. Dextran T in the same concentrations increased the adherence ratio 5.7 to 6.5 times; dextran of this molecular weight was able to restore adherence ratios to levels found under conditions for endogenous dextran production. Effects of exogenous dextran of endogenous origin on adherence. Adherence of S. sanguis grown in a sucrose-free medium was restored to the levels found when the bacteria were grown under sucrose-containing conditions by exogenous dextran of endogenous origin (Table 3). Concentrations of 5 mg of glucans obtained from S. sanguis and S. mutans per ml were able to restore adherence to levels obtained under sucrose conditions. These data on the similarity in adherence ratio-restoring ability of the exogenous commercial dextran T-2000 and exogenous glucans of endogenous origin suggest that these glucans may have similar molecular weights. Effects of preexposure to exogenous dextran on adherence. Adherence of S. sanguis to damaged valve sections was studied when the S. sanguis bacterial cells or the damaged valve sections were preincubated for 30 min with exogenous dextran. 3

4 4 RAMIREZ-RONDA TABLE 2. Effects of molecular weight and concentration of dextran on adherence of S. sanguis CR-100 to damaged aortic valve leaflets Adherence ratio (xio5; mean ± standard error)b Dextran concn FMC' + 5% su- FMC + dex- FMC + dextran FMC + dextran FMC + dextran FMC + dextran (mg/ml) crose control tran T-10 T-40 T-70 T-500 T ,890 ± ± ± ± ± ± ,690 ± ± ± 38 1,390 ± 111 3,108 ± 200 4,600 ± ,990 ± ± ± 32 1,406 ± 98 3,000 ± 300-4,790 ± ,795 ± ± ± 49 1,432 ± 111 3,001 ± 290 5,108 ± ,890 ± 232 1,112 ± 40 1,099 ± 51 1,702 ± 89 3,502 ± 300 5,202 ± ,794 ± 251 1,142 ± 50 1,106 ± 48 1,700 ± 201 3,801 ± 199 4,980 ± ,869 ± 199 1,102 ± 72 1,201 ± 100 1,690 ± 99 3,798 ± 198 4,842 ± 296 Bacterial suspensions in all experiments were standardized at absorbency at 590 num = 0.3. Determinations of viable counts of all standardized suspensions were performed and varied from 3.8 x 108 to 4.7 x 108 CFU/ml. b Adherence ratios presented were calculated from viable counts (four observations per experiment). 'All cells were grown in FMC, and then the exogenous dextran was added, except for cells in the control grown in FMC plus 5% sucrose. TABLE 3. Effects of dextran and glucans (5 mg/ml) of various sources on adherence of unlabeled and 5'Cr-labeled S. sanguis CR-1(X grown in FMC to damaged canine valves Adherence ratio (xlo; mean ± Dextran or glucan standard error) source Unlbcellse Labeled cells' Dextran T ,108 ± 302 5,098 ± 192 S. sanguis dextran 4,999 ± 196 S. sanguis glucan 5,091 ± 190 S. mutans dextran 5,200 ± 404 S. mutans glucan 5,238 ± 301 a Bacterial suspensions in all experiments were standardized at absorbency at 590 nm = 0.3. Determinations of viable counts of all standardized suspensions of unlabeled cells were performed and varied from 3.8 x 10" to 4.8 x 108 CFU/ml. Determinations of radioactivity of all standardized suspensions of labeled cells were performed and varied from 495,000 to 515,000 cpm/ml. b,c As in Table 1, except that there were three observations per experiment. The adherence of S. sanguis grown in FMC with damaged heart valves was compared in two adherence media, PBS and PBS with 5 mg of dextran T-2000 per ml (Table 4). The bacterial cells or the valve leaflet sections, or both, were preincubated with dextran T Preincubation of bacterial cells or valve leaflets did not alter significantly the adherence when the adherence medium contained dextran. If there was no dextran in the adherence medium, unsupplemented PBS, the adherence of S. sanguis to damaged valves was lower than the adherence found when the incubation medium contained dextran. Preincubation of bacterial cells with dextran T-2000 and adherence determined in PBS increased the adherence threefold compared with that found when the cells were grown INFECT. IMMUN. in FMC; when the adherence was determined in PBS-dextran, the increase was fivefold. The data suggest that preexposure to high-molecularweight dextran of exogenous origin increases the adherence of the bacterial cells and platelet-fibrin-covered valve leaflet sections, and supports the concept that dextran is one of the factors that mediate adherence. Receptors are involved in the bacterial cell surface; whether receptors are involved in the platelet-fibrin-covered valve leaflet needs to be ascertained. The effects of preexposure of S. sanguis or damaged heart valve sections to dextran T-70 on adherence are shown in Table 5. Comparison of the adherence ratios of FMC-grown bacterial cells preexposed to 5 mg of dextran T-70 per ml with untreated damaged valve leaflet sections and with valve leaflet sections preexposed to dextran T-70 and dextran T-2000 showed that the adherence-restoring ability resided in the high-molecular-weight dextran. As long as highmolecular-weight dextran was present in the adherence medium, whether by preexposure or at the moment of adherence, the adherence-restoring ability was preserved. This suggests a role of high-molecular-weight dextran deposited in the surface of the bacterial cell or valve section as the mediator of adherence. In the presence of dextran T-70 (5 mg/ml) in the adherence medium, there were no significant variations in the adherence ratio as compared with the ratios seen in PBS. When dextran T-2000 at 5 mg/ml was present in the adherence medium, the adherence ratios were increased under the three experimental conditions and restored to values seen when the adherence ratios were determined with cells grown in FMC-sucrose. The adherence of bacterial cells grown in FMC-sucrose, and therefore covered with endogenous glucans, to valve sections pretreated with dextran T-70 and T-2000 is shown in Table

5 VOL. 29, 1980 TABLE 4. Effects ofpreincubation of bacterial cells and valve leaflet sections with dextran T-2000 on the adherence of S. sanguis CR-400 in PBS and PBS with dextran T-20XKa Adherence ratio (x10; mean + standard error) Experimental condition PBS + dex- PBS tran T-2000 (5 mg/ml) Cells grown in FMC 894 ± 101 5,103 ± 401 Cells grown in FMC + sucrose 5,102 ± 203 5,108 ± 302 Cells grown in FMC, then 2,890 ± 196 4,948 ± 322 preincubation of bacterial cells for 30 min with dextran T-2000 (5 mg/ml) Cells grown in FMC, then 4,172 ± 193 5,096 ± 306 preincubation of valve leaflets sections with dextran T Cells grown in FMC, then 4,979 ± 294 4,704 ± 301 preincubation of both bacterial cells and valve leaflets sections with dextran T-2000 'Bacterial suspensions in all experiments were standardized at absorbency at 590 nm = 0.3. Determinations of viable counts of all standardized suspensions were performed and varied from 3.2 x 105 to 3.9 x 105 CFU/ml. 'As in Table 1, except that there were three observations per experiment. 5. Pretreatment of valve sections with dextran T-70 decreased the adherence ratio to 3, , and pretreatment of the valve sections with dextran T-2000 increased adherence to 5,219 ± 194. These data suggest also that pretreatment of valve leaflet sections with exogenous dextran of low molecular weight partially blocks the adherence of endogenous glucan-covered bacterial cells. The partial block was unchanged by exogenous dextran T-70 in the medium but was reversed by dextran T EFFECTS OF DEXTRAN ON ADHERENCE OF S. SANGUIS DISCUSSION Originally we reported that gram-positive cocci adhere better than gram-negative bacilli to heart valves (7). The adherence of glucan-positive streptococci to damaged heart valves (18) and to platelet-fibrin matrix (19) depends on the capacity of the streptococci to synthetize glucans. Dextran plays a role in the adherence of these organisms to heart valves and may be a significant factor in the pathogenesis of endocarditis. The source of dextran has been shown not to be important in the mediation of adherence, but the molecular weight of dextran plays a role in the mediation of adherence of S. mutans to A. viscosus and smooth glass surfaces (2). We previously demonstrated that exogenous dextran T-2000 (5 mg/ml) was able to restore adherence to values found when the bacteria were grown in a sucrose-containing medium (18). The data presented demonstrate that the adherence of S. sanguis to damaged heart valves is dependent on the molecular weight of dextran. Dextrans of molecular weights below 70,000 were unable to restore the adherence to values found in controls; dextran of molecular weight 500,000 increased the adherence four times, but only dextran T-2000 and endogenous dextrans from S. sanguis and S. mutans were able to restore adherence ratios to control values. Dextran-induced aggregation results from dextran cross-linking of cells, whereas sucroseinduced aggregation depends on the synthesis of dextran by dextransucrase and subsequent cellular binding of the product (8). Dextran binding of S. mutans may be mediated by two classes of cell-associated proteins, dextransucrase and a nonenzyme protein receptor (12-14). Extracellular glucosyltransferase (GTF) can bind to the binding site or sites, synthetize cellbound glucan from sucrose, and produce in vitro adherence (10, 13, 14, 20). There is evidence that cell surface glucan participates in the binding of GTF (8, 13, 14). The existence of binding sites on the S. mutans surface which are capable of binding dextran and causing agglutination has been proposed (6, 8, 12, 15, 25). There might be more than one binding site for glucans on the cell surface of S. mutans, but the nature of the second site is unknown (25). The binding site for glucan is a glycoprotein (25) or a protein with (20) or without (21) glucan. The data present evidence that there are dextran receptors in the cell surface of S. sanguis, but their nature was not characterized. It has been shown that levels of GTF from S. sanguis vary according to the growth medium utilized (3), and our experiments support this concept. Whether receptors are involved in the endothelial surface covered with fibrin and platelets remains to be fully elucidated. The effect of dextran T-70 on adherence is interesting. Pretreatment of either the bacterial cells or valve section leaflets with dextran T-70 decreased the adherence of S. sanguis to the damaged valve leaflet. Dextran T-70 interfered with aggregation even when the bacterial cells were grown in FMC plus 5% sucrose or when the cells were preexposed to dextran T The interference was more marked when there was no dextran or low-molecular-weight dextran present in the adherence medium. Other authors demonstrated that preincubation of cells with dextran T-70 did not reduce the quantity of glucan bound, and the converse was also true (26). Low-molecular-weight dextran may bind to a nonenzyme receptor in the cell surface (4) and interfere with binding of the dextrans of higher molecular weight. This binding of dextran T-70 to the receptor sites occupies the receptor site, 5

6 6 RAMIREZ-RONDA INFECT. IMMUN. TABLE 5. Effects ofpreincubation of bacterial cells grown in FMC and valve leaflet sections with dextran T- 70 and bacterial cells grown in FMC-sucrose on adherence of unlabeled and 51Cr-labeled S. sanguis CR- 100 to damaged heart valves in PBS, PBS with dextran T- 70, and PBS with dextran T-2000' Adherence ratio (x10'; mean ± standard error) PBS PBS + dextran T-70 (5 PBS + dextran F-2000 (5 Experimental condition mg/mil) mg/ml) Unlabeled Labeled Unlabeled Labeled cells Unlabeled Labeled cells cells' cells' cells cells Bacterial cells preincubated with Dextran T-70 Untreated valves 1,190 ± 196 1,099 ± 192 1,396 ± 217 1, ,072 ± 4,121 ± Valves preincubated with dextran 1,095 ± 196 1,100 ± 190 1,120 ± 208 1, ,894 ± 3,944 ± 256 T Valves preincubated with dextran 4,094 ± 306 4,150 ± 291 4,111 ± 197 4,203 ± 192 4,296 ± 4,401 ± 295 T Valve sections preincubated with dextran T-70 Untreated bacteria 1,015 ± 176 1,040 ± 159 1, , ,316 _ 4,291 ± Bacteria preincubated with dex- 1,174 ± 176 1,098 ± 167 1,311 ± 89 1,345 ± 109 3,799 ± 3,817 ± 309 tran T Bacteria preincubated with dex- 4,094 ± 306 4,129 ± 292 4,413 ± 193 4,508 ± 206 4,592 ± 4,646 ± 211 tran T Bacterial cells grown in FMC-sucrose Untreated valves 5,102 ± 203 5,219 ± 200 5,103 ± 241 5, ,120 ± 5,219 ± Valves preincubated with dextran 3,503 ± 196 3,392 ± 196 3,622 ± 199 3,421 ± 178 4,196 ± 4,000 ± 317 T Valves preincubated with dextran 4,219 ± 194 4,119 ± 190 4,601 ± 209 4,590 ± 196 4,520 ± 4,491 ± 269 T a Bacterial suspensions in all experiments were standardized at absorbency at 590 nm = 0.3. Determinations of viable counts of all standardized suspensions of unlabeled cells were performed and varied from 3.7 X 10i to 4.1 X 108 CFU/ml. Determinations of radioactivity of all standardized suspensions of labeled cells were performed b,c and varied from 491,000 to 514,000 cpm/ml. As in Table 1, except that there were three observations per experiment. and the polymer size of dextran T-70 might be insufficient to bridge two or more bacterial cells (6). A second possibility is that low-molecularweight dextran interferes with the bridging of high-molecular-weight dextrans. The data suggest a possible mechanism by which in vivo adherence of bacterial cells during an episode of bacteremia might be blocked from binding to a damaged valve leaflet, using low-molecularweight dextran. The data presented help clarify the role of dextran in the adherence of S. sanguis to damaged canine heart valves, support the concept of a receptor for glucans in the surface of the bacterial cell, and suggest a possible receptor for glucans in the damaged endothelium. Adherence of S. sanguis to damaged heart valves is dependent on high-molecular-weight dextrans; low-molecular-weight dextrans can interfere with this adherence. ACKNOWLEDGMENTS This project was supported by the Veterans Administration Research Service and by a Puerto Rico Heart Association grant. The technical assistance of Minerva Nevarez and Doris Vera and the secretarial expertise of Consuelo Lopez are gratefully acknowledged. LITERATURE CITED 1. Bernstein, L, and W. Weatherell Chance differences, p In Statistics for medical and other biological students. E & S Livingston Ltd., Edinburgh. 2. Bourgeau, G., and B. C. McBride Dextran-mediated interbacterial aggregation between dextran-synthesizing streptococci and Actinomyces viscosus. Infect. Immun. 13: Carlsson, J., and B. Elander Regulation of dextran sucrase formation by Streptococcus sanguis. Caries Res. 7: Germaine, G. R., and C. F. Schachtele Streptococcus mutans dextran sucrase: modest interaction with high molecular weight dextran and role in either aggregation. Infect. Immun. 13: Gibbons, R. J., and S. Banghart Synthesis of extracellular dextran by cariogenic bacteria and its presence in human dental plaque. Arch. Oral Biol. 12: Gibbons, R. J., and R. J. Fitzgerald Dextran induced agglutination of Streptococcus mutans, and its potential role in the formation of microbial dental plaques. J. Bacteriol. 98: Gould, K., C. H. Ramirez-Ronda, R. K. Holmes, and J. P. Sanford Adherence of bacteria to heart

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