Streptococcus sanguis Endocarditis in Rabbits

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1 ANTIMICROBIAL AGNTS AND CHMOTHRAPY, Feb. 1984, p /84/ $02.00/O Copyright 1984, American Society for Microbiology Vol. 25, No. 2 Role of Granulocytes in the Prevention and Therapy of xperimental Streptococcus sanguis ndocarditis in Rabbits M. J. M. MDDNS, J. THOMPSON, H. MATTI, AND R. VAN FURTH* Department of Infectious Diseases, University Hospital, 2333AA Leiden, The Netherlands Received 15 March 1983/Accepted 28 September 1983 The contributions of granulocytes to the prevention and therapy of Streptococcus sanguis endocarditis with procaine benzylpenicillin (PBP) was investigated in rabbits. Depletion of granulocytes by treatment with mechlorethamine appeared to have no significant effect on either the prophylactic or therapeutic activities of PBP. Administration of 3,000 IU of PBP before inoculation with S. sanguis retarded the course of the endocarditis for only 24 h whether granulocytes were normal or depressed in numbers. Prophylaxis with either 15,000 or 30,000 IU of PBP was equally effective in non-granulocytopenic and granulocytopenic rabbits. Treatment of established infections with PBP at doses of 3,000 to 300,000 IU of PBP at 12-h intervals for 48 h was equally effective in rabbits with normal and depressed numbers of granulocytes. The effect of 3,000 IU of PBP was equivalent, however, to that of granulocytes alone, as shown by the fact that the numbers of CFU per gram of vegetation in the granulocytopenic rabbits treated with this dose of PBP and in the non-pbp-treated control rabbits were not significantly different. In an earlier study, we showed that granulocytes have a significant influence on the course of left-sided Streptococcus sanguis endocarditis in rabbits (10). This was investigated by depleting the circulation of these cells by treatment with mechlorethamine (HN2). It was found that granulocytes exerted a protective effect, as indicated by the numbers of streptococci in endocardial vegetation and in the circulatory system during the course of the endocarditis. Other authors have reported a similar effect during rightsided streptococcal endocarditis in rabbits (15). Selective depletion of monocytes had no effect on the induction and course of the S. sanguis endocarditis (13). The present study was performed to find out whether granulocytes play a role in the prevention of S. sanguis endocarditis and the sterilization of infected endocardial vegetation by procaine benzylpenicillin (PBP). MATRIALS AND MTHODS xperimental animals. One hundred and fourteen male chinchilla rabbits, weighing 1.8 to 2.3 kg, raised in the Central Institute for Breeding of Laboratory Animals, Zeist Austerlitz, The Netherlands, were used in this study. In the prophylactic experiments, 30 control and 28 granulocytopenic rabbits were used; in the therapeutic experiments, 31 control and 25 granulocytopenic rabbits were used. Rabbits belonging to the respective experimental groups were numbered randomly (5), and each step in the experimental procedure was carried out in this sequence. Induction of granulocytopenia. Mechlorethamine (HN2, mustine hydrochloride; ACF, Chemie Farma B.V., Maarssen, The Netherlands) was used to induce granulocytopenia as described elsewhere (4, 10). Briefly, 10 mg of HN2 was dissolved in 10 ml of saline just before use. A single dose of 5 mg was injected into a marginal ear vein immediately after catheterization. For monitoring the effect of HN2 on the numbers of blood leukocytes, blood samples (approximately 1 ml) of 14 out of 31 control and 12 out of 28 HN2-treated rabbits used in the therapeutic experiments were taken from * Corresponding author. the central ear artery 1 day before catheterization (i.e., before the injection of the drug), at the time of inoculation of the streptococci, and at each day during infection. These samples were collected in plastic vials containing 10 mg of crystalline potassium DTA and were diluted 1:20 in leukocyte pipettes with Turk solution containing 6% acetic acid. Total leukocyte counts were done in duplicate in a Burker hemocytometer. The total numbers of granulocytes, mopocytes, and lymphocytes per microliter were calculated from the total number of leukocytes per microliter and differential counts of 400 leukocytes in four blood smears. Characteristics of the infecting strain. The strain used in this study, S. sanguis NCTC 7864, was the same as that employed in previous studies (10, 13). Bacteria from an overnight culture in Todd-Hewitt broth were diluted with saline to 105 CFU/ml. The minimal inhibitory concentration of penicillin G for this strain, the lowest penicillin concentration with which no growth was observed after 18 h of culture at 37 C in Isosensitest broth (Oxoid Ltd., London, United Kingdom), was 0.06,ug/ml. The same type of test in broth was carried out for measurement of the minimal bactericidal concentration. The inoculum consisted of 105 CFU/ml, and the test was done in Isosensitest broth too. After incubation for 18 h at 37 C, a standard volume of 10,ul from each tube that did not show growth was cultured on blood agar. The minimal bactericidal concentration was regarded as the concentration at which 99.9% of the bacteria were killed. This value was found to be -16,ug of penicillin per ml. Since the ratio of minimal inhibitory concentration to minimal bactericidal concentration exceeded 1/32, this strain can be regarded tolerant. Induction of endocarditis. Nonbacterial thrombotic endocarditis was induced by inserting a plastic catheter into the left ventricle via the left carotid artery by the method of Durack and Beeson (2). Three days later, 1 ml of a s-uspension containing 105 CFU of S. sanguis was injected into a marginal ear vein, an inoculum that was found to cause infection in 100% of both control and HN2-treated rabbits (10). The catheter was left in situ during the experiments. Penicillin regimens. For prophylaxis, single doses of 3,000, 15,000, or 30,000 IU of PBP (Depocillin, 300,000 IU/ml of 263

2 264 MDDNS T AL. aqueous suspension, kindly donated by Gist-Brocades N.V., Delft, The Netherlands) was administered subcutaneously 30 min before inoculation of both HN2-treated rabbits and controls. Animals were killed 46 h later, and the vegetations were removed for counting S. sanguis in these lesions. For therapy, four doses of 3,000, 30,000, 100,000, or 300,000 IU of PBP were administered subcutaneously at 12-h intervals starting 20 h after inoculation of the rabbits with streptococci. Animals were killed 12 h after the last injection so that the bacteria in the vegetations could be counted. Monitoring of concentrations of penicillin in blood. Blood samples (25 to 100,ul) collected from the central ear artery at various times during prophylaxis and therapy were added immediately to 200 to 500,ul of sterile distilled water to lyse the blood cells. These samples were tested within 24 h or after storage at -20 C for no more than 4 weeks. Concentrations of penicillin in blood were determined by a cup plate method (3, 7). The test microorganism used was Bacillus stearothermophilus subsp. calidolactis (optimal growth temperature, 56 C). Serial dilutions of a blood sample were compared with a standard in diluted rabbit blood by determining the zone diameter after 18 h of incubation at 56 C (6). The detection limit of this assay is 0.01,ug of penicillin per ml. The concentration was expressed as micrograms of penicillin per milliliter of plasma, after correction for the hematocrit value. Measurement of intensities of bacteremias and heart valve infections. The methods used were generally the same as those described in detail earlier (12). Briefly, endocardial vegetations were isolated aseptically, weighed, and homogenized in 2 ml of glucose broth. Tenfold serial dilutions of this homogenate in saline were made, and 100-,ul samples were plated on sheep blood agar and incubated for 24 to 48 h at 37 C. The degree of infection of the vegetations was expressed as CFU per gram of vegetation. For quantitative blood cultures, 2-ml blood samples were taken from the ear artery or, immediately after the rabbit was killed, from the inferior vena cava. These samples were added to 1 ml of Liquoid (Oxoid; 0.75% [wt/vol] and 0.18 mm para-amino benzoic acid in saline), plated on 5 ml of glucose broth agar, and incubated for 24 to 48 h at 37 C. Bacteremias were expressed as CFU per milliliter of blood. Residual penicillin in homogenates of vegetation or in blood cultures was inactivated by the addition of penicillinase (lot 79D 27, Gist-Brocades N.V.) to a final concentration of 75 IU/ml. Statistical analysis. ffects of HN2 treatment and PBP on the number of CFU per gram of vegetation or per milliliter of blood were compared by a two-sided Student t test. To analyze more than two parameters simultaneously, an analysis of covariance was used. ffects of HN2 and PBP on the incidence of infected vegetations and on the incidence of positive blood cultures were assessed with the chi-square test (1). RSULTS Levels of granulocytopenia and monocytopenia. When HN2 was injected immediately after catheterization, the number of granulocytes per microliter of blood decreased significantly to less than 100 on day 1 (Table 1). The number of granulocytes per microliter of blood in HN2-treated rabbits was significantly lower than that in control rabbits at the time of injection of the streptococci and at all times thereafter. The circulation was depleted of monocytes and lymphocytes simultaneously. Concentration of penicillin in blood. The concentrations of ANTIMICROB. AGNTS CHMOTHR. TABL 1. ffect of HN2 treatment on the number of granulocytes and monocytes in the circulatory system Leukocytes per,ul (mean ± SD) Leukocytes Daya in rabbits': p Control HN2 treated Granulocytes -4 3,086 ± 2,126 2,124 ± ,999 ± 1, ± 291 < ,547 ± 1, ± 28 < ,188 ± 2, ± 59 < ,724 ± 2,314 1,057 ± 757 <0.05 Monocytes ± ± ± 181 7± 9 < ± ± 23 < ± ± ± ± a Day relative to time of inoculation of rabbits with 105 streptococci. b Data are reported for 12 HN2-treated rabbits and 14 controls. Both groups were used in the therapy experiments. penicillin in blood during prophylaxis are given in Fig. 1. The highest concentration was found 30 min after the administration of PBP, i.e., at the time of injection of the streptococci. At no time was there a significant difference between concentrations in control and in HN2-treated rabbits (P > 0.15). The concentrations of penicillin maintained in blood during therapy are given in Fig. 2. Rabbits in which endocarditis was induced received four subcutaneous injections of PBP at 12-h intervals, starting 20 h after the injection of 105 streptococci. Four dosage levels were investigated, as indicated in Fig. 2; concentrations of penicillin in blood were measured at 6-h intervals. These concentrations did not fluctuate significantly at any given dose %4- (U C- C CFU of S.sanguis I I I hours 8 after 30,000 U PBP,15,000 IUPBP,3,000 IUPBP 22 s,c. injection of PBP FIG. 1. Concentrations of penicillin in the blood of 23, 18, and 17 rabbits at various times after injection of 3,000, 15,000, or 30,000 IU of PBP. All data points represent combined data of control and HN2-treated rabbits. Bars show standard errors of the mean. 46

3 VOL. 25, PBP V o q g g c c ro < PBP PBP PBP V V1 V 4." o o- -o-o- /- \4 300,000 IU PBP A hours after iv injection of 105 CFU of S sanguis 6 100,000 iu PBP 30,000 IU PBP -o 0 3,000 IU PBP FIG. 2. Concentrations of penicillin in the blood during therapy of S. sanguis endocarditis in 13, 7, 6, and 9 rabbits given doses between 3,000 and 300,000 IU of PBP. Concentrations at 32, 44, and 56 h were determined before injection of PBP. Concentrations at 26, 38, 50, and 62 h were determined 6 h after dosage. All points represent combined data of control and HN2-treated rabbits. Bars show standard error of the mean. ffect of prophylaxis with PBP in control and HN2-treated rabbits. After the injection of 105 streptococci, the incidences of infected vegetations did not differ significantly between control and HN2-treated rabbits with prophylaxis at any of the dosage levels investigated (Table 2). Also, the number of CFU per gram of vegetation in rabbits that became infected despite prophylaxis with 3,000 IU of PBP did not differ significantly between the control and HN2- treated rabbits. With the higher dosages of PBP, infected vegetations were found only incidentally, which made statistical evaluation of differences impossible. After 3,000 IU of PBP, two HN2-treated rabbits had sterile vegetations, and therefore these animals were excluded from the statistical evaluation of the incidence of positive blood cultures. At 22 h, 3 of 12 control rabbits and 6 of 8 HN2-treated rabbits had positive blood cultures. This difference is significant (X2 = 4.85, P < 0.03). At 46 h, the incidence of positive blood cultures in the two groups of rabbits did not differ significantly (X2 = 0.16, P > 0.5). The number of CFU/ml of blood in the positive blood cultures at 22 h did not differ significantly in the control and HN2- treated rabbits (data not shown; P > 0.8), but at 46 h it was significantly higher in the treated group than in the controls (mean log10 CFU/ml of blood, [n = 6] and [n = 8], respectively; P < 0.05). After prophylaxis with 15,000 or 30,000 IU of PBP, positive blood cultures only occurred occasionally. GRANULOCYTS AND S. SANGUIS NDOCARDITIS 265 ffect of therapy with PBP on infection of the vegetations and intensity of bacteremias. Immediately before the start of PBP therapy, i.e., 20 h after the injection of 105 streptococci, numbers of CFU per gram of vegetation in control and HN2- treated rabbits did not differ significantly. At 68 h, these numbers were significantly higher in HN2-treated animals than in controls (Table 3). After 3,000 IU of PBP, the numbers of CFU per gram of vegetation in the control rabbits were not significantly different from those in the HN2-treated rabbits. At this time, these numbers did not differ significantly from those in the non-pbp-treated control rabbits. In the HN2-treated rabbits, therapy with 3,000 IU of PBP effected a sevenfold difference of the numbers of CFU tper gram of vegetation (P < 0.001). Therapy with higher doses of PBP effected a significant reduction in the numbers of CFU per gram of vegetation (P < 0.001) in both control and HN2-treated rabbits. The magnitude of this reduction was not influenced by either HN2 treatment or the dose of PBP (Table 3). In the non-pbp-treated rabbits and in those treated with 3,000 IU of PBP, the numbers of CFU per milliliter of blood were significantly higher in the HN2-treated rabbits than in the controls at all times (Table 3). There were no significant differences in these numbers in rabbits with or without 3,000 IU of PBP in either the control or HN2 treatment groups (P > 0.20). Therapy with doses ranging from 30,000 to 300,000 IU of PBP led to a significant decrease in the numbers of CFU per milliliter (P < 0.001), but there was no significant difference among the three dosage levels. At 32 h, the combined data for the three dosage levels showed a significantly higher incidence of positive cultures in the HN2- treated rabbits than in the controls, but such a difference was not found at later times (data not shown). DISCUSSION In the present study, administration of HN2 led to both granulocytopenia and monocytopenia. Since an earlier study showed that selective depletion of monocytes has no effect on the induction or course of S. sanguis endocarditis (13), effects of HN2 treatment in the current study may be attributed to the absence of granulocytes. Prophylaxis with PBP was equally effective in non-granulocytopenic and granulocytopenic rabbits. A dose of 3,000 IU of PBP had no effect in either control or granulocytopenic rabbits, and the numbers of bacteria in infected vegetations did not differ significantly between these two groups. The situation at 46 h in rabbits with endocarditis after prophylaxis with 3,000 IU of PBP is similar to that at 20 h in rabbits without prophylaxis, which means that a single dose of 3,000 IU of PBP only retards the course of S. sanguis endocarditis. Compared with 3,000 IU of PBP, prophylaxis with 15,000 IU of PBP resulted in a significantly higher number of rabbits with sterile vegetations, but the incidence of infected vegeta- TABL 2. ffect of prophylaxis with PBP on bacteria in infected vegetations Incidence of infected vegetations Log1o CFU/g of infected vegetations PBP dose (IU) in rabbitsa: X2 p (mean ± SD) in rabbitsb: Control HN2 treated Control HN2 treated 3,000 12/12 9/ > ± ± ,000 0/10 2/ >0.15 Sterile 7.28, ,000 1/8 3/ > ± 2.84 a Number of rabbits with infected vegetations/total number of rabbits investigated after injection of 10' streptococci. b For rabbits given 3,000 IU of PBP for prophylaxis, P > For rabbits given 15,000 and 30,000 IU, numbers with infected vegetations were too small for statistical evaluation.

4 266 MDDNS T AL. ANTIMICROB. AGNTS CHMOTHR. TABL 3. ffect of therapy with PBP on infection of vegetations Incidene of ifectedlogl0 CFU/g (mean ± SD) in Samples vegetations or positive rabbits: pc examined PBP dose (IU)a Time (h) cultures in rabbitsb p Control HN2 Control HN2 Vegetation /6 4/ ± ± 0.99 > /5 6/ ± ± 0.32 <0.03 3, /7 6/ ± ± 0.32 > , /4 3/ ± ± 1.07 > , /4 2/ ± ± , /5 3/ ± ± 0.52 Blood culture /5 5/ ± ± 0.99 < /5 6/ ± ± /5 6/ ± ± , /7 6/ ± ± 0.46 < / 6/ ± ± /7 6/ ± ± 0.19 a Four subcutaneous injections of PBP were given at 12-h intervals, starting 20 h after the injection of 105 streptococci. Vegetations were isolated 12 h after the last PB3P injection. Blood cultures were drawn at the times indicated (at 20 h immediately before the start of therapy). b Number of rabbits with infected vegetations or with positive blood cultures/total number of rabbits investigated. The incidence of infected vegetations was not significantly different in control and HN2-treated rabbits at 20 or 68 h either without PBP or after injection of any dosage (P > 0.50). P values for vegetations in groups without PBP and with 3,000 IU of PBP were obtained by comparing control and HN2-treated rabbits by the Student t test. P values for numbers of bacteria in the blood were obtained by an analysis of covariance using the combined data of control and IN2-treated rabbits at the three time points. In the groups treated with 30,000, 100,000, and 300,000 IU of PBP, the P was obtained from an analysis of covariance using the combined data of control and HN2-treated rabbits at these three dosage levels. tions did not differ significantly between control and granulocytopenic rabbits. Doubling of the dose to 30,000 IU of PBP did not lead to a larger number of rabbits with sterile vegetations in either group. The results of the present study show that during 48 h of therapy with PBP at any of the dosage levels investigated, granulocytes had no effect on the numbers of CFU in the vegetations. Therapy with 3,000 IU of PBP did not significantly reduce the number of streptococci in the vegetations of either control or granulocytopenic rabbits, but doses of 30,000 to 300,000 IU of PBP did. Therapy with 3,000 IU of PBP in granulocytopenic rabbits had an effect on the number of streptococci in the vegetations similar to the effect of granulocytes alone in control rabbits without therapy. However, in the control rabbits, the combination of granulocytes with 3,000 IU of PBP did not further reduce the number of CFU in the vegetations. Apparently, granulocytes are able to keep the number of CFU in check but have difficulty in eradicating them from the vegetations. After 30,000 IU or more of PBP, the number of CFU in the vegetations of granulocytopenic rabbits did not differ significantly from that in control rabbits. This makes an effect of granulocytes in combination with these dosages of PBP unlikely. In contrast to its effect on the vegetations, treatment with 3,000 IU of PBP had no effect on the number of streptococci in the blood of either control or granulocytopenic rabbits. During therapy with the higher dosages of PBP, the streptococci disappeared from the blood, although sterilizing the circulation took a little longer in granulocytopenic rabbits than in controls. This might have been due to the already higher number of streptococci in the circulation of granulocytopenic rabbits at the start of the therapy, or to a longer period of seeding of the streptococci from the vegetations into the circulation. Two explanations can be suggested for the different effects of granulocytes on the numbers of microorganisms in vegetations and in blood during low-dose therapy. Streptococci are continuously seeded from the infected vegetations into the circulation (8-10). These streptococci can resettle on the vegetational surface, become covered by fibrin and platelets, and form new colonies by multiplication. This process might be kept in check by granulocytes, which could phagocytose streptococci on the vegetational surface. It seems likely that during low-dose therapy, altered surface properties (11, 14) would cause fewer streptococci to become attached to the vegetations, and therefore, a protective effect of granulocytes could no longer be measured. Alternatively, as suggested earlier (9, 14), granulocytes might produce and release antibacterial substances that could curb the proliferation of streptococci, but these substances do not potentiate the effect of penicillin, and therefore, their effect on the number of viable bacteria in the vegetations would resemble the effect of low-dose penicillin. Nevertheless, in both situations, streptococci regaining access to the vegetational surface from inside the vegetations would not be retained there by granulocytes in granulocytopenic rabbits (10), which would lead to a more severe bacteremia in these animals than in control rabbits, despite treatment with 3,000 IU of PBP. In summary, an enhancement by granulocytes of the effect of PBP in the prophylaxis or therapy of S. sanguis endocarditis cannot be measured at the level of the vegetations, but at low dosages such a contribution is measurable in the circulatory system. ACKNOWLDGMNTS The skillful technical assistance of W. C. Bauer and R. P. Demeijer is gratefully acknowledged. This investigation was supported by grant from the Dutch Heart Association, The Hague, The Netherlands.

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