Staphylococcus aureus

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1993, p /93/ $2./ Copyright X 1993, American Society for Microbiology Vol. 37, No. 11 Uptake of Azithromycin by Human Monocytes and Enhanced Intracellular Antibacterial Activity against Staphylococcus aureus A. P. MEYER, C. BRIL-BAZUIN, H. MATFIE, AND P. J. VAN DEN BROEK* Department of Infectious Diseases, University Hospital Leiden, Building 1, CS-P42, P. O. Box 96, 23 RC Leiden, The Netherlands Received 29 March 1993/Returned for modification 17 May 1993/Accepted 17 August 1993 The uptake of azithromycin by human monocytes and the intracellular antibacterial activity of azithromycin against Staphylococcus aureus were investigated. With an extracellular ph of 6.9, the maximum intracellular concentration of azithromycin in monocytes was about six times the extracellular concentration. The half-life for diffusion was 44 min. The results support the view that no active transport is involved in the intracellular accumulation of azithromycin. In cell-free medium, the maximum effect of azithromycin on S. aureus was bacteriostasis, which was achieved at a concentration of 5 mg/liter. In contrast, concentrations greater than 1.5 mg of azithromycin per liter were bactericidal for S. aureus ingested by monocytes. The difference in maximum growth inhibition on S. aureus for the two conditions was.168 h-1 (95% confidence interval,.128 to.28). The concentration of the drug that achieved 5%o of the maximum effect was.434 mg/liter for both conditions. The enhancement of the effect on S. aureus ingested by monocytes suggests that the intracellular environment in human monocytes favors the antibacterial action of azithromycin. Enhancement of the antibacterial activity of azithromycin was not observed with granulocytes. Azithromycin is an antimicrobial drug which binds extensively to cells. For granulocytes and macrophages, cellularto-extracellular ratios (C/E ratios) of up to 3 have been described (1-3, 8). Like erythromycin, azithromycin enters cells by nonionic diffusion (3, 1). Accumulation of the drug is caused by ion trapping in lysosomes (2, 3). Only the nonionized form of the drug has antibacterial activity (11, 12). Under steady-state conditions, the concentration of nonionized azithromycin in lysosomes is equal to that in the cytoplasm, which in turn is equal to the extracellular concentration of nonionized drug. As a consequence, the C/E ratio for nonionized drug is always 1 under steady-state conditions. Therefore, high total intracellular drug concentrations do not imply high intracellular antimicrobial activity of the drug. This activity can be evaluated only by direct measurement. The aim of the present study was to compare the antimicrobial activity of azithromycin against Staphylococcus aureus phagocytosed by human monocytes with that against S. aureus in cell-free medium. MATERIALS AND METHODS Azithromycin. Stock solutions of 25 mg of azithromycin (Pfizer, Rotterdam, The Netherlands) per liter were freshly prepared in phosphate buffer (ph 6.) before each experiment. Lysostaphin. A stock solution of 1 U of lysostaphin (Sigma Chemical Co., St. Louis, Mo.) per ml in phosphatebuffered saline (PBS) was prepared and was stored at -2 C. For each experiment the activity of lysostaphin was checked by incubating 16 S. aureus organisms per ml in the presence of.5 U of lysostaphin per ml for 5 min at 4 C and then determining the number of viable bacteria microbiologically. According to the criteria applied, 99.5% of the bacteria had to be killed during this incubation (16). * Corresponding author Serum. After clotting of blood from healthy human type AB blood donors at room temperature, serum was prepared by centrifugation at 1,1 x g for 2 min and was stored at -7C. Medium. Hanks' balanced salt solution with.1% (wt/vol) gelatin and 2 mm HEPES (N-2-hydroxyethylpiperazine-N'- 2-ethanesulfonic acid) as buffer (ph 7.) was used as the medium in all experiments. Monocytes and granulocytes. Monocytes were isolated from recalcificated buffy coats of blood from healthy adult donors by differential centrifugation on Ficoll-Isopaque gradients. The interphase layer containing monocytes and lymphocytes was washed four times with PBS containing.5 U of heparin per ml (heparin-pbs) and was then suspended in medium to a concentration of 17 monocytes per ml. This suspension contained about 3.5 x 17 lymphocytes per ml and fewer than 2 x 15 granulocytes per ml. Granulocytes were obtained from the pellet of the Ficoll- Isopaque gradient by sedimentation of erythrocytes with a 5% solution of dextran (molecular weight, 2,) in PBS. Erythrocytes were lysed by incubation for 1 min in distilled water, the solution was then made isotonic by adding 1.8% saline solution. The granulocytes were suspended in medium to a concentration of 17 cells per ml. S. aureus. An overnight culture of S. aureus (type 42 D) in Nutrient Broth No. 2 (Oxoid Ltd., London, United Kingdom) was washed twice with PBS before preopsonization by incubation of 17 bacteria per ml in medium with 1% (vol/vol) serum for 3 min under rotation (4 rpm) at 37 C. Next, the suspension was washed twice with medium and was resuspended at a concentration of 17 S. aureus organisms per ml. The MIC of azithromvcin for S. aureus determined on solid medium with 5 x 1 S. aureus organisms per spot (multipoint inoculator) was.5 mg/liter. Measurement of the effect of azithromycin on preopsonized S. aureus in suspension. Suspensions of preopsonized S. aureus were diluted in medium to 5 x 15 bacteria per ml,

2 VOL. 37, 1993 and the mixture was incubated with various concentrations of azithromycin under rotation (4 rpm) at 37 C. Samples were taken at, 6, 12, and 18 min and were diluted 1-fold in medium. The number of viable bacteria was determined by plating 1-fold serial dilutions on diagnostic sensitivity test agar (Oxoid Ltd.) and counting the colonies after incubation for 18 h at 37 C. Measurement of the effect of azithromycin on preopsonized S. aureus phagocytosed by monocytes. Equal volumes of a suspension of 17 monocytes per ml and 17 preopsonized S. aureus per ml were incubated for 3 min under rotation (4 rpm) at 37 C; phagocytosis was then stopped by cooling the test tubes in crushed ice. Noningested bacteria were removed by differential centrifugation (11 x g, 4 min) and by washing the cells twice. Next, the suspension of monocytes with ingested S. aureus was adjusted to a volume of 2.5 x 16 cells per ml and was divided over separate tubes, one tube for each time point. Azithromycin was added and reincubation under rotation (4 rpm) at 37 C was started. At, 6, 12, and 18 min, the number of viable cell-associated bacteria was determined microbiologically after separation of the cells from the medium by centrifugation at 11 x g for 4 min and disruption of the monocytes in distilled water containing.1% (wt/vol) bovine serum albumin. During reincubation, no serum was added to prevent the killing of S. aureus by the monocytes (4). Therefore, the decrease in the number of viable cell-associated bacteria is a measure of the effect of the antibiotic. The viability of the monocytes was checked by trypan blue dye exclusion and was greater than 9% during all experiments. Calculations. For comparison of the antibacterial activity of azithromycin against S. aureus ingested by monocytes and S. aureus in cell-free medium, the effect of azithromycin on the numbers of S. aureus (EN) at time t was expressed as the logarithm of the ratio of the number of viable bacteria in the control (no antibiotic) at time point t (Nc,) to the number present during incubation with the antibiotic at the same time point (Nat) and calculated by the equation EN, = log Nc, - log Nat (equation 1). The contribution of monocytes and incubation time t to the variation of EN was calculated by multiple regression analysis by the equation EN = a1t + a2tm + a3m + a4 (equation 2), in which m is a dummy variable with a value of for cell-free medium and a value of 1 for ingested S. aureus, and a, to a4 are constants of the equation. The slope, a1 for cell-free medium and a1 + a2 for ingested S. aureus, indicates the growth inhibition (ER) achieved with a given concentration of azithromycin. The concentration-effect relationship was then calculated by nonlinear regression analysis (Systat, Systat Inc., Evanston, Ill.) by the Hill equation (17), as follows: ER = ER,maX x {CV[(EC5)s + CS]} (equation 3), in which ER,ma., is the maximum value of ER estimated from the experimental results, C is the concentration of the drug, s is a parameter reflecting the steepness of the concentration-effect curve, and EC5 is the concentration of the drug that achieves 5% of the maximum effect. Differentiation between extracellular and intracellular localization of monocyte-associated S. aureus. Differentiation between intracellular and extracellular localization of monocyte-associated S. aureus was determined as described elsewhere (13). In short, the monocytes in samples taken during the assay of the effect of azithromycin on preopsonized S. aureus phagocytosed by monocytes were spun down by centrifugation at 11 x g for 4 min. One of the samples was incubated with.5 U of lysostaphin per ml for 5 min at 4 C; cytospin preparations were then made and HUMAN MONOCYTES AND AZITHROMYCIN 2319 stained with Giemsa. As a control the second sample was incubated with PBS and was processed similarly. The numbers of bacteria and monocytes were counted in 5 fields of view under a microscope (magnification, x1,). The results were expressed as the number of bacteria per 1 monocytes. The lysostaphin-treated samples reflect the intracellular bacteria. The difference between the number of bacteria in the nontreated and the lysostaphin-treated samples reflects the number of extracellular bacteria. Determination of the cell-associated concentration of azithromycin. Human monocytes were further purified from the interphase layer of the centrifugation of the buffy coat on Ficoll-Isopaque by elutriation centrifugation in a Beckman J2-21 centrifuge. The cell suspension obtained by this procedure contained 9% monocytes with viability exceeding 93%, as determined by trypan blue exclusion. For determination of the cell-associated concentration of azithromycin, 2.5 x 17 monocytes or granulocytes per ml were incubated in Hanks' balanced salt solution with.1% (wt/vol) gelatin and 2 mm HEPES as buffer (ph 7.) in the presence of 5 mg of azithromycin per liter under rotation (4 rpm) at 37 C. At various time points, samples were taken and the cells were separated from the medium by centrifugation at 1, x g for 45 s over a silicone oil layer. The cells were lysed in distilled water, and the concentration of azithromycin was determined by an agar diffusion method described by Mattie et al. by using Sarcina lutea at ph 8.. The standard was made with distilled water. Control experiments showed no difference between standards based on monocyte lysate and distilled water. The cellular concentration (C) was calculated by the equation C = (V1 x C1)I(G x Vc) (equation 4), in which V1 is the volume of the cell lysate, C1 is the antibiotic concentration in the cell lysate, G is the total number of cells present during incubation, and Vc is the volume of a single cell. The volume of a human monocyte is 421 fl; that of a granulocyte 334 fl (7). To calculate the maximum intracellular concentration, the following equation was fitted by nonlinear regression analysis to the values of C: C = Cma (1 - e/ct) (equation 5), in which Cm. is the extrapolated intracellular concentration at steady state, and k is a diffusion rate constant. RESULTS Effect of azithromycin on preopsonized S. aureus in cell-free medium and on preopsonized S. aureus ingested by monocytes. In cell-free medium, azithromycin showed a concentration-dependent effect in the concentration range.1 to 5 mg/liter (Fig. 1). The maximum effect was bacteriostasis. The effect of azithromycin on ingested S. aureus was concentration dependent in the same concentration range. Concentrations greater than 1.5 mg/liter led to a decrease in the number of viable bacteria (Fig. 1). The antibacterial activities were compared by calculating the ER,ma,,, according to equation 3, obtained under the two conditions (Fif. 2). ER,max for S. aureus in cell-free medium was.26 h-, and for ingested S. aureus it was.428 h-', the difference being.168 (95% confidence interval,.128 to.28). The EC5 was.434 mg/liter for both conditions. Differentiation between intra- and extracellular localization of monocyte-associated S. aureus. Intracellular localization of the bacteria was proven with the help of lysostaphin on the basis of the condition that lysostaphin lyses only extracellular S. aureus (13). At min, i.e., after 3 min of phagocytosis and two washes to remove noningested bacteria, about 8% of the S. aureus was located intracellularly, confirming

3 232 MEYER ET AL. ANTIMICROB. AGENTS CHEMOTHER. A cell-free medium B monocytes azithronycin (mg/i) O o azithromycin (mg/l) i e~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.1.25 co 1.5 cn ~~~~~~~~~~~~~~~~~ > ~~~~~~~~~~~~~~~~~1. cn~~~~~~~~~~~~.25 z '. o ~~~~~~~~~~~~~~~ O min FIG. 1. (A) Effect of azithromycin on preopsonized S. aureus. (I monocytes Effect of azithromycin on preopsonized S min aureus ingested by earlier findings (4, 14) (Fig. 3). In the absence of azithromycin, the number of intracellular bacteria remained about the same during incubation. In the presence of azithromycin, the number of extracellular as well as intracellular S. aureus decreased, indicating that azithromycin indeed affects intracellular bacteria (Fig. 3). Effect of azithromycin on preopsonized S. aureus ingested by granulocytes. The antibacterial effect of azithromycin against preopsonized S. aureus ingested by human granulocytes was determined by the same method used for preopsonized bacteria phagocytosed by monocytes. The ER values achieved with 5 and 1 mg of azithromycin per liter on S. il concentration (mg/l) FIG. 2. Concentration-effect relationship for azithromycin against monocyte-associated S. aureus () and S. aureus in cell-free medium (). See text for calculations. aureus ingested by granulocytes were.235 and.231 h-1, respectively. These effects were the same as those in cellfree medium, i.e.,.254 h-1 for 5 mg/liter and.261 h-1 for 1 mg/liter (P =.5). ER on S. aureus ingested by monocytes was.411 h-1 with azithromycin at 5 mg/liter and.398 h-' with azithromycin at 1 mg/liter (P =.1). Cell-associated concentration of azithromycin. The concentration of cell-associated antibiotic was determined under the same conditions that were used to measure the antibacterial activity of the drug. The results show that 3 h of ụ@ 2 C CU C> IU, E 1-. *u Eu 1 - s ci no azithromycin azithronycin ( 5 mg/l ) min FIG. 3. Number of S. aureus organisms per 1 monocytes at, 6, 12, and 18 min of incubation of monocytes with ingested S. aureus in the absence or presence of 5 mg of azithromycin per liter. The height of a column with the standard error of the mean line above the column represents the mean number of S. aureus in the control incubated without lysostaphin (extracellular and intracellular bacteria). The dotted part of the column with the standard error of the mean line within the column represents the mean number of S. aureus after incubation with lysostaphin (intracellular bacteria). The values in parentheses indicate the number of experiments.

4 VOL. 37, 1993 Time (min) TABLE 1. Cellular concentration of azithromycin Cellular concn (mg/liter) C/E ratio Monocytes Granulocytes Monocytes Granulocytes incubation of both monocytes and granulocytes in the presence of 5 mg of azithromycin per liter leads to a cellassociated concentration of between 25 and 3 mg/liter (Table 1). The best fit for these concentrations was calculated by equation 5 as C = x (1 - e--95'). The extracellular ph of the medium was 6.8 to 7.. The cellassociated concentration of 1 mg/l at min indicates a rapid association between drug and cells during the few minutes required to separate the cells from the medium after the addition of azithromycin to the cell suspension at the beginning of the experiment. DISCUSSION The results of the present investigation show that the antibacterial activity of azithromycin against S. aureus ingested by human monocytes is enhanced compared with the effect on S. aureus in cell-free medium. As expected, in cell-free medium azithromycin is bacteriostatic for S. aureus. However, the antibiotic caused a decrease in the number of viable S. aureus phagocytosed by monocytes. This enhancement of the antibacterial activity was not observed with granulocytes. The activity of azithromycin against bacteria located intracellularly in human monocytes has not been reported earlier. Milatovic (6) reported a lack of intracellular activity of azithromycin against S. aureus ingested by human granulocytes, in contrast to the intracellular activities of erythromycin and roxithromycin. Milatovic (6) suggested that the low intracellular ph inhibited the intracellular activity of azithromycin. However, Milatovic (6) used lysostaphin in the microbiological assay, which is questionable and might have influenced the results (13, 15, 16). The equal activities of azithromycin, erythromycin, and clarithromycin against S. aureus phagocytosed by human granulocytes during up to 4 h of incubation was shown by Paulsen et al. (9). The minimal effective concentration of all three antibiotics was.1 mg/liter, and an increase in the concentration did not result in greater antibacterial activity. The minimal effective concentration of roxithromycin was 1.2 mg/liter; at that concentration the drug was as effective as the other macrolides. The growth-inhibiting effects of azithromycin and roxithromycin lasted for 24 h (9). Monocytes and granulocytes accumulate azithromycin to the same extent. Therefore, differences in intracellular concentrations do not explain the difference in intracellular antibacterial activities between the two cell types. In the present investigation, a rather low level of uptake of azithromycin was found compared with data from the literature (1-3, 8). In those investigations, RPMI medium was used at ph 7.4, often with serum added. Using RPMI medium, our assay yielded C/E ratios of about 3 to 45 after 3 h of incubation (data not shown). The uptake of azithromycin depends markedly on the extracellular ph (3). The ph of the HUMAN MONOCYTES AND AZITHROMYCIN 2321 medium used in our assays of the antibacterial activity of azithromycin was 6.8 to 7., which explains the low level of uptake that we found. The extrapolated maximum intracellular concentration of mg/liter is about six times the extracellular concentration. Assuming two pky values for azithromycin of 8.8 and 8.1, it can be calculated that, when the extracellular ph is 6.9, the intracellular ph should be 6.5 to account for this amount of ion trapping. The value of.95 h-' for k corresponds to a half-life for diffusion of 44 min. These results support the view that no active transport is involved in the intracellular accumulation of azithromycin. 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