Experim ental Chem otherapy Chemotherapy 1992;38:185-190
Department of Paediatrics and Department of Medical Statistics, University of Kiel, FRG
Key Words Azithromycin Clarithromycin Erythromycin Roxithromycin Macrolide Polymorphonuclear Leucocyte Chediak-Higashi syndrome
Influence of Azithromycin and Other Macrolides on the Intracellular Killing of Staphylococcus aureus by Human Polymorphonuclear Leucocytes of Healthy Donors and a Patient with Chediak-Higashi Syndrome
Abstract A mixture of human blood phagocytes from healthy donors and opsonized staphylococci was incubated in vitro for 30 min. After that time all the bacteria were phagocytosed. The test tubes were further incubated for 2, 4 and 24 h with or without addition of a macrolide (erythromycin, azithromycin, clarith romycin, roxithromycin) and the effect of these drugs on the survival of intracellular staphylococci (Staphylococcus aureus ATCC 25923) was measured. The minimal effective concentra tion of the antibiotic which killed 80-90% of the bacteria after a 4-hour incubation was 0.1 mg/1 for erythromycin, azithromy cin and clarithromycin and 1.2 mg/1 for roxithromycin. The per centage of surviving bacteria after 2 and 4 h incubation was not significantly different between these macrolides at the minimal effective concentration. Increasing the concentration of each antibiotic above the minimal effective concentration did not alter the killing rate of intracellular staphylococci. The bacte rial activity of polymorphonuclear leucocytes (PMNL) from a patient with Chediak-Higashi syndrome was less in compari son to PMNL from healthy donors, but was improved in vitro by the addition of erythromycin or azithromycin.
Prof C Simon University Children’s Hospital Schwancnweg 20 D-W-2300 Kiel (FRG)
© 1992 S. Karger AG, Basel 0009-3157/92/ 0383-0185 $ 2.75/0
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P. Paulsen C. Simon O. Peters J. Hedderich P. Heim
Intracellular killing of bacteria in polymor phonuclear leucocytes (PNL) can be en hanced by antibiotics penetrating into blood cells. With the new macrolide azithromycin, it is well known that intracellular and tissue lev els of the antibiotic during treatment are much higher than serum levels [1, 2], Clari thromycin [3-5], roxithromycin [4, 6-8] and erythromycin [4,6,7,9-11] also accumulated in cells, and may be very active against intracellu lar bacteria, but no comparative studies have been done between these 4 drugs under the same test conditions. It would also be interest ing to compare the antibacterial activity of erythromycin and azithromycin in PMNL from healthy donors and a patient suffering from Chediak-Higashi syndrome (CHS). CHS is a rare and fatal autosomal recessive disease. It is clinically characterized by partial oculo cutaneous albinism, photophobia, nystagmus, an accelerated lymphohistiocytic phase and recurrent severe pyogenic infections at any lo cation [12]. The pathological hallmark of CHS is the presence of abnormal large granules in PMNL and other granule-containing cells. PMNL from CHS are defective in chcmotaxis, degranulation and intracellular killing of bac teria.
Material and Methods Antimicrobial Agents Erythromycin base and clarithromycin were ob tained from Abbott, Wiesbaden, FRG, azithromycin from Pfizer, Karlsruhe, FRG, and roxithromycin from Roussel Laboratories, Paris, France. The antibiotics (except azithromycin) were dissolved in methanol, azithromycin in acetonitril (stock solution 1 mg/ml) and then diluted in Hank’s balanced salt solution (HBSS, Gibco). Final antibiotic concentrations in the test tubes (after adding polymorphonuclear leuco cytes, pooled AB serum and bacteria) were 0.6,0.3,0.1, 0.05 and 0.01 mg/1. In studies with roxithromycin 1.2 mg/1 was also tested.
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Bacteria The Staphylococcus aureus strain ATCC 25923 was used for test bacteria. Staphylococci from an overnight broth culture were centrifuged, washed and resus pended in 5 ml HBSS. After further dilution with HBSS (1:100) the staphylococcal suspension was son icated for 15 s at 70 W on an ultrasonicator immediately before use, to disrupt bacterial clumps. The bacterial count in this suspension was 3 x 105-6 x 105 cfu/ml. Patient From birth a 9-year-old girl had suffered from re current severe infections of the upper and lower respi ratory tract, and numerous episodes of high fever of unknown origin. At the age of 3 months, nystagmus, photophobia and albinism were recognized for the first time. At 2 years of age CHS was diagnosed at the Uni versity of Rostock, FRG, and later confirmed in our hospital. Laboratory tests then revealed neutropenia and a slightly hypochromic normocytic anaemia. Giant lysosomal peroxidase-positive granules of mature neu trophils, monocytes and lymphocytes were detected in blood smears. The same feature was seen in bone mar row preparations, which also showed increased granu lopoiesis and atypical cytoplasmic vacuoles in the granulocytes. Random and stimulated migration of granulocytes (chemotaxis) were reduced to 70 and 50% of normal, respectively. Bactericidal activity was significantly di minished whereas superoxide generation and the nitroblue tetrazolium test were normal. Lymphocyte typ ing presented a normal distribution, but lymphocyte stimulation was clearly diminished. Immunoglobulins, complement (CH50, APH50, C3, C4, C3d), liver and kidney parameters were in the normal range. Examina tions on admission established oculocutaneous albi nism, cholesteatoma, Candida stomatitis and vaginitis. An intelligence test revealed slight mental retardation (IQ of 80). Polymorphonuclear Leucocytes Fresh human blood was obtained by venepuncture of healty donors and the patient suffering from CHS. Heparin was used as an anticoagulant. For the pre paration of granulocytes, Percoll (Pharmacia) was tak en as medium for density gradient centrifugation with polyvinyl-pyrrolidone-coated silica particles. Stock iso tonic Percoll was prepared by dissolving solid NaCl in Percoll to a final concentration of 0.15 M. This mixture was referred to as 100% Percoll and had a density of 1.136 g/ml. A discontinuous gradient was prepared as follows: 71% isotonic Percoll (4 ml; p = 1.1026 g/ml) was poured into a 14-ml polystyrol centrifuge tube. This so
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Introduction
lution was then ovcrlayercd with 55% isotonic Percoll solution (4 ml; p = 1.0776 g/ml). Undiluted human blood (4 ml) was applied to the top of the gradient and the tube was centrifuged for 25 min at 350 g in an MSE Super Minor centrifuge. After centrifugation the gran ulocyte band was removed with a Pasteur pipette. The granulocytes obtained by this procedure were washed once in phosphate-buffered saline (PBS) and centri fuged at 300 g for 10 min. Contaminating red cells were removed by addition of 20 ml of cold sterile distilled water for 20 s. 5 ml of a 3.5% saline solution were then added to obtain an isotonic solution. After washing again in PBS and centrifuging at 300 g for 10 min the cells (from the pellet) were resuspended in 1 ml HBSS and the cell count was adjusted to 4 x 106 PMNL/ml. Opsonization o f Bacteria and Phagocytosis In polystyrol test tubes 0.5 ml of the PMNL suspen sion, 0.4 ml of the bacterial suspension and 0.1 ml of pooled AB serum were mixed and incubated in a wa ter-bath for 30 min. After that time all the bacteria were phagocytosed and no extracellular bacteria could be seen under the ultraviolet microsope (after staining a sample with 0.001% acridine orange solution). After removal of 0.1 ml from each tube to determine the in tracellular bacterial count, 0.1 ml of the antibiotic was added for further incubation. Determination o f the Bacterial Count After 2, 4 and 24 h of incubation 0.1 ml from each tube was removed (after thorough mixing), 0.9 ml icecold distilled water added and the mixture sonicated for 15 s to disrupt the cells. In none of these samples ex tracellular bacteria were detectable microscopically (also after 24 h of incubation). The bacterial count (number of colony forming units) was determined by the dilution and pour-plate technique.
Table 1. Median percentage of surviving intracel lular staphylococci after 2, 4 and 24 h incubation at the minimal effective concentration of erythromycin, azithromycin, clarithromycin and roxithromycin Drug
n
Cone. mg/1
Erythromycin
7
0.1
Azithromycin
10
0.1
Clarithromycin
10
0.1
Roxithromycin
8
1.2
Control without drug
22
-
Percentage of surviving bacteria after 2h
4h
24 h
24.1 (118) 27.3 (122) 24.7 (144) 18.2 (90)
11.6 (95) 11.9 (99) 12.2 (121) 8.8 (85)
27.8 (70) 7.9 (67) 23.0 (85) 6.3 (40)
42.0
25.3
456.2
Values in parentheses are percentages in control tubes without PMNL. All these results with drugs are significantly different (p < 0.05) from the correspond ing values in tubes without an antibiotic.
test is suitable for the comparison of independent sam ples. The test variable H allows the evaluation of statistically significant differences between groups (p < 0.05). Pairwise comparisons were done by a mul tiple-comparison procedure.
The minimal effective concentration of added antibiotic for killing 70-80% of staphy lococci phagocytosed by PMNL after 2 h in cubation was 0.1 mg/1 for erythromycin, azith romycin and clarithromycin and 1.2 mg/1 for roxithromycin. After 4 h incubation 80-90% of bacteria were killed by these antibiotics (table 1). This was the lowest antibiotic concentra tion giving significant differences in compari son to the control tubes (without an anti biotic). After 24 h incubation the percentages
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Results Statistics The statistical analysis, based on comparisons of relative survival rates of bacteria between the antibiot ics at different concentrations, were performed using the BMDP programs [13]. Central location values giv en in the text and tables are median values (50th per centiles) calculated from at least 7 (usually more) ob servations in each sample. The range is characterized by minimum and maximum values in table 2. Because a normal distribution could not be assumed, the nonparametric Kruskal-Wallis test was performed. This
Table 2. Percentages of surviving intracellular staphylococci after 2, 4, and 24 h incubation at different con centrations of erythromycin, azithromycin, clarithromycin and roxithromycin. Percentage of surviving bacteria after
Cone. mg/1
Drug
0.01
A C E R
45.3 48.9 43.8 44.2
(37-52) (36-57) (25-63) (26-58)
23.1 28.4 27.5 26.3
(12-38) (18-33) (10-32) (11-31)
145.4 244.3 218.8 254.7
0.05
A C E R
27.5 34.2 27.5 41.6
(6-35) (20-40 (10-32) (21-54)
16.9 13.3 14.1 25.8
(2-18) (10-20) (9-24) (12-38)
52.5 (10-65) 111.0 (16-177) 89.1 (66-133) 262.0 (98-421)
0.1
A C E R
27.3 24.7 24.1 42.2
(9-34) (9-33) (12-40) (38-46)
11.9 12.2 11.6 27.8
(1-21) (1-20) (8-18) (23-32)
7.9 (1-24) 23.0 (16-58) 27.8 (15-65) 333.0 (134-773)
0.3
A C E R
17.9 28.5 20.5 40.0
(5-36) (0-36) (12-36) (29-57)
10.4 17.5 10.9 22.5
(1-16) (4-23) (8-20) (17-31)
3.0 (1-6) 5.0 (2-6) 8.4 (0.2-12) 69.2 (27-98)
0.6
A C E R
17.9 24.5 21.0 25.8
(10-52) (8-48) (14-42) (13-52)
11.3 (3-18) 10.5 (4-38) 13.5 (3-21) 15.2 (6-40)
0.4 (0.2-3) 3.4 (2-13) 3.0 (1-17) 51.3 (19-70)
1.2
R
18.2 (13-31)
8.8 (6-11)
6.3 (1-11)
none
42.0 (24-73)
25.3 (7^14)
456.2 (147-800)
4h
2h
24 h (65-262) (157—400) (85-441) (105-410)
of surviving bacteria were still low, varying be tween 6 and 27%, despite the fact that nearly 50% of the granulocytes were no longer alive (shown by staining with acridine orange and UV microscopy). Under the influence of azithromycin (at 0.1 mg/1) and roxithromycin (at 1.2 mg/1) the colony count was significantly lower than with the other macrolides (at the same concentration). In the control tubes
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without an antibiotic the bacterial count after 24 h incubation had increased 4- to 5-fold due to the disruption of many granulocytes by nu merous intracellular bacteria. In the control tubes without PMNL but with antibiotic (table 1) the bacterial count af ter 24 h incubation was reduced to 40-85% of the initial values (at 0.1 mg/1 resp. 1.2 mg/1). Comparing the percentage of surviving
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Values are median (range). A = azithromycin; C = clarithromycin; E = erythromycin; R = roxithromycin.
Discussion Intracellular killing of staphylococci in the presence of azithromycin, clarithromycin and erythromycin was observed at a minimal add ed concentration of 0.1 mg/1, with roxithromy cin at 1.2 mg/1. This has also been shown by other authors of azithromycin [14], roxithro mycin [15,16], clarithromycin [3] and erythro mycin [10], In the studies of Anderson et al. [3] clarithromycin had nearly the same intracellu lar bactericidal activity for Listeria monocyto genes as erythromycin but was more active than erythromycin for S. aureus. Roxithromy cin was comparable to erythromycin with re-
Table 3. Median percentage of surviving intracel lular staphylococci after 2, 4, 24 h with and without erythromycin or azithromycin in PMNL from healthy donors and from a child with CHS. Drug
Cone. Percentage of surviving mg/1 bacteria in PMNL from Healthy persons
CHS
_
_
E A
0.3 0.3
2h
4h
24 h
2h
4h
24 h
78 50 49
49 42 18
580 6 14
42 20 18
25 10 10
456 8 3
E = erythromycin; A = azithromycin
spect to the bacteriostatic effect on phagocy tosed S. aureus [17]. Milatovic [17] also com pared the influence of azithromycin and roxithromycin on the intracellular killing of staphylococci and found that roxithromycin was more active against the used test strain than azithromycin. From the studies of Craig et al. [18] it is well known that several extracel lular factors can influence the staphylococcicidal capacity of human PMNL, and that small differences in methodology may explain con tradictory results in the literature. In the patient with CHS the bactericidal ac tivity of PMNL after 2 and 4 h was less than that of PMNL from healthy persons. By addi tion of erythromycin or azithromycin after 2 and 4 h incubation, the intracellular killing rate of bacteria was improved but was still dif ferent from that in PMNL from healthy per sons. After 24 h incubation the percentage of surviving bacteria in the PMNL of the patient was as low as in the PMNL from healthy per sons. This may be useful for the treatment of staphylococcal infections in patients with CHS.
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bacteria at 0.1, 0.3 and 0.6 mg/1 no significant differences were found between erythromy cin, azithromycin and clarithromycin after 2 and 4 h incubation (table 2). Roxithromycin at 0.6 mg/1 seemed to produce a similar killing rate of intracellular staphylococci after 2 and 4 h as the other macrolides at 0.1 mg/1, but this was not significantly different in comparison to the control tubes until 24 h. At 1.2 mg/1 roxitromycin was as active as the other anti biotics after 2 and 4 h incubation. Intracellular killing of staphylococci in PMNL from the 9-year-old child with CHS was less in comparison to that in PMNL from healthy donors (table 3). In PMNL of this child about 50% of phagocytosed staphylo cocci (without addition of an antibiotic) were killed after 4 h incubation (versus 75% of bac teria in normal PMNL). In the presence of 0.3 mg/1 erythromycin the killing rate in PMNL from this patient after 4 h incubation was 58% in comparison to 90% in PMNL from healthy donors, but after 24 h the killing rate was simi lar. Azithromycin (0.3 mg/1) reduced the per centage of intracellular bacteria in the PMNL of the patient to 18 and 14% after 4 and 24 h, respectively.
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1 Gladue RP. Bright, GM, Isaacson RE, Newborg MF: In vitro and in vivo uptake of azithromycin (CP-62, 993) by phagocytic cells: Possible mechanism of delivery and release at sites of infection. Antimicrob Agents Chemother 1989;33:277-282. 2 Laufen H, Wildfeuer A, Lach P: Mechanism of azithromycin uptake in human polymorphonuclear leu kocytes. Arzneimittelforschung 1990;40: 686-689. 3 Anderson R, Joone G, van Rensburg CEJ: An in-vitro evaluation of the cellular uptake and intraphagocytic bioactivity of clarithromycin (A-56268, TE-031 ), a new macrolide antimicrobial agent. J Antimicrob Chemother 1988;22:923-933. 4 Ishiguro M, Koga H, Kohno S, Hayashi T, Yamaguchi K, Hirota M: Penetration of macrolides into hu man polymorphonuclear leucocytes. J Antimicrob Chemother 1989;24: 719-729. 5 Masaki M: Polymorphonuclear leu cocyte (PMN) penetration of mac rolides. Chemotherapy 1987;35:430. 6 Anderson R, van Rensburg CE], Joone G, Lukey PT: An in vitro com parison of the intraphagocytic bio activity of erythromycin and roxith romycin. J Antimicrob Chemother 1987;20(suppl B):57-68.
7 Carlier MB, Zenebergh A, Tulkens PM: Cellular uptake and subccllular distribution of roxithromycin and erythromycin in phagocytic cells. J Antimicrob Chemother 1987;20 (suppl B):47-56. 8 Hand WL, King-Thompson NL. Holman JW: Entry of roxithromycin (RU 965), imipenem, cefotaxime, trimethoprim and metronidazole into human polymorphonuclear leu kocytes. Antimicrob Agents Chemo ther 1987;31:1553-1557. 9 Madgwick L, Mayer S, Keen P: Pen etration of antibiotics into bovine neutrophils and their activity against intracellular Siaphycoccus aureus. J Antimicrob Chemother 1989;24: 709-718. 10 Miller MF: Erythromycin uptake and accumulation by human poly morphonuclear leucocytes and effi cacy of erythromycin in killing in gested Legionella pneumophila. J In fect Dis 1984;149:714-718. 11 Prokcsch RC, Hand WL: Antibiotic entry into human polymorphonu clear leucocytes. Antimicrob Agents Chemother 1982;21:373-380. 12 Stolz W, Graubncr U, Gerstmeier J, Burg G. Belohradsky BH: ChediakHigashi syndrome: Approaches in diagnosis and treatment. Curr Probl Dermatol 1989;18:93-100.
13 Dixon WJ: Biomedical Computer Programs. Statistical Software Man ual, vol 1. Berkeley, University of California Press, 1988. 14 Wildfeucr A, Laufcn H. MullerWcning D, Haferkamp O: Interac tion of azithromycin and human phagocytic cells. Uptake of the anti biotic and the effect on the survival of ingested bacteria in phagocytes. Arzneimittelforschung 1989;39:755758. 15 Fietta A, Mangiarotti P. Bersani C, De Rose V. Gialdroni Grassi G: In vitro effect of RU 28965 and other macrolide antibiotics on human neutrophil functions; in Butzler JP (cd): Makrolides. Amsterdam, Excerpta Medica, 1986, pp 114-117. 16 Labro MT. Amit N, Babin-Chevaye C, Hakim J. Synergy between RU 28965 (roxithromycin) and human neutrophils for bactericidal activity in vitro. Antimicrob Agents Chemo ther 1986;30:137-142. 17 Milatovic D: Intraphagocytic activ ity of erythromycin, roxithromycin and azithromycin. Eur J Clin Micro biol Infect Dis 1990;1:33-35. 18 Craig CP, Suter E: Extracellular fac tors influencing staphylocidal ca pacity of human polymorphonu clear leucocytes. J Immunol 1966; 97:287-2%.
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References
Department of Paediatrics and Department of Medical Statistics, University of Kiel, FRG
Key Words Azithromycin Clarithromycin Erythromycin Roxithromycin Macrolide Polymorphonuclear Leucocyte Chediak-Higashi syndrome
Influence of Azithromycin and Other Macrolides on the Intracellular Killing of Staphylococcus aureus by Human Polymorphonuclear Leucocytes of Healthy Donors and a Patient with Chediak-Higashi Syndrome
Abstract A mixture of human blood phagocytes from healthy donors and opsonized staphylococci was incubated in vitro for 30 min. After that time all the bacteria were phagocytosed. The test tubes were further incubated for 2, 4 and 24 h with or without addition of a macrolide (erythromycin, azithromycin, clarith romycin, roxithromycin) and the effect of these drugs on the survival of intracellular staphylococci (Staphylococcus aureus ATCC 25923) was measured. The minimal effective concentra tion of the antibiotic which killed 80-90% of the bacteria after a 4-hour incubation was 0.1 mg/1 for erythromycin, azithromy cin and clarithromycin and 1.2 mg/1 for roxithromycin. The per centage of surviving bacteria after 2 and 4 h incubation was not significantly different between these macrolides at the minimal effective concentration. Increasing the concentration of each antibiotic above the minimal effective concentration did not alter the killing rate of intracellular staphylococci. The bacte rial activity of polymorphonuclear leucocytes (PMNL) from a patient with Chediak-Higashi syndrome was less in compari son to PMNL from healthy donors, but was improved in vitro by the addition of erythromycin or azithromycin.
Prof C Simon University Children’s Hospital Schwancnweg 20 D-W-2300 Kiel (FRG)
© 1992 S. Karger AG, Basel 0009-3157/92/ 0383-0185 $ 2.75/0
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P. Paulsen C. Simon O. Peters J. Hedderich P. Heim
Intracellular killing of bacteria in polymor phonuclear leucocytes (PNL) can be en hanced by antibiotics penetrating into blood cells. With the new macrolide azithromycin, it is well known that intracellular and tissue lev els of the antibiotic during treatment are much higher than serum levels [1, 2], Clari thromycin [3-5], roxithromycin [4, 6-8] and erythromycin [4,6,7,9-11] also accumulated in cells, and may be very active against intracellu lar bacteria, but no comparative studies have been done between these 4 drugs under the same test conditions. It would also be interest ing to compare the antibacterial activity of erythromycin and azithromycin in PMNL from healthy donors and a patient suffering from Chediak-Higashi syndrome (CHS). CHS is a rare and fatal autosomal recessive disease. It is clinically characterized by partial oculo cutaneous albinism, photophobia, nystagmus, an accelerated lymphohistiocytic phase and recurrent severe pyogenic infections at any lo cation [12]. The pathological hallmark of CHS is the presence of abnormal large granules in PMNL and other granule-containing cells. PMNL from CHS are defective in chcmotaxis, degranulation and intracellular killing of bac teria.
Material and Methods Antimicrobial Agents Erythromycin base and clarithromycin were ob tained from Abbott, Wiesbaden, FRG, azithromycin from Pfizer, Karlsruhe, FRG, and roxithromycin from Roussel Laboratories, Paris, France. The antibiotics (except azithromycin) were dissolved in methanol, azithromycin in acetonitril (stock solution 1 mg/ml) and then diluted in Hank’s balanced salt solution (HBSS, Gibco). Final antibiotic concentrations in the test tubes (after adding polymorphonuclear leuco cytes, pooled AB serum and bacteria) were 0.6,0.3,0.1, 0.05 and 0.01 mg/1. In studies with roxithromycin 1.2 mg/1 was also tested.
186
Bacteria The Staphylococcus aureus strain ATCC 25923 was used for test bacteria. Staphylococci from an overnight broth culture were centrifuged, washed and resus pended in 5 ml HBSS. After further dilution with HBSS (1:100) the staphylococcal suspension was son icated for 15 s at 70 W on an ultrasonicator immediately before use, to disrupt bacterial clumps. The bacterial count in this suspension was 3 x 105-6 x 105 cfu/ml. Patient From birth a 9-year-old girl had suffered from re current severe infections of the upper and lower respi ratory tract, and numerous episodes of high fever of unknown origin. At the age of 3 months, nystagmus, photophobia and albinism were recognized for the first time. At 2 years of age CHS was diagnosed at the Uni versity of Rostock, FRG, and later confirmed in our hospital. Laboratory tests then revealed neutropenia and a slightly hypochromic normocytic anaemia. Giant lysosomal peroxidase-positive granules of mature neu trophils, monocytes and lymphocytes were detected in blood smears. The same feature was seen in bone mar row preparations, which also showed increased granu lopoiesis and atypical cytoplasmic vacuoles in the granulocytes. Random and stimulated migration of granulocytes (chemotaxis) were reduced to 70 and 50% of normal, respectively. Bactericidal activity was significantly di minished whereas superoxide generation and the nitroblue tetrazolium test were normal. Lymphocyte typ ing presented a normal distribution, but lymphocyte stimulation was clearly diminished. Immunoglobulins, complement (CH50, APH50, C3, C4, C3d), liver and kidney parameters were in the normal range. Examina tions on admission established oculocutaneous albi nism, cholesteatoma, Candida stomatitis and vaginitis. An intelligence test revealed slight mental retardation (IQ of 80). Polymorphonuclear Leucocytes Fresh human blood was obtained by venepuncture of healty donors and the patient suffering from CHS. Heparin was used as an anticoagulant. For the pre paration of granulocytes, Percoll (Pharmacia) was tak en as medium for density gradient centrifugation with polyvinyl-pyrrolidone-coated silica particles. Stock iso tonic Percoll was prepared by dissolving solid NaCl in Percoll to a final concentration of 0.15 M. This mixture was referred to as 100% Percoll and had a density of 1.136 g/ml. A discontinuous gradient was prepared as follows: 71% isotonic Percoll (4 ml; p = 1.1026 g/ml) was poured into a 14-ml polystyrol centrifuge tube. This so
Paulscn/Simon/Peters/Hedderich/Hcim
Macrolides and PMNL
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Introduction
lution was then ovcrlayercd with 55% isotonic Percoll solution (4 ml; p = 1.0776 g/ml). Undiluted human blood (4 ml) was applied to the top of the gradient and the tube was centrifuged for 25 min at 350 g in an MSE Super Minor centrifuge. After centrifugation the gran ulocyte band was removed with a Pasteur pipette. The granulocytes obtained by this procedure were washed once in phosphate-buffered saline (PBS) and centri fuged at 300 g for 10 min. Contaminating red cells were removed by addition of 20 ml of cold sterile distilled water for 20 s. 5 ml of a 3.5% saline solution were then added to obtain an isotonic solution. After washing again in PBS and centrifuging at 300 g for 10 min the cells (from the pellet) were resuspended in 1 ml HBSS and the cell count was adjusted to 4 x 106 PMNL/ml. Opsonization o f Bacteria and Phagocytosis In polystyrol test tubes 0.5 ml of the PMNL suspen sion, 0.4 ml of the bacterial suspension and 0.1 ml of pooled AB serum were mixed and incubated in a wa ter-bath for 30 min. After that time all the bacteria were phagocytosed and no extracellular bacteria could be seen under the ultraviolet microsope (after staining a sample with 0.001% acridine orange solution). After removal of 0.1 ml from each tube to determine the in tracellular bacterial count, 0.1 ml of the antibiotic was added for further incubation. Determination o f the Bacterial Count After 2, 4 and 24 h of incubation 0.1 ml from each tube was removed (after thorough mixing), 0.9 ml icecold distilled water added and the mixture sonicated for 15 s to disrupt the cells. In none of these samples ex tracellular bacteria were detectable microscopically (also after 24 h of incubation). The bacterial count (number of colony forming units) was determined by the dilution and pour-plate technique.
Table 1. Median percentage of surviving intracel lular staphylococci after 2, 4 and 24 h incubation at the minimal effective concentration of erythromycin, azithromycin, clarithromycin and roxithromycin Drug
n
Cone. mg/1
Erythromycin
7
0.1
Azithromycin
10
0.1
Clarithromycin
10
0.1
Roxithromycin
8
1.2
Control without drug
22
-
Percentage of surviving bacteria after 2h
4h
24 h
24.1 (118) 27.3 (122) 24.7 (144) 18.2 (90)
11.6 (95) 11.9 (99) 12.2 (121) 8.8 (85)
27.8 (70) 7.9 (67) 23.0 (85) 6.3 (40)
42.0
25.3
456.2
Values in parentheses are percentages in control tubes without PMNL. All these results with drugs are significantly different (p < 0.05) from the correspond ing values in tubes without an antibiotic.
test is suitable for the comparison of independent sam ples. The test variable H allows the evaluation of statistically significant differences between groups (p < 0.05). Pairwise comparisons were done by a mul tiple-comparison procedure.
The minimal effective concentration of added antibiotic for killing 70-80% of staphy lococci phagocytosed by PMNL after 2 h in cubation was 0.1 mg/1 for erythromycin, azith romycin and clarithromycin and 1.2 mg/1 for roxithromycin. After 4 h incubation 80-90% of bacteria were killed by these antibiotics (table 1). This was the lowest antibiotic concentra tion giving significant differences in compari son to the control tubes (without an anti biotic). After 24 h incubation the percentages
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Results Statistics The statistical analysis, based on comparisons of relative survival rates of bacteria between the antibiot ics at different concentrations, were performed using the BMDP programs [13]. Central location values giv en in the text and tables are median values (50th per centiles) calculated from at least 7 (usually more) ob servations in each sample. The range is characterized by minimum and maximum values in table 2. Because a normal distribution could not be assumed, the nonparametric Kruskal-Wallis test was performed. This
Table 2. Percentages of surviving intracellular staphylococci after 2, 4, and 24 h incubation at different con centrations of erythromycin, azithromycin, clarithromycin and roxithromycin. Percentage of surviving bacteria after
Cone. mg/1
Drug
0.01
A C E R
45.3 48.9 43.8 44.2
(37-52) (36-57) (25-63) (26-58)
23.1 28.4 27.5 26.3
(12-38) (18-33) (10-32) (11-31)
145.4 244.3 218.8 254.7
0.05
A C E R
27.5 34.2 27.5 41.6
(6-35) (20-40 (10-32) (21-54)
16.9 13.3 14.1 25.8
(2-18) (10-20) (9-24) (12-38)
52.5 (10-65) 111.0 (16-177) 89.1 (66-133) 262.0 (98-421)
0.1
A C E R
27.3 24.7 24.1 42.2
(9-34) (9-33) (12-40) (38-46)
11.9 12.2 11.6 27.8
(1-21) (1-20) (8-18) (23-32)
7.9 (1-24) 23.0 (16-58) 27.8 (15-65) 333.0 (134-773)
0.3
A C E R
17.9 28.5 20.5 40.0
(5-36) (0-36) (12-36) (29-57)
10.4 17.5 10.9 22.5
(1-16) (4-23) (8-20) (17-31)
3.0 (1-6) 5.0 (2-6) 8.4 (0.2-12) 69.2 (27-98)
0.6
A C E R
17.9 24.5 21.0 25.8
(10-52) (8-48) (14-42) (13-52)
11.3 (3-18) 10.5 (4-38) 13.5 (3-21) 15.2 (6-40)
0.4 (0.2-3) 3.4 (2-13) 3.0 (1-17) 51.3 (19-70)
1.2
R
18.2 (13-31)
8.8 (6-11)
6.3 (1-11)
none
42.0 (24-73)
25.3 (7^14)
456.2 (147-800)
4h
2h
24 h (65-262) (157—400) (85-441) (105-410)
of surviving bacteria were still low, varying be tween 6 and 27%, despite the fact that nearly 50% of the granulocytes were no longer alive (shown by staining with acridine orange and UV microscopy). Under the influence of azithromycin (at 0.1 mg/1) and roxithromycin (at 1.2 mg/1) the colony count was significantly lower than with the other macrolides (at the same concentration). In the control tubes
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without an antibiotic the bacterial count after 24 h incubation had increased 4- to 5-fold due to the disruption of many granulocytes by nu merous intracellular bacteria. In the control tubes without PMNL but with antibiotic (table 1) the bacterial count af ter 24 h incubation was reduced to 40-85% of the initial values (at 0.1 mg/1 resp. 1.2 mg/1). Comparing the percentage of surviving
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Values are median (range). A = azithromycin; C = clarithromycin; E = erythromycin; R = roxithromycin.
Discussion Intracellular killing of staphylococci in the presence of azithromycin, clarithromycin and erythromycin was observed at a minimal add ed concentration of 0.1 mg/1, with roxithromy cin at 1.2 mg/1. This has also been shown by other authors of azithromycin [14], roxithro mycin [15,16], clarithromycin [3] and erythro mycin [10], In the studies of Anderson et al. [3] clarithromycin had nearly the same intracellu lar bactericidal activity for Listeria monocyto genes as erythromycin but was more active than erythromycin for S. aureus. Roxithromy cin was comparable to erythromycin with re-
Table 3. Median percentage of surviving intracel lular staphylococci after 2, 4, 24 h with and without erythromycin or azithromycin in PMNL from healthy donors and from a child with CHS. Drug
Cone. Percentage of surviving mg/1 bacteria in PMNL from Healthy persons
CHS
_
_
E A
0.3 0.3
2h
4h
24 h
2h
4h
24 h
78 50 49
49 42 18
580 6 14
42 20 18
25 10 10
456 8 3
E = erythromycin; A = azithromycin
spect to the bacteriostatic effect on phagocy tosed S. aureus [17]. Milatovic [17] also com pared the influence of azithromycin and roxithromycin on the intracellular killing of staphylococci and found that roxithromycin was more active against the used test strain than azithromycin. From the studies of Craig et al. [18] it is well known that several extracel lular factors can influence the staphylococcicidal capacity of human PMNL, and that small differences in methodology may explain con tradictory results in the literature. In the patient with CHS the bactericidal ac tivity of PMNL after 2 and 4 h was less than that of PMNL from healthy persons. By addi tion of erythromycin or azithromycin after 2 and 4 h incubation, the intracellular killing rate of bacteria was improved but was still dif ferent from that in PMNL from healthy per sons. After 24 h incubation the percentage of surviving bacteria in the PMNL of the patient was as low as in the PMNL from healthy per sons. This may be useful for the treatment of staphylococcal infections in patients with CHS.
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bacteria at 0.1, 0.3 and 0.6 mg/1 no significant differences were found between erythromy cin, azithromycin and clarithromycin after 2 and 4 h incubation (table 2). Roxithromycin at 0.6 mg/1 seemed to produce a similar killing rate of intracellular staphylococci after 2 and 4 h as the other macrolides at 0.1 mg/1, but this was not significantly different in comparison to the control tubes until 24 h. At 1.2 mg/1 roxitromycin was as active as the other anti biotics after 2 and 4 h incubation. Intracellular killing of staphylococci in PMNL from the 9-year-old child with CHS was less in comparison to that in PMNL from healthy donors (table 3). In PMNL of this child about 50% of phagocytosed staphylo cocci (without addition of an antibiotic) were killed after 4 h incubation (versus 75% of bac teria in normal PMNL). In the presence of 0.3 mg/1 erythromycin the killing rate in PMNL from this patient after 4 h incubation was 58% in comparison to 90% in PMNL from healthy donors, but after 24 h the killing rate was simi lar. Azithromycin (0.3 mg/1) reduced the per centage of intracellular bacteria in the PMNL of the patient to 18 and 14% after 4 and 24 h, respectively.
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1 Gladue RP. Bright, GM, Isaacson RE, Newborg MF: In vitro and in vivo uptake of azithromycin (CP-62, 993) by phagocytic cells: Possible mechanism of delivery and release at sites of infection. Antimicrob Agents Chemother 1989;33:277-282. 2 Laufen H, Wildfeuer A, Lach P: Mechanism of azithromycin uptake in human polymorphonuclear leu kocytes. Arzneimittelforschung 1990;40: 686-689. 3 Anderson R, Joone G, van Rensburg CEJ: An in-vitro evaluation of the cellular uptake and intraphagocytic bioactivity of clarithromycin (A-56268, TE-031 ), a new macrolide antimicrobial agent. J Antimicrob Chemother 1988;22:923-933. 4 Ishiguro M, Koga H, Kohno S, Hayashi T, Yamaguchi K, Hirota M: Penetration of macrolides into hu man polymorphonuclear leucocytes. J Antimicrob Chemother 1989;24: 719-729. 5 Masaki M: Polymorphonuclear leu cocyte (PMN) penetration of mac rolides. Chemotherapy 1987;35:430. 6 Anderson R, van Rensburg CE], Joone G, Lukey PT: An in vitro com parison of the intraphagocytic bio activity of erythromycin and roxith romycin. J Antimicrob Chemother 1987;20(suppl B):57-68.
7 Carlier MB, Zenebergh A, Tulkens PM: Cellular uptake and subccllular distribution of roxithromycin and erythromycin in phagocytic cells. J Antimicrob Chemother 1987;20 (suppl B):47-56. 8 Hand WL, King-Thompson NL. Holman JW: Entry of roxithromycin (RU 965), imipenem, cefotaxime, trimethoprim and metronidazole into human polymorphonuclear leu kocytes. Antimicrob Agents Chemo ther 1987;31:1553-1557. 9 Madgwick L, Mayer S, Keen P: Pen etration of antibiotics into bovine neutrophils and their activity against intracellular Siaphycoccus aureus. J Antimicrob Chemother 1989;24: 709-718. 10 Miller MF: Erythromycin uptake and accumulation by human poly morphonuclear leucocytes and effi cacy of erythromycin in killing in gested Legionella pneumophila. J In fect Dis 1984;149:714-718. 11 Prokcsch RC, Hand WL: Antibiotic entry into human polymorphonu clear leucocytes. Antimicrob Agents Chemother 1982;21:373-380. 12 Stolz W, Graubncr U, Gerstmeier J, Burg G. Belohradsky BH: ChediakHigashi syndrome: Approaches in diagnosis and treatment. Curr Probl Dermatol 1989;18:93-100.
13 Dixon WJ: Biomedical Computer Programs. Statistical Software Man ual, vol 1. Berkeley, University of California Press, 1988. 14 Wildfeucr A, Laufcn H. MullerWcning D, Haferkamp O: Interac tion of azithromycin and human phagocytic cells. Uptake of the anti biotic and the effect on the survival of ingested bacteria in phagocytes. Arzneimittelforschung 1989;39:755758. 15 Fietta A, Mangiarotti P. Bersani C, De Rose V. Gialdroni Grassi G: In vitro effect of RU 28965 and other macrolide antibiotics on human neutrophil functions; in Butzler JP (cd): Makrolides. Amsterdam, Excerpta Medica, 1986, pp 114-117. 16 Labro MT. Amit N, Babin-Chevaye C, Hakim J. Synergy between RU 28965 (roxithromycin) and human neutrophils for bactericidal activity in vitro. Antimicrob Agents Chemo ther 1986;30:137-142. 17 Milatovic D: Intraphagocytic activ ity of erythromycin, roxithromycin and azithromycin. Eur J Clin Micro biol Infect Dis 1990;1:33-35. 18 Craig CP, Suter E: Extracellular fac tors influencing staphylocidal ca pacity of human polymorphonu clear leucocytes. J Immunol 1966; 97:287-2%.
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References
