30
New Antimicrobial Agents
Comparative in Vitro Activity of the New Erythromycin Derivative Dirithromycin against GramPositive Bacteria Isolated from Cancer Patients K. V. Rolston*, D. H. Ho, B. L e B l a n c , G.P. B o d e y The in vitro activity of dirithromycin (LY237216), a new macrolide erythromycin derivative, was compared to that of four other agents (clarithromycin, erythromycin, roxithromycin, clindamycin) against 334 gram-positive isolates obtained from cancer patients. Dirithromycin was similar in potency and antimicrobial spectrum to the other agents tested. It was very active against beta-haemolytic streptococci and Streptococcus pneumoniae, and moderately active against penicillin and methicillin susceptible Staphylococcus aureus, Bacillus spp., Listeria monocytogenes and Corynebacterium jeikeium. Erythromycin resistant organisms were also resistant to dirithromycin. Macrolide antibiotics such as erythromycin, which was introduced in 1952, are active against various microorganisms including streptococcal species and methicillin-susceptible staphylococci, and are useful alternatives to the penicitlins in penicillin-allergic patients. In recent years several new macrolide compounds have been developed with improved chemical, biological or pharmacokinetic properties when compared to erythromycin (1). Dirithtomycin (LY237216) is a new 9-N-11-0-oxazine derivative of erythromycin formed from the condensation of 9Serythromycylamine with (2-methoxyethoxy)acetaldehyde. Pharmaeokinetic studies in animals have shown dirithromycin to have a longer half-life with higher and more persistent serum and tissue levels than erythromycin (F. T. Counter et al., and V. Busch et al., 28th Interscience Conference on Antimicrobial Agents and Chemotherapy, Los Angeles, 1988, Abstracts No. 920 and 922). We evaluated the in vitro activity of dirithromycin against 334 grampositive bacterial isolates obtained from blood culture specimens of cancer patients, a patient Section of Infectious Diseases (Box 47), Department of Medical Specialties, University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd., Houston, Texas 77030, USA.
Eur. J. Clin. Microbiol. Infect Dis.
population in which these organisms are emerging as significant pathogens (2). The activity of dirithromycin was compared to that of erythromycin, clindamycin and two newer macrolides roxithromycin and clarithromycin. Materials and Methods. The antimicrobial agents tested were erythromycin and clarithromycin (Abbott Laboratories, USA), roxithromycin (Hoechst-Roussel Pharmaceuticals, USA), clindamycin (Upjohn, USA) and dirithromycin (Lilly Research Laboratories, Indianapolis, IN, USA). Standard laboratory powders of known potency were used for susceptibility testing and kept frozen at -70°C prior to being used. All 334 isolates representing 14 bacterial species were obtrained from recent blood cultures of patients admitted to The University of Texas M. D. Anderson Cancer Center. Such isolates are stored in our laboratory by ultrafreezing methods and older strains are discarded as newer ones become available. Susceptibility testing was performed using a previously described microtiter broth dilution method, and in accordance with guidelines established by the National Committee for Clinical Laboratory Standards (3, 4). Briefly, MICs were determined in disposable plastic microtiter plates with 96 wells (Cooke Laboratory Products, USA). MIC plates were freshly prepared before each susceptibility testing experiment. Antibiotics were prepared in serial twofold dilutions in concentrations ranging from 64.0-0.03 I~g/ml and dispensed automatically using an MIC-2000 apparatus (Dynatech Laboratories, USA). The test medium used was cationsupplemented (Ca z+ 50 mg/1, and Mg2+ 25 mg/1) Mueller-Hinton broth (MHB; Difco Laboratories, USA) for all organisms except Corynebacterium jeikeium, which was tested in brainheart infusion broth with 5 % rabbit serum, and streptococci, which were tested in cationsupplemented MHB with 2 % lysed horse blood. Organisms were inoculated into broth and incubated at 37 °C for 18 h. Appropriate dilutions were then made so that the final inoculum of organisms tested was 105 CFU/ml. The MIC was recorded after incubation for 1620 h at 35 °C and defined as the lowest concentration of each drug required to inhibit visible bacterial growth. Two independent investigators performed each susceptibility test run and Staphylococcus aureus ATCC 25923 was included as the control strain for each procedure to ensure the validity of the results. Results and Discussion. Results of the susceptibility studies are shown in Table 1. Dirithro-
Vol. 1, 1990
31
Table 1: C o m p a r a t i v e in vitro activity of d i r i t h r o m y c i n a n d o t h e r antimicrobial a g e n t s a g a i n s t g r a m positive bacteria. M I C (mg/l)
O r g a n i s m s (n)
Antimicrobial agent
Bacillus spp.
dirithromycin clarithromycin erythromycin roxithromycin clindamycin
0.06 0.12 0.25 0.25 0.5
0.25 0.12 2.0 0.5 1.0
dirithromycin clarithromycin erythromycin roxithromycin clindamycin
0,25 0.5 2.0 4.0 > 64.0
8.0 4.0 > 64.0 > 64.0 > 64.0
Listeria monocytogenes
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
1.0 0.25 0.5 1.0 2.0
1.0 0.25 0.5 1.0 2.0
0.5 0.12 0.5 0.5 1.0-
Staphylococcus aureus
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
1.0 0.25 0.5 1,0 0.12
2.0 0.5 0.5 1.0 0.12
0.06 - 2.0 0.06 - 0.25 0.12 - 0.5 0.25 - 1.0 0.06-1.0
dirithromycin clarithromycln erythromycin roxithromycin clindamycin
0.25 0,12 0.12 0,25 0,25
> 64.0 >64.0 > 64.0 > 64.0 > 64.0
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
32.0 8.0 16.0 32.0 64.0
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
dirithromycin clarithromycm erythromycin roxithromycin clindamycin dirithromycin clarithromycm erythromycin roxithromycin clindamycin
32.0 32.0 64.0 64.0 > 64,0 0,12 0.06 0.12 0.06 0.12
> 64.0 > 64,0 > 64.0 > 64.0 > 64.0 0.12 0.12 0.25 0.12 0.12
0.5 0.06 64,0 > 64.0 > 64.0 > 64.0 > 64.0 0.12 0,12 0.25 0.12 0.12
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
0.25 0.12 0.03 0.06 0.03
0.25 0.25 0.12 0.12 0.06
0.12 0.06 64.0 0.06 - > 64,0 0.12 - > 64.0 > 64.0 > 64.0 > 64.0 > 64.0 > 64.0 0.5 0.06 0.12 0.25 0.06-
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
32
Eur. J. Clin. Microbiol. Infect Dis.
Table 1 continued
Organisms (n)
Antimicrobial agent
Streptococcus pneumoniae
dirithromycin clarithromycin erythromycin roxithromycin elindamyein dirithromycin elarithromyein erythromycin roxithromycin clindamycin dirithromycin clarithromycin erythromycin roxithromycin elindamycin dirithromycin clarithromycin erythromyein roxithromycin elindamycin
(25)
Streptococcus sanguis (35)
Streptococcus m i t i s (26)
Enterococcus faecalis (20)
mycin was e x t r e m e l y active against the betah a e m o l y t i c s t r e p t o c o c c i and Streptococcus pneumoniae, inhibiting all isolates at a concentration of 0.5 gg/ml. T h e activity of the other agents tested against these organisms was similar to that of dirithromycin. A l p h a - h a e m o l y t i c s t r e p t o c o c c a l isolates (Streptococcus sanguis and Streptococcus mitis) w e r e much less susceptible to all agents tested than o t h e r streptococcal isolates. However, there was a bimodal p a t t e r n of susceptibility for these organisms, 5 5 70 % being relatively susceptible and the rest having much higher MICs. D i r i t h r o m y c i n was active against penicillin susceptible Staphylococcus aureus isolates and had m o d e r a t e activity against penicillin resistant b u t methicillin susceptible Staphylococcus aureus isolates. All isolates of methicillin resistant Staphylococcus aureus and most isolates of c o a g u l a s e negative staphyloccocaI species w e r e resistant to all agents tested. D i r i t h r o mycin was also active against Bacillus spp. and Listeria monocytogenes. D i r i t h r o m y c i n and clarithromycin were more active against Corynebacterium ]eikeium than the other agents tested. The spectrum of activity and potency of dirithromycin w e r e t h e r e f o r e generally similar to those of the other antibiotics tested with only a few minor differences, erythromycin resistant strains also being resistant to dirithromycin. Interest in macrolide antimicrobial agents has been rekindled with the d e v e l o p m e n t of several newer compounds. Most of these offer no ad-
.... MIC 50 0.12 0.06 0.12 0.06 0.12 2.0 0.5 0.12 0.06 0.12 1.0 0.5 0.12 0.5 0.06 4.0 8.0 4.0 4.0 16.0
MIC (mg/1) MIC 90 0.12 0.12 0.25 0.25 0.25 16.0 4.0 4.0 4.0 2.0 32.0 16.0 8.0 16.0 8.0 > 64.0 > 64.0 > 64.0 > 64.0 > 64.0
Range
0.12 - 0.5 0.06 - 0.5 0.06 - 0.5 < 0.03 - 1.0 < 0.03 - 0.5 0.5- 64.0 0.06-16.0 0.12 - 8.0 0.06 8.0 0.12- 16.0 0.5 - 64.0 0.5- 32.0 0.06- 16.0 0.12 - 32.0 - 64.0 0.06- > 64.0 0.12 - > 64.0 0.12 - > 64.0 4.0 - > 64.0 -
v a n t a g e s over e r y t h r o m y c i n in t e r m s of spectrum or potency (1, 3, 5). Our data indicate that the activity of dirithromycin is similar to that of e r y t h r o m y c i n against isolates o b t a i n e d f r o m cancer patients. Investigators who tested isolates f r o m a variety of other sources have similar data, indicating that a l t h o u g h g r a m - p o s i t i v e infections m a y b e m o r e f r e q u e n t in c a n c e r patients, the organisms causing these infections do not generally possess a g r e a t e r degree of resistance than those isolated f r o m other patient p o p u l a t i o n s (1; K. W. Yu et al., 29th I n t e r science C o n f e r e n c e on A n t i m i c r o b i a l A g e n t s and C h e m o t h e r a p y , H o u s t o n , Texas, 1989, Abstract No. 1035). Clinical trials will d e t e r m i n e w h e t h e r n e w e r macrolides (including dirithromycin), which have better pharmacokinetic properties, are m o r e effective than erythromycin in the t r e a t m e n t of h u m a n infections.
References 1. Hardy JJ, Hensey DM, Beyer JM, Vojiko C, McDonald E J, Fernandez PB: Comparative in vitro activities of new 14-, 15- and 16-membered maerolides. Antimicrobial Agents and Chemotherapy 1988, 32:
1710-1719. 2. Viscoli C, Van der Auwera P, Meunier F: Grampositive infections in granulocytopenic patients: an
important issue? Journal of Antimicrobial Chemotherapy 1988, 21, Suplement C: 149-156.
Vol. 1, 1990
3. Rolston KVI, LeBlanc B, Ho DH: In vitro activity of RU-28965, a new macrolide, compared to that of erythromyein. Journal of Antimicrobial Chemotherapy 1986,17: 161-163. 4. National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard M7-A. NCCLS, Villanova, PA, 1985. 5. Rolston K, Gooch G, Ho D: In vitro of clarithromycin (A-56268; TE-031) against gram-positive bacteria. Journal of Antimicrobial Chemotherapy 1989, 23: 455-457.
Intraphagocytic Activity of Erythromycin, Roxithromycin and Azithromycin D. Milatovic The intraphagocytic activity of erythromycin, roxithromycin and azithromycin against phagocytosed Staphylococcus aureus was compared. Erythromycin and roxithromycin both acted bacteriostatically at concentrations corresponding to 10 × MIC. Azithromycin, however, did not prevent intracellular proliferation of the staphylococci. On comparison of the pH dependency of the antibacterial activity of the three drugs, azithromycin was found to be inactivated earlier in an acidic milieu. Intraphagocytic activity of an antibiotic is an important property when treating infections due to bacteria which are able to survive intracellularly such as legionella, mycobacteria, listeria, brucella and Staphylococcus aureus. Erythromycin, a well established antibiotic, has regained clinical importance as the drug of choice in Legionnaire's disease. Parallel to its clinical efficacy, erythromycin has been shown to accumulate in phagocytic cells (1, 2, 3) and to exhibit intracellular activity against legionella (4, 5, 6). However, data published on the intracellular activity of erythromycin against Staphylococcus aureus are contradictory (1, 7, 8). Newly developed macrolide antibiotics such as roxithromycin and azithromycin have been reported to reach even higher intracellular concentrations compared to erythromycin (1, 2, 3, 9). Furthermore, the data obtained on in vitro evaluation of these drugs point towards an expanded spectrum which might include other Department of Medical Microbiology, Technical University Munich, Trogerstral]e 32, 8000Munich 80, FRG.
33
intracellular microorganisms such as M y c o bacterium avium complex or ToxopIasma gondii (10, 11). The purpose of this study was to compare the intraphagocytic activity of the new macrolides roxithromycin and azithromycin with that of erythromycin. Materials and Methods. Human polymorphonuclear leukocytes (PML) were isolated from heparinized blood by dextran sedimentation and F i c o l l - H y p a q u e density gradient as previously described (12). S t a p h y l o c o c c u s aureus strain 502 A opsonized in 10 % pooled human serum was exposed to the PML for 30 min at 37 °C (ratio of bacteria to PML 4:1). Extracellular bacteria were removed by adding 1 U/ml lysostaphin. After incubation for 15 min at 37 °C the P M L with the intracellular staphylococci were washed in Hank's Balanced Salt Solution (5 min, 4 °C, 160 x g) and res u s p e n d e d in Eagles Minimum Essential Medium (MEM) containing 10 % fetal calf serum (FCS) and various concentrations (10 x MIC, lx MIC, 1/3 MIC) of erythromycin ( A b b o t t , F R G ) , roxithromycin (Roussel, France) and azithromycin (Pfizer, UK) respectively. The MIC of the antibiotics for Staphylococcus aureus 502 A was 0.5 mg/1, 2 mg/1 and 0.5 mg/1 respectively. The control PML were suspended in medium without addition of antibiotics. Each experimental set consisted of three identical tubes. The tubes were incubated at 37 °C and rotated end over end. The number of intracellular surviving bacteria was determined at 0, 3 and 18 h. For this purpose at the indicated times one of the three identical tubes was washed twice with ice-cold PBS (5 min, 4 °C, 160 x g) and the pellet resuspended in an equal volume of distilled water to lyse the PML and release intracellular bacteria. From serial tenfold dilutions 100 I.tl samples were transferred onto blood agar plates. After incubation for 18 h at 37 °C colonies were counted. Furthermore, antibiotic killing of Staphylococcus aureus in the absence of PML was evaluated using an antibiotic concentration of 10 × MIC. In these experiments the pH of the medium was modified, ranging from 4 to 8. Results and Discussion. During incubation for 3 h the number of intracellularly surviving bacteria in the control assay was reduced to 2 1 % ( S D + 7 % ) , whereas after 18h proliferation of the bacteria was observed (Figure 1, closed symbols). Addition of any of the antibiotics tested did not enhance the intracellular killing of the bacteria after 3 h. After incubation for 18 h none of the three macrolides inhibited intraphagocytic multiplication of the bacteria at concentrations equal to the MIC or higher (results with 1/3 MIC not shown in
New Antimicrobial Agents
Comparative in Vitro Activity of the New Erythromycin Derivative Dirithromycin against GramPositive Bacteria Isolated from Cancer Patients K. V. Rolston*, D. H. Ho, B. L e B l a n c , G.P. B o d e y The in vitro activity of dirithromycin (LY237216), a new macrolide erythromycin derivative, was compared to that of four other agents (clarithromycin, erythromycin, roxithromycin, clindamycin) against 334 gram-positive isolates obtained from cancer patients. Dirithromycin was similar in potency and antimicrobial spectrum to the other agents tested. It was very active against beta-haemolytic streptococci and Streptococcus pneumoniae, and moderately active against penicillin and methicillin susceptible Staphylococcus aureus, Bacillus spp., Listeria monocytogenes and Corynebacterium jeikeium. Erythromycin resistant organisms were also resistant to dirithromycin. Macrolide antibiotics such as erythromycin, which was introduced in 1952, are active against various microorganisms including streptococcal species and methicillin-susceptible staphylococci, and are useful alternatives to the penicitlins in penicillin-allergic patients. In recent years several new macrolide compounds have been developed with improved chemical, biological or pharmacokinetic properties when compared to erythromycin (1). Dirithtomycin (LY237216) is a new 9-N-11-0-oxazine derivative of erythromycin formed from the condensation of 9Serythromycylamine with (2-methoxyethoxy)acetaldehyde. Pharmaeokinetic studies in animals have shown dirithromycin to have a longer half-life with higher and more persistent serum and tissue levels than erythromycin (F. T. Counter et al., and V. Busch et al., 28th Interscience Conference on Antimicrobial Agents and Chemotherapy, Los Angeles, 1988, Abstracts No. 920 and 922). We evaluated the in vitro activity of dirithromycin against 334 grampositive bacterial isolates obtained from blood culture specimens of cancer patients, a patient Section of Infectious Diseases (Box 47), Department of Medical Specialties, University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd., Houston, Texas 77030, USA.
Eur. J. Clin. Microbiol. Infect Dis.
population in which these organisms are emerging as significant pathogens (2). The activity of dirithromycin was compared to that of erythromycin, clindamycin and two newer macrolides roxithromycin and clarithromycin. Materials and Methods. The antimicrobial agents tested were erythromycin and clarithromycin (Abbott Laboratories, USA), roxithromycin (Hoechst-Roussel Pharmaceuticals, USA), clindamycin (Upjohn, USA) and dirithromycin (Lilly Research Laboratories, Indianapolis, IN, USA). Standard laboratory powders of known potency were used for susceptibility testing and kept frozen at -70°C prior to being used. All 334 isolates representing 14 bacterial species were obtrained from recent blood cultures of patients admitted to The University of Texas M. D. Anderson Cancer Center. Such isolates are stored in our laboratory by ultrafreezing methods and older strains are discarded as newer ones become available. Susceptibility testing was performed using a previously described microtiter broth dilution method, and in accordance with guidelines established by the National Committee for Clinical Laboratory Standards (3, 4). Briefly, MICs were determined in disposable plastic microtiter plates with 96 wells (Cooke Laboratory Products, USA). MIC plates were freshly prepared before each susceptibility testing experiment. Antibiotics were prepared in serial twofold dilutions in concentrations ranging from 64.0-0.03 I~g/ml and dispensed automatically using an MIC-2000 apparatus (Dynatech Laboratories, USA). The test medium used was cationsupplemented (Ca z+ 50 mg/1, and Mg2+ 25 mg/1) Mueller-Hinton broth (MHB; Difco Laboratories, USA) for all organisms except Corynebacterium jeikeium, which was tested in brainheart infusion broth with 5 % rabbit serum, and streptococci, which were tested in cationsupplemented MHB with 2 % lysed horse blood. Organisms were inoculated into broth and incubated at 37 °C for 18 h. Appropriate dilutions were then made so that the final inoculum of organisms tested was 105 CFU/ml. The MIC was recorded after incubation for 1620 h at 35 °C and defined as the lowest concentration of each drug required to inhibit visible bacterial growth. Two independent investigators performed each susceptibility test run and Staphylococcus aureus ATCC 25923 was included as the control strain for each procedure to ensure the validity of the results. Results and Discussion. Results of the susceptibility studies are shown in Table 1. Dirithro-
Vol. 1, 1990
31
Table 1: C o m p a r a t i v e in vitro activity of d i r i t h r o m y c i n a n d o t h e r antimicrobial a g e n t s a g a i n s t g r a m positive bacteria. M I C (mg/l)
O r g a n i s m s (n)
Antimicrobial agent
Bacillus spp.
dirithromycin clarithromycin erythromycin roxithromycin clindamycin
0.06 0.12 0.25 0.25 0.5
0.25 0.12 2.0 0.5 1.0
dirithromycin clarithromycin erythromycin roxithromycin clindamycin
0,25 0.5 2.0 4.0 > 64.0
8.0 4.0 > 64.0 > 64.0 > 64.0
Listeria monocytogenes
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
1.0 0.25 0.5 1.0 2.0
1.0 0.25 0.5 1.0 2.0
0.5 0.12 0.5 0.5 1.0-
Staphylococcus aureus
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
1.0 0.25 0.5 1,0 0.12
2.0 0.5 0.5 1.0 0.12
0.06 - 2.0 0.06 - 0.25 0.12 - 0.5 0.25 - 1.0 0.06-1.0
dirithromycin clarithromycln erythromycin roxithromycin clindamycin
0.25 0,12 0.12 0,25 0,25
> 64.0 >64.0 > 64.0 > 64.0 > 64.0
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
32.0 8.0 16.0 32.0 64.0
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
dirithromycin clarithromycm erythromycin roxithromycin clindamycin dirithromycin clarithromycm erythromycin roxithromycin clindamycin
32.0 32.0 64.0 64.0 > 64,0 0,12 0.06 0.12 0.06 0.12
> 64.0 > 64,0 > 64.0 > 64.0 > 64.0 0.12 0.12 0.25 0.12 0.12
0.5 0.06 64,0 > 64.0 > 64.0 > 64.0 > 64.0 0.12 0,12 0.25 0.12 0.12
dirithromycin clarithromycm erythromycin roxithromycin clindamycin
0.25 0.12 0.03 0.06 0.03
0.25 0.25 0.12 0.12 0.06
0.12 0.06 64.0 0.06 - > 64,0 0.12 - > 64.0 > 64.0 > 64.0 > 64.0 > 64.0 > 64.0 0.5 0.06 0.12 0.25 0.06-
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
> 64.0 > 64.0 > 64.0 > 64.0 > 64.0
32
Eur. J. Clin. Microbiol. Infect Dis.
Table 1 continued
Organisms (n)
Antimicrobial agent
Streptococcus pneumoniae
dirithromycin clarithromycin erythromycin roxithromycin elindamyein dirithromycin elarithromyein erythromycin roxithromycin clindamycin dirithromycin clarithromycin erythromycin roxithromycin elindamycin dirithromycin clarithromycin erythromyein roxithromycin elindamycin
(25)
Streptococcus sanguis (35)
Streptococcus m i t i s (26)
Enterococcus faecalis (20)
mycin was e x t r e m e l y active against the betah a e m o l y t i c s t r e p t o c o c c i and Streptococcus pneumoniae, inhibiting all isolates at a concentration of 0.5 gg/ml. T h e activity of the other agents tested against these organisms was similar to that of dirithromycin. A l p h a - h a e m o l y t i c s t r e p t o c o c c a l isolates (Streptococcus sanguis and Streptococcus mitis) w e r e much less susceptible to all agents tested than o t h e r streptococcal isolates. However, there was a bimodal p a t t e r n of susceptibility for these organisms, 5 5 70 % being relatively susceptible and the rest having much higher MICs. D i r i t h r o m y c i n was active against penicillin susceptible Staphylococcus aureus isolates and had m o d e r a t e activity against penicillin resistant b u t methicillin susceptible Staphylococcus aureus isolates. All isolates of methicillin resistant Staphylococcus aureus and most isolates of c o a g u l a s e negative staphyloccocaI species w e r e resistant to all agents tested. D i r i t h r o mycin was also active against Bacillus spp. and Listeria monocytogenes. D i r i t h r o m y c i n and clarithromycin were more active against Corynebacterium ]eikeium than the other agents tested. The spectrum of activity and potency of dirithromycin w e r e t h e r e f o r e generally similar to those of the other antibiotics tested with only a few minor differences, erythromycin resistant strains also being resistant to dirithromycin. Interest in macrolide antimicrobial agents has been rekindled with the d e v e l o p m e n t of several newer compounds. Most of these offer no ad-
.... MIC 50 0.12 0.06 0.12 0.06 0.12 2.0 0.5 0.12 0.06 0.12 1.0 0.5 0.12 0.5 0.06 4.0 8.0 4.0 4.0 16.0
MIC (mg/1) MIC 90 0.12 0.12 0.25 0.25 0.25 16.0 4.0 4.0 4.0 2.0 32.0 16.0 8.0 16.0 8.0 > 64.0 > 64.0 > 64.0 > 64.0 > 64.0
Range
0.12 - 0.5 0.06 - 0.5 0.06 - 0.5 < 0.03 - 1.0 < 0.03 - 0.5 0.5- 64.0 0.06-16.0 0.12 - 8.0 0.06 8.0 0.12- 16.0 0.5 - 64.0 0.5- 32.0 0.06- 16.0 0.12 - 32.0 - 64.0 0.06- > 64.0 0.12 - > 64.0 0.12 - > 64.0 4.0 - > 64.0 -
v a n t a g e s over e r y t h r o m y c i n in t e r m s of spectrum or potency (1, 3, 5). Our data indicate that the activity of dirithromycin is similar to that of e r y t h r o m y c i n against isolates o b t a i n e d f r o m cancer patients. Investigators who tested isolates f r o m a variety of other sources have similar data, indicating that a l t h o u g h g r a m - p o s i t i v e infections m a y b e m o r e f r e q u e n t in c a n c e r patients, the organisms causing these infections do not generally possess a g r e a t e r degree of resistance than those isolated f r o m other patient p o p u l a t i o n s (1; K. W. Yu et al., 29th I n t e r science C o n f e r e n c e on A n t i m i c r o b i a l A g e n t s and C h e m o t h e r a p y , H o u s t o n , Texas, 1989, Abstract No. 1035). Clinical trials will d e t e r m i n e w h e t h e r n e w e r macrolides (including dirithromycin), which have better pharmacokinetic properties, are m o r e effective than erythromycin in the t r e a t m e n t of h u m a n infections.
References 1. Hardy JJ, Hensey DM, Beyer JM, Vojiko C, McDonald E J, Fernandez PB: Comparative in vitro activities of new 14-, 15- and 16-membered maerolides. Antimicrobial Agents and Chemotherapy 1988, 32:
1710-1719. 2. Viscoli C, Van der Auwera P, Meunier F: Grampositive infections in granulocytopenic patients: an
important issue? Journal of Antimicrobial Chemotherapy 1988, 21, Suplement C: 149-156.
Vol. 1, 1990
3. Rolston KVI, LeBlanc B, Ho DH: In vitro activity of RU-28965, a new macrolide, compared to that of erythromyein. Journal of Antimicrobial Chemotherapy 1986,17: 161-163. 4. National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard M7-A. NCCLS, Villanova, PA, 1985. 5. Rolston K, Gooch G, Ho D: In vitro of clarithromycin (A-56268; TE-031) against gram-positive bacteria. Journal of Antimicrobial Chemotherapy 1989, 23: 455-457.
Intraphagocytic Activity of Erythromycin, Roxithromycin and Azithromycin D. Milatovic The intraphagocytic activity of erythromycin, roxithromycin and azithromycin against phagocytosed Staphylococcus aureus was compared. Erythromycin and roxithromycin both acted bacteriostatically at concentrations corresponding to 10 × MIC. Azithromycin, however, did not prevent intracellular proliferation of the staphylococci. On comparison of the pH dependency of the antibacterial activity of the three drugs, azithromycin was found to be inactivated earlier in an acidic milieu. Intraphagocytic activity of an antibiotic is an important property when treating infections due to bacteria which are able to survive intracellularly such as legionella, mycobacteria, listeria, brucella and Staphylococcus aureus. Erythromycin, a well established antibiotic, has regained clinical importance as the drug of choice in Legionnaire's disease. Parallel to its clinical efficacy, erythromycin has been shown to accumulate in phagocytic cells (1, 2, 3) and to exhibit intracellular activity against legionella (4, 5, 6). However, data published on the intracellular activity of erythromycin against Staphylococcus aureus are contradictory (1, 7, 8). Newly developed macrolide antibiotics such as roxithromycin and azithromycin have been reported to reach even higher intracellular concentrations compared to erythromycin (1, 2, 3, 9). Furthermore, the data obtained on in vitro evaluation of these drugs point towards an expanded spectrum which might include other Department of Medical Microbiology, Technical University Munich, Trogerstral]e 32, 8000Munich 80, FRG.
33
intracellular microorganisms such as M y c o bacterium avium complex or ToxopIasma gondii (10, 11). The purpose of this study was to compare the intraphagocytic activity of the new macrolides roxithromycin and azithromycin with that of erythromycin. Materials and Methods. Human polymorphonuclear leukocytes (PML) were isolated from heparinized blood by dextran sedimentation and F i c o l l - H y p a q u e density gradient as previously described (12). S t a p h y l o c o c c u s aureus strain 502 A opsonized in 10 % pooled human serum was exposed to the PML for 30 min at 37 °C (ratio of bacteria to PML 4:1). Extracellular bacteria were removed by adding 1 U/ml lysostaphin. After incubation for 15 min at 37 °C the P M L with the intracellular staphylococci were washed in Hank's Balanced Salt Solution (5 min, 4 °C, 160 x g) and res u s p e n d e d in Eagles Minimum Essential Medium (MEM) containing 10 % fetal calf serum (FCS) and various concentrations (10 x MIC, lx MIC, 1/3 MIC) of erythromycin ( A b b o t t , F R G ) , roxithromycin (Roussel, France) and azithromycin (Pfizer, UK) respectively. The MIC of the antibiotics for Staphylococcus aureus 502 A was 0.5 mg/1, 2 mg/1 and 0.5 mg/1 respectively. The control PML were suspended in medium without addition of antibiotics. Each experimental set consisted of three identical tubes. The tubes were incubated at 37 °C and rotated end over end. The number of intracellular surviving bacteria was determined at 0, 3 and 18 h. For this purpose at the indicated times one of the three identical tubes was washed twice with ice-cold PBS (5 min, 4 °C, 160 x g) and the pellet resuspended in an equal volume of distilled water to lyse the PML and release intracellular bacteria. From serial tenfold dilutions 100 I.tl samples were transferred onto blood agar plates. After incubation for 18 h at 37 °C colonies were counted. Furthermore, antibiotic killing of Staphylococcus aureus in the absence of PML was evaluated using an antibiotic concentration of 10 × MIC. In these experiments the pH of the medium was modified, ranging from 4 to 8. Results and Discussion. During incubation for 3 h the number of intracellularly surviving bacteria in the control assay was reduced to 2 1 % ( S D + 7 % ) , whereas after 18h proliferation of the bacteria was observed (Figure 1, closed symbols). Addition of any of the antibiotics tested did not enhance the intracellular killing of the bacteria after 3 h. After incubation for 18 h none of the three macrolides inhibited intraphagocytic multiplication of the bacteria at concentrations equal to the MIC or higher (results with 1/3 MIC not shown in
