Department of Clinical Microbiology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previously, in vitro and in vivo synergy between ß-lactam antibiotics, such as penicillin, ampicillin, piperacillin and cefuroxime, has been demonstrated when combined with gentamicin against S. pneumoniae.5,6 Antagonism in vitro has been demonstrated for combinations such as ampicillin and chloramphenicol against Haemophilus influenzae7 and against group B streptococci.8 Clinically important antagonism has been reported between penicillin and tetracycline against pneumococci,9,10 penicillin and erythromycin against group A streptococci11 and ampicillin, streptomycin and chloramphenicol in acute bacterial meningitis.12 Theoretically, the empirical initial treatment of pneumonia with a combination of a ß-lactam agent and a macrolide may also be inexpedient in the case of pneumococcal infection, as the bacteriostatic macrolide may antagonize the bactericidal effect of the ß-lactam.
In the present study we investigated the possible interaction between penicillin and erythromycin in vitro against four clinical isolates of pneumococci with various susceptibilities to penicillin and erythromycin.6,13 We studied interactions in vivo in the mouse peritonitis model.6
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Four clinical isolates of pneumococci were used [isolate numbers 73 (68062), 75 (68098), 86 (68122) and 93 (493/ 73)].13 The MICs of penicillin for these isolates ranged from 0.016 to 0.5 mg/L and those of erythromycin from 0.25 to >128 mg/L (Table).
|
The drugs used were penicillin G (Leo Pharmaceutical Co., Ballerup, Denmark) and erythromycin (Sigma Chemical Co.). Penicillin G was diluted in phosphate-buffered saline pH 6.5 ± 0.1, and erythromycin was diluted in 9 mL sterile water and 1 mL 96% alcohol; the pH of the erythromycin solution was adjusted to between 6.7 and 7.3.
MICs and MBCs
MICs were determined by the broth macrodilution method in glass tubes. All tests were done in duplicate and results were read after 20 h of incubation at 35°C. The broth macrodilution method in glass tubes was performed with MuellerHinton broth (Statens Serum Institut) to which 5% sheep blood was added; an inoculum of 106 cfu/mL was used. Penicillin G was diluted in two-fold steps in Mueller Hinton broth to give concentrations of 0.00464 mg/L. The lowest concentration of antibiotic at which there was no visible growth was taken as the MIC. We used S. pneumoniae ATCC 49619 as a control strain for the MIC tests.
The MBC was determined by subculture of tubes with no visible growth after MIC determination. From each tube, 100 µL was cultured on agar plates containing penicillinase (Leo Pharmaceutical Co.) 1000 IU/plate and colonies were counted after 1824 h incubation at 35°C. The MBC was defined as the lowest concentration of penicillin that reduced the inoculum by 99.9%. All assays were performed in duplicate.
Timekill curves
To study possible interactions between penicillin and erythromycin, timekill experiments were performed with clinically relevant penicillin and erythromycin concentrations of 10 and 1 mg/L, respectively. Before timekill experiments, isolate 86 (erythromycin resistant) was grown on 5% blood agar plates containing erythromycin 4 mg/L (induction of erythromycin resistance) and on plates without erythromycin (no induction of erythromycin resistance). Pneumococci (106 cfu/mL) were incubated in 20 mL of MuellerHinton broth at 35°C with shaking. To ensure exponential growth of the bacteria, antibiotics were first added after 1 h of incubation. Samples were taken before the addition of antibiotics and 1, 2, 3 and 5 h later. Timekill curves were not extended further, since autolysis started to occur after 56 h for all isolates. The numbers of cfu/mL were determined after making appropriate dilutions, and 100 µL was spread on to 5% blood agar plates. For undiluted samples, agar plates containing penicillinase (as above) were used. Colonies were counted after 20 h of incubation at 35°C. All timekill experiments were performed in duplicate. The variation in colony counts at the same time points for repeated experiments was <0.5 log10 per mL. Antagonism was defined as a significantly decreased killing effect (i.e. >0.5 log10 cfu/mL from 1 to 5 h after adding drugs) of the combination of penicillin and erythromycin as compared with penicillin alone.14
In the timekill experiments, we measured possible pH changes in the flasks with pH test strips (pH range 4.510.0; graduated in 0.5 pH units; Sigma Chemical Co.).
Animal experiments (mouse peritonitis model)
All animal experiments were approved by the Danish national animal ethical committee. Outbred female ssc CF-1 mice (Statens Serum Institut) aged 812 weeks, weight 2830 g were used, in groups of five to 36 mice. Overall, we used 88 mice for isolate 73, 89 mice for isolate 75, 88 mice for isolate 86 (without induction), 94 mice for isolate 86 (induced with erythromycin as for timekill studies) and 89 mice for isolate 93. The mice were kept in cages with five to seven mice per cage; they were allowed free access to food and water. Pneumococcal suspension (0.5 mL) was inoculated intraperitoneally via a 25-gauge syringe. The inoculum contained 106 cfu/mL with 5% (w/v) mucin in MuellerHinton broth. Using such inocula, there is c. 100% mortality in untreated mice, which succumb 3648 h after inoculation. Antibiotics were administered subcutaneously in the neck region in a volume of 0.25 mL per dose.13 The following schedule was used: erythromycin was given 90 min after bacterial inoculation and in the combination group erythromycin was given 90 min after bacterial inoculation and penicillin 60 min later. Penicillin was given alone 150 min after bacterial inoculation. Control mice were given sterile saline 90 min after bacterial inoculation.
Doses of penicillin and erythromycin were chosen according to the Hill equation sigmoid doseeffect curves, in such a way that penicillin alone would be expected to result in c. 95% survival, while erythromycin alone was expected to prevent mortality in 10% of the mice. Mice infected with isolate 73 were treated with penicillin 10 mg/ mouse and/or erythromycin 100 µg/mouse. Mice infected with isolate 75 were treated with penicillin 2 mg/mouse and/or erythromycin 100 µg/mouse. Mice infected with isolate 86 (not induced) were treated with penicillin 150 µg/ mouse and/or erythromycin 100 µg/mouse. Mice infected with isolate 86 (grown in the presence of sub-inhibitory concentrations of erythromycin before inoculation) were treated with penicillin 150 µg/mouse and/or erythromycin 100 µg/mouse. Mice infected with isolate 93 were treated with penicillin 400 µg/mouse and/or erythromycin 100 µg/ mouse.
Blood samples were obtained through orbital cuts after anaesthetizing the mice with CO2. Mice were killed and peritoneal washes were then performed by injecting 2 mL of sterile saline intraperitoneally, massaging the abdomen and opening the peritoneum to collect the fluid.15 Blood and peritoneal fluid samples were immediately diluted, and 0.1 mL was plated on to 5% blood agar plates.
In vivo timekill curves were constructed for one of the pneumococcus isolates (number 93), which showed significant antagonism in vivo. Pneumococci in the peritoneum and blood were obtained after inoculating 36 mice intraperitoneally with 5.0 x 106 cfu/mL of the bacterial suspension. Ten minutes, 80 min, 140 min, 3 h and 5 h after challenge, groups of three control mice (inoculated with sterile saline) were killed. At 140 min, 3 h and 5 h after challenge, groups of three mice treated with erythromycin 90 min after challenge were killed. At 3 h and 5 h after challenge, groups of three mice treated either with the combination of penicillin and erythromycin or penicillin alone 150 min after intraperitoneal infection were killed. Blood and peritoneal washings were sampled for quantitative culture of pneumococci.
Determination of ED50 of penicillin for individual pneumococci
The ED50, the single dose giving protection to 50% of the mice, for each pneumococcus isolate was determined by treating groups of five mice with doubling doses of the antibiotics; survival of the mice was observed for 7 days. A group of five mice treated with 0.9% NaCl was included in each experiment as a control for the lethality of the infection. For each isolate the ED50 was calculated by the method of Reed & Muench16 and from the Hill equation (GraphPad Prism; GraphPad Software, Inc., San Diego, CA, USA). We did not perform any experiments in order to calculate the erythromycin ED50s since this has been done previously for strains with similar MICs in our laboratory.17 We chose penicillin doses that would be expected to result in an estimated 95% survival of the mice and erythromycin doses that would be expected to result in an estimated 10% survival of the mice. For combination therapy, the antibiotics were given in separate injections.
Statistics
Fisher's exact test for categorical data was used, with a two-sided 5% level of significance.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of the timekill experiments are shown in Figure 1. By this method, all isolates showed in vitro antagonism for the combination of penicillin and erythromycin (Figure 1ac and e
) because erythromycin almost completely inhibited the bactericidal effect of penicillin. However, when erythromycin resistance was induced by growing the erythromycin resistant isolate (number 86) on blood agar plates containing erythromycin 4 mg/L before the timekill study, erythromycin antagonism was neutralized (Figure 1d
).
|
In the mouse peritonitis model, we found that the combined treatment of penicillin and erythromycin resulted in antagonism in mice challenged with isolates 75 and 93 (Figure 2b and e). There was significantly higher mortality in the mice treated with erythromycin 60 min before penicillin than in mice treated with penicillin alone (isolate 75: mortality 32/36 and 3/12, respectively, P < 0.05; isolate 93: mortality 24/36 and 3/12, respectively, P < 0.05) (Figure 2
). In the remaining two isolates, mortality in mice given both antibiotics was similar to that in mice given penicillin alone (Figure 2a and d
). All control mice died when infected intraperitoneally with pneumococci, except mice infected with isolate 86, for which the mortality was 8090%. Even when the bacterial inoculum of isolate 86 was increased to 108 cfu/mL, there was not 100% mortality.
|
The in vivo timekill curves for isolate 93 showed a 12 log increase in growth of bacteria in blood 80 min after challenge compared with 10 min after challenge in the control mice (Figure 3), whereas the number of pneumococci in peritoneal washes at 80 min was the same as that 10 min after inoculation. The number of pneumococci cultured from blood and peritoneal washes at different time points in the different treatment groups were almost parallel, with 12 log higher cfu/mL in peritoneal washes than in blood. Killing of bacteria was most efficient in the penicillin-treated group, with a decrease in bacterial growth of 3 logs and 4 logs in blood and peritoneal washes, respectively. The combination therapy showed a similar effect on bacterial growth as when erythromycin was administered alone (antagonism).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In order to evaluate the possible interaction between the two drugs in vivo, we chose the mouse peritonitis model, which has previously been useful in demonstrating interaction between antibiotics,6 and which also enables one to study drug action on bacteria by performing in vivo timekill experiments on organisms in the peritoneum and/or blood.17 Doses of penicillin and erythromycin were chosen according to the Hill equation sigmoid doseeffect curves, in such a way that penicillin alone would be expected to result in c. 95% survival, while erythromycin alone would only be able to prevent mortality in 10% of the mice. With these doses, an effect of the erythromycin dose with no influence of a subsequent penicillin dose would result in a mortality determined solely by the activity of the macrolide. The antagonistic activity of the two drugs was subsequently confirmed, by timekill curves in vivo, to be due to the same growth-inhibitory activity of erythromycin as demonstrated in vitro. The importance of induction of erythromycin resistance in the erythromycin-resistant isolate (number 86) was also demonstrated in vivo: induction with erythromycin significantly decreased the survival in spite of erythromycin treatment as compared with survival following erythromycin treatment of uninduced pneumococci.
With one erythromycin-susceptible isolate (number 73), it was not possible to show antagonism in vivo with the method used. There may be several explanations for this. The possibility that not all pneumococci respond in a similar way to the two drugs used seems unlikely, especially when the activity was equally demonstrable in vitro. It is more likely that the situation in vivo is different for the different strains, e.g. the virulence of the pneumoccci depends on a number of factors which are difficult to standardize, such as the type and size of the capsule, other virulence factors connected with the membrane or toxins, or the growth behaviour in vivo, which is very variable for pneumococci. It is likely that we could have demonstrated antagonism in vivo with isolate 73 if we had titrated the inoculum and the timing of the two drugs in relation to each other, but this would require an excessive number of animals. The clear-cut demonstration of the interaction between the two drugs against at least two pneumococcal strains both in vitro and in vivo, which was demonstrated by the increase in mortality as well as by the changes in in vivo timekill curves, is ample evidence for an effect, and should warn clinicians against the use of these two drugs together to treat pneumococcal infections.
The clinical significance of antagonism between a bactericidal drug such as penicillin or ampicillin and a bacteriostatic protein synthesis inhibitor, e.g. tetracycline, erythromycin or chloramphenicol, has been demonstrated in several studies.712,18 Antagonism has been confirmed in vivo in an experimental study with penicillin and chloramphenicol against pneumococcal meningitis in dogs.19 In spite of this early experience with such drug combinations, the combination of penicillin and erythromycin is recommended even in standard textbooks as empirical treatment in pneumonia of unknown aetiology.4 The drug combination is particularly chosen to encompass pneumococci and Legionella spp., which are considered to be important and deadly aetiological agents in pneumonia. It is difficult to know whether the results of the present study are directly applicable to the clinical situation. In the clinical situation, sub-optimal doses of erythromycin would not usually be used, but with inhibited action of penicillin one may have to rely upon the bacteriostatic effect of erythromycin, if the pneumococcus is erythromycin susceptible. It is not known if the inducible effect of erythromycin demonstrated in the present study also takes place in the pneumonic focus in humans.
In conclusion, the present study has demonstrated antagonism between penicillin and erythromycin against three erythromycin-sensitive isolates of pneumococci in vitro. This antagonism also occurred with two of the isolates in vivo in a simple experimental model with mice. When erythromycin resistance was not induced, antagonism was also demonstrated in vitro for the erythromycin-resistant pneumococcal isolate, but induction with erythromycin before inoculation obliterated the antagonistic effect.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Bangsborg, J. M. (1997). Antigenic and genetic characteriztion of Legionella proteins: contributions to taxonomy, diagnosis and pathogenesis. Acta Pathologica Microbiologica et Immunologica Scandinavica 70, 153.
3 . Rechnitzer, C. (1994). Pathogenic aspects of legionnaires' disease. Acta Pathologica Microbiologica et Immunologica Scandinavica 43, 143.
4 . Donowitz, G. R. & Mandell, G. L. (1995). Acute pneumonia. In Principles and Practice of Infectious Diseases, 4th edn (Mandell, G. L. & Raphael, D., Eds), pp. 6312. Churchill Livingstone, Edinburgh.
5 . Darras-Joly, C., Bédos, J.-P., Sauve, C., Moine, P., Vallée, E., Carbon, C. et al. (1996). Synergy between amoxicillin and gentamycin in combination against a highly penicillin-resistant and -tolerant strain of Streptococcus pneumoniae in a mouse pneumonia model. Antimicrobial Agents and Chemotherapy 40, 214751.[Abstract]
6 . Frimodt-Møller, N. & Thomsen, V. F. (1987). Interaction between ß-lactam antibiotics and gentamycin against Streptococcus pneumoniae in vitro and in vivo. Acta Pathologica Microbiologica et Immunologica Scandinavica Section B 95, 26975.[ISI]
7 . Lapointe, J. R., Lavallee, C., Michaud, A., Chicoine, L. & Joncas J. H. (1986). In vitro comparison of ampicillinchloramphenicol and ampicillincefotaxime against 284 Haemophilus isolates. Antimicrobial Agents and Chemotherapy 29, 5947.[ISI][Medline]
8 . Weeks, J. L., Mason, E. O. & Baker, C. J. (1981). Antagonism of ampicillin and chloramphenicol for meningeal isolates of group B streptococci. Antimicrobial Agents and Chemotherapy 20, 2815.[ISI][Medline]
9 . Lepper, H. M. & Dowling, H. F. (1951). Treatment of pneumococci meningitis with penicillin compared with penicillin plus aureomycin. Archives of Internal Medicine 88, 48994.
10 . Olsson, R. A., Kirby, J. C. & Romansky, M. J. (1961). Pneumococcal meningitis in the adult. Clinical, therapeutic, and prognostic aspects in forty-three patients. Annals of Internal Medicine 55, 5459.[ISI]
11 . Ström, J. (1961). Penicillin and erythromycin singly and in combination in scarlatina therapy and the interference between them. Antibiotics and Chemotherapy 11, 6947.[ISI][Medline]
12 . Mathies, A. W., Leedom, J. M., Ivler, D., Wehrle, P. F. & Portnoy, B. (1967). Antibiotic antagonism in bacterial meningitis. Antimicrobial Agents and Chemotherapy 7, 21824.
13 . Knudsen, J. D., Frimodt-Møller, N. & Espersen, F. (1995). Experimental Streptococcus pneumoniae infection in mice for studying correlation of in vitro and in vivo activities of penicillin against pneumococci with various susceptibilities to penicillin. Antimicrobial Agents and Chemotherapy 39, 12538.[Abstract]
14 . Eliopoulos, G. M. & Moellering, R. C. (1996). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 33096. Williams & Wilkins, Baltimore, MD.
15 . Frimodt-Møller, N., Sebbesen, O. & Thomsen, V. F. (1983). The pneumococcus and the mouse protection test: importance of the lag phase in vivo. Chemotherapy 29, 12834.[ISI][Medline]
16 . Reed, I. J. & Muench, H. (1938). A simple method of estimating fifty percent endpoints. American Journal of Hygiene 27, 4937.
17
.
den Hollander, J. G. D., Knudsen, J. D., Mouton, J. W., Fuursted, K., Frimodt-Møller, N., Verbrugh, H. A. et al. (1998). Comparison of pharmacodynamics of azithromycin and erythromycin in vitro and in vivo. Antimicrobial Agents and Chemotherapy 42, 37782.
18 . Li, R. C., Schentag, J. J. & Nix, D. E. (1993). The fractional maximal effect method: a new way to characterize the effect of antibiotic combinations and other nonlinear pharmacodynamic interactions. Antimicrobial Agents and Chemotherapy 37, 52331.[Abstract]
19 . Wallace, J. F., Smith, R. H., Garcia, M. & Petersdorf, R. G. (1967). Studies on the pathogenesis of meningitis. VI. Antagonism between penicillin and chloramphenicol in experimental pneumococcal meningitis. Journal of Laboratory and Clinical Medicine 70, 40818.[ISI][Medline]
Received 21 February 2000; returned 4 June 2000; revised 13 July 2000; accepted 19 August 2000