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In-vitro and In-vivo Efficacy of Zinc Acetate against Propionibacteria Alone and in Combination with Erythromycin
Zent.bl. Bakteriol. 289, 445-456 (1999) © Urban & Fischer Verlag http://www.urbanfischer.de/j ournals/ zblbakteriol
Zentralblatt fur
In-vitro and In-vivo Efficacy of Zinc Acetate against Propionibacteria Alone and in Combination with Erythromycin Joachim W. Fluhr!, Bettina Bosch!, Max Gloor!, and Ulrich Hoffler2 1 2
Dept. of Dermatology, Klinikum Karlsruhe, Germany Institute of Medical Microbiology and Hygiene, Klinikum Ludwigshafen, Germany
Received January 29,1998· Revision received April 13, 1999· Accepted May 12, 1999
Summary Some studies have been published about the in vitro activity of zinc acetate (ZA), eryth romycin (E) and their combination (ZAIE) against Propionibacterium spp., especially erythromycin resistant strains. The efficacy of topical ZAIE combination has been reported as well, but a comparison to ZA monotherapy is missing. Therefore, the MIC values of ZA, E and the ZA/E combination were determined for 15 erythromycin-re sistant and 12 erythromycin-sensitive Propionibacterium strains using the agar dilu tion method and the checkerboard technique. Furthermore, the antimicrobial efficacy of ZA (1,2 %) vs. the ZA/E (1,2 %/4 %) combination in an alcoholic solution was test ed in a 7-day treatment administered to 32 acne patients. The MIC 100 for ZA was 1024 Ilg ZNml for both, erythromycin resistant and erythromycin sensitive Propion ibacterium strains. The ZA, as well as the ZAIE solution showed efficacy reducing both the Propionibacterium spp., and the Micrococcaceae in the sebaceous gland infundi bula of acne patients. There was no significant difference between the two treatments. As the MIC 100 of ZAIE was equal to the MIC 100 of ZA, the decrease of the erythromycin MIC of the ZAIE combination in erythromycin-resistant strains may be partly attributed to the addition of ZA to E. The in vivo antibacterial efficacy on 32 acne patients supports the hypothesis that the antibacterial effect of ZNE in short-term treatment can be mostly attributed to ZA.
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J. W. Fluhr et a!.
Introduction Several studies have shown an in vivo efficacy of topical treatment of acne vul garis using zinc acetate in different combinations (12, 24, 25, 27). Some reports are available regarding the in vitro activity of zinc acetate (ZA), erythromycin (E) and their combination (ZAJE) especially against erythro mycin-resistant strains of Propionibacterium acnes (1, 8, 17). Other data have shown better therapeutical results of the combination of erythromycin with zinc acetate (ZAJE) in acne patients than erythromycin alone (12). However, no controlled study has been performed that would have shown a superiority of the ZAIE combination vs. ZA alone. In the present study, therefore, the in vitro activities of erythromycin, zinc acetate and their combination against propioni bacteria were determined using the checkerboard technique (18). In the second part of the study, the in vivo efficacy of zinc acetate versus the combination of erythromycin and zinc acetate in an alcoholic solution was tested. Within the sebaceous gland infundibulum of acne patients propionibacteria and Micro coccaceae counts were determined using the cyanoacrylate method (16).
Materials and Methods In vitro study Cultivation and identification of propionibacteria. The bacterial flora of the infundi bulum was removed using the cyanoacrylate method (16) and inoculated onto RCM agar plates (Oxoid-Unipath GmbH, Wesel, Germany). The agar was supplemented with 1.0 ml Tween 80 (Merck-Schuchardt, Hohenbrunn, Germany) and 0.06 g Furox ine (Fabry, Deinze, Belgium) per 1000 m!. Subsequently, the cultivated strains were identified by standard methods, including colony morphology after 48 hours of incu bation under anaerobic conditions (Gas-Pak-method, Becton-Dickinson, Heidelberg, Germany) on sheep blood agar, as well as on Wilkins-Chalgren agar (Oxoid GmbH, Wesel, Germany). Further methods were Gram stain reaction, microscopic morpho logy, catalase test, indole production from tryptophane, and exclusion of aerobic growth. Propionibacterium strains were stored at -20°C in a Microbank system (Mast Diagnostics, Hamburg, Germany). Antimicrobial susceptibility testing. All strains were subcultivated on sheep blood agar for 48-72 hours at 37°C under anaerobic conditions. Subsequently, they were transferred to Wilkins-Chalgren broth and incubated for another 48 hours. These broth cultures were used as inoculum. Based on duplicate counts of colony forming units (CFU) for three strains of each species, the mean final inoculum was calculated to be 8 x 10 7 CFU. Aerobically cultivated Pseudomonas aeruginosa, DSM 1107, Staphylococcus aureus, DSM 1104 =ATCC 29213, and Escherichia coli, DSM 1103 = ATCC 25922, were used as antibiotic activity controls according to the recommenda tions of DIN 58940 (3). Growth controls on drug-free medium, and negative controls on agar plates inoculated with propionibacteria broth culture, which were aerobical ly cultivated, were included in each assay. The MICs for erythromycin and zinc acetate were determined using the agar dilu tion method. Freshly prepared drug Wilkens-Chalgren agar mixtures were poured into
Zinc Acetate and Erythromycin Against Propionibacteria
447
Petri dishes and dried for 2 hours after solidification. Subsequently the agar plates were inoculated automatically using the Mastascan Multipoint Inoculator (Mast Diagnos tics, Hamburg, Germany). After incubation for 48 hours at 3rC using the Gas-Pak method, the results were read. MIC was considered to be the lowest drug concentra tion at which no visible growth occurred. Faint growth, and less than four point-like colonies were rated negative. All tests were done in duplicate. Checkerboard technique. The MIC values for the combination of zinc acetate and erythromycin were determined using the agar dilution method in the checkerboard arrangement (18). The checkerboard consisted of columns in which each agar plate contained the same amount of erythromycin which was diluted along the x-axis. Rows in which each agar plate contained the same amount of zinc acetate were diluted on the y-axis. The inoculation of the strains was performed as mentioned above.
In vivo study Patients. The in vivo study involved 32 patients with acne papulopustulosa according to Plewig and Kligman (23). Four additional patients were excluded before the begin ning of the study, as the amount of propionibacteria was less than 100 CFU/cm 2 on one of the two test sites. 23 of the subjects were women (71,9%) and 9 were men (28,1 %). The average age was 28.8 years (women 27.7 years [range: 20-41), men 27.9 years [range 19-53)). Except for acne, the patients showed no dermatoses or system ic diseases. Four weeks before the beginning of the study, no medication was adminis tered. Previous systemic treatment with isotretinoin was regarded as an exclusion cri terium. During the study, the subjects were urged to avoid using cleansing products on the forehead. The aims of the study had been explained to the patients, and they had given their written informed consent. They could withdraw from the study at any time, without giving reasons. Cultivation and identification of bacteria. The methods for harvesting, cultivation, and identification of propionibacteria have been described above. Harvesting of Mi crococcaceae was also done by the cyanoacrylate method. Cultivation (sheep blood agar) and identification (colony morphology, Gram staining and coagulase test) were done using standard methods. Antibacterial efficacy: The following two formulations were compared: 1. Zinc acetate 1.2 % (Zinc acetate dihydrate from Sigma-Aldrich Chemie, Steinheim, Germany) Erythromycin 4.0 % (Erythromycin from Abbott, Wiesbaden, Germany) Alcoholic solution (ethanol 55.2 %, diisopropyle sebacate 25.1 %, water ad 100.0, alcoholic base of Zineryt®, Hermal, Reinbeck, Germany [before mixing the zinc acetate and erythrom ycin containing powder with the alcoholic base)) 2. Zinc acetate 1.2 % Alcoholic solution (ethanol 55.2 %, diisopropyle sebacate 25.1 %, water ad 100.0, alcoholic base of Zineryt®, Hermal, Reinbeck, Germany [before mixing the zinc acetate and erythrom ycin containing powder with the alcoholic base)) The formulations were prepared, blinded and randomised by Drais-Pharmacy, Karls ruhe, Germany (Manager:]. Zybowski). The antibacterial efficacy was measured
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J. W. Fluhr et al.
based on the reduction of bacteria (propionibacteria and Micrococcaceae) after a 7days treatment. 0.1 ml each of test products were applied on an area of 2.0 X 2.0 cm size (0.025 mllcm2) twice daily on the right and left site of the forehead. The formula tions were tested in double-blind method, as neither the patients nor the investigators knew the randomisation of the test products. The cyanoacrylate method (Permabond technique, Holland et al. 1974) was used to obtain samples of the infundibulum bac terial flora (for cultivation see above). Bacteria were removed before (t 0) and after a 7-day treatment (t7) with the two formulations. Statistical analysis (in vivo) The colony forming units per cm 2 (CFU/cm2) were determined and the log 10 was cal culated. Since a normal distribution could not be assumed, Wilcoxon's distribution free rank test for paired samples was used to test the antibacterial efficacy of the 2 for mulations in the course of time (to vs. t7) (question 1a for propionibacteria, and ques tion 1b for Micrococcaceae) (2-tailed). In the event of established efficacy (question 1a and 1b answered positively), the delta (tl-tO) of the two formulations were compared (question 2a and 2b) with Wilcoxon's rank test for paired samples (2-tailed). The test ing of the two (a+b) questions was carried out in accordance with the method of a priory-ordered hypothesis (21). The results were considered significant at a level of p < 0.05. The above mentioned tests were performed with Prism 2, GraphPad®, USA.
Results The results of the in vitro study are shown in Tables 1-3. The MIC 100 val ue for zinc acetate alone was 1024 Ilg/ml (Table 1) for erythromycin-resistant (ER) strains. The MIC 100 values for erythromycin sensitive (ES) strains, was 512 Ilg/ml for P.acnes, and 1024 Ilg/ml for P.granulosum. The MIC 100 val ues for erythromycin alone was in ES P. acnes: 0.125 Ilg/ml and in ES P. gran ulosum: 0.125 Ilg/ml (Table 2). The MIC 100 value for erythromycin alone was in ER > 512 Ilg/ml for both P. acnes and P. granulosum (Table 3). With increasing concentration of ZA (starting at 16 Ilg/ml up to 256 Ilg/ml) the combination of ZA and E showed a slight decrease of the MIC values for erythromycin in the ER-group for some strains of P. acnes. At a zinc acetate concentration of 1024 Ilg/ml a dramatic decrease of the MIC 100 values for erythromycin was detected for both P. acnes and P. granulosum in ER. Further increase of the ZA concentration (2048 Ilg/ml) confirmed the low MIC values for erythromycin (0.125 Ilglml) for both P. acnes and P. granulosum in all ER. The values and statistical differences of the in vivo study are given in Figs. 1 and 2. Zinc acetate at a concentration of 12 000 Ilglml (1.2 %) significantly reduced the propionibacteria (p < 0.0001) and the Micrococcaceae counts (p < 0.0001) in the infundibulum after a topical treatment during 7 days. The combination of 12000 Ilglml (1.2 %) zinc acetate and erythromycin 40000 Ilglml (4.0 %) significantly reduced the propionibacteria (p < 0.0001) and the Micrococcaceae counts (p < 0.0001) in the infundibulum after a top ical treatment during 7 days. Thus, questions la (propionibacteria) and Ib
Zinc Acetate and Erythromycin Against Propionibacteria
449
(Micrococcaceae) concerning an infundibular antibacterial effect could be an swered positively for ZA and for the ZAJE combination. The bacterial reduction of the two test formulations (delta) was slightly, but not significantly, higher for the combination ZAJE than the monotherapy ZA (propionibacteria (p =0.6988) and Micrococcaceae (p =0.0965)). Therefore, questions 2a (propionibacteria) and 2b (Micrococcaceae) regarding a possible superiority of the antibacterial effect of ZAJE combination in comparison to the mono therapy with ZA was not statistically significant.
Discussion The combination of zinc acetate and erythromycin is known to be effective in the topical therapy of acne vulgaris (12, 24, 25, 27). The literature about the effect of a combination of zinc and erythromycin has been extensively revie wed by Gollnick et al. (11). Pierard et al. showed a sebosuppressive effect by the combination of 1.2 % zinc acetate and 4 % erythromycin in comparison to an untreated area at the forehead (22). An antiandrogen activity of zinc ac etate inhibiting the Sa-reductase has been shown in vitro (26, 28). An anti inflammatory activity of zinc has been demonstrated (2). Bojar et al. showed for the combination of 1.2 % zinc acetate/4 % erythromycin and 4 % eryth romycin alone, after 8 weeks of therapy, a significant reduction of total pro pionibacteria, as well as a reduction of erythromycin-resistant propionibacte ria (1). However, after 12 weeks, erythromycin-resistant propionibacteria Table 1. MIC values (!tg/ml) of zinc acetate against propionibacteria. Agar dilution test. (E. S. '" erythromycin sensitive; 'E. R. '" erythromycin resistant) Type Species
Cumulative percentages of strains inhibited by MIC values (!tg/ml) of zinc acetate n
E.S.
64.0
128.0 256.0 512.0 1024.0 2048.0
P. acnes P. granulosum total
0.0 0.0 0.0 33.3 0.0 8.3
11.1 66.7 25.0
E.R. P. acnes P. granulosum total
0.0 0.0 0.0 0.0 33.3 0.0 6.7
33.3 6.7
66.7 100.0 100.0 66.7 100.0 100.0 66.7 100.0 100.0
Total P. acnes P. granulosum total
0.0 0.0 0.0
9.5 33.3 14.8
81.0 100.0 100.0 66.7 100.0 100.0 77.8 100.0 100.0
4.8 16.7 7.4
100.0 100.0 66.7 100.0 91.7 100.0
100.0 100.0 100.0
100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
88.9 100.0 100.0 100.0 100.0 100.0
100.0 100.0 55.6 100.0 100.0 100.0 100.0 100.0 100.0 100.0
88.9 100.0
100.0 100.0 100.0 100.0 66.7 100.0
P.acnes P. granulosum
P. acnes P. granulosum
P.acnes P. granulosum
P.acnes P. granulosum
P. acnes P. granulosum
P.acnes P. granulosum
321lg/ml
641lg/ml
2561lg/ml
10241lg/ml
20481lg/ml
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0.25
100.0 100.0
0.125
100.0 100.0
n
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64.0
100.0 100.0
100.0 100.0
100.0 100.0
100.0 100.0 100.0 100.0
100.0 100.0
100.0 100.0 100.0 100.0 100.0 100.0
100.0 100.0
100.0 100.0
100.0 100.0 100.0 100.0
100.0 100.0
100.0 100.0 100.0 100.0
100.0 100.0
100.0 100.0
100.0 100.0 100.0 100.0 100.0 100.0
16.0
256.0
512
100.0 100.0 100.0 100.0
100.0 100.0 100.0 100.0
100.0 100.0
100.0 100.0
100.0 100.0
100.0 100.0
100.0 100.0
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100.0 100.0
100.0 100.0 100.0 100.0
100.0 100.0
100.0 100.0
100.0 100.0
100.0 100.0
100.0 100.0 100.0 100.0
100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
100.0 100.0
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128.0
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Cumulative percentages of strains inhibited by MIC values (Ilg/ml) of Erythromycin
100.0 100.0
Species
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o Ilg/ml
Concentration of zinc acetate
Table 2. MIC values (Ilg/ml) of erythromycin against erythromycin-sensitive propionibacteria strains in combination with different zinc acetate concentrations. Agar dilution test
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2048 ftg/ml
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100.0 100.0
100.0 66.7
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64.0
33.3
0.0
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8.3 8.3 8.3 0.0 0.0
8.0
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33.3 0.0
33.3 0.0
0.0 0.0
0.25
33.3 50.0 50.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0
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o ftg/ml
n
Cumulative percentages of strains inhibited by MIC values (ftg/ml) of Erythromycin
Species
Concentration of zinc acetate
Table 3. MIC values (ftg/ml) of erythromycin against erythromycin resistant propionibacteria strains in combination with different zinc acetate concentrations. Agar dilution test
J. W. Fluhr et al.
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were reacquired or appeared de novo. The reduction of Micrococcaceae were slight and after 12 weeks erythromycin-resistant strains were predominant. The in vitro sensitivity was the same for erythromycin-resistant and erythrom ycin-sensitive propionibacteria (MIC 32-64 !-tg/ml zinc acetate). For eryth romycin resistant and erythromycin sensitive coagulase-negative staphylococ ci, an MIC 128-512 !-tg/ml zinc acetate was established (1). The MIC values for erythromycin in erythromycin-sensitive and erythro mycin resistant propionibacteria strains were comparable with the MIC 100 values given by Bajar et al. (1). In contrast our MIC 100 values for both groups of bacteria when analysing zinc acetate were far higher than the val ues given by Bajar et al. (1): 1024 !-tg/ml vs. 64 !-tg/ml which corresponds to 4 dilution steps. Farmery et al. showed an MIC for zinc acetate of 3264 !-tg/ml irrespective to resistance of propionibacteria against erythromycin or tetracycline (8). Holland et al. showed a delayed growth of erythromycin resistant propionibacteria in vitra with 96 !-tg/ml, while 300 !-tg/ml prevented the growth of these propionibacteria (17). To insure the macroscopic growth
Zinc Acetate and Erythromycin Against Propionibacteria
453
in two days culturing of propionibacteria, we inoculated 8 X 10 7 CFU, onto the Wilkins-Chalgren-agar internationally accepted for testing of anaerobes, while Bojar et al. (1), Farmery et al. (8) and Holland et al. (17) inoculated 105 CFU onto brain heart infusion agar or RCM-broth. This could explain the higher MIC values detected in our in vitro study for propionibacteria. MIC values show a strong antibacterial effect on propionibacteria using a zinc acetate concentration of 1024 !-tg/ml or higher. The inhibition of propi onibacteria growth in vitro by the erythromycin-zinc acetate combination, especially on erythromycin-resistant strains, is mostly due to the antibacteri al effect of zinc acetate. To our knowledge, the in vivo part of our study was the first clinical trial to analyse the infundibular efficacy flora (propionibacteria and Micrococca ceae) of the ZAiE complex in comparison to ZA in the same alcoholic solu tion. We could show a significant reduction of bacteria for both products, and both bacterial groups, after 7 days of topical treatment. Thus the ZA concen tration within the infundibula using an alcoholic vehicle is supposed to be higher than the MIC 100 value of 1024 !-tg/ml for both groups of bacteria. A
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454
]. W. Fluhr et a!.
treatment with the ZNE combination lacked to show an antibacterial efficacy in the infundibulum superior to the monotherapy by ZA. The results of our studies provide evidence supporting the hypothesis that a decrease of the erythromycin MIC in ER propionibacteria under a treat ment with the combination of zinc acetate/erythromycin may be mainly due to the antibacterial effect of zinc acetate. As we could not show an in vivo superiority of the ZNE combination in comparison to the mono therapy by ZA, both in the same alcoholic solution for propionibacteria and Micrococ caceae, an acne treatment only with ZA may be nearly as effective as the ZAIE combination, considering the antibacterial efficacy in the infundibu lum. The clinical impact of paraantibiotic erythromycin effects (e. g. antiin flammatory and inhibition of ectoenzymes) has remained unknown. There fore clinical studies on a larger cohort and a long-term treatment are neces sary to prove the efficacy of a ZA monotherapy due to antibacterial and anti inflammatory effects of ZA. A wider use of antimicrobial agents not belon ging to the group of antibiotics could be an interesting issue in the light of the rapidly increasing problem of antibiotic resistance (4, 5, 6, 7, 9, 10, 13, 14, 15, 19,20). Acknowledgement. The authors wish to cordially thank Mrs. Barbara Wasik and Mrs. Sibylle E. Franke for their patient and helpful technical assistance and to Jochen Hohberg for his assistance performing the statistical analysis.
References 1. Bojar, R. A., E. A. Eady, C. E.Jones, w.]. Cunliffe, and K. T. Holland: Inhibition of erythromycin-resistant propionibacteria on the skin of acne patients by topical erythromycin with and without zinc. Br.].Dermato!. 130 (1994) 329-336 2. Chvapil, M.: Effect of zinc on cells and biomembranes. Med. Clin. North Am. 60 (1976) 799-812 3. DIN 58940IDIN 58944. In: DIN-Taschenbuch Medizinische Mikrobiologie und Im munologie. Beuth Verlag, Berlin (1992) 4. Eady, E. A., J. H. Cove, J. Blake, K. T. Holland, and w.]. Cunliffe: Recalcitrant acne vulgaris. Clinical, biochemical and microbiological investigation of patients not re sponding to antibiotic treatment. Br.J.Dermato!. 118 (1988) 415-423 5. Eady, E. A., J. H. Cove, K. T. Holland, and w.]. Cunliffe: Erythromycin resistant propionibacteria in antibiotic treated acne patients: association with therapeutic failure. Br.].Dermato!. 121 (1989) 51-57 6. Eady, E. A., C. E.Jones, K.J. Gardner, ]. P. Taylor, ]. H. Cove, and w.]. Cunliffe: Tetracycline-resistant propionibacteria from acne patients are cross-resistant to doxycycline, but sensitive to minocycline. Br.]. Dermato!' 128 (1993) 556-560 7. Eady, E. A., C. E.Jones, S. Vyarkman,]. F. Cove, and w.J. Cunliffe: Increasing pre valence of antibiotic resistant propionibacteria on the skin of acne patients: Results of a five year study. Br.]. Dermato!' 137 (1997) Supp!. 50, 27-28 8. Farmery, M. R., C. E.Jones, E. A. Eady, and w.]. Cunliffe: In vitro activity of azela ic acid, benzoyl peroxide and zinc acetate against antibiotic-resistant propionibac teria from acne patients.]. Dermato!' Treat. 5 (1994) 63-65
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9. Fluhr, ]. w., M. Gloor, P. Dietz, and U. Hoffler: In vitro activity of 6 antimicrobi als against propionibacteria isolates from untreated acne papulopustulosa. Zent. bl. Bakteriol. - Int. J. Med. Microbiol. 289 (1999) 53-61 10. Forssman, Th., M. Gloor und W. Gehring: Antibiotikaresistenzen bei Acne vul garis - Eine retrospektive Untersuchung an einem antibiotisch vorbehandelten und einem unbehandelten Kollektiv. Z. Hautkr. 69 (1994) 828-832 11. Gollnick, H. und A. Melzer: Erythromycin und eine Erythromycin-Zink-Kom bination in der topischen Aknetherapie. Z. f. Dermatologie 183 (1997) 8-18 12. Habbema, L., B. Koopmanss, H. E. Menke, S. Doornweerd, and K. de Boule: A 4 % erythromycin and zinc combination (Zineryt®) versus erythromycin (Ery derm®) in acne vulgaris: a randomized, double-blind comparative study. Br. J. Der matol. 121 (1989) 497-502 13. Hoffler, U., H. L. Ko, and G. Pulverer: Antimicrobial susceptibility of Propionibac terium acnes and related microbial species. Antimicrob. Agents Chemother. 10 (1976) 387-394 14. Hoffler, U., W. Niederau, and G. Pulverer: Susceptibility of cutaneous propionibac teria to newer antiobiotics. Chemother. 26 (1980) 7-11 15. Hoffler, U.: Resistenzinduktion nach topischer antimikrobieller Akne-Therapie? Hautarzt 36 (1985) Suppl. VIII, 175-177 16. Holland, K. T., C. D. Roberts, w.]. Cunliffe, and M. Williams: A technique for sampling micro-organisms from the pilo-sebaceous ducts. J. appl. Bact. 37 (1974) 289-308 17. Holland, K. T., R. A. Bojar, w.]. Cunliffe, A. G. Cucliffe, E. A. Eady, L. Farooq, A. M. Farrell, E. M. Gribbon, and D. Taylor: The effect of zinc and erythromycin on the growth of erythromycin-resistant and erythromycin-sensitive isolates of Propionibacterium acnes: an in-vitro study. Br. J. Dermatol. 126 (1992) 505-509 18. Krogstadt, D.]. and R. C. Moellering: Combination of antibiotics, mechanisms of interaction against bacteria. In: Antibiotics in laboratory medicine. Ed.: Lorian V., Williams & Wilkins, Baltimore, London (1986) pp.537-594 19. Kurokawa, I., S. Nishijima, and Y. Asada: The antibiotic susceptibility of Propioni bacterium acnes: a 15 year bacteriological study and retrospective evaluation: J. Dermatol. 15 (1988) 149-154 20. Leyden, ].]., K.]. Mc Ginley, S. Cavalieri, G. F. Webster, o. H. Mills, and A. M. Kligman: Propionibacterium acnes resistance to antibiotics in acne patients. J. Am. Acad. Dermatol. 8 (1983) 41-45 21. Maurer, w., L. Hothorn, and W. Lehmacher: Multiple comparison in drug clinical trials and preclinical assays. In: Vollmar, J.: Biometrie in der chemisch-pharma zeutischen Industrie. Vol. 6, p. 3-21, Fischer, Stuttgart 1995 22. Pierard, G. E. and C. Pierard-Franchimont: Effect of a topical erythromycin-zinc formulation on sebum delivery. Evaluation by combined photometric-multi-step samplings with Sebutape®. Clin. Exp. Dermatol. 18 (1993) 410-413 23. Plewig, G. und M. Kligman: Akne und Rosazea. Springer-Verlag, Berlin, Heidel berg, (1994) p. 36 24. Schachner, L., W. Eaglstein, C. Kittles, and P. Mertz: Topical erythromycin and zinc therapy for acne. J. Am. Acad. Dermatol. 22 (1990) 253-260 25. Schachner, L., A. Pestana, and C. Kittles: A clinical trial comparing the savety and efficacy of a topical erythromycin-zinc formulation with a topical clindamycin for mulation. J. Am. Acad. Dermatol. 22 (1990) 489-495 26. Stamatiadis, D., M. C. Bulteau-Portois, and I. Mowszowics: Inhibition of 5 alpha reductase activity in human skin by zinc and azelaic acid. Br. J. Dermatol 119 (1988) 627-632
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27. Strauss, J. S. and A. M. Stranieri: Acne treatment with topical erythromycin and zinc: Effect on propionibacterium acnes and free fatty acid composition. J. Am. Acad. Dermatol. 11 (1984) 86-89 28. Sugimoto, Y., I. Lopez-Solache, and F. Labrie: Cations inhibit specifically type I5a reductase found in human skin. J. Invest. Derm. 104 (1995) 775-778
Corresponding author: Dr. Joachim Fluhr, Dept. of Dermatology, Klinikum Karlsruhe, MoltkestraBe 120, D-76133 Karlsruhe, Germany, Tel.: 0049-721-974-0, Fax: 0049721-974-2609, E-mail: [email protected]
Zentralblatt fur
In-vitro and In-vivo Efficacy of Zinc Acetate against Propionibacteria Alone and in Combination with Erythromycin Joachim W. Fluhr!, Bettina Bosch!, Max Gloor!, and Ulrich Hoffler2 1 2
Dept. of Dermatology, Klinikum Karlsruhe, Germany Institute of Medical Microbiology and Hygiene, Klinikum Ludwigshafen, Germany
Received January 29,1998· Revision received April 13, 1999· Accepted May 12, 1999
Summary Some studies have been published about the in vitro activity of zinc acetate (ZA), eryth romycin (E) and their combination (ZAIE) against Propionibacterium spp., especially erythromycin resistant strains. The efficacy of topical ZAIE combination has been reported as well, but a comparison to ZA monotherapy is missing. Therefore, the MIC values of ZA, E and the ZA/E combination were determined for 15 erythromycin-re sistant and 12 erythromycin-sensitive Propionibacterium strains using the agar dilu tion method and the checkerboard technique. Furthermore, the antimicrobial efficacy of ZA (1,2 %) vs. the ZA/E (1,2 %/4 %) combination in an alcoholic solution was test ed in a 7-day treatment administered to 32 acne patients. The MIC 100 for ZA was 1024 Ilg ZNml for both, erythromycin resistant and erythromycin sensitive Propion ibacterium strains. The ZA, as well as the ZAIE solution showed efficacy reducing both the Propionibacterium spp., and the Micrococcaceae in the sebaceous gland infundi bula of acne patients. There was no significant difference between the two treatments. As the MIC 100 of ZAIE was equal to the MIC 100 of ZA, the decrease of the erythromycin MIC of the ZAIE combination in erythromycin-resistant strains may be partly attributed to the addition of ZA to E. The in vivo antibacterial efficacy on 32 acne patients supports the hypothesis that the antibacterial effect of ZNE in short-term treatment can be mostly attributed to ZA.
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Introduction Several studies have shown an in vivo efficacy of topical treatment of acne vul garis using zinc acetate in different combinations (12, 24, 25, 27). Some reports are available regarding the in vitro activity of zinc acetate (ZA), erythromycin (E) and their combination (ZAJE) especially against erythro mycin-resistant strains of Propionibacterium acnes (1, 8, 17). Other data have shown better therapeutical results of the combination of erythromycin with zinc acetate (ZAJE) in acne patients than erythromycin alone (12). However, no controlled study has been performed that would have shown a superiority of the ZAIE combination vs. ZA alone. In the present study, therefore, the in vitro activities of erythromycin, zinc acetate and their combination against propioni bacteria were determined using the checkerboard technique (18). In the second part of the study, the in vivo efficacy of zinc acetate versus the combination of erythromycin and zinc acetate in an alcoholic solution was tested. Within the sebaceous gland infundibulum of acne patients propionibacteria and Micro coccaceae counts were determined using the cyanoacrylate method (16).
Materials and Methods In vitro study Cultivation and identification of propionibacteria. The bacterial flora of the infundi bulum was removed using the cyanoacrylate method (16) and inoculated onto RCM agar plates (Oxoid-Unipath GmbH, Wesel, Germany). The agar was supplemented with 1.0 ml Tween 80 (Merck-Schuchardt, Hohenbrunn, Germany) and 0.06 g Furox ine (Fabry, Deinze, Belgium) per 1000 m!. Subsequently, the cultivated strains were identified by standard methods, including colony morphology after 48 hours of incu bation under anaerobic conditions (Gas-Pak-method, Becton-Dickinson, Heidelberg, Germany) on sheep blood agar, as well as on Wilkins-Chalgren agar (Oxoid GmbH, Wesel, Germany). Further methods were Gram stain reaction, microscopic morpho logy, catalase test, indole production from tryptophane, and exclusion of aerobic growth. Propionibacterium strains were stored at -20°C in a Microbank system (Mast Diagnostics, Hamburg, Germany). Antimicrobial susceptibility testing. All strains were subcultivated on sheep blood agar for 48-72 hours at 37°C under anaerobic conditions. Subsequently, they were transferred to Wilkins-Chalgren broth and incubated for another 48 hours. These broth cultures were used as inoculum. Based on duplicate counts of colony forming units (CFU) for three strains of each species, the mean final inoculum was calculated to be 8 x 10 7 CFU. Aerobically cultivated Pseudomonas aeruginosa, DSM 1107, Staphylococcus aureus, DSM 1104 =ATCC 29213, and Escherichia coli, DSM 1103 = ATCC 25922, were used as antibiotic activity controls according to the recommenda tions of DIN 58940 (3). Growth controls on drug-free medium, and negative controls on agar plates inoculated with propionibacteria broth culture, which were aerobical ly cultivated, were included in each assay. The MICs for erythromycin and zinc acetate were determined using the agar dilu tion method. Freshly prepared drug Wilkens-Chalgren agar mixtures were poured into
Zinc Acetate and Erythromycin Against Propionibacteria
447
Petri dishes and dried for 2 hours after solidification. Subsequently the agar plates were inoculated automatically using the Mastascan Multipoint Inoculator (Mast Diagnos tics, Hamburg, Germany). After incubation for 48 hours at 3rC using the Gas-Pak method, the results were read. MIC was considered to be the lowest drug concentra tion at which no visible growth occurred. Faint growth, and less than four point-like colonies were rated negative. All tests were done in duplicate. Checkerboard technique. The MIC values for the combination of zinc acetate and erythromycin were determined using the agar dilution method in the checkerboard arrangement (18). The checkerboard consisted of columns in which each agar plate contained the same amount of erythromycin which was diluted along the x-axis. Rows in which each agar plate contained the same amount of zinc acetate were diluted on the y-axis. The inoculation of the strains was performed as mentioned above.
In vivo study Patients. The in vivo study involved 32 patients with acne papulopustulosa according to Plewig and Kligman (23). Four additional patients were excluded before the begin ning of the study, as the amount of propionibacteria was less than 100 CFU/cm 2 on one of the two test sites. 23 of the subjects were women (71,9%) and 9 were men (28,1 %). The average age was 28.8 years (women 27.7 years [range: 20-41), men 27.9 years [range 19-53)). Except for acne, the patients showed no dermatoses or system ic diseases. Four weeks before the beginning of the study, no medication was adminis tered. Previous systemic treatment with isotretinoin was regarded as an exclusion cri terium. During the study, the subjects were urged to avoid using cleansing products on the forehead. The aims of the study had been explained to the patients, and they had given their written informed consent. They could withdraw from the study at any time, without giving reasons. Cultivation and identification of bacteria. The methods for harvesting, cultivation, and identification of propionibacteria have been described above. Harvesting of Mi crococcaceae was also done by the cyanoacrylate method. Cultivation (sheep blood agar) and identification (colony morphology, Gram staining and coagulase test) were done using standard methods. Antibacterial efficacy: The following two formulations were compared: 1. Zinc acetate 1.2 % (Zinc acetate dihydrate from Sigma-Aldrich Chemie, Steinheim, Germany) Erythromycin 4.0 % (Erythromycin from Abbott, Wiesbaden, Germany) Alcoholic solution (ethanol 55.2 %, diisopropyle sebacate 25.1 %, water ad 100.0, alcoholic base of Zineryt®, Hermal, Reinbeck, Germany [before mixing the zinc acetate and erythrom ycin containing powder with the alcoholic base)) 2. Zinc acetate 1.2 % Alcoholic solution (ethanol 55.2 %, diisopropyle sebacate 25.1 %, water ad 100.0, alcoholic base of Zineryt®, Hermal, Reinbeck, Germany [before mixing the zinc acetate and erythrom ycin containing powder with the alcoholic base)) The formulations were prepared, blinded and randomised by Drais-Pharmacy, Karls ruhe, Germany (Manager:]. Zybowski). The antibacterial efficacy was measured
448
J. W. Fluhr et al.
based on the reduction of bacteria (propionibacteria and Micrococcaceae) after a 7days treatment. 0.1 ml each of test products were applied on an area of 2.0 X 2.0 cm size (0.025 mllcm2) twice daily on the right and left site of the forehead. The formula tions were tested in double-blind method, as neither the patients nor the investigators knew the randomisation of the test products. The cyanoacrylate method (Permabond technique, Holland et al. 1974) was used to obtain samples of the infundibulum bac terial flora (for cultivation see above). Bacteria were removed before (t 0) and after a 7-day treatment (t7) with the two formulations. Statistical analysis (in vivo) The colony forming units per cm 2 (CFU/cm2) were determined and the log 10 was cal culated. Since a normal distribution could not be assumed, Wilcoxon's distribution free rank test for paired samples was used to test the antibacterial efficacy of the 2 for mulations in the course of time (to vs. t7) (question 1a for propionibacteria, and ques tion 1b for Micrococcaceae) (2-tailed). In the event of established efficacy (question 1a and 1b answered positively), the delta (tl-tO) of the two formulations were compared (question 2a and 2b) with Wilcoxon's rank test for paired samples (2-tailed). The test ing of the two (a+b) questions was carried out in accordance with the method of a priory-ordered hypothesis (21). The results were considered significant at a level of p < 0.05. The above mentioned tests were performed with Prism 2, GraphPad®, USA.
Results The results of the in vitro study are shown in Tables 1-3. The MIC 100 val ue for zinc acetate alone was 1024 Ilg/ml (Table 1) for erythromycin-resistant (ER) strains. The MIC 100 values for erythromycin sensitive (ES) strains, was 512 Ilg/ml for P.acnes, and 1024 Ilg/ml for P.granulosum. The MIC 100 val ues for erythromycin alone was in ES P. acnes: 0.125 Ilg/ml and in ES P. gran ulosum: 0.125 Ilg/ml (Table 2). The MIC 100 value for erythromycin alone was in ER > 512 Ilg/ml for both P. acnes and P. granulosum (Table 3). With increasing concentration of ZA (starting at 16 Ilg/ml up to 256 Ilg/ml) the combination of ZA and E showed a slight decrease of the MIC values for erythromycin in the ER-group for some strains of P. acnes. At a zinc acetate concentration of 1024 Ilg/ml a dramatic decrease of the MIC 100 values for erythromycin was detected for both P. acnes and P. granulosum in ER. Further increase of the ZA concentration (2048 Ilg/ml) confirmed the low MIC values for erythromycin (0.125 Ilglml) for both P. acnes and P. granulosum in all ER. The values and statistical differences of the in vivo study are given in Figs. 1 and 2. Zinc acetate at a concentration of 12 000 Ilglml (1.2 %) significantly reduced the propionibacteria (p < 0.0001) and the Micrococcaceae counts (p < 0.0001) in the infundibulum after a topical treatment during 7 days. The combination of 12000 Ilglml (1.2 %) zinc acetate and erythromycin 40000 Ilglml (4.0 %) significantly reduced the propionibacteria (p < 0.0001) and the Micrococcaceae counts (p < 0.0001) in the infundibulum after a top ical treatment during 7 days. Thus, questions la (propionibacteria) and Ib
Zinc Acetate and Erythromycin Against Propionibacteria
449
(Micrococcaceae) concerning an infundibular antibacterial effect could be an swered positively for ZA and for the ZAJE combination. The bacterial reduction of the two test formulations (delta) was slightly, but not significantly, higher for the combination ZAJE than the monotherapy ZA (propionibacteria (p =0.6988) and Micrococcaceae (p =0.0965)). Therefore, questions 2a (propionibacteria) and 2b (Micrococcaceae) regarding a possible superiority of the antibacterial effect of ZAJE combination in comparison to the mono therapy with ZA was not statistically significant.
Discussion The combination of zinc acetate and erythromycin is known to be effective in the topical therapy of acne vulgaris (12, 24, 25, 27). The literature about the effect of a combination of zinc and erythromycin has been extensively revie wed by Gollnick et al. (11). Pierard et al. showed a sebosuppressive effect by the combination of 1.2 % zinc acetate and 4 % erythromycin in comparison to an untreated area at the forehead (22). An antiandrogen activity of zinc ac etate inhibiting the Sa-reductase has been shown in vitro (26, 28). An anti inflammatory activity of zinc has been demonstrated (2). Bojar et al. showed for the combination of 1.2 % zinc acetate/4 % erythromycin and 4 % eryth romycin alone, after 8 weeks of therapy, a significant reduction of total pro pionibacteria, as well as a reduction of erythromycin-resistant propionibacte ria (1). However, after 12 weeks, erythromycin-resistant propionibacteria Table 1. MIC values (!tg/ml) of zinc acetate against propionibacteria. Agar dilution test. (E. S. '" erythromycin sensitive; 'E. R. '" erythromycin resistant) Type Species
Cumulative percentages of strains inhibited by MIC values (!tg/ml) of zinc acetate n
E.S.
64.0
128.0 256.0 512.0 1024.0 2048.0
P. acnes P. granulosum total
0.0 0.0 0.0 33.3 0.0 8.3
11.1 66.7 25.0
E.R. P. acnes P. granulosum total
0.0 0.0 0.0 0.0 33.3 0.0 6.7
33.3 6.7
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Total P. acnes P. granulosum total
0.0 0.0 0.0
9.5 33.3 14.8
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4.8 16.7 7.4
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88.9 100.0 100.0 100.0 100.0 100.0
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88.9 100.0
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P. acnes P. granulosum
P.acnes P. granulosum
P.acnes P. granulosum
P. acnes P. granulosum
P.acnes P. granulosum
321lg/ml
641lg/ml
2561lg/ml
10241lg/ml
20481lg/ml
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16.0
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100.0 100.0
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were reacquired or appeared de novo. The reduction of Micrococcaceae were slight and after 12 weeks erythromycin-resistant strains were predominant. The in vitro sensitivity was the same for erythromycin-resistant and erythrom ycin-sensitive propionibacteria (MIC 32-64 !-tg/ml zinc acetate). For eryth romycin resistant and erythromycin sensitive coagulase-negative staphylococ ci, an MIC 128-512 !-tg/ml zinc acetate was established (1). The MIC values for erythromycin in erythromycin-sensitive and erythro mycin resistant propionibacteria strains were comparable with the MIC 100 values given by Bajar et al. (1). In contrast our MIC 100 values for both groups of bacteria when analysing zinc acetate were far higher than the val ues given by Bajar et al. (1): 1024 !-tg/ml vs. 64 !-tg/ml which corresponds to 4 dilution steps. Farmery et al. showed an MIC for zinc acetate of 3264 !-tg/ml irrespective to resistance of propionibacteria against erythromycin or tetracycline (8). Holland et al. showed a delayed growth of erythromycin resistant propionibacteria in vitra with 96 !-tg/ml, while 300 !-tg/ml prevented the growth of these propionibacteria (17). To insure the macroscopic growth
Zinc Acetate and Erythromycin Against Propionibacteria
453
in two days culturing of propionibacteria, we inoculated 8 X 10 7 CFU, onto the Wilkins-Chalgren-agar internationally accepted for testing of anaerobes, while Bojar et al. (1), Farmery et al. (8) and Holland et al. (17) inoculated 105 CFU onto brain heart infusion agar or RCM-broth. This could explain the higher MIC values detected in our in vitro study for propionibacteria. MIC values show a strong antibacterial effect on propionibacteria using a zinc acetate concentration of 1024 !-tg/ml or higher. The inhibition of propi onibacteria growth in vitro by the erythromycin-zinc acetate combination, especially on erythromycin-resistant strains, is mostly due to the antibacteri al effect of zinc acetate. To our knowledge, the in vivo part of our study was the first clinical trial to analyse the infundibular efficacy flora (propionibacteria and Micrococca ceae) of the ZAiE complex in comparison to ZA in the same alcoholic solu tion. We could show a significant reduction of bacteria for both products, and both bacterial groups, after 7 days of topical treatment. Thus the ZA concen tration within the infundibula using an alcoholic vehicle is supposed to be higher than the MIC 100 value of 1024 !-tg/ml for both groups of bacteria. A
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454
]. W. Fluhr et a!.
treatment with the ZNE combination lacked to show an antibacterial efficacy in the infundibulum superior to the monotherapy by ZA. The results of our studies provide evidence supporting the hypothesis that a decrease of the erythromycin MIC in ER propionibacteria under a treat ment with the combination of zinc acetate/erythromycin may be mainly due to the antibacterial effect of zinc acetate. As we could not show an in vivo superiority of the ZNE combination in comparison to the mono therapy by ZA, both in the same alcoholic solution for propionibacteria and Micrococ caceae, an acne treatment only with ZA may be nearly as effective as the ZAIE combination, considering the antibacterial efficacy in the infundibu lum. The clinical impact of paraantibiotic erythromycin effects (e. g. antiin flammatory and inhibition of ectoenzymes) has remained unknown. There fore clinical studies on a larger cohort and a long-term treatment are neces sary to prove the efficacy of a ZA monotherapy due to antibacterial and anti inflammatory effects of ZA. A wider use of antimicrobial agents not belon ging to the group of antibiotics could be an interesting issue in the light of the rapidly increasing problem of antibiotic resistance (4, 5, 6, 7, 9, 10, 13, 14, 15, 19,20). Acknowledgement. The authors wish to cordially thank Mrs. Barbara Wasik and Mrs. Sibylle E. Franke for their patient and helpful technical assistance and to Jochen Hohberg for his assistance performing the statistical analysis.
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Corresponding author: Dr. Joachim Fluhr, Dept. of Dermatology, Klinikum Karlsruhe, MoltkestraBe 120, D-76133 Karlsruhe, Germany, Tel.: 0049-721-974-0, Fax: 0049721-974-2609, E-mail: [email protected]