Scand J Plast Reconstr Hand Surg 24: 193-197, 1990
ANTIBACTERIAL EFFECT OF ZINC OXIDE IN VITRO
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Thor A. Soderberg,] Bo Sunzel,* Stig Holm,3 Theodor E l m r o ~ , ~ Goran Hallmans' and Staffan Sjoberg6 From the Departments of 'Hand Surgery, 'Clinical Bacteriology, and 4Derrnatology, University Hospital of Ume& Departments of 20raI Surgery, 5Nutritional Research and Pathology, and 'Inorganic Chemistry, University of Uined, Umed, Sweden
(Submitted for publication March 16, 1990)
Abstract. Antibacterial activity, zinc concentrations and pH were measured in Miiller-Hinton broth containing different amounts of zinc oxide and inoculated with Staphylococcus aureus (lo6colony forming units/ml). The minimum inhibitory concentrations (MIC) of zinc oxide to different clinical isolates were determined using the Muller-Hinton agar dilution tests. Gram-positive bacteria were most susceptible. Gram-negative aerobic bacteria and streptococci were usually not inhibited even at the highest concentrations used (1 024 pg/ml), but staphylococciparticularly some isolates of Staphylococcus epidermidis -were sensitive enough to allow determination of their MIC. Key words: zinc oxide, zinc, antibacterial effect.
The antibacterial effects of adhesive zinc tapes on streptococci and staphylococci were described by Gilje as early as 1949 (2). Later Henriksen & Gilje (6) reported that this antibacterial effect was caused mainly by an antioxidant present in the tape. No antibacterial effect was observed when zinc oxide was tested. In 1968 Marples & Kligman (7) reported an antibacterial effect on the resident skin flora and Staphylococcus aureus that had been inoculated beneath occlusive tapes applied on the skin of volunteers. The antibacterial effect was ascribed to an unidentified substance in the adhesive mass. Hallmans & Elmros (5) found that an adhesive tape containing zinc oxide (Mezinc@)had an antibacterial effect on resident Staph. aureus and Pseudomonas aeruginosa inoculated into third degree burns in guinea pigs. The antibacterial effect in vitro of zinc oxide in a phosphate buffer on Staph. aureus, Streptococcus pyogenes and Ps. aerugiRosa, was also noted but no effect on Escherichia coli was seen. In a comparative study (12) of different dressings applied to excisional wounds on rats, the number of bacteria in the granulation tissue was 14-908223
significantly lower under a zinc oxide adhesive tape than under a gauze and hydrocolloid dressing. In vitro the tape was found to have antibacterial effects on Staph. aureus and E. coli, but no inhibition of growth of Ps. aeruginosa was found. In dentistry the antibacterial effects of zinc oxide have been reported (8, 9, 13). Zinc oxide is extensively used as a component of cements and periodontal dressings, and as a filler in endodontic gutta-percha cones. To try and elucidate these partly contradictory findings about the effect of zinc oxide on different bacteria, we set out to investigate the in vitro antibacterial effects of zinc oxide using different methods. MATERIAL AND METHODS "Adhesive tapes" A cotton web coated with polyvinyl chloride (PVC) on one side (ICG Inc., TN, USA) was covered on the opposite side with different concentrations of zinc oxide powder (>99% purity) or titanium dioxide powder (>99% purity) supplied by E. Merck, Darmstadt, FRG. Titanium dioxide was used as an inert reference substance. Various amounts of the substances were put into acid washed glass tubes containing acetone, shaken vigorously for five minutes, and poured onto the cotton web. The web was dried in air at room temperature for 24 hours, and circular 9 mm pieces were punched in the web. Bacterial strains Staph. aureus (25 strains), Staphylococcus epidermidis (24 strains), Streptococcus faecalis (18 strains), Streptococcus haemolyticus (group A-3 strains; group B-5 strains; group G-2 strains), P s . aeruginosa (10 strains), Proteus mirabilis (14 strains), Klebsiella pneumoniae (12 strains), and E. coli (18 strains) were isolated from clinical specimens at the Department of Clinical Bacteriology, Umei, Sweden. Strains were identified according to the Scand J Plast Reconstr Hand Surg 24
194 T. A . Soderberg et al.
Scand J Plast Surg Recontr Surg Hand Surg Downloaded from informahealthcare.com by University of Leeds on 09/15/13 For personal use only.
method of Cowan & Steel (1) or by immunological techniques (streptococci), alternatively the Analytical Profile Index system was used. Staph. aureus 209-P was a gift from Statens Bakteriologiska Laboratorium, Stockholm, Sweden, and was used as a reference strain. Measurements of zinc concentrations To measure the zinc concentration in the test solutions, samples were passed through a 0.22 ym filter before analysis to prevent the zinc oxide particles from passing through. In a pilot study no differences in zinc concentrations were found in filtrated (0.22 and 0.1 ym) or centrifuged samples at 1OOOOxg. These findings indicate that no larger zinc oxide particles passed through the 0.22 pm filter. Samples for zinc measurements were therefore filtrated through a 0.22 ym filter (Millipore AB, Vastra Frolunda, Sweden) and assays carried out on the filtrate. The samples were collected in acid-washed borosilicate glass tubes and dissolved in 0.6 M hydrochloric acid. The concentrations of zinc were then measured using an atomic absorption spectrophotometer (Varian AA-66DB) as described by Hallmans (4). pH measurements pH was measured using a standard pH-meter calibrated at 37°C. Bacreriul viability Three different methods were used to evaluate the antibacterial effects of zinc oxide: disc diffusion in agar, agar dilution, and broth dilution. Muller-Hinton broth and agar were used as standards (Kebo Lab AB, Stockholm, Sweden). Muller-Hinton-agar and 0.5% horse blood was used for streptococci. Disc diffusion test3 Circular (9 mm diameter) pieces of cotton web coated with test substances (zinc oxide or titanium dioxide 1.2 or 5.0 mg/cm2)were placed on Miiller-Hinton agar (pH=7.2) with the “adhesive” substance facing the agar. The zones of inhibition were defined as the mean value (mm) of two perpendicular diagonals of the circular zone of no visible bacterial growth surrounding the discs, thus including the diameter of the tape (9 mm). The agar plate had earlier been seeded with an overnight culture (1 x lo6 CFU/ml) of Staph. aureus, 209-P, P s . ueruginosa (2 strains), P . mirabilis (2 strains), K . pneumoniae (2 strains) and E . coli (2 strains). The plates were incubated at 37°C for 18 hours and the diameters of the zones of inhibition were measured with Vernier callipers. Agar dilution tests Zinc oxide powder was dissolved in I % acetic acid, diluted to the following concentrations: 2, 4,8, 16, 32, 64, 128, 256, 512, and I024 ygiml in Miiller-Hinton agar (pH=7.2) at 50°C and poured into a Petri dish. With multipoint inoculation equipment, lo6 bacteridspot of the bacterial strains tested were inoculated on the agar plates containing zinc oxide. In a pilot study no difference in antibacterial activity was noted using two different bacterial inocula (10‘ and lo6 CFU/ml), consequently only one inocuScaitd J Plnst
Reconstr Hand
Surg 24
lum (lo6 CFUlml) at the logarithmic growth phase was used. After incubation for 18 hours at 37°C the minimum inhibitory concentration (MIC) of zinc oxide was calculated. The end point was defined as the concentration of dissolved zinc oxide in the agar plate where there was no visible growth of bacteria. Controls contained 1 % acetic acid. Broth dilution tests Zinc oxide powder (1790, 179, 17.9, and 1.79 mg) was added to 1000 ml Miiller-Hinton broth (pH=7.2) in four different vials. The suspensions were stirred for 24 hours at 37°C. Zinc concentrations and pH were registered at the start of the experiment, after I , 4, 6, 8, 10, 12, 14, 16, and 24 hours. After 24 hours, roughly lo6 CFU/ml of Staph. aureus 209-P were inoculated into each suspension and zinc concentrations, and the pH and bacterial viability were measured at the time intervals described above. By withdrawing 10 PI samples from the cultures, bacterial viability was measured on blood agar plates. Simultaneously 3 ml samples were recovered to analyse the pH and zinc concentration.
RESULTS Disc diffsion tests The zones of inhibition around tapes covered with zinc oxide (1.2 mg/cm2) on agar seeded with Staph. aureus 209-P were 10 mm in diameter and a higher concentration of zinc oxide (5.0 mg/cm2) resulted in a larger zone of inhibition (11.5 mm). Titanium dioxide and acetone had no antibacterial effect. No zones of inhibition were seen on any plate seeded with Ps. aeruginosa ( 2 strains), P . mirabilis ( 2 strains), K . pneumoniae ( 2 strains), or E . coli ( 2 strains). Agar dilution tests The MIC of zinc oxide dissolved in acetic acid on the bacterial strains tested are shown in Table I . Staph. epidermidis and Staph. aureus were the most susceptible bacteria, whereas most strains of enterococci, Ps. aeruginosa and P . mirabilis were resistant to the zinc concentrations tested. Inhibition of growth of streptococci, K . pneumoniae, and E . coli were noted only in the highest concentration tested. The measured zinc concentrations in the dilution series were as expected. Broth dilution tests there were no important differences in zinc concentrations between Miiller-Hinton broth containing no bacteria and broth inoculated with Staph. aureus 209-P, only one figure is shown (Fig. 1). As
Antibacterial effect of zinc oxide in vitro
195
i:m
-z 150
5 Scand J Plast Surg Recontr Surg Hand Surg Downloaded from informahealthcare.com by University of Leeds on 09/15/13 For personal use only.
5
100 15
g
U
z
5 0
A
A
0 1
4
z
z -
A
A
z 6
z 8
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A
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A
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A
z
t
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12
14
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Fig. 1 . Zinc concentrations in filtrates of Muller-Hinton broth inoculated with lo6 CFU/ml of Staph. aureus 209-P. The following amounts of zinc oxide were added: 1790 pgiml (A),179 pgiml (m),17.9 pg/ml (0)and 1.79 pgiml (+). Controls (*). Mean values of 3 experiments (SEM not exceeding ?lo%).
can be seen, the dissolution process took about an hour after which there was a steady state condition for up to 24 hours. Furthermore, the zinc concentration in solution increased as increasing amounts of zinc oxide were added. The zinc concentrations in the broths containing 1.79 and 17.9 pg/ml of zinc oxide implied that the zinc oxide had been totally dissolved. The zinc concentrations in the broths containing 179 and 1790 pg/ml of zinc oxide were 100-110 pg/ml and 150-180 pg/ml, indicating that in these suspensions considerable amounts of zinc oxide still remained undissolved. The high concentrations of dissolved zinc were mainly due to the pH-
buffering effect of the Miiller-Hinton broth as well as its contents of potential complexing agents (for example, peptides) towards zinc. The increased solubility of zinc oxide in the broth was also reflected in the measured pH-values. A small increase in pH was noted as increasing amounts of the base zinc oxide were dissolved. In addition the pH of the different broth solutions remained constant after one hour's equilibration. The addition of bacteria to the broths caused slight increases in pH after 12 hours, finally reaching values of 7.9 in the controls and 7.8 in broths containing 1.79 and 17.9 pg/ml zinc in solution (no
Table I. MICs of zinc in Miiller-Hinton agar plates towards Staph. aureus (25 strains), Staph. epidermidis (24 strains), Strep. faecalis (18 strains), Strep. huemolyticus (group A-3 strains; group B-5 strains; group G-2 strains), Ps. aeruginosa (10 strains), P . mirabilis (14 strains), K . pnecrmoniae (12 struins), and E . coli (18 strains) MIC (pgirnl) Bacteria (No. of strains)
Staph. aureus (25) Staph. epidermidis (24) Sirep. faecalis (18) Strep. pyogenes (10) Ps. aeruginosa (10) P. mirabilis (14) K . pneumoniae (12) E. coli (18)
8
16
32
1
1
I 1 2 1
64
128
256
512
1 6 1
9 2
14
1
1
1
1
1 1 1
1024
> I 024
1
2 1 3 11 13
17 5 9 10 3
Scand J Plast Reconstr Hand Surg 24
Scand J Plast Surg Recontr Surg Hand Surg Downloaded from informahealthcare.com by University of Leeds on 09/15/13 For personal use only.
196 T . A . Soderberg et al.
t , , 0 1
4
6
8
10
12
14
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TIME (HOURS)
Fig. 2. Bacterial counts (CFUlml) in Miiller-Hinton broth inoculated with lo6 CFUiml of Staph. aureus 209-P, containing 1790 pg/ml (A),179 pgiml (m), 17.9 yg/ml(O) and 1.79 pg/ml(+) of zinc oxide and controls (*). Mean values of 3 experiments (SEM not more than f0.5log" CFU).
undissolved zinc oxide). In these solutions there was no inhibition of bacterial growth (Fig. 2 ) . In the most concentrated zinc oxide suspensions, however, (179 and 1790 pg/ml of zinc oxide) a clear antibacterial effect could be seen (Fig. 2 ) . In these broths pH remained constant throughout the 24 hour period. DISCUSSION The results of the present study show that zinc oxide has an antibacterial effect, mainly on some Gram positive bacteria. When using the agar dilution method Staph. epidermidis and Staph. aureus were most susceptible, whereas most strains of enterococci, Ps. aeruginosa and P . mirabilis were resistant to the zinc concentrations tested. MICs of most streptococci, K . pneumoniae and E. coli were 512-1024 kg/ml, even though some strains were sensitive in lower concentrations. To evaluate the antibacterial effect of zinc oxide further, and to measure the zinc concentration in a filtrate of an overnight suspension of zinc oxide, the broth dilution method was used. The inhibitory effect on Staph. aureus 209-P using this method was similar to that seen when the agar dilution method (128 p.g/ml) was used. Bacterial growth was inhibited to the same extent in the two most concentrated zinc oxide suspensions, indicating that Scand J Plast Reconstr Hand Surg 24
the inhibitory concentration of zinc to Staph. aureus 209-P in Miiller-Hinton broth was lower than 100-110 p.g/ml. No inhibition of bacterial growth was seen in broths containing only dissolved zinc oxide (zinc concentration about 14 pg/ml), the inhibitory concentration of zinc must therefore have been higher. The inhibition of bacterial growth in the broth dilution system was thus related to the amount of dissolved zinc. During this experiment the pH remained constant when the two highest concentrations of zinc oxide were used. This might be explained by the pH-buffering capacity of the undissolved zinc oxide in these broths as well as by the reduced bacterial growth. The agar diffusion technique, applying tapes on agar surfaces, was assumed to reflect the clinical procedure for the treatment of wounds. The method is based on diffusion and thus the water solubilities of the substances studied are important. Even though zinc oxide is almost insoluble in water, inhibitory zones on agar plates were found around tapes impregnated with zinc oxide powder indicating that zinc was present in an inhibitory concentration in the agar outside the tapes. A slow release of zinc ions from zinc oxide in vitro as well as in vivo is dependent on many different physical and chemical factors-for example, pH, temperature, phosphates (lo), proteins, amino acids ( l l ) , and different combinations of these as seen in broths, serum,
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Antibacterial effect of zinc oxide in vitro
and wound exudates. The release of zinc ions from zinc oxide also depends on what substances have been mixed with it-for example, rosin, rubber, antioxidants in adhesive tapes, and periodontal dressings. These factors may explain the unexpectedly high zinc concentrations (22 mmol/l) that Hallmans (3) found beneath an adhesive zinc tape when treating burns in humans. There are several possible mechanisms for the antibacterial action of zinc. It has been suggested that zinc binds to the membranes of micro-organisms in a similar way as to mammalian cells. There are also several theories about how zinc affects bacteria when zinc binds to the membranes, and it has been observed that it modulates membrane associated enzymes, impairs calcium uptake, and changes the fluidity of the membrane (11). In conclusion we have shown that zinc oxide in Muller-Hinton broth and agar has antibacterial effects on staphylococci, some streptococci, and occasionally on Gram negative bacteria, although high concentrations of zinc oxide were needed to inhibit bacterial growth.
ACKNOWLEDGEMENTS This research was supported by grants from the Bo Rydin Foundation, SCA, Sweden. We gratefully acknowledge Miss K. Olofsson and Mrs I. Sjostrom for skilful technical assistance and Mr S. Granstrom and Mr R. Sjostrom for valuable advice and support.
197
REFERENCES 1. Cowan ST, Steel KJ. Manual for the identification of medical bacteria. London: Cambridge University Press, 1974. 2. Gilje 0. Ulcus cruris in venous circulatory disturbances. Acta Derm Venereol (Stockh) 1949. Suppl 22. 3. Hallmans G. Treatment of burns with zinc tape. A study of local absorption in humans. Scand J Plast Reconstr Surg 1977; 11: 155-161. 4. Hallmans G. Absorption of topically applied zinc and changes in zinc metabolism during wound healing. Acta Derm Venereol 1978; 58 (Suppl 80): 1-36. 5 . Hallmans G, Elmros T. Zinc tape treatment of burns infected by Pseudomonas aeruginosa. Scand J Plast Reconstr Surg 1980; 14: 221-227. 6. Henriksen SD, Gilje 0. Adhesive tape-Its bacteriostatic effect and its use in the treatment of ulcus cruris. Acta Derm Venereol 1965; 45: 471478. 7. Marples RR, Kligman AM. Growth of bacteria under adhesive tapes. Arch Dermatol 1969; 99: 107-110. 8. Moorer WR, Genet JM. Antibacterial activity of gutta-percha cones attributed to the zinc oxide component. Oral Surg 1982; 53: 508-517. 9. Paetzold OH, Wiese A. Experimentelle Untersuchungen uber die antimikrobielle Wirkung von Zinkoxid. Arch Derm Res 1975; 253: 151-159. 10. Spear PA. Zinc in the aquatic environment: Chemistry, distribution and toxicology. National Research Council of Canada, NRCC Associate Committee on Scientific Criteria for Environmental Quality, NRCC 1981: No. 17589. 11. Sugarman B. Zinc and infection. Rev Inf Dis 1983; 5 : 138- 147. 12. Soderberg TA, a g r e n M , Tengrup 1, Hallmans G, Banck G. The effects of an occlusive zinc medicated dressing on the bacterial flora in excised wounds in the rat. Infection 1989; 17: 81-85. 13. Turkheim HJ. Bacteriological investigations on dental filling materials. Br Dent J 1953; Vol XCV: 1-7.
Scand J Plast Reconstr Hand Surg 24
ANTIBACTERIAL EFFECT OF ZINC OXIDE IN VITRO
Scand J Plast Surg Recontr Surg Hand Surg Downloaded from informahealthcare.com by University of Leeds on 09/15/13 For personal use only.
Thor A. Soderberg,] Bo Sunzel,* Stig Holm,3 Theodor E l m r o ~ , ~ Goran Hallmans' and Staffan Sjoberg6 From the Departments of 'Hand Surgery, 'Clinical Bacteriology, and 4Derrnatology, University Hospital of Ume& Departments of 20raI Surgery, 5Nutritional Research and Pathology, and 'Inorganic Chemistry, University of Uined, Umed, Sweden
(Submitted for publication March 16, 1990)
Abstract. Antibacterial activity, zinc concentrations and pH were measured in Miiller-Hinton broth containing different amounts of zinc oxide and inoculated with Staphylococcus aureus (lo6colony forming units/ml). The minimum inhibitory concentrations (MIC) of zinc oxide to different clinical isolates were determined using the Muller-Hinton agar dilution tests. Gram-positive bacteria were most susceptible. Gram-negative aerobic bacteria and streptococci were usually not inhibited even at the highest concentrations used (1 024 pg/ml), but staphylococciparticularly some isolates of Staphylococcus epidermidis -were sensitive enough to allow determination of their MIC. Key words: zinc oxide, zinc, antibacterial effect.
The antibacterial effects of adhesive zinc tapes on streptococci and staphylococci were described by Gilje as early as 1949 (2). Later Henriksen & Gilje (6) reported that this antibacterial effect was caused mainly by an antioxidant present in the tape. No antibacterial effect was observed when zinc oxide was tested. In 1968 Marples & Kligman (7) reported an antibacterial effect on the resident skin flora and Staphylococcus aureus that had been inoculated beneath occlusive tapes applied on the skin of volunteers. The antibacterial effect was ascribed to an unidentified substance in the adhesive mass. Hallmans & Elmros (5) found that an adhesive tape containing zinc oxide (Mezinc@)had an antibacterial effect on resident Staph. aureus and Pseudomonas aeruginosa inoculated into third degree burns in guinea pigs. The antibacterial effect in vitro of zinc oxide in a phosphate buffer on Staph. aureus, Streptococcus pyogenes and Ps. aerugiRosa, was also noted but no effect on Escherichia coli was seen. In a comparative study (12) of different dressings applied to excisional wounds on rats, the number of bacteria in the granulation tissue was 14-908223
significantly lower under a zinc oxide adhesive tape than under a gauze and hydrocolloid dressing. In vitro the tape was found to have antibacterial effects on Staph. aureus and E. coli, but no inhibition of growth of Ps. aeruginosa was found. In dentistry the antibacterial effects of zinc oxide have been reported (8, 9, 13). Zinc oxide is extensively used as a component of cements and periodontal dressings, and as a filler in endodontic gutta-percha cones. To try and elucidate these partly contradictory findings about the effect of zinc oxide on different bacteria, we set out to investigate the in vitro antibacterial effects of zinc oxide using different methods. MATERIAL AND METHODS "Adhesive tapes" A cotton web coated with polyvinyl chloride (PVC) on one side (ICG Inc., TN, USA) was covered on the opposite side with different concentrations of zinc oxide powder (>99% purity) or titanium dioxide powder (>99% purity) supplied by E. Merck, Darmstadt, FRG. Titanium dioxide was used as an inert reference substance. Various amounts of the substances were put into acid washed glass tubes containing acetone, shaken vigorously for five minutes, and poured onto the cotton web. The web was dried in air at room temperature for 24 hours, and circular 9 mm pieces were punched in the web. Bacterial strains Staph. aureus (25 strains), Staphylococcus epidermidis (24 strains), Streptococcus faecalis (18 strains), Streptococcus haemolyticus (group A-3 strains; group B-5 strains; group G-2 strains), P s . aeruginosa (10 strains), Proteus mirabilis (14 strains), Klebsiella pneumoniae (12 strains), and E. coli (18 strains) were isolated from clinical specimens at the Department of Clinical Bacteriology, Umei, Sweden. Strains were identified according to the Scand J Plast Reconstr Hand Surg 24
194 T. A . Soderberg et al.
Scand J Plast Surg Recontr Surg Hand Surg Downloaded from informahealthcare.com by University of Leeds on 09/15/13 For personal use only.
method of Cowan & Steel (1) or by immunological techniques (streptococci), alternatively the Analytical Profile Index system was used. Staph. aureus 209-P was a gift from Statens Bakteriologiska Laboratorium, Stockholm, Sweden, and was used as a reference strain. Measurements of zinc concentrations To measure the zinc concentration in the test solutions, samples were passed through a 0.22 ym filter before analysis to prevent the zinc oxide particles from passing through. In a pilot study no differences in zinc concentrations were found in filtrated (0.22 and 0.1 ym) or centrifuged samples at 1OOOOxg. These findings indicate that no larger zinc oxide particles passed through the 0.22 pm filter. Samples for zinc measurements were therefore filtrated through a 0.22 ym filter (Millipore AB, Vastra Frolunda, Sweden) and assays carried out on the filtrate. The samples were collected in acid-washed borosilicate glass tubes and dissolved in 0.6 M hydrochloric acid. The concentrations of zinc were then measured using an atomic absorption spectrophotometer (Varian AA-66DB) as described by Hallmans (4). pH measurements pH was measured using a standard pH-meter calibrated at 37°C. Bacreriul viability Three different methods were used to evaluate the antibacterial effects of zinc oxide: disc diffusion in agar, agar dilution, and broth dilution. Muller-Hinton broth and agar were used as standards (Kebo Lab AB, Stockholm, Sweden). Muller-Hinton-agar and 0.5% horse blood was used for streptococci. Disc diffusion test3 Circular (9 mm diameter) pieces of cotton web coated with test substances (zinc oxide or titanium dioxide 1.2 or 5.0 mg/cm2)were placed on Miiller-Hinton agar (pH=7.2) with the “adhesive” substance facing the agar. The zones of inhibition were defined as the mean value (mm) of two perpendicular diagonals of the circular zone of no visible bacterial growth surrounding the discs, thus including the diameter of the tape (9 mm). The agar plate had earlier been seeded with an overnight culture (1 x lo6 CFU/ml) of Staph. aureus, 209-P, P s . ueruginosa (2 strains), P . mirabilis (2 strains), K . pneumoniae (2 strains) and E . coli (2 strains). The plates were incubated at 37°C for 18 hours and the diameters of the zones of inhibition were measured with Vernier callipers. Agar dilution tests Zinc oxide powder was dissolved in I % acetic acid, diluted to the following concentrations: 2, 4,8, 16, 32, 64, 128, 256, 512, and I024 ygiml in Miiller-Hinton agar (pH=7.2) at 50°C and poured into a Petri dish. With multipoint inoculation equipment, lo6 bacteridspot of the bacterial strains tested were inoculated on the agar plates containing zinc oxide. In a pilot study no difference in antibacterial activity was noted using two different bacterial inocula (10‘ and lo6 CFU/ml), consequently only one inocuScaitd J Plnst
Reconstr Hand
Surg 24
lum (lo6 CFUlml) at the logarithmic growth phase was used. After incubation for 18 hours at 37°C the minimum inhibitory concentration (MIC) of zinc oxide was calculated. The end point was defined as the concentration of dissolved zinc oxide in the agar plate where there was no visible growth of bacteria. Controls contained 1 % acetic acid. Broth dilution tests Zinc oxide powder (1790, 179, 17.9, and 1.79 mg) was added to 1000 ml Miiller-Hinton broth (pH=7.2) in four different vials. The suspensions were stirred for 24 hours at 37°C. Zinc concentrations and pH were registered at the start of the experiment, after I , 4, 6, 8, 10, 12, 14, 16, and 24 hours. After 24 hours, roughly lo6 CFU/ml of Staph. aureus 209-P were inoculated into each suspension and zinc concentrations, and the pH and bacterial viability were measured at the time intervals described above. By withdrawing 10 PI samples from the cultures, bacterial viability was measured on blood agar plates. Simultaneously 3 ml samples were recovered to analyse the pH and zinc concentration.
RESULTS Disc diffsion tests The zones of inhibition around tapes covered with zinc oxide (1.2 mg/cm2) on agar seeded with Staph. aureus 209-P were 10 mm in diameter and a higher concentration of zinc oxide (5.0 mg/cm2) resulted in a larger zone of inhibition (11.5 mm). Titanium dioxide and acetone had no antibacterial effect. No zones of inhibition were seen on any plate seeded with Ps. aeruginosa ( 2 strains), P . mirabilis ( 2 strains), K . pneumoniae ( 2 strains), or E . coli ( 2 strains). Agar dilution tests The MIC of zinc oxide dissolved in acetic acid on the bacterial strains tested are shown in Table I . Staph. epidermidis and Staph. aureus were the most susceptible bacteria, whereas most strains of enterococci, Ps. aeruginosa and P . mirabilis were resistant to the zinc concentrations tested. Inhibition of growth of streptococci, K . pneumoniae, and E . coli were noted only in the highest concentration tested. The measured zinc concentrations in the dilution series were as expected. Broth dilution tests there were no important differences in zinc concentrations between Miiller-Hinton broth containing no bacteria and broth inoculated with Staph. aureus 209-P, only one figure is shown (Fig. 1). As
Antibacterial effect of zinc oxide in vitro
195
i:m
-z 150
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5
100 15
g
U
z
5 0
A
A
0 1
4
z
z -
A
A
z 6
z 8
g
A
g
A
g
A
z
t
10
12
14
16
24
A
TIME (HOURS)
Fig. 1 . Zinc concentrations in filtrates of Muller-Hinton broth inoculated with lo6 CFU/ml of Staph. aureus 209-P. The following amounts of zinc oxide were added: 1790 pgiml (A),179 pgiml (m),17.9 pg/ml (0)and 1.79 pgiml (+). Controls (*). Mean values of 3 experiments (SEM not exceeding ?lo%).
can be seen, the dissolution process took about an hour after which there was a steady state condition for up to 24 hours. Furthermore, the zinc concentration in solution increased as increasing amounts of zinc oxide were added. The zinc concentrations in the broths containing 1.79 and 17.9 pg/ml of zinc oxide implied that the zinc oxide had been totally dissolved. The zinc concentrations in the broths containing 179 and 1790 pg/ml of zinc oxide were 100-110 pg/ml and 150-180 pg/ml, indicating that in these suspensions considerable amounts of zinc oxide still remained undissolved. The high concentrations of dissolved zinc were mainly due to the pH-
buffering effect of the Miiller-Hinton broth as well as its contents of potential complexing agents (for example, peptides) towards zinc. The increased solubility of zinc oxide in the broth was also reflected in the measured pH-values. A small increase in pH was noted as increasing amounts of the base zinc oxide were dissolved. In addition the pH of the different broth solutions remained constant after one hour's equilibration. The addition of bacteria to the broths caused slight increases in pH after 12 hours, finally reaching values of 7.9 in the controls and 7.8 in broths containing 1.79 and 17.9 pg/ml zinc in solution (no
Table I. MICs of zinc in Miiller-Hinton agar plates towards Staph. aureus (25 strains), Staph. epidermidis (24 strains), Strep. faecalis (18 strains), Strep. huemolyticus (group A-3 strains; group B-5 strains; group G-2 strains), Ps. aeruginosa (10 strains), P . mirabilis (14 strains), K . pnecrmoniae (12 struins), and E . coli (18 strains) MIC (pgirnl) Bacteria (No. of strains)
Staph. aureus (25) Staph. epidermidis (24) Sirep. faecalis (18) Strep. pyogenes (10) Ps. aeruginosa (10) P. mirabilis (14) K . pneumoniae (12) E. coli (18)
8
16
32
1
1
I 1 2 1
64
128
256
512
1 6 1
9 2
14
1
1
1
1
1 1 1
1024
> I 024
1
2 1 3 11 13
17 5 9 10 3
Scand J Plast Reconstr Hand Surg 24
Scand J Plast Surg Recontr Surg Hand Surg Downloaded from informahealthcare.com by University of Leeds on 09/15/13 For personal use only.
196 T . A . Soderberg et al.
t , , 0 1
4
6
8
10
12
14
16
24
TIME (HOURS)
Fig. 2. Bacterial counts (CFUlml) in Miiller-Hinton broth inoculated with lo6 CFUiml of Staph. aureus 209-P, containing 1790 pg/ml (A),179 pgiml (m), 17.9 yg/ml(O) and 1.79 pg/ml(+) of zinc oxide and controls (*). Mean values of 3 experiments (SEM not more than f0.5log" CFU).
undissolved zinc oxide). In these solutions there was no inhibition of bacterial growth (Fig. 2 ) . In the most concentrated zinc oxide suspensions, however, (179 and 1790 pg/ml of zinc oxide) a clear antibacterial effect could be seen (Fig. 2 ) . In these broths pH remained constant throughout the 24 hour period. DISCUSSION The results of the present study show that zinc oxide has an antibacterial effect, mainly on some Gram positive bacteria. When using the agar dilution method Staph. epidermidis and Staph. aureus were most susceptible, whereas most strains of enterococci, Ps. aeruginosa and P . mirabilis were resistant to the zinc concentrations tested. MICs of most streptococci, K . pneumoniae and E. coli were 512-1024 kg/ml, even though some strains were sensitive in lower concentrations. To evaluate the antibacterial effect of zinc oxide further, and to measure the zinc concentration in a filtrate of an overnight suspension of zinc oxide, the broth dilution method was used. The inhibitory effect on Staph. aureus 209-P using this method was similar to that seen when the agar dilution method (128 p.g/ml) was used. Bacterial growth was inhibited to the same extent in the two most concentrated zinc oxide suspensions, indicating that Scand J Plast Reconstr Hand Surg 24
the inhibitory concentration of zinc to Staph. aureus 209-P in Miiller-Hinton broth was lower than 100-110 p.g/ml. No inhibition of bacterial growth was seen in broths containing only dissolved zinc oxide (zinc concentration about 14 pg/ml), the inhibitory concentration of zinc must therefore have been higher. The inhibition of bacterial growth in the broth dilution system was thus related to the amount of dissolved zinc. During this experiment the pH remained constant when the two highest concentrations of zinc oxide were used. This might be explained by the pH-buffering capacity of the undissolved zinc oxide in these broths as well as by the reduced bacterial growth. The agar diffusion technique, applying tapes on agar surfaces, was assumed to reflect the clinical procedure for the treatment of wounds. The method is based on diffusion and thus the water solubilities of the substances studied are important. Even though zinc oxide is almost insoluble in water, inhibitory zones on agar plates were found around tapes impregnated with zinc oxide powder indicating that zinc was present in an inhibitory concentration in the agar outside the tapes. A slow release of zinc ions from zinc oxide in vitro as well as in vivo is dependent on many different physical and chemical factors-for example, pH, temperature, phosphates (lo), proteins, amino acids ( l l ) , and different combinations of these as seen in broths, serum,
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Antibacterial effect of zinc oxide in vitro
and wound exudates. The release of zinc ions from zinc oxide also depends on what substances have been mixed with it-for example, rosin, rubber, antioxidants in adhesive tapes, and periodontal dressings. These factors may explain the unexpectedly high zinc concentrations (22 mmol/l) that Hallmans (3) found beneath an adhesive zinc tape when treating burns in humans. There are several possible mechanisms for the antibacterial action of zinc. It has been suggested that zinc binds to the membranes of micro-organisms in a similar way as to mammalian cells. There are also several theories about how zinc affects bacteria when zinc binds to the membranes, and it has been observed that it modulates membrane associated enzymes, impairs calcium uptake, and changes the fluidity of the membrane (11). In conclusion we have shown that zinc oxide in Muller-Hinton broth and agar has antibacterial effects on staphylococci, some streptococci, and occasionally on Gram negative bacteria, although high concentrations of zinc oxide were needed to inhibit bacterial growth.
ACKNOWLEDGEMENTS This research was supported by grants from the Bo Rydin Foundation, SCA, Sweden. We gratefully acknowledge Miss K. Olofsson and Mrs I. Sjostrom for skilful technical assistance and Mr S. Granstrom and Mr R. Sjostrom for valuable advice and support.
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