Bactericidal Effectivities of Zinc, Copper, Silver, and Stainless Steel on Staphylococcus aureus.
Sean Loh, Natalie Karapetian, and Wei Gan.
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
With antibiotic resistant bacteria on the rise, alternative methods of sterilization, such as those utilizing theoligodynamic effect, are being researched with the aim of reducing the spread of disease without fostering antibiotic resistant bacteria, which is of particular importance to hospitals. The investigators predicted zinc would exhibit the highest level of antibacterial activity and that zinc, copper and silver would all be more antibacterial than stainless steel. Cultures of Staphylococcus aureus were grown on nutrient agar while exposed to samples of the four different metals for 24 hours (n=10). The zone of inhibition was measured to determine which metal was the most effective in inhibiting bacterial growth. Of the four metals tested, zinc (7.343 mm ± 0.232, ± SEM) was shown to statistically have a significant zone of inhibition (p=0.00, ANOVA; p<0.05, Bonferroni post-hoc) incomparison to stainless steel (0.166 mm ± 0.071,±SEM), copper (0.229 mm ± 0.075,±SEM), and silver(0.350 mm ± 0.010,±SEM). The investigators concluded that zinc was the only bactericidal metal due to the highly oxidative nature of zinc, thereby contributing to a strong oligodynamic effect.
Introduction
The primary objective of the proposed project is to determine which materials best inhibit or terminate Staphylococcus aureus growth via the oligodynamic effect. In consideration of the ease in which bacteria pass from surface to surface and the increasing commonality of antibiotic resistant bacteria, it is important to determine which types of surfaces are most effective in preventing bacterial contamination, especially for use in medical facilities. This knowledge will aid in the development of more sanitary surface options to be used in the construction of commonly touched surfaces such as door knobs, countertops, and bathroom fixtures to aid in the combat against the spread of bacterial diseases.
Certain metals exhibit bactericidal properties in low concentrations, known as the oligodynamic effect, via mechanisms of oxidation-reduction reactions and intracellular accumulation of ions (Kraemer, 1905 and Miyano et al., 2010). Metal ions are transported into the cell through the cell membrane, where the ions accumulate and become toxic to the cell by slowing or terminating cellular metabolism (Rentz et al., 2003 and Khalid et al., 2014). Additionally, oxidation-reduction reactions between metal ions and molecules in the bacterial cell walls can cause the metal ions to bind to the cellular proteins, rendering the protein non-functional and causing the cell wall to rupture (Rentz, 2003 and Miyano, 2010). While current understanding of the oligodynamic effect is primarily focused around research done on metallic solutions, the investigators of this project aim to lend insight into the bactericidal action of solid metals to be applied on fixtures such as doorknobs. Similar studies found copper and silver to exhibit high antibacterial properties, leading the investigators of this project to expect that copper and silver metals will be effective in inhibiting the growth of Staphylococcus aureus(Heidenauet al., 2005 and Miyano, 2010). However, the highly oxidative characteristics of zinc lead the project investigators to expect that zinc metal will exhibit the highest level of bactericidal activity while zinc, copper, and silver will all be more antibacterial than stainless steel (control).
Materials and Methods
Pieces of copper, silver, and zinc, and stainless steel were obtained. On 28 October 2015, ten pieces of each metal type were cut out to the dimensions 1.0 cm by 0.7 cm using tin snips and the surface of piece of metal was sanded with 220 grit sand paper and then autoclaved. Using aseptic technique, 150 µl of Staphylococcusaureus inoculum was then dispensed onto each of the 10 Petri dishes with a P200 micropipette and spread with a cotton swab to create a bacterial lawn on the surface of each plate of agar. One piece of each type of sterilized metal was then pressed into each corner of the agar with sterilized forceps. The lid was then replaced, inverted, and incubated in an air incubator at 37°C. After 24 hours of incubation, the Petri dishes were moved to a refrigerator. The zone of inhibition from the edge of each of the four sides of each metal piece was measured for all metal samples in all Petri dishes on 2 November 2015. An average zone of inhibition was then recorded for each of the 10 samples of each metal by averaging the zone of inhibition measurements for the 4 edges of each sample. Measurements for samples that had fallen from the agar during the incubation period were labeled “F” for fallen and the data for that sample was not used in calculations.
Results
An analysis of variance (ANOVA) was run on the measured average zone of inhibition for the metal samples of each metal type. According to the results of the ANOVA, the p-value was reported to be 0.00, thereby indicating a significant difference between the zones of inhibition for the different types of metal. The Bonferroni correction post-hoc test was run to reveal a p-value of less than 0.05 for all comparisons tests that included zinc but p-values greater than 0.05 for all comparisons that did not include zinc. This indicates that zinc was the only significantly antibacterial metal tested and thereby exhibited higher bactericidal activity than stainless steel (control), copper, and silver (Figure 1).
The average zone of inhibition for the Petri dishes containing Staphylococcus aureus and stainless steel (control) was 0.166 mm ± 0.071 (mean ± SEM, n=8), average zone of inhibition with zinc was 7.343 mm ± 0.232 (mean ± SEM, n=10), average zone of inhibition with copper was 0.229 mm ± 0.075 (mean ± SEM, n=7), and the average zone of inhibition with silver was 0.350 mm ± 0.010 (mean ± SEM, n=10).
Figure 1: Mean zone of inhibition (mm) for each type of metal tested. Mean values for stainless steel, zinc, copper and silver are 0.166 mm ± 0.071, 7.343 mm ± 0.232, 0.229 mm ± 0.075, and 0.350 mm ± 0.010, respectively. Analysis of variance (ANOVA) indicated p=0.00 and Bonferroni post-hoc test indicated p < 0.5 for zinc but p > 0.5 for all other metals tested, signifying zinc is the only significantly antibacterial metal, as indicated by the asterisks. Error bars are mean ± SEM.
Discussion
The results of our experiment were consistent with our hypothesis to a limited degree. Experimental data suggests that zinc is the most antibacterial metal of those tested, as predicted by our hypothesis. However the data produced by this experiment also suggests silver and copper lack the bactericidal properties found in other studies (Heidenauet al., 2005 and Miyano, 2010), as the results of this experiment did not indicate that copper and silver are any more antibacterial than stainless steel. The average measurement of the zone of inhibition of metals showed that zinc was had the greatest antimicrobial properties, which was theorized was due to zinc’s higher rate of oxidation when compared to the other metals being tested (Figure 1). It should be noted that while the results indicate that zinc has greater antibacterial properties than more common materials, there have been reported cases and research done on people who have allergies to zinc oxide. Reactions to zinc oxide range can anywhere from mild swelling at the point of contact to breathing difficulties (Hiroshi et al., 2006). However, such allergies are also extremely rare and are found in less than one percent of the population (Achouet al., 2010). The lower rates of oxidation of the other metals tested may have resulted is a weaker oligodynamic effect compared to zinc, as evidenced by the low p-value calculated.This is consistent with previous research that have shown easily oxidized metals to have greater antibacterial properties than more conventional materials used in construction (Kraemer, 1905 and Miyanoet al., 2010).
One of the fastest growing concerns in modern hospitals is how to contain the spread of bacteria and diseases in hospitals. With so many diseases in such close proximity, being able to minimize human exposure is a top priority. One of the ways that bacteria can travel is through contact with infected surfaces. The rise of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (Ayliffe, 1997) as well as the ease of transferring bacteria from surface to surface makes research into more effective antimicrobial surfaces highly important. The purpose of this experiment was to determine which of four different metallic compounds was the most effective at inhibiting microbial growth on its surface. The results of this research can result in more sanitary work conditions, particularly in places like hospitals.
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