The Effect of Common Bactericidal Agents on Staphylococcus Epidermidis

Introduction

Staphylococcus epidermidis is a facultative anaerobic bacterium. It is part of the normal human flora and is found on the skin. Colonies of these bacteria can produce a protective slime called a hydrophobic biofilm. Staphylococcus epidermidis is usually not pathogenic unless it enters the human body. One of the most common places for infections are hospitals where people often have weakened immune systems, open wounds or medical devices implanted in their bodies.

Staphylococcus epidermidis can be fatal because of the protective biofilm and the bacteria’s resistance to common antibiotics makes the infection difficult to treat. This stresses the importance for hospitals to prevent infection by keeping a sterile environment. There are many chemical agents that can be used to kill bacteria, but a hospital’s choices are limited by availability and cost of the agents. In this experiment the effectiveness of three common bactericidal agents, bleach, hydrogen peroxide, and isopropyl alcohol, were tested.

This was done by creating aqueous solutions of staphylococcus epidermidis bacteria and a chemical agent. The solutions were then incubated on an agar petri dish for 24 hours. The quantity of staphylococcus epidermidis bacteria colonies that were grown were used as an indicator of how effective each agent was in killing the bacteria. Each bactericidal agent has its own mechanism for killing bacteria. Both bleach and hydrogen peroxide are strong oxidizing agents, although hydrogen peroxide is a stronger oxidizing agent than hypochlorous acid, the oxidizing agent found in bleach.

Oxidizing agents are very reactive compounds that take electrons away from other molecules to obtain a more stable form. When the oxidizing agents react with the bacteria they create free radicals which react with the lipids of the cell membrane. This reaction changes the molecular composition of the cell membrane causing an important part of the cell to cease functioning. The damage done to the bacteria is amplified by the products produced by the reaction. As the lipids are oxidized, they produce more free radicals and highly reactive compounds like aldehydes.

These compounds along with the free radicals denature proteins and break apart DNA. The reaction results in cell death and destroys the bacteria’s ability to replicate. The isopropyl alcohol is less reactive than the oxidizing agents, but still has the potential to kill bacteria cells. When the bacteria are exposed to an alcohol/ water mixture, the lipids in the cell membrane become soluble in water. This causes the cell membrane to fall apart and allows the alcohol to enter the cell. Once inside the cell, the alcohol can denature the proteins.

Alcohol kills bacteria in a similar way as the oxidizing agents, by destroying the functionality of the cell membrane and proteins. However, isopropyl alcohol does not have the same damaging affect on DNA, nor does it have the ability to destroy bacterial spores like the strong oxidizing agents. In this experiment in order to accurately determine the most effective bactericidal agent, the solutions that were compared were prepared containing the same concentration of the different chemical agents.

The oxidizing agents are more reactive than the isopropyl alcohol , so if the reactivity(or the how easily the substance reacts with other substances)of the chemical agent determines the effectiveness in which the chemical agent in kills staphylococcus epidermidis, it was could be conjectured that the strong oxidative agents would be more effective than the isopropyl alcohol and the hydrogen peroxide would be the most effective bactericidal agent. Methods and Results

In order to determine which chemical agent is most effective in killing staphylococcus epidermidis, solutions were made with the same concentration per volume. The chemical agents used were bleach, hydrogen peroxide, and isopropyl. The original stock solutions of the chemical reagents were regular household grade. The solutions were prepared by first creating a chemical stock solution at the desired concentration. The first set of stock solutions was prepared with a concentration of 10% chemical agent per volume.

The stock solutions were then added to a solution containing aqueous staphylococcus epidermidis and the new solutions were diluted to a plated dilution of 1X10-5. The three solutions were mechanically mixed using the vortex for approximately 10 seconds. The mixed solutions were poured onto an agar petri dish and the solutions were distributed evenly by using glass beads. Once the glass beads were removed the solutions were incubated for twenty four hours. The bacteria colonies were then counted and data was collected.

The results showed that the strong oxidative solutions did not allow for the growth of bacterial colonies where the solution containing alcohol allowed for the growth of 264 colonies. Since there was no growth in either of the petri dishes containing hydrogen peroxide or bleach, it could not be concluded which, if either, chemical agent was the superior disinfectant. (See figure 1). In order to determine which strong oxidative agent was more effective, a second trial was run. The purpose of this second trial was to find an upper limit on the concentration where the bacteria would grow in the presence of bleach and hydrogen peroxide.

Also in this second trial additional isopropyl alcohol solutions were prepared in an attempt to find a concentration where the alcohol would prevent colony growth. In the second trial solutions were prepared in the same fashion as the first trial except the concentration of the chemical agent in the stock solutions were reduced to 1 % and 5% , with the same plated dilutions of 1X10-5. The additional alcohol solutions were prepared with a concentration of 15% and 20% per volume with a plated dilution of 1X10-5. The 5% solutions showed the same trend as the as the 10% solution.

The concentration of the bleach and hydrogen peroxide showed no colony growth while the 5% solution of isopropyl alcohol produced a little over three times the amount of bacteria colonies as it did in the 10% concentrated solution. The 1% bleach solution still showed no growth and the the petri dish containing the 1% solution of isopropyl alcohol produced 161 more colonies than it did in the 5% solution of alcohol (figure 1). During the 1% trial the hydrogen peroxide petri dish became contaminated and produced more colonies than could be counted.

In figure 1 the uncountable amount of colonies was represented by having the number of colonies reach the y- axis’ maximum value. The contaminated solution showed long oval shaped bacteria which was different from the bacteria in the other trials (picture 1 and picture 2). Picture 1: This is a picture of the contaminated 1% hydrogen peroxide solution at 1X10-5 plated dilution. There are long green oval shaped bacteria colonies in addition to the circular colonies. Picture 2: This is a picture of a noncontaminated solution of 15% isopropyl alcohol at a plated dilution of 1X10-5 .

All of colonies of staphylococcus epidermidis are round and white. The 15% and 20% solution produced about 864 colonies The increased concentration of the 15% and 20% isopropyl alcohol by volume produced the same amount of colonies as the 5% solution. Figure 1:The bar graphs above represents the number of staphylococcus epidermidis colonies produced in the presents of the different concentrations of the different bactericides hydrogen peroxide, bleach and isopropyl alcohol. Hydrogen peroxide and bleach did not allow for any colony growth and the amount of colonies increased with the decrease in concentration of isopropyl alcohol.

The hydrogen peroxide at 1% concentration represents an uncountable number of colonies which grew due to contamination in the solution. Discussion Based on the results shown in figure 1, both the hydrogen peroxide and bleach solutions (excluding the contaminated solution) were more effect bactericides than isopropyl alcohol. The alcohol solutions allowed for the growth of staphylococcus epidermidis colonies and the hydrogen peroxide and bleach solutions inhibitied colony growth by either completely killing all of the bacteria or significantly impeded the bacteria’s ability to reproduce during the 24 hour incubation period.

This supports the first part of the original hypothesis that the strong oxidative agents would be more effective bactericides than the isopropyl alcohol. However, the data is not completely conclusive that reactivity is an indicator for how effective a chemical agent will be at killing bacteria. The more reactive compounds were more effective , but there is not enough data to support a trend especially because both hydrogen peroxide and bleach solutions completely stopped colony growth so in this case there is no way to tell if the stronger oxidizing agent would kill more bacteria.

Other experiments have shown that bleach is actually more effective at killing bacteria including, staphylococcus epidermidis, than hydrogen peroxide. This however does mean that reactiveness does not play a role in the abilities of the bactericidal agent it just means that there are other factors involved. When an oxidative agent is denaturing a protein the bacteria has defense mechanism that can resist the change in conformation. The hypochlorous acid in bleach reacts faster than the hydrogen peroxide giving the bacteria less time to defend itself. Bleach also has the ability to cause important proteins in the bacterium to irreversibly aggregate together rendering them useless. Bleach is a more effective bactericidal agent than hydrogen peroxide because of its additional properties this does not mean the reactiveness is not an important factor in determining the strength of the bactericidal agent. There is also other evidence from other studies that supports the idea that more reactive compounds make better disinfecting agents. Iodine is another oxidative chemical agent commonly used to kill bacteria.

In an article published in The journal of Antimicrobial Chemotherapy it was said that iodine is a weaker bactericidal agent than hydrogen peroxide. This would support the link between the idea that more reactive a compound make better bactericidal agents because hydrogen peroxide is a stronger oxidative agent than iodine. The oxidative agents in this experiment are better at killing staphylococcus epidermidis, but this does not necessarily mean the results are related to reactivity of the chemical agent .

Recent studies have shown that alcoholic ingredients in skin disinfectants increase biofilm expression of staphylococcus epidermidis. This could explain why in trial two increasing the concentration of isopropyl alcohol from 10% to 15% and 20% caused an increase in the amount of bacteria colonies. Instead of the number of colonies decreasing with an increase of alcohol concentration as they did from 1% to 10%, it is possible the that there is a lower threshold where increasing the concentration of alcohol triggers a defense mechanism.

Since the alcohol cannot penetrate the hydrophobic biofilm the solution cannot react with the bacteria cells. This would mean that isopropyl alcohol is a less effective bactericidal because the defense mechanisms of this particular strand of bacteria is resistant to alcohol treatments and not because of alchol’s weaker reactive strength. The results of the experiment were relatively inconclusive due to time constrains which did not allow for further trials to refine the procedure and produce better results.

Before any serious suggestion about which common bactericidal agent hospitals should use to kill staphylococcus epidermidis certain parts of the experiment should be revisited and certain parts of the procedure should be revised. The contaminated solution of 1% hydrogen peroxide should be repeated to see if any bacteria colonies would grow. If colonies form in a 1% hydrogen peroxide solution it could be concluded that bleach is the most effective bactericide out of the three chemical agents.

If hydrogen peroxide does not produce any colonies then the experiment should be run again with even lower concentrations of the chemical agents in order to find the point where the strong oxidative agents do not kill off all the bacteria and colonies can form. It would also be important to repeat the whole experiment multiple times to get new sets of data for each set of chemical agent at all the concentrations. This would strengthen any trends the data shows. The procedure would also have to change in a way that would decrease the chances of the samples becoming contaminated.

One particular revision that would greatly decrease the chance of contamination is to cover the tops of the test tubes and beakers while the solutions are not in use to stop air born microbes from landing in the solutions. In the revised experiment additional chemical agent should be included. The chemicals should have different levels of reactiveness and different mechanisms to kill bacteria to provide a broader range in the data. This would produce more accurate trends in the data, and allow for a conclusion to be drawn about the effect that a chemical compound’s reactiveness has on the strength of the bactericidal agent.

Currently the data only supports using strong oxidizing agents to sanitize hospital equipment in order to reduce the risk of staphylococcus epidermidis infections. The conclusions from similar future experiments could be used to find or create new chemicals that could be more effective and readily available than current bactericides. References Cabiscol, Elisa, Jordi Tamarit, and Joaquim Ros. “Oxidative Stress in Bacteria and Protein Damage by Reactive Oxygen Species. ” INTERNATL MICROBIOL (2000). Print. Chaieb, Kamel, Tarek Zmantar, Yosra Souiden, Kacem Mahdouani, and Amina Bakhrouf. XTT Assay for Evaluating the Effect of Alcohols, Hydrogen Peroxide and Benzalkonium Chloride on Biofilm Formation of Staphylococcus Epidermidis. ” Microbial Pathogenesis 50 (2011). Print. Johannes K. -M. Knobloch, Matthias A. Horstkotte, Holger Rohde,Paul-Michael Kaulfers, and Dietrich Mack, Alcoholic ingredients in skin disinfectants increase biofilm expression of Staphylococcus epidermidis J. Antimicrob. Chemother. (2002) 49(4): 683-687 doi:10. 1093/jac/49. 4. 683 McDonnell, Gerald, and A. D. Russell. “Antiseptics and Disinfectants: Activity, Action, and Resistance. Clinical Microbiology Reviews 12 (1999): 147-79. Print. “Oxidizing and Reducing Agents. ” Purdue University College of Science Welcome. Web. 28 Apr. 2011. . Presterl, E. , M. Suchomel, M. Eder, S. Reichmann, A. Lassnigg, W. Graninger, and M. Rotter. “Effects of Alcohols, Povidone-iodine and Hydrogen Peroxide on Biofilms of Staphylococcus Epidermidis. ” Journal of Antimicrobial Chemotherapy 60. 2 (2007): 417-20. Print. Winter, J. , M. Ilbert, P. C. F Graf, D. O? Zcelik, and U. Jakob. “Bleach Activates a Redox-Regulated Chaperone by Oxidative Protein Unfolding. ” Cell (2008). Print.