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Home , Bactericide, Bacteriostatic agent, Serial dilution

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Introduction

The use of antibacterial agents has become commonplace in daily life in many countries. Numerous and antibacterial agents are now used extensively and are found in products ranging from cosmetics to surgical sutures to fabrics, with use expanding daily. One of the most commonly used antibacterial compounds is , which is frequently found in mouthwashes, cosmetics, and soaps. It is a concern that in some the use of triclosan may accelerate the development of resistant . Numerous studies have been conducted to determine the effects of triclosan on antibiotic resistance. In this study

I attempted to find Minimum Inhibitory (MIC) and Minimum

Bactericidal Concentration (MBC) values for the bacteria Escherichia coli,

Staphylococcus aureus, and Pseudomonas aeruginosa using the antibacterial compound triclosan.

An antibacterial agent is a general term for a compound that affects growth and viability of bacteria. are a subset of antibacterial agents, which are produced by other microorganisms. An antibacterial agent works specifically against bacteria. Triclosan acts as an antibacterial agent, slowing of the growth of or, if the concentration is high enough, killing the bacteria present and acting as a bactericidal agent. Antibiotics are widely used in the medical field. Antibacterial agents are frequently prescribed to treat bacterial illnesses and to sanitize surfaces in health care practices.

Concerns over triclosan use are widespread, and range from concerns over the impact on hormone regulation, to environmental concerns, including the 3 perceived risk of selecting for antibiotic resistance [5]. For those in the medical field, alarm has been raised about the phenomenon in which some bacteria that develop resistance to triclosan also develop resistance to antibiotic agents. The FDA published an article entitled “Triclosan: What Consumers Should Know” in 2010. At a superficial level, the article assured American consumers that there was nothing to fear from the use of triclosan in their household products. Yes, animal-testing studies had shown that triclosan alters hormone regulation. No, the agency did not have evidence that using triclosan provided an advantage in antibacterial activity over using regular soap and water. The article did not mention antibiotic resistance in any form. The FDA was correct; triclosan has not been proven to be directly harmful to human health. However, there are additional concerns surrounding antibacterial agent use that may not have entered public awareness yet.

Antibiotic resistance is a naturally occurring phenomenon as species adapt to the use of antibiotics over time. However, antibiotic resistance can be accelerated in some cases. For instance, we could devise a hypothetical situation in which George, an average man, has a severe cold. George goes to a hospital, and even though the cold is viral, he is prescribed an antibiotic regimen to take. George, not knowing that antibiotics have no effect on viruses, takes the prescribed antibiotics. The antibiotics do their job and kill bacteria in George’s body. But maybe George doesn’t complete the antibiotic regimen as instructed, resulting in only the weakest, most susceptible bacteria being killed off. This not only leaves the more resistant organisms alive and well in George’s body, it eliminates any competition that they may experience, 4 leaving them ample room and resources for growth. Now, instead of a just a cold,

George has a large population of antibiotic resistant bacteria living in his body.

Why should we care about hypothetical George and his antibiotic resistant bacteria? Well, maybe George feels better and goes back home, out into the community, and starts spending time with family and friends. As he interacts with people, he spreads the antibiotic resistant bacteria, not knowing what he carries. As more people fall ill with the , they go to a hospital for treatment. Instead of the doctor being able to easily prescribe an antibiotic to kill the offending bacteria and treat the illness, the medical staff must now struggle to find an antibiotic that is effective against this evolved resistant bacteria, increasing the cost and length of the hospital stay.

A Brief History of Triclosan

The chemical triclosan has been in use for several decades. Originally introduced to the marketplace as a in 1969, use of the chemical quickly expanded to use as a surgical scrub in the 1970’s and is currently used as an antibacterial and antifungal agent in cosmetics, soaps, fabrics and textiles, as well as in cleaning and sterilization of surgical equipment [27]. Triclosan is also known by its chemical name, 2,4’-trichloro-2’-hydroxydiphenyl ether, and is a lipophilic and phenolic compound [2]. A member of the bisphenol family, triclosan belongs to a class of chemicals that possess a wide range of antibiotic activity. In fact, triclosan has grown to be the most common bisphenol is use over the past 30 years [3]. 5

Figure 1: Chemical structure of Triclosan

With a molecular weight of 289.5, the white crystalline powder has a pKa of 8.14.

Triclosan does not readily dissolve in water, with a of 0.001 to 0.004 grams triclosan per 100 g of water, which can make it a challenging chemical to work with. In most studies, triclosan is dissolved in acetone, , or isopropanol

(Table 1) [26].

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Table 1: Solubility of triclosan in selected and chemicals

Solvent Solubility at 25 degrees Celsius (g triclosan / 100 g ) Distilled water (20 C) 0.001

Distilled water (50 C) 0.004

1 N caustic soda 31.7

1 N sodium 0.40 carbonate 1 N ammonium 0.30 hydroxide Triethanolamine >100

Acetone >100

Ethanol 70% or 95% >100

Isopropanol >100

Propylene glycol >100

Polyethylene glycol >100

Dipropylene glycol ~40

Glycerine 0.15

N-Hexane 8.5

Petroleum jelly ~0.5 (white, USP) Olive oil ~60

Castor oil ~90

Antibiotic resistance is an increasing problem for the medical community and those concerned about public health. As the number of resistant bacteria 7 increases, so does the need for greater understanding of resistance, how it develops over time, and the mechanisms through which organisms develop resistance.

Resistance is a naturally occurring process and a form of natural selection, but there are factors that can affect the rate at which resistance develops. Studies concerning the incidence of antibiotic resistance show a trend toward increasing frequency of antibiotic resistance in bacteria. Diagnoses of antibiotic resistant bacterial strains also appear to be on the rise (8).

Review Aim

The goal of this literature review is to give a clear picture of the issues concerning triclosan use. Discussion will encompass general public awareness, the uses of triclosan, mechanisms of resistance, and factors that influence the development and spread of the associated antibiotic resistance. At the conclusion of this literature review, the reader should have a clear understanding of the healthcare and antibiotic resistance implications of triclosan use.

Triclosan

As a synthetic, non-ionic antimicrobial agent, triclosan was thought to act on bacteria non-specifically in a biocidal role through interference with cell membrane function [16]. Until 1998, this was assumed to be the case. In studies conducted independently by McMurry, McDermott, and Levy as well as Heath, Yu, Shapiro,

Olsen, and Rock, triclosan was shown to act on a specific target in the biosynthetic 8 pathway involved with fatty synthesis [15] [14]. Triclosan acts as a pseudo substrate in a directed manner on a specific site acting as an inhibitor of enoyl-acyl carrier reductase (also referred to as ACP reductase, or the ENR enzyme)

[14]. More specifically, triclosan acts by altering the NADH and NADH-dependent

ACP reductase, causing the inhibition of fatty acid synthesis in bacteria [20].

Realization of the mechanism allows clarification of the earlier beliefs of how triclosan works on bacterial cell walls. It has been shown that triclosan affects membrane structure and functions, but with further study, it was shown that this was a secondary effect due to the inhibition of biosynthesis of the fatty that make up the membranes.

At low levels, triclosan acts as a . As the concentration increases, the chemical becomes a bactericidal agent [21]. These varying concentrations depend largely on the characteristics of the bacteria exposed to triclosan. When working below lethal concentrations, triclosan is thought to work by inhibiting the critical enzyme in fatty acid synthesis, enoyl-acyl carrier protein reductase (ACP reductase). Enoyl-acyl carrier protein (ACP) reductase is the product of the fabI gene. At higher concentrations, triclosan acts as a through multiple proposed nonspecific mechanisms thought to interfere further with the cellular membrane integrity [13].

When biocide agents act on their target bacteria, their depends on a variety of factors both extrinsic and intrinsic. The intrinsic and extrinsic factors proposed to induce cross-resistance are listed in the following table. Scientists also know that biocides act in a nonspecific manner on multiple targets in the cell [21]. 9

Because of this nonspecific mode of attack, it can be reasonably assumed that bacteria that have developed resistance to a biocide may have developed a cellular system for lowering intracellular concentrations of the chemical to tolerable, non- toxic levels [21]. The proposed mechanisms of resistance ranges from changes in membrane permeability to changes in the cellular envelope to efflux and degradation of the substrate, functioning to impede the efficacy of triclosan in the cell.

Table 2: Bacterial mechanisms inducing potential cross-resistance [21]

Mechanism Nature Level of Cross- susceptibility to resistance biocides reported Change in Intrinsic/acquired No Yes bacterial envelope Expression of Intrinsic/acquired Reduced Yes efflux pumps Enzymatic Intrinsic/acquired Reduced No modification Target Site Acquired Reduced No Mutation Phenotypic Following Reduced Yes Change exposure

Of the mechanisms listed and described in Table 2 and Table 3, changes in enoyl acyl carrier reductase, efflux, and by-pass of metabolic blockage are the most likely contributors in most cases of antibacterial resistance to triclosan. Changes in enoyl acyl carrier reductase, an enzyme necessary for fatty acid synthesis in bacteria, play a role in resistance to sub-lethal concentrations of triclosan resistance. 10

[21]. Studies have shown that at low concentrations, triclosan acts to inhibit bacterial fatty acid synthesis through interference with fabI, a gene necessary for membrane homeostasis, specifically in Escherichia coli and Staphylococcus aureus.

Mutations in fabI serve to lower the ability of triclosan to interact with the ENR-

NAD+ binding, which in turn causes a decrease in minimum inhibitory concentration

(MIC) of triclosan [21].

Other mechanisms of note are those that enable the bacteria to bypass any metabolic pathway blockage. The prime example of bypassing metabolic pathways inhibited by the presence of triclosan is found in Salmonella enterica. S. enterica has a set of 9 frequently altered that are involved in pyruvate and fatty acid synthesis. These proteins are able to form an alternative pathway for creation of fatty acids that functions in the presence of triclosan, allowing the organism to survive conditions that would normally result in lethality [30]. Another example of organisms possessing mutations that serve to help triclosan resistant strains survive is S. aureus. S. aureus is able to modify the lipid composition of the cellular membrane, which is associated with altered gene expression of the genes involved in of fatty acids [25]. When bacteria are able to bypass the metabolic impairments caused by triclosan to survive and reproduce successfully, the bacteria are considered resistant to the chemical.

The final mechanism of note, an efflux pump, uses triclosan as a substrate.

Bacteria that contain the genes for active efflux pumps are able to use active transport to “pump” materials out of the cell almost as quickly as they enter, preventing fatal buildup of from biocides and other chemicals. Specifically, 11 studies show that triclosan acts as a substrate of the AcrAB efflux pump naturally occurring in several E. coli species. This pump is similar to pumps seen in other

Gram-negative bacteria, which indicates triclosan may show lower efficacy in these bacteria as well [21]. Bacteria that have developed these efflux pumps show a decreased susceptibility to several chemical agents, as this defense is non-specific, giving them broad-spectrum resistance to many antibacterial agents. The effect of efflux pumps in antibiotic and antibacterial resistance bacteria is considered the largest contributing factor to the development of resistance.

Table 3: Mechanisms of Bacterial Resistance to Triclosan at the Cellular Level [21]

Mechanisms Effects Changes in Cell Lowering concentration that reaches Permeability target sites Gram negative outer membranes -Protein porins -Fatty Acid Changes in surface Lowering binding and interaction of the properties biocide and the cell surface

Efflux mechanisms Lower cellular concentration of biocide

Enzymatic Lowers intracellular and exocellular modification concentrations of the biocide Target mutation FabI gene mutation

Bypass Metabolic Raising pyruvate synthesis using altered Blockage metabolic pathways

Efflux Pumps

Up regulation and adaptation to existing efflux pumps are known strategies of resistance to triclosan [20]. Current research shows that resistance involves cells 12 acquiring the genes to create an efflux pump. These genes are usually transferred from other bacteria through transformation or conjugation. Efflux pumps are used throughout for transporting a variety of substrates, from inorganic ions to xenobiotic and chemotherapeutic [11]. There are five main families of efflux pumps; the major facilitators (MFS), ATP-binding cassettes (ABC), small multidrug resistance (SMR), resistance-nodulation-cell division (RND), and the multi antimicrobial extrusion protein (MATE) families [19]. The ABC efflux pump family is most extensively studied in the context of antibiotic resistance. Efflux pumps are highly conserved across organisms, and are found in eukaryotes and prokaryotes.

Coded for by highly specific genes, ABC efflux pumps are naturally present in many bacterial cells, but not always active. The gene for function can be turned on by a variety of situations, including the presence of a useable substrate like triclosan

[19].

Proposed Mechanism

In bacteria, triclosan acts to inhibit fatty acid production and elongation by competitively inhibiting the active site of the bacterial enzyme enoyl-acyl carrier protein reductase (ENR). By preventing the enzyme from binding, the process is significantly slowed as the triclosan molecules bind to the ENR active sites. The more active sites the triclosan binds to, the greater the negative effect on the cell wall of the bacteria. By stopping the production of fatty acids, the bacteria cannot produce this essential component of their cell wall, and lethality occurs. 13

The actual process of cell wall synthesis requires multiple enzymes to carry out to completion. Specifically, in E. coli, the fabI gene encodes for the ENR enzyme.

Triclosan acts in E. coli to competitively inhibit the active site of the ENR enzyme and create a complex with fabI and NAD+. The complex is the mechanism by which triclosan prevents the continuation of fatty acid synthesis and stops elongation of the fatty acid chains, weakening the integrity of the cellular membrane and leading to cell lysis (41). Humans do not have the ENR enzyme, though the process of triclosan inhibition is analogous in eukaryotic and bacterial cells.

The basic components of an ABC efflux pump are a dimer of dimers, or two identical molecules linked together with another set of two identical molecules. In this case, two transmembrane substrate-binding domains form a channel and two

ATP-binding cassettes bind and hydrolyze ATP to provide energy for the efflux function [11]. The Switch model is considered to be the most accurate, and is applied to other antibiotic resistance research concerning ABC efflux pumps. In

Figure 2, the Switch model is illustrated.

In the endoplasmic reticulum of eukaryotic cells, embedded TAP1 and TAP2 proteins, which are dimer components of the ABC efflux pump, form a channel. As the TAP nucleotide binding domains (NBDs) are open, the peptide-binding cavity is accessible to the cytosol of the cell. When peptides bond, a conformational change occurs, leading to the hydrolysis of ATP into ADP using TAP2. This allows for the

NBDs to dimerize, or combine with a similar molecule, and open the peptide-binding cavity to the endoplasmic reticulum lumen, allowing the peptide to be released. The

ATP hydrolysis deteriorates the bonds separating the NBDs, making further 14 hydrolysis unnecessary and letting TAP1 and TAP2 return to their resting position

[17]. ATP-binding cassette (ABC) systems are widely distributed in many organisms, and have a remarkably similar function in bacterial . The mechanism of bacterial efflux pumps is very similar to those of eukaryotic cell models (43).

Figure 2: Switch Model Mechanism of ABC efflux pumps [17]

Passing on Transfer of Resistance

Some of the genes for these mechanisms can be transferred from bacterium to bacterium horizontally; in fact is the leading cause of spreading antibiotic resistance, second only to mutagenesis of the efflux genes with selection of the new function. There are three main mechanisms for horizontal gene transfer, transformation, transduction, and conjugation. Transformation involves uptake of genetic material from the environment and incorporation into the bacterial genome. In transduction, a virus moves genetic material from one bacterium to another. Conjugation uses plasmids to transfer genetic material from a donor bacterium to a recipient during direct contact of the cells. Depending on the 15 concentration of triclosan, the horizontal gene transfer of resistance is increased or decreased. As concentration of triclosan increases, so does cellular membrane damage, lowering the competency of the cell to uptake new genetic material [18].

Perceived Gaps in Knowledge and Research

Despite the volume of research carried out, there remain substantial gaps in knowledge and scholarship. To begin with, the incidence of triclosan use at sub- lethal concentrations causing antibiotic resistance has only been significantly seen in select bacterial genera. In furthering this, while there is in vitro evidence detailing triclosan and its effect on emerging antibiotic resistance, there is a lack of adequate studies performed detailing the importance of this phenomenon from a public health standpoint. While antibiotic resistance may develop and be observed in these studies, what is the significance of these findings for the general public? Finally, there is also a significant lack of information concerning the maintenance and transferability of the resistance and determining the pathogenicity of resistant bacteria [21]. The lack of scientific information calls into question the validity of public concern over triclosan’s influence in creating antibiotic-resistant bacteria. As a result of completing this literature review, several research questions have been formed. I would like to further my research on the subject by looking into the different concentrations of triclosan and how they affect the development of resistance on different strains of common bacteria. To this end, I will determine the

MIC and MBC of triclosan when testing Escherichia coli, Staphylococcus aureus, and 16

Pseudomonas aeruginosa. To ensure that the methods are sound, I will also test the methods using ampicillin, a common antibiotic.

Ampicillin

Figure 3: Ampicillin structure

Ampicillin is a common antibiotic that is listed on the World Health

Organization’s list of Essential (36). Introduced to the medical market in

1961, ampicillin is a member of the family, which is a group of beta-lactam antibiotics. With a molecular weight of 349.4 grams, ampicillin is commonly seen as a white crystalline powder or as white needle-like crystals that are odorless.

Ampicillin dissolves easily in water, and is practically insoluble in ether and benzene

(35). Ampicillin is taken up by and can act upon both gram positive and gram- negative bacteria by binding to penicillin-binding proteins that are essential components of many bacterial cell walls (44). In particular, ampicillin is known to have in vitro activity against gram-positive and gram-negative aerobic as well as anaerobic bacteria. However, ampicillin has no effect on existing bacterial cell walls, 17 therefore, bacteria must be multiplying and reproducing for the ampicillin to have a bactericidal affect (35). By interfering with the cell wall process, ampicillin compromises the bacterial cell wall and causes cell lysis. Because of its mechanism of action, ampicillin is used for a wide variety of infectious diseases.

Bacteria Utilized

Escherichia coli is a common gram-negative bacterium that is frequently found as a natural and normal component of the human gastrointestinal tract. Under normal circumstances, E. coli works in the human gastrointestinal tract to aid in food breakdown and absorption throughout digestion (34). In abnormal conditions,

E. coli can cause gastrointestinal issues, such as intestinal cramps, vomiting, and diarrhea. A facultative rod-shaped bacteria, E. coli grows best at 37 degrees Celsius, close to body temperature. The bacterium is frequently used in the lab as it has a rapid growth rate and does not sporulate, enabling easy sterilization. Consisting of a single circular chromosome and often a circular plasmid, the genome of E. coli has been completely sequenced. The rod-shaped E. coli have fimbriae that extend from a cell wall containing an outer membrane of , a peptidoglycan layer, and a cytoplasmic membrane on the inside (37). Escherichia coli was selected as a bacterium for this study to serve as a classic example of gram-negative bacteria.

Staphylococcus aureus is a gram-positive bacterium that forms grape-like groups that divide in two planes, and have characteristic yellow colonies when grown on plates which gives the bacterium its name. The gram-positive characteristic means that the bacterium has a thick layer of peptidoglycan within its 18 cell walls. S. aureus is a facultative anaerobe that uses aerobic respiration or fermentation for metabolic processes (37). Staphylococcus aureus is commonly heard during discussions concerning “super-bugs”, specifically considering MRSA, or Methycillin-Resistant Staphylococcus aureus. Staphylococcus aureus is considered to be one of the most common hospital associated (39). As a result, S. aureus is a common cause of skin infections and wound complications, such as secondary infections. Staphylococcus aureus was selected for this study to serve as a characteristic gram-positive bacteria that has an associated with antibiotic-resistant bacteria.

Pseudomonas aeruginosa is a gram-negative rod with single flagella. P. aeruginosa is an obligate aerobe, preferring to perform respiration for metabolism and forms characteristic pellicles when grown in test tubes. P. aeruginosa is able to catabolize many different organic molecules giving a flexible metabolism (37). P. aeruginosa does not normally infect human hosts, as it is largely a soil organism.

Infections by this bacterium are most likely to be observed in immunocompromised patients. P. aeruginosa is known for its characteristic blue-green growth, and has a tendency to form biofilms that are difficult to eliminate (38). Pseudomonas aeruginosa was selected for this study because of its flexible metabolism, which poses a challenge to antibiotic agents.

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Materials and Methods

Six tests were carried out to determine the minimum inhibitory concentration (MIC) of triclosan. BHI agar, or brain heart infusion agar, was used in each test. Plates containing E. coli, S. aureus, and P. aeruginosa were streaked for isolation and allowed to incubate overnight, then used to make overnight suspensions in BHI medium.

Test 1: Serial of triclosan using ethanol as a solvent:

1 gram of triclosan in powder form was dissolved in 10 ml of ethanol to give a 10% triclosan stock . Test tubes containing 5 ml of BHI agar were inoculated with 100 microliters of the chosen bacteria, with 7 tubes containing E. coli, 7 with S. aureus, and 7 with P. aeruginosa. The bacterial strains were then exposed to varying concentrations of the triclosan, beginning with .1% and increasing to .5% in .1% increments. Two control tubes were preserved, one with

5ml BHI medium and ethanol, the other with BHI medium and sterile water. The tubes were then incubated at 37 degrees Celsius overnight.

Test 2: Disc diffusion assay of triclosan using ethanol as a solvent:

100 microliters of E. coli, S. aureus, and P. aeruginosa were evenly spread on three BHI plates. A sterile disc was soaked in ethanol and placed on each of the plates. A second sterile disc was soaked in the stock solution of 10% triclosan in ethanol and placed on each plate. The plates were then incubated overnight at 37 degrees Celsius. 20

Test 3: Disc diffusion assay of triclosan using acetone as a solvent:

1 gram of triclosan was dissolved in 10 ml of acetone to create a stock solution of 10% triclosan in acetone. 100 microliters of E. coli, S. aureus, and P. aeruginosa were evenly spread on three BHI plates. A sterile disc was soaked in acetone and placed on each of the plates. A second sterile disc was soaked in the stock solution of 10% triclosan in acetone and placed on each plate. The plates were then incubated overnight at 37 degrees Celsius.

Test 4: Disc diffusion assay of ampicillin using sterile water as a solvent:

A stock solution of ampicillin was prepared at a concentration of 100 mg/ml in sterile water. 100 microliters of E. coli, S. aureus, and P. aeruginosa were evenly spread on three BHI plates. A sterile disc was soaked in sterile water and placed on each of the plates. A second sterile disc was soaked in the ampicillin stock solution and placed on each plate. The plates were then incubated overnight at 37 degrees

Celsius.

Test 5: Serial dilution of triclosan using acetone as a solvent:

Test tubes containing 5 ml of BHI agar were inoculated with 100 microliters of the chosen bacteria, with 6 tubes containing E. coli. The bacterial strains were then exposed to varying concentrations of the triclosan, beginning with .1% and increasing to .5% in .1% increments. A control tube of E. coli was preserved with 21

5ml BHI medium and acetone. The tubes were then incubated at 37 degrees Celsius overnight. The tubes were sampled and re-plated post incubation (Figure 6).

Test 6: Serial dilution of ampicillin using sterile water as a solvent:

Test tubes containing 5ml of BHI agar were inoculated with 100 microliters of the chosen bacteria E. coli, with 6 tubes containing the chosen E. coli. The bacterial strains were then exposed to varying concentrations of the ampicillin, beginning with 0.0 and ranging to 10 mcg/ml, increasing at 1 mcg/ml increments. A control tube was preserved containing only 5 ml of the BHI medium and sterile water.

Results

Test 1: Serial dilution of triclosan using ethanol as a solvent:

Immediately upon adding the triclosan-ethanol solution, turbidity was observed. The medium bubbled and became cloudy. Post incubation period, growth was observed at all concentrations of triclosan. The triclosan failed to show antibacterial activity or act as a growth deterrent agent in any of the tubes at any of the tested concentrations.

Test 2: Disc diffusion assay of triclosan using ethanol as a solvent:

The zones of inhibition observed on these plates varied widely (Figure 4). On the E. coli plate, a clear zone of inhibition was observed around the triclosan-ethanol disc. Bacterial growth was observed all around the ethanol disc, with no 22 interruption or inhibition observed. The P. aeruginosa plate showed lysing of the cells surrounding the triclosan-ethanol disc, in a smaller and more symmetrical area than the E. coli and S. aereus plates. Again, no inhibition was observed around the ethanol disc. On the S. aureus disc, the zone of inhibition around the triclosan- ethanol disc reached to the disc containing ethanol alone, much larger and more pronounced than the E. coli zone. The disc containing ethanol alone showed to inhibition of bacterial growth, with S. aureus growth present all around the disc.

Figure 4: Zones of Inhibition for E. coli, P. aeruginosa, and S. aureus when exposed to

Triclosan in Ethanol Solution

Test 3: Disc diffusion assay of triclosan using acetone as a solvent:

Two of the three tested bacteria had large zones of inhibition when exposed to the triclosan-acetone solution (Figure 5). On the E. coli plate, the observed zone of inhibition large and encompassed both the disc containing triclosan-acetone and acetone alone. Zones of inhibition were not observed on the P. aeruginosa plate around the acetone-soaked disc. Around the triclosan-acetone disc, a small area of lysed cells was observed. On the S. aureus plate, the zone of inhibition 23 was observed to encompass almost the entire plate, including the area near the acetone-soaked disc.

Figure 5: Zones of Inhibition for E. coli, P. aeruginosa, and S. aureus when exposed to

Triclosan in Acetone Solution

Test 4: Disc diffusion assay of ampicillin using sterile water as a solvent:

A large range of inhibition was observed on the plates for this assay (Figure

6). On the E. coli plate, a symmetrical and clearly defined zone of inhibition was observed around the ampicillin-soaked disc, with no inhibition observed around the sterile water disc. The plate containing P. aeruginosa showed a very small zone of inhibition around the ampicillin disc, with no visible inhibition around the sterile water disc. On the S. aureus plate, a large zone of inhibition was observed surrounding the ampicillin disc, touching the disc soaked in sterile water alone.

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Figure 6: Zones of Inhibition for E. coli, P. aeruginosa, and S. aureus when exposed to

Ampicillin in Sterile Water Solution

Test 5: Serial dilution of triclosan using acetone as a solvent:

As the triclosan-acetone solution was added to the tubes, turbidity was observed. Turbidity increased with the increased concentration of triclosan. The turbidity impeded visual reading of bacterial growth. 100 microliters of bacteria were taken from each of the test tubes and re-plated on LB plates to measure the growth of each concentration (Figure 8). Interestingly, no colonies were observed growing at the .1% concentration, and varying levels of growth were observed on plates with the .2% and .3% triclosan in acetone concentrations. Very small colonies were observed growing on the plate containing .4% triclosan in acetone. No colonies were observed growing on the plate with .5% triclosan in acetone concentration.

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Figure 8: Plate growth following serial dilution test of triclosan in acetone solution

for .1%, .2%, .3%, .4%, and .5% concentrations.

Test 6: Serial dilution of ampicillin using sterile water as a solvent:

When examining the tubes, there was a clear difference in cloudiness between the tube containing 5 and the tube containing 6, indicating a cessation of bacterial growth in tube 5 (Figure 9). 26

Figure 9: Serial dilution of ampicillin in sterile water solution in decreasing

concentrations from left to right.

Discussion

It is unclear why the addition of the triclosan to the BHI medium caused such a violent and turbid reaction. It was expected that the initial test, test 1, would yield a definitive cessation of growth in one of the tubes, enabling a MIC determination.

The results of each experiment involving triclosan were consistently inconsistent with expected outcomes from literature. The disc assays performed showed that triclosan does act as an antimicrobial in varying ways on each of the tested bacteria, which made the results of the multiple dilution assays all the more unexpected. I found it most interesting that in both serial dilution assays, using the acetone as the solvent and ethanol as the solvent, yielded such similar results visually. The development of turbidity in both of these tests was unexpected and had not been encountered in the literature. 27

When the contents of the triclosan-acetone assay were plated to examine growth, the results were equally confounding. The plate with the lowest concentration of triclosan and the plate with the highest concentration saw no growth of bacterial colonies, while the .2%, .3%, and .4% concentrations saw bacterial growth. Growth on the lower concentrations decreasing as concentration increased was the expected result for each of the assays performed.

A possible explanation for the results of the assays performed could be the formation of micelles around the triclosan in solution. A micelle is a lipid molecule that forms in a sphere when exposed to . The unique shape serves to reduce entropy, and has the added affect of sequestering molecules of substances within the micelle. Molecules can aggregate and form micelle structures that can lead to poor outcomes with the use of triclosan and other antimicrobial agents.

Molecules of triclosan can be sequestered inside these micelles and be effectively inactivated (40). If micelles did form in the test tube , this would explain the lack of antimicrobial effects of triclosan, and would allow for growth at all concentration of triclosan, as observed in the tubes.

Another possible explanation of the unusual results could be the difficulty and challenges associated with the solubility of triclosan, as seen in Table 1.

Choosing a solvent for the triclosan that would have a minimal effect, if any effect at all, on bacterial growth was a challenge for this experiment. The use of controls throughout testing ruled out the possibility of the bacteria being killed or otherwise affected by the choice of solvents. One of the reasons triclosan is so challenging to work with is its high hydrophobicity. The presence of the aromatic –OH group, 28 which is ionizable at pH values above 10, allows for better solubility of the molecule in alkaline conditions. However, alkaline conditions are not compatible with the majority of pharmaceutical applications (42). The acetone solvent is polar aprotic, and is frequently used as a solvent in pharmaceutical engineering. The 95% ethanol used as a solvent has a hydroxyl group, making it very polar from the large electronegativity of the molecule. Both of these chemicals allow for great solubility of triclosan, and were tested as controls to confirm they have no effect on bacterial viability.

Pairing the triclosan tests with assays using ampicillin allowed for verification of the efficacy of testing methods. The results of the ampicillin assays were consistent and within expected ranges. This confirms that the protocol and procedure for the experiment were viable and could be used to determine the MIC of bacteria tested.

Trends in Current Research

As interest in triclosan grows, so do the number and variants of studies conducted. Recently, many have concerned the growth of particularly resistant bacteria when exposed to triclosan, as well as on the potential uses of triclosan in cancer prevention and treatment. Several articles have been published detailing the use of triclosan as a potential option to treat MRSA, Methicillin-Resistant

Staphylococcus aureus, a bacterium that is renowned for difficulty to eliminate (32,

33). Concerns have also arisen over the accumulation of triclosan in the environment, with studies being conducted to determine accrual in the 29 groundwater sources, as well as concerns over triclosan levels found in human breast milk (28, 2).

Antibiotic resistance remains a hot topic within the scientific community, as those working in health care fields gain understanding of mechanisms of action, and the general public gains awareness of issues associated with the widespread use of antibiotics. Looking ahead, it is likely that triclosan will be removed from many more products before this controversy is decided. Already, companies like Proctor &

Gamble and Colgate, as well as hospital chains in California have banned the use of triclosan in their products.

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Works Consulted

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