Triclosan exposure and antibiotic resistance gene transfer efficiency

Efficiency of Antibiotic Resistance Gene Transfer Mechanisms Upon Exposure to Triclosan

Triclosan has become the latest buzz word in the grocery store. It is being hailed as the ultimate biocide and finds its way into many everyday products such as toothpaste and hand soap. Mass media produced a great amount of hype and convinced the general public that this was necessary to protect them from potentially harmful or even fatal bacteria. Now the use of antibacterial products is being widely questioned by the medical community as it is now known that bacteria can develop resistance to antibacterial agents and that we may be producing a type of super-bacteria. The proposed research will explore the efficiency of antibiotic gene transfer mechanisms upon exposure to triclosan. It is expected that the research will empirically demonstrate that exposure to triclosan significantly increases rate and efficiency of antibiotic gene transfer mechanisms.

Efficiency Of Antibiotic Resistance Gene Transfer Mechanisms Upon Exposure To Triclosan

Introduction

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is a broad-spectrum antibacterial agent which targets the cytoplasmic membrane of both Gram-negative and Gram-positive microorganisms. Tricloson has found its way into many common products on the grocery store shelf. For many years, mass media promoted the use of antibacterial products in everything from toothpaste to toilet bowl cleaners. Now the use of these products has sparked one of the hottest controversies in the scientific world in many years [3] We know that bacteria have evolved to adjust to environmental changes, just as any other living organism that has managed to survive on earth. The ability to adapt to change is the key to the survival of a species. Bacteria are particularly good at this adaptation process. They have evolved to adapt to antibacterials in the environment [3]. This ability to adapt has caused many to fear the by the excessive use of antibacterial products we are promoting the evolution of increasingly resistant bacteria and that this will lead to the evolution of "superbacteria" that could cause an epidemic in the human population.

Rationale for Study

Humans live in constant contact with microbes, the vast majority of which do not cause disease. Pathogenic commensal bacteria have frequent contact with bacteria from many sources found in nature. These commensal bacteria, which often provide a benefit to the host, can serve as reservoirs for resistance genes. Collecting them and holding them for future transmission of other organisms [19]. Ultimately, one of the recipients for this genetic largesse can be a disease causing bacterium.

Bacteria in every environment are constantly evolving, aided in part by the exchange of genetic material. Evidence is growing that extensive horizontal transfer of antibiotic resistance genes occur in nature between clinical and nonclinical bacteria [23]. Hence the commensal reservoir bacteria may be important players in the spread of antibiotic resistant genes. Methods of DNA transfer between organisms include transformation by naked DNA, viral transduction, and bacterial conjugation.

All mechanisms of DNA transfer involve the cell membrane. Since triclosan disrupts the microbial cell membrane, it is important to examine whether triclosan affects the acquisition of antibiotic resistance genes. Experiments would measure the efficiency of gene transfer between different classes of bacteria upon exposure of triclosan.

In the proposed experiment. plasmids carrying marker genes such as those coding for tetracycline and kanamycin resistance will be introduced into several hosts including the bacterium, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa. The efficiency of transformation by naked DNA and gene transfer between bacteria vial bacterial conjugation can be examined upon exposure to various levels of triclosan. Likewise the ability of triclosan to inhibit bacteriophage infection, another common method of gene transfer will be analysed. Our focus on the alterations in the efficiencies of gene transfer mechanisms upon exposure to triclosan may elucidate novel physiological effects.

Significance of Study

In light of the recent media hype concerning the " overuse of anitbacterial agents" including triclosan and the overuse of antibiotics by the medical profession. The question of whether tricloson actually improves the antibiotic resistance abilities of bacteria could have an significant economic and health impact on the general public. If studies show that triclosan actually helps to promote the development of super bacteria, then the companies who have spent so much to promote its use in their products could stand to lose billions of dollars.

In addition, if triclosan does indeed prove to improve the efficiency of the antibiotic resistance capabilities of bacteria, it could have serious health implications for the general public. This research is expected to mirror and confirm previous studies on the subject, which conclude the triclosan does cause bacteria to become antibiotoc resistant. The confirmation of these studies could lead to a policy change regarding the use and promotion of triclosan as an antibacterial agent.

The results of studies on the effects of triclosan and other agents like it could have a major impact on the production and use of antibacterial agents in common household products. Companies will have to re-develop their formulas and some may have to drop product lines altogether. This could have major impacts on some very large companies. It will also cause a change in the way the public thinks about antibacterial products and their use in the home.

Literature Review

The mechanism of how bacteria work and infect the body have been common knowledge for many years, so have the effects of antibodies in the ability to limit the ability of the bacteria to do harm and cause disease in the human body. Recent media attention to the question of whether antibacterial agents, including triclosan, limited the effectiveness of antibiotics in curing disease, has led to a rash of experiments to test these ideas. The jury is still out and there are no sweeping conclusive results. However, there is sufficient evident to provide background on which to base this research that will be the subject of this study.

The Mechanism of Triclosan

Triclosan works by inhibiting an enzyme that is important to the growth of bacteria. This would seem harmless, except that it trips another genetic switch called the multiple antibiotoc resistance (mar) operon. When this switch is turned on, a pump mechanism in the cell wall expels a wide variety of unwanted chemicals including antibiotics[3] Triclosan essentially allows a bacteria to spit out antibiotics. Sometimes these pumps do not turn off, and when the bacteria replicates, we then have a bacteria that is resistant to antibiotics. This pump mechanism is referred to as the efflux receptor [3].

Triclosan is widely used an advertised, however, there are some who question its efficacy in controlling bacteria. In an experiment by Gilbert, [7], four-day non-brushing studies were used to demonstrate the short-term plaque efficacy of toothpaste containing triclosan, This study showed that there was a statistically significance in the ability of triclosan containing toothpaste in the ability to limit bacterial action in the sample subjects [7]. Other studies confirm these results [1,21]. These results would seem reproducible under the circumstances, but there were many confounding variables that neither were nor identified in all three of these studies. For instance, baselines were not measures to determine the natural level of bacteria in the sample subjects. In addition, nothing is known about the subjects prior to the study as far as dental hygiene and the presence of disease prior to the study.

Mechanism For The Development Of Resistant Bacteria

Natural genetic transformation is believed to be the primary and most commmon mechanism for the acquisition of genetic adaptability bacteria. During bacterial evolution, the ability of Bacteria to adapt to new environments often results from the acquisition of new genes through horizontal transfer, rather than by the alteration of gene functions through numerous naturally occuring processes call point mutations [18]. Horizontal gene transfer is the movement of genetic material between bacteria, other than by descent in which information travels through the generations as the cell divides. Horizontal gene transfer differxs from inherited characteristics of genes as it is commonly described [5]. It is most often considered to be a sexual process in that that requires transfer of chromosomal DNA among two bacterial cells[19].

One of the most common methods for bacteria to adapt to different environmental conditions is through the acquisition of mosaic genes through contact with other bacteria. A mosaic gene is an acquired allele that is obtained through the transformation and combination with the original allele. The result is the formation of a new species of bacteria, that has the characteristics of both parents. It has characteristics of both of the parents, but also some characteristics that are uniquely its own [20].

Often this new bacteria contains a genetic marker that can be selected, such as that used for the antibiotic resistance to tetracycline and kanamycin, or another such marker. In this way horizontal exchange through transformation permits the movement of alleles in bacterial generations. There are many examples of horizontal genetic exchange through both transformation and conjugation in bacteria species that have enabled them to better adapt to their environment [19]. This is the basis for the argument concerning the formation of "superbacteria" and the process through which ti may occur.

What is Known about Tetracycline?

Tetracyclines and its derivatives are antibiotics which inhibit the bacterial growth by stopping protein synthesis in the bacteria. Bacteria must synthesis proteins into energy in order to survive, mush in the same way we synthesize fats, carbohydrates, and proteins into energy that can be used in our body's various systems. Teetracycline compounds have been widely used for the past forty years as therapeutic agent in medicine. The emergence of bacterial resistances to these antibiotics has n limited their use in recent times. Three different specific mechanisms of tetracycline resistance have been identified: tetracycline efflux, ribosome protection and tetracycline modification [23].These are common mechanisms found in other situations and will be discussed in detail.

Tetracycline efflux is acccomplished through thhe use of an export protein from the major facilitator family [23]. The export protein functions as an electroneutral antiport system that promoted and enables the exchange of a tetracycline cation complex for a proton [26]. The difference between the mechanisms in Gram-negative and Gram-positive bacteria is in the chemical used to achieve thiis effect. In Gram-negative bacteria the export protein contains transmembrane fragments (12 TMS), whereas in Gram-positive bacteria it displays another transmembrane fragmetn (14 TMS). Ribosome protection is mediated by a soluble protein which shares homolgy with the GTPases participating in protein synthesis [22]. The third mechanism involves a cytoplasmic protein that chemically changes tetracycline. This reaction takes only place in very specific environemtnal conditions and does not function in the natural host [28].

Several tetracycline resistance markers are currently being used in molecular biology. This feature has also been exploited to construct tightly regulated expression systems by using the regulatory elements of the Tn10 tetracycline operon. The tetM gene from Tn916 which can be expressed both in Gram-positive and Gram-negative bacteria is also frequently used [23]. These genes provide a measurement system and tools for the measurement of genetic material transfer between bacteria.

Many studies have been conducted that concluded that the environment of the bacteria may help influence the ultimate host range of specific tet genes. If the trend is reversed towards the development of inreasingly antibiotic-resistant bacteria, it would lead us to consider the need to change how antibiotics are used in both human and animal health as well as food production [22].

This is the general opinion, that has been held by many scientists who are in support of lessened usage of antibacterial compounds. However there are those who disagree and consider them to be a valuable asset to society. These persons continue to promote their use. Some feel that the effects have not been studied extensively enough to make a conclusion that their use leads to genetic mutations making the bacteria resistant antibiotics [31]. Yet these same persons still express that we should limit the use of antibacterial agents until more studies have been conducted.

Hypothesis and Research Questions

This study will attempt to answer the following research questions: (1) Does triclosan improve the efficiency of acquisition of antibiotic resistance genes in Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa to tetracycline and kanamycin? (2) Does triclosan result in an antibiotic resistant bacteria? (3) Does this effect elicit a dose/response relation ship to increasing levels of triclosan? And (4) Does triclosan limit the ability of a bacteriophage to infect a cell?

The research will empirically prove the following hypotheses:

H1 - Triclosan will improve the efficiency of acquisition of antibiotic resistance genes in Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa to tetracycline and kanamycin.

H0 - Th null hypothesis will show that improvement in the efficiency of acquisition of antibiotic resistance genes in Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa to tetracycline and kanamycin will be statistically insignificant and no difference will be found.

H2 - The second hypothesis is that triclosan does result in antibiotic resistant bacteria.

H20 - The null hypothesis will show that triclosan does not produce an antibiotic resistant bacteria at statistically significant levels.

H3 - The third hypothesis will be that this effect does elicit a dose/response relationship to increasing levels of triclosan in the ability of Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa to acquire gene mechanism to resist the antibiotics tetracycline and kanamycin.

H30 - The null hypothesis will state that a statistically significant dose response will not be found to increasing levels of triclosan in the ability of Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa to acquire gene mechanism to resist the antibiotics tetracycline and kanamycin.

H4 - The fourth hypothesis will be that triclosan does limit the ability of a bacteriophage to infect a cell.

H40 - The null hypothesis will state the ability of the bacteriophage will not be statistically significant to show that triclosan limits its ability to infect a cell.

These four hypothesis will answer the research questions and give incite as to the potential hazards and advantages to continued use of triclosan in consumer products. It will act as confirmation to previous research that concludes these effects to be significant. This research will lead to a set of recommendations regarding the use of triclosan in consumer products as it relates to the development of antibiotic resistance in bacteria. It may not answer all of the questions revealed in the literature review regarding the social implications of this policy. Bu tit will confirm previous research and will have a wider applicability than most previous studies.

Sample

The purpose of this study is to measure the efficiency of gene transfer between different classes of bacteria upon exposure of triclosan. There are many different families of bacteria and it is the goal of this study to be able to apply the results to make generalizations about the overall effect of triclosan on a wide variety of common bacteria that would be found in a typical human environment. For this reason the sample will be repeated on four species of bacteria: Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa, all of which are common in the human environment, especially in the typical household. These bacteria will receive plasmids carrying marker genes such as those coding for tetracycline and kanamycin resistance.

Tetracycline and kanamycin are two of the oldest and most widely used antibiotics prescribed in bacterial infections. Much is known about their mechanisms and the effects of various variables that may effect their efficacy. Because there is a wide array of knowledge about these two particular antibiotics, they were chosen to test the ability of the bacteria to resist them.

The selection of common bacteria and a common antibiotic will make the result more applicable to a real human environment and allow is to make some real inferences to a human population. There are many confounding variables that could effect the applicability to the research results to a real human population, such as the effects of individual immune system, general health and genetic predispositions that may effect results. However, we can infer some applicability to a real human population by using common bacteria and antibiotics rather than one that are rarely encountered in the real world.

Methodology

The research will consist of several groups of bacteria. For each species there will be a control group that does not receive any treatment, a control group with the plasmid carrying marker genes for tetracycline and kanamycin. This will help to rule out a technical error in the application and testing methods of the technician. The other groups will consist of the same two groups, only these will receive increasing levels of triclosan. The sample groups will be summarized as follows.

Control without plasmid

Control with plasmid

Plasmid with low triclosan levels

Plasmid with moderate triclosan levels

Plasmid with high triclosan levels

Escherichia coli

Staphylococcus aureus

Salmonella typhimurium

Pseudomonas aeruginosa

These sample groups will be assigned appropriate labels.

Control groups, both with and without the plasmid added would act as the independent variable against which to measure to effects of the dependent variable. The dependent variable in this case will be the groups that have the triclosan added. The control groups are expected to have no effect on the ability of the bacteria to require gene resistance to tetracycline and kanamycin. All groups will consists of the same number of sample subjects in order to make the analysis more valid. Control groups will get the exact same treatment as the other groups in order to rule out an error in technique or other procedural variables.

Data Analysis

Data will be analyzed using descriptive statistics. A p-value of

The data will be expected to show no statistical significance between the two control groups, both with and without the plasmid introduction. If a statistical significance is found between these two groups the experiment must be repeated as any other statistically significant results cannot be attributed to the introduction of the independent variable. It is expected that the results will be statistically significant between the control groups and the other groups. It is expected that the statistical significance will increase between the control groups and the groups receiving the higher dose of triclosan as compared to those receiving low dosages. These results will be statistically significant from the control groups

It is also expected that there will be a statistically significant correlation between the efficiency of the bacteria's ability to achieve gene resistance to the two antibiotics chosen and the increasing dose of triclosan. The rate will increase in proportion to the dose administered and demonstrate a dose/response effect.

If the differences in efficiency between these groups does not demonstrate the stated criteria, then the results will not prove the hypotheses and will support the null hypothesis. Each hypothesis will be tested separately to determine if the criteria are met.

Conclusions

The use of triclosan in consumer products has become accepted by the American public, widely due to a mass media campaign by the companies who developed it. Scare tactics have been the primary method to achieve this widespread acceptance by the public and the medical profession as well. Drug manufacturers and makers of these products and stand to make considerable profit from the promotion of these items.

The use of triclosan as an agent to eliminate the "dangerous" biological agents that could potentially endanger our health has come under controversy. The use of triclosan has been shown in previous studies to turn on the mechanism that allows bacteria to reject antibiotics and make them resistant to our only defense against them. The idea of "Superbacteria" is still has not been scientifically proven. It is not known if triclosan produces a permanent change in the bacteria, and in addition, it is not known if this change is passed on to the next generation. We only know, at this time about the current generation of bacteria to which triclosan was introduced.

It is expected that this research will confirm and expand on previous studies, it is expected that the result swill be reliable in making inferences about the use of triclosan in common household products., it is also expected that this research will pave the road for further research into the applicability of this research in a real human immune system. The results of this study will help to formulate opinions and policies regarding the use of triclosan in products for consumers.

The results of this study will confirm all four hypotheses and disprove the null hypotheses in all cases. They will also help to answer the original research questions: 1) Does triclosan improve the efficiency of acquisition of antibiotic resistance genes in Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa to tetracycline and kanamycin? (2) Does triclosan result in antibiotic resistant bacteria? (3) Does this effect elicit a dose/response relation ship to increasing levels of triclosan? And (4) Does triclosan limit the ability of a bacteriophage to infect a cell? IT is expected that the answers to all of these questions will be a resounding "yes."

The results of this study are expected to lead to the conclusion that triclosan should be limited in use in common household products. It will suggest that triclosan may have some uses that are appropriate, but that in most cases, its widespread use should be limited. It will cause us to reexamine the difference between beliefs that are a result of empirical evidence and those that are a result of mass marketing by large corporations, set to protect their own interests, possibly at the expense of public health.