Efficiency of Antibiotic Resistance Gene Transfer Mechanisms

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.

The Reason for all the Media Hype

Levy [11,12,13,14,15,16] attributes the increased occurrence of resistant strains of bacteria to the overusee of antibiotics by physicians and the public at large. Levy has been a key promoter of the movement to limit the use of antibiotics an antibiotic agents. He feels that there are many factors, not attributable to science that have led to this phenomenon. Levy feels that these reasons are [11,12,16] that the fear of litigation and the perception of patient expectations are a contributing factor to antibiotic misuse and, therefore, bacterial resistance. The increased consumer demand for and stockpiling of antibiotics plays a larger role in promoting the use of products that increase bacterial resistance. Travel has increased spread of resistant bacteria.

Levy also blames some advances in medical science for the spread of resistant bacteria including advances in treatment of such conditions as cancer and transplant procedures that leave patients vulnerable to infections with bacteria bearing intrinsic antibiotic resistance. Often immunosuppreseants are used in these procedures.

Levy states that there have been many new strains of bacteria appear that were previously not clinically significant in the past. Some previously benign bacteria have mutated to become harmful. In addition, some harmful strains are becoming more difficult to treat with conventional antibiotics. This means that as bacteria become more resistant, we will have to develop new compounds to combat them and they will continue to change. This will lead to a constant race between scientists and bacteria in order to have effective treatments. This is essentially the basis behind the "superbacteris" phenomenon that has been the subject of the media hype in recent years. The works of Levy are primarily responsible for this publicity.

Bacteria have developed the resistance to multiple antibiotic compounds and these bacteria have become an increasing problem in hospitals[25]. An intersting phenomenon has been found in farm workers that use subtherapeutic levels of drug given to animals to promote growth. The workers and animals all contain multidrug-resistant strains of Escherichia coli, a bacteria that could have significant clinical results in the workers [15].

Throughout his many published works, in both popular and peer-reviewed magazines and journal, Levy draws several generalizations about the use of antibiotics and antibacterial agents by the general public and the health professions. The first is that the numbers of outpatient antibiotic prescriptions and over-the-counter availability of antibiotics in developing countries promots drug resistant strains. This availability to the public creates a situation where even if healthcare professionals see a need to limit their use, they are not in a position to have an impact. The public would have to be convinced, and presently there is a massive media campaign by the manufacturers to promote their use. This exaggerated use of antibiotics and antibacterials in the home will increase the incidence of resistant bacteria in other members of the household[16].

Levy points to other problems in the continued overuse of these products. The disposal and excretion of antibiotics into the environment can change the microbiology of a region [15]. This could result in localized epidemics of resistant bacterial strains that could then begin to spread to other regions. When triclosan, is added to antibacterial cleaning products for the home, leaves residues and remains in sewers and on household surfaces, allowing resistant bacteria to emerge [15]. One incidence of a bacteria where this has happened in that the occurrence of multidrug-resistant Staphylococcus aureus is increasing worldwide, in the community as well as the hospital. Antibacterial products in homes may contribute to the problem [15]. This is the bacteria responsible for the widely publicized flesh-eating skin disease that had been sensationalized by the media.

Levy also explores teht duration of the resistance to antibiotics. He concluded that resistance may be transient with short courses of antibiotics. However, long courses of therapy encourage the development of permanently resistant strains [30]. Once resistance to a drug appears, it cannot be eradicated. However, measures can be taken to minimize the spread of resistant bacteria and promote repopulation with susceptible bacteria [24,27,29].

Some bacteria are inherently resistant to antibiotics, whereas others accumulate genetic resistance in addition to their intrinsic resistance. Changes in bacterial phenotypes are relatively simple to make, since these organisms have only one chromosome [4]. In addition, plasmids replicate independently of the chromosome but transfer resistance through the genes they carry. Bacteriophages can infect cells and introduce resistance genes. Plasmids can also move from one baterial species to another for example from Salmonella to Escherichia coli [6,9, 17].

The wide spread use of compound that promote antibiotic resistant bacteria is staggering, emphasizing the urgency in making a determination of results and recommendations for usage. Hooten makes the following statements regarding the severity of this overuse

Plant and animal farming may account for twice as much antibiotic use as in humans in the U.S. Other data indicate that 35 to 50 million pounds of antibiotics are produced annually in the United States. Hundreds of thousands of pounds are sprayed onto fruit trees each year, causing a large geographic spread of drugs and therefore drug-resistant bacteria. In animals, as much as 80% of antibiotics is given to promote growth. Mash made from carcasses containing bacteria that produce tetracycline for human use was accidentally discovered to promote growth through traces of tetracyclines left after extraction. After this finding, companies began to add tetracycline and penicillin to feed intentionally. The levels of drug given are subtherapuetic but sufficiently high to select for resistance. As a result, in the 1970s, farm workers and animals were found to harbor multidrug-resistant E. coli. Levels of resistance were once thought to correlate directly with dose, but this has been disproved, since for many antibiotics, resistance is mediated by genes conferring high levels of resistance" [8, p. 1089-1090].

The concern over this problem stems from the occurrence of multi-drug resistant bacteria in the community at large. They used to be confined to certain situations and locations, such as in hospitals and on farms [3,11, 16]. This has been especially common regarding E.coli bacteria. Clinicians and hospitals now have little recourse in treating certain types of bacteria and the problem seems to be spreading regionally and it seems to be occurring with an increasing number of bacteria.

The problem continues to grow at an alarming rate. An illustration of the extent of the problem, can be found in a report on the incidence of the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) found insamples of blood and cerebrospinal fluid of hospitalised patients and monitored over theperiod 1989-95. The incidence remained stable from 1989-91; after which it rose to 13.2% in 1995. The resistance to six other commonly-used antibiotics increased significantly over the same period and there was a trend for methicillin-resistance to be linked to resistance to other antibiotics [29].

It is known that triclosan has an effect on the efficiency of resistnat genetic material in E. coli [2] and Porphyromonas gingivalis [2] cells. These mechanisms need higher drug concentrations than those needed to stop the growth of the bacteria. This may suggest that we do not need to stop the use of anitbacterial agents altogether, but need to place limits on their use. Some mutations offer protection against triclosan-mediated mutation in bacteria [4]. Therefore it cannot be said that antibacterial agents cause mutations in every circummstance. They level fo triclosan must be high enough to cause mutation and the presence of certain genes that limit the ability of the agent to cause mutation must not be present. This research has been largely ignored by Levy and others who promote the discontinuance of antibacterial agents in the current wide spread use that we see today. This research will make the regulation and proper level and usage establishment difficult, because the same results cannot be obtained in every situation.

Our results show that organisms that are intrinsically resistant to triclosan may contain triclosan-insensitive enoyl.

A review of current literature on the effects of bacteria resistance in response to antibacterial agents has been limited and there are many conflicting studies. The fact that resistance does occur and the mechanisms that lead to this are accepted as fact in the scientific community. However, the controversy surrounds the extent of the problem and its implications on public health.

The effects of wide spread antibacterial agent use ahs been studied in isolated populations such as hospitals and on animal and produce farms. There was found to be a considerable negative effect on these communities. However, it is not known whether these studies can be applied to the community at large and further, what the potential consequence might be. Studies have not been conflicting as to the mechanism of antibacterial resistance, only the full extent of the problem and the potential effects on humans. Early studies focused on the mechanism of the development of resistance, whereas later studies have focused on the implications.

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.