Microbial resistance to triclosan: a case study in natural selection.
Article Type: Case study
Subject: Natural selection (Case studies)
Escherichia coli (Case studies)
Staphylococcus aureus (Case studies)
Drug resistance in microorganisms (Case studies)
Triclosan (Case studies)
Authors: Serafini, Amanda
Matthews, Dorothy M.
Pub Date: 11/01/2009
Publication: Name: The American Biology Teacher Publisher: National Association of Biology Teachers Audience: Academic; Professional Format: Magazine/Journal Subject: Biological sciences; Education Copyright: COPYRIGHT 2009 National Association of Biology Teachers ISSN: 0002-7685
Issue: Date: Nov-Dec, 2009 Source Volume: 71 Source Issue: 9
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 246348914
Full Text: [ILLUSTRATION OMITTED]

Charles Darwin's theory of evolution by natural selection is a cornerstone concept in biology (White, 2007). Natural selection is the mechanism of evolution caused by the environmental selection of organisms most fit to reproduce, sometimes explained as "survival of the fittest" (Mader, 2004). An example of evolution by natural selection is the development of bacteria that are resistant to antimicrobial agents as a result of exposure to these agents (Yazdankhah et al., 2006). Antimicrobials kill off susceptible members of a population, hut cells that have some resistance from the start or that acquire it later through mutation or gene exchange may survive. These survivors are "best fit" in that particular environment where they proliferate (Levy, 2007).

While acquisition of knowledge of evolution by natural selection is a seminal goal of science education (NABT, 2008), it is difficult for students to observe this phenomenon directly in their own lives. Perhaps the reason for this is that humans have a generation time of about 25 years. It takes 100 years--a period of time beyond the life expectancy of most people--for four generations of progeny to be traced from the original parents (National Oceanic and Atmospheric Administration, 2008). This sharply contrasts with bacteria that have shorter generation times, in some cases as little as 20 minutes (Tortora, Funke & Case, 2010). Theoretically, that means that over 100 years, about 2,500,000 generations of bacterial descendents could be produced from an original cell. This huge reproductive potential makes bacteria especially well-suited for use in the study of natural selection and, as genetic differences accumulate to produce major transformations, to clearly illustrate evolution.

This article describes research on the resistance of wild clonal populations of Escherichia coli and Staphylococcus aureus to triclosan and the subsequent reversion of these resistant bacteria back to wild-type when triclosan is removed from their environment. These experiments can serve as apractical, timely, and engaging model for the study of natural selection in the biology classroom and can be performed either as a long-term open inquiry (Welden & Hossler, 2003) or as a teacher-guided inquiry.

* Background Information

Triclosan (2, 2, 4'-trichloro-2'-hydroxydiphenyl ether) is a broad-spectrum antimicrobial agent that is effective against bacteria (Perencevich et al., 2001), fungi (McMurry et al., 1998), and viruses (Schweizer, 2001). See Figure 1 for a diagram of triclosan.

[FIGURE 1 OMITTED]

Invented at Ciba, triclosan is the generic name of the chemical that Ciba sells as Irgasan[R] (Ciba.com, 2008). Triclosan is also used in plastics and clothing by other manufacturers under the name Microban[R], and used in acrylic fibers as Biofresh[R] (Glaser, 2004). It was introduced as a surgical scrub in 1972, typically at 0.3% bactericidal concentrations, and used primarily to limit the spread of infections in health care settings. Since the mid-1990s, triclosan has been marketed to the general consumer, typically at 0.1% bacteriostatic concentrations, and is now a ubiquitous presence in our lives. Triclosan is used in many personal care products such as toothpaste, shower gels, deodorant soaps, hand lotions and creams, mouthwashes, underarm deodorants, and hand soap. Eighty-four percent of antibacterial bar soaps and 100% of antibacterial liquid soaps contain triclosan. It is also infused into many household items such as cutting boards, counter tops, mops, paint, floor tiles, wallpaper, and even toys (Levy, 2000; Schweizer, 2001).

This practice is not restricted to the U.S., but is a worldwide phenomenon. One billion dollars are spent annually on antimicrobial household products (Glaser, 2004) and the rate is rising at 3-7% per year (Jagger, 2008). Concerns about the Influenza A virus subtype H1N1 during 2009 heightened the importance of hand washing to infection control (CDC, 2009) and will likely contribute to even greater use of antimicrobial products.

While it is promoted as an antimicrobial agent (i.e., a substance toxic to bacteria, fungi and protists, and viruses), there is no evidence that the use of triclosan in household products prevents infection in humans. There is the suspicion, however, that its overuse may actually be harmful (Larson et al., 2004). While triclosan has not been found to have any carcinogenic or teratogenic effects in humans (Bhargava & Leonard, 1996), a number of recent studies raise concern that the extensive use of triclosan-containing products can select for bacteria that are resistant to this chemical as well as to other antimicrobials (Birosova & Mikulasova, 2009; Larson et al., 2004). For this reason, widespread use of triclosan may represent a potential public health risk with regard to development of concomitant resistance to clinically-important antimicrobials (Yazdankhah et al., 2006).

* How Does Triclosan Work?

At bactericidal concentrations, triclosan appears to act upon multiple cell membrane and cytoplasmic targets, while at lower concentrations, triclosan affects specific targets such as the enoyl-acyl carrier protein reductase (ENR) enzyme (Yazdankhah et al., 2006). Bacteria, unlike humans, use the ENR enzyme to synthesize their fatty acids (Levy, 2000; Russell, 2004; Schweizer, 2001). Triclosan binds irreversibly to the active site of the ENR enzyme, thereby inhibiting fatty acid synthesis. Many bacteria, such as Staphylococcus aureus and Escherichia coli, use the fabI gene to code for the ENR enzyme (Ling et al., 2004). When these bacteria are exposed to triclosan, variants with mutations in the fabI gene are favored. A single mutation infabI may result in these bacteria requiring up to 100 times more triclosan than wild-type cells to show even minimal inhibition (Perencevich et al., 2001).

Some bacteria, like Pseudomonas aeruginosa, use the fabK gene to synthesize their fatty acids and are very resistant to triclosan. If the fabK gene is acquired by other bacteria, either through genetic engineering or through normal horizontal transfer of genes from one cell to another, the resultant recombinant bacterium will be resistant to triclosan (Health & Rock, 2000).

Another mode of action of triclosan is alteration of efflux pumps. Efflux pumps are systems in the cell membrane of bacteria that act like "sump pumps." Bacteria use efflux pumps to actively pump antibiotics or other hazardous chemicals out of their cells before they can have an effect (Schweizer, 2001). Triclosan may turn on multi-drug efflux systems (Levy, 2000; McMurry et al., 1998), causing many different types of chemicals, including triclosan, to be pumped out of a cell without causing harm to the cell (Braoudaki & Hilton, 2004).

* Research Questions

This research focused on the dynamics between bacteria and triclosan and raised two questions. The first question asked: Can common bacteria, like Staphylococcus aureus and Escherichia coli, develop resistance to triclosan? Previous work (Welden & Hossler, 2003) addressed this question for Escherichia coli (a Gram-negative bacterium) alone. This article expands the work of Welden and Hossler to include S. aureus, a Gram-positive organism. It was hypothesized that if S. aureus and E. coli are grown in the presence of triclosan, these bacteria may develop resistance to this agent. The second question asked: Is bacterial resistance to triclosan a reversible phenomenon? If triclosan resistance is reversible, then by removing triclosan from unnecessary products, triclosan-resistant microbes may regain previous sensitivity levels. It was hypothesized that if S. aureus- and E. coli-resistant populations were grown without further exposure to triclosan, then descendants of these bacteria may revert back to the wild-type and regain sensitivity to triclosan.

* Materials & Methods

Experiment 1: Triclosan Resistance Experiment

Experiment 1 investigated whether triclosan could induce clonal populations of S. aureus and E. coli to become resistant.

Establishment of Clonal Populations

Stock cultures of S. aureus and E. coli were obtained from the culture supply in the microbiology laboratory. To eliminate the possibility that variants already existed in the stock culture, clonal populations of S. aureus and E. coli were first established. Quadrant streak plates of each organism were made on nutrient agar plates (NAP) (Benson, 2002) and isolated colonies were produced. One colony of each organism was randomly selected and cultured in nutrient broth and served as the original clonal population for the study.

Teacher tip: These bacteria are inexpensive and are readily available from biological suppliers.

Triclosan Solution

One hundred milliliters (100 mL) of a 0.1% solution of triclosan was made by placing 0.1 g of powdered triclosan (99% USP [KIC Chemicals, Inc.] purchased from Sigma Aldrich) in a beaker to which 100 mL of 70% ethanol was added.

Teacher tip: If pure triclosan is not available or too expensive to purchase, one can substitute a liquid antibacterial soap. Antibacterial soaps typically contain 0.1% triclosan.

Disk-Diffusion Method

The Disk-Diffusion Method (Benson, 2002) was used in this experiment. This involved soaking a sterile disk of filter paper in triclosan and placing it on an agar plate that had been previously inoculated with a test organism using a sterile swab. After incubation, if the chemical is antimicrobial, a clear zone representing inhibition of growth can be seen around the disk. Details of this experiment are presented below.

1. Bacteria from the clonal broth cultures were swabbed onto nutrient agar plates using the Kirby-Bauer method (Benson, 2002). This swabbing method produces a solid "lawn" of bacteria. Ten plates were swabbed with S. aureus and ten with E. coli.

Teacher tip: If resources are limited, one can use E. coli alone and achieve satisfactory results.

2. Ten 13 mm paper disks were soaked with the triclosan solution and ten 13 mm sterile paper disks were soaked with 70% ethanol to act as a control.

Teacher tip: We purchased pre-made sterile paper disks. These were expensive and often difficult to obtain. You can make your own by cutting filter paper with a 1/2" hole punch and sterilizing the disks in an oven or autoclave.

3. Each disk was grasped with sterile forceps and soaked in triclosan. Each triclosan-soaked disk was pressed in the center of the inoculated plates and pressed lightly down onto the agar.

Teacher tip: If liquid antibacterial soap is used as the source of triclosan, its viscous properties cause poor absorption. To overcome this, hold the disk vertically and pipet 1 drop of soap on the disk. Then lay the disk in the center of the swabbed plate.

4. All plates were incubated at 37 [degrees]C for 24 hours. The zone of inhibition was measured in millimeters from the edge of the disk to the outside edge of bacterial growth. These plates were designated "Subculture One."

5. At the edge of the zone of inhibition, a fuzzy halo of growth existed. The halo contained some tiny colonies of bacteria that could grow in the presence of triclosan and represented mutant variants from the original population. See Figure 2 for a diagram of this plate.

6. To establish "Subculture Two," a sterile swab was used to pick up bacteria from the halo region of each of the plates and was used to swab two new plates of nutrient agar. This resulted in 20 plates of S. aureus and 20 plates of E. coli. Triclosan-soaked disks were placed in the center of these plates, and incubated at 37 [degrees]C for 24 hours. The zone of inhibition was measured, and the colonies at the halo region were used to create the next subculture, "Subculture Three."

7. This procedure was repeated until bacteria that grew right up to the edge of the triclosan-soaked disks were produced. Such a growth pattern meant that the bacteria were not inhibited by the triclosan solution and were deemed to be resistant to 0.1% triclosan. Seven plates of S. aureus and 17 plates of E. coli were established in all.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

* Experiment 2: Reversion to WildType Experiment

Question 2 asked whether triclosan-resistant bacteria could revert back to wild-type sensitivity if they were repeatedly grown in the absence of triclosan. The 17 plates of triclosan-resistant E. coli and seven plates of triclosan-resistant S. aureus produced in Experiment 1 were used as "Culture One" for this experiment. The following procedure was performed for each of these plates of bacteria until the zone of inhibition was equal to that of "Subculture One" from Experiment 1.

1. Bacteria from "Culture One" were swabbed onto two new separate nutrient agar plates, coded "1A" and "1B." Paper disks were not placed on plates labeled "A," so the bacteria on plates "A" grew in the absence of triclosan. Disks soaked with triclosan, however, were pressed onto plates labeled "B."

2. Plates 1A and 1B were incubated at 37 [degrees]C for 24 hours. The zone of inhibition for plate 1B was measured. Plate 1B was then eliminated from the experiment. Only plate 1A was used in the next step of this experiment.

3. Bacteria from plate 1A were swabbed onto two new nutrient agar plates which were labeled 2A and 2B and constituted "Subculture Two."

4. This procedure was repeated until the zone of inhibition for the subcultured plate B bacteria were equal to that of the original, wild-type starting populations. See Figure 3.

* Results

Experiment 1: Triclosan Resistance Experiment

Question 1 investigated whether triclosan could induce clonal populations of S. aureus and E. coli to become resistant. Our hypothesis was supported since 17 plates of E. coli and seven plates of S. aureus were produced that were completely resistant to triclosan.

An analysis of variance (ANOVA), performed using SPSS, was used to determine if differences in the sensitivity of S. aureus and E. coli to triclosan existed. While S. aureus and E. coli both became resistant to triclosan, these bacteria differed in their sensitivity to triclosan in a statistically significant way (F=20.634; df =1, 48; p<.05). This comparison showed that S. aureus (mean zone of inhibition = 5.7 mm) was more sensitive to triclosan than was E. coli (mean zone of inhibition = 2.4 mm). Perhaps the reason for this difference in sensitivity is because E. coli is a Gram-negative organism with a thinner but more complex cell wall than S. aureus; triclosan may be able to enter S. aureus cells more easily than Gram-negative cells.

A statistically significant difference in resistance to triclosan was also found for the variable subculture (F=32.7; df =1, 48; p<.05). This means that the greater the number of times bacteria were subcultured, the more resistant they became to triclosan. It appears that continual exposure to triclosan selects for mutants that are more and more resistant to triclosan than the original wild-type population. This experiment indicates that triclosan acts as an agent of natural selection favoring the survival of variants that are resistant to it.

Presented in Figure 4 is the graphical representation of these results. Each point on the graph represents the mean of 20 sets of data, and visually shows a decrease in size of the zones of inhibition as these organisms developed resistance to triclosan. Although the graph does not show this specifically, by the 5th subculture, some E. coli plates contained organisms that had developed total resistance to triclosan. This means E. coli can become resistant to triclosan in less than one week.

* Reversion to Wild-Type Experiment

Question 2 investigated whether triclosan-resistant bacteria could revert back to wild-type when grown in an environment that lacks triclosan. These results indicated that both organisms reverted back to wild-type when grown in the absence of triclosan, and some differences in the ability to do so were observed.

An ANOVA was used to analyze the difference in the ability of triclosan-resistant bacteria to revert to wild-type. This analysis indicates that these organisms differed from one another in their ability to revert to wild-type (F=7.1; df =1, 43; p<.05). S. aureus (mean zone of inhibition = 10.5 ram) reverted to wild-type more quickly than did E. coli (mean zone of inhibition = 11.9 mm). S. aureus completely reverted to wild-type after 25 subcultures; some E. coli populations had not reverted back to wild-type by the 44th subculture. This means that although E. coli developed resistance to triclosan more readily than S. aureus, once it became resistant, it took longer for E. coli to revert back to wild-type when triclosan was removed from its environment.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Presented in Figure 5 is a graphical representation of reversion of subcultures to wild-type triclosan sensitivity.

* Conclusions & Suggestions

We are living in a world that is inundated with antimicrobials and relies on such chemicals to treat pathogens. The paradox is: The more chemicals we use, the more antimicrobial variants appear. The most important message to be taken away from this experiment may be that if we use chemicals more sparingly, we may avoid the potential scenario of producing a plethora of "superbugs" that are resistant to treatment with traditional antimicrobials.

As well as having ecological implications, this experiment can serve as a practical, timely, and engaging model for the study of natural selection in the biology classroom. While natural selection is a key idea in biology, it is a difficult concept to teach. Students often come to the classroom with pre-existing anti-evolution attitudes. We argue that the experiment described in this article provides compelling evidence for natural selection that may lead students to experience discomfort with their pre-existing nonscientific notions. This "case study" may contribute to student development of contemporary scientific understandings of evolution.

Biology education strives to enable students to develop understandings about the processes of science as well as the content of science. The ease with which this experiment can be accomplished may lead to a positive regard for scientific inquiry. We suggest that this experiment be performed over the course of a semester as a teacher-guided inquiry. This will allow the teacher not only to facilitate classroom discussions about natural selection and environmental issues, but also to present these ideas within the context of the "experiment" which will enable students to learn about the processes of science as well.

* Acknowledgments

The authors thank Dr. Kristine Santilli, Michael Matthews, and Linda Thorburn for reviewing this manuscript and Cliff Williams for help with SPSS. We also thank The Sage Colleges and Walter Robb for their financial support.

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BIO

AMANDA SERAFINI is an R.N. Public Health Sanitarian, Rensselaer County Health Department, Troy, NY 12180; e-mail: ajes08@gmail.com. DOROTHY. M MATTHEWS is Associate Professor of Biology at The Sage Colleges, Troy, NY 12180; e-mal: matthd@sage.edu.
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