Bacterial Resistance of Serratia marcescens to Hand Sanitizer
Parinaz Malakzadeh, Patrick Mahoney, and Paige Organ
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Overuse of hand sanitizers is a growing concern due to predictions of sanitizer-resistant bacterial strains. Bacteria cultures exposed to antibacterial agents in sanitizers over multiple generations are expected to undergo a significant shrinking of the zones of inhibition. Petri dishes of nutrient agar were divided into separate sections for plain, original formula Purell sanitizer, aloe-scented sanitizer, and sterile water before being inoculated with cultures of Serratia marcescens. After 48 hours of incubation, the zone of inhibition radii were measured and recorded before samples of bacteria were taken from the zone of inhibition’s edge. These samples were placed in separate test tubes of nutrient broth and autoclaved for 48 hours before being plated again. The procedure was repeated for a total of three generations. The single factor ANOVA test suggested there is a significant difference between generations for plain and aloe Purell (p = 1.54x10-10 and 4.49x10-9, respectively). There was no significant difference between generations for sterile water (p = 0.43). Thus, the data suggest that bacteria build a resistance to Purell of both the plain and aloe variety. More generations may reveal the extent to which bacteria can build a resistance to the sanitizers.
Introduction
The marketing of hand sanitizer in the United States is a big business. Sales of hand sanitizer grew over 70% between 2007 and 2009, where the peak of hand sanitizer sales grew to over $300 million (Fottrell, 2013). Hand sanitizers are a constant presence - from public bathrooms, to the checkout line at grocery stores. Due to its portability, using hand sanitizer can be easier and more convenient than washing with soap and water. It can also be more effective (1). In a controlled study comparing hand sanitizer to soap and water, hand sanitizer proved to be more than twice as effective as soap and water (Liu et al., 2010).
It has been theorized that continued use of alcohol-based sanitizer could lead to strains of bacteria that are resistant to the protein-denaturing effects of the sanitizer (Aiello et al., 2005). This resistance would be caused by selective pressure being placed on the bacteria by the sanitizers. One study by Reynoldset al. (2006) has suggested that scented hand sanitizers may be less effective due to additives; thus different additives to alcohol based hand sanitizers may increase or decrease the effectiveness of the product.Purell, a common sanitizer brand, has both a plain, original formula as well as an aloe-scented formula. It is hypothesized that there will be a significant difference in zone of inhibition radius between generations for aloe-scented and plain Purell, though more so for the plain Purell.
Methods and Materials
The experiment took place at Saddleback College, Mission Viejo in Room SM 244 and was conducted on Mondays, Wednesdays, and Fridays from 4 November 2013 to 18 November 2013. A sample of Serratia marcescens was obtained from the Saddleback Biology Department. One liter of nutrient agar (Criterion lot # : 11339) and one liter of nutrient rich broth (Criterion lot # : 07037) were prepared and autoclaved for three hours. Using sterile Petri dishes, ten nutrient agar plates were prepared and inoculated with Serratia marcescensusing the lawn spread method. Each of the ten plates was divided into three equal sections using lines drawn on the base of the plate. Each section was then treated with one of three methods: The first method was placing a 10 µL drop of plain, original formula Purell hand sanitizer (70% ETOH by volume) directly onto the inoculated plate. The second method was placing a 10 µL drop of aloe-scented Purell hand sanitizer (70% ETOH by volume) directly on the plate. The third method was to place 10 µL drop of sterile water on top of a sterile paper chad which was then placed on the inoculated agar. All of the measurements were done using a calibrated micropipette and sterile techniques. The plates were then placed in an incubator at 30 °C for 48 hours.
After the plates had incubated, the radii of the zone of inhibition around the spots were measured using a ruler. The surviving bacteria from the inner edges of the zones of inhibition were then collected(4) and placed into 30 separate test tubes of nutrient broth and(2) kept in an incubator to grow for 48 hours. All test tubes were labeled with both a number and group. The bacteria grown from these cultures were used to inoculate the next generation of Petri dishes. Using a sterile cotton swab, the three sections of the Petri dishes were inoculated with bacteria grown from each of the three respective groups. For each new generation(4) the zone of inhibition was measured and a new sample of bacteria was collected using the same techniques. Nutrient agar and broth was(3) prepared as needed. This procedure was repeated for a total of three generations.
The radius of the zone of inhibition was averaged for the control (sterile water), plain Purell, and aloe-scented Purell groups for each generation and an ANOVA was(3) run on the data to compare each group.
Results
The mean radius of the zone of inhibition for the first generation of plain Purell was 7.45mm ± 0.78 (n=10, ± S.E.M.). The mean radius for the last generation of plain Purell was 0.95mm ± 0.23 (n=10, ± S.E.M.). An ANOVA run using data from all generations of plain Purell showed that there was a statistical difference between generations (p=1.54x10-10).
The mean radius of the zone of inhibition for the first generation of aloe Purell was 6.95mm ± 0.45 (n=10, ± S.E.M.). The mean radius for the last generation of aloe Purell was 1.53mm ± 0.26 (n=10, ± S.E.M.). An ANOVA run using data from all generations of aloe Purell showed that there was a statistical difference between generations (p=4.49x10-9).
The mean radius of the zone of inhibition for the first generation of the control group (Sterile Water) was 0.10mm ± 0.015 (n=10, ± S.E.M.). The mean radius for the last generation of control group (Sterile Water) was 0.08mm ± 0.028 (n=10, ± S.E.M.). An ANOVA run using data from all generations of sterile water showed that there was not a statistical difference between generations (p=0.43).
Figure 1. Average zone of inhibition radius of each group per generation (n = 10). The average radius significantly differs between generations for both aloe and plain Purell (p = 4.49x10-9 and p = 1.54x10-10 respectively, single factor ANOVA test). There was no statistical difference between generations for sterile water (p = 0.43, single factor ANOVA test). Error bars are ± S.E.M.
Discussion
The data collected supports the hypothesis that the ring of inhibition would become smaller over generations. There is a statistical difference between generations with both the original Purell as well as the aloe(5) version. However, there was no statistical difference in the effectiveness of the original version versus the aloe version of the Purell product. This is most likely due to there being equal amounts of the active ingredient (70 percent ETOH by volume) of.(5) There was also no statistical difference between any of the generations of the control (sterile water) group which also supports our hypothesis. The experiment could be continued for several more generations to ensure the accuracy of the data as well as (5)test the limits of the bacteria’s ability to resist the sanitizers.
The active ingredient in Purell, ethyl alcohol, works by denaturing bacterial proteins, leading to death (Aiello et al., 2005). This may change the effectiveness of the product on different species, such as coliform bacteria, as they have different proteins. This may also mean that buildup of sanitizer-resistant genes varies from species to species as well. Pan et al. (2006) and Reynolds et al. (2006) conducted studies using sanitizers with mixed peroxides(4) and 33 percent isopropanol as their respective active ingredients and found that the effectiveness of each product varied. Treatments of peroxides resulted in a resistance to the sanitizer, while the 33 percent isopropanol was(5) ineffective concentration to produce resistance. Thus the impact of sanitizers on bacteria cannot be determined by one species or one type of sanitizer alone. It is suggested that additional tests using different species and sanitizers with different active ingredients should be conducted.
Literature Cited
Aiello, Allison E., et al. 2005. “Antibacterial cleaning products and drug resistance.” Emerg Infect Dis11 (10): p 1565-1570.
Fottrell, Quentin. "Hand Sanitizer Spread."Wall Street Journal.15 Jan. 2013. Web.
Liu, Pengbo, et al. 2010. “Effectiveness of Liquid Soap and Hand Sanitizer Against Norwalk Virus on Contaminated Hands”. Applied and Environmental Microbiology 76 (2): p 394.
Pan, Y., F. Breidt, and S. Kathariou. 2006. "Resistance of Listeria Monocytogenes Biofilms to Sanitizing Agents in a Simulated Food Processing Environment." Applied and Environmental Microbiology 72 (12): p 771.
Reynolds, Scott A; Foster, Levy; Walker, Elane S. 2006. Journal of Environmental Health 69 (4): p 48, 51.
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s):___Patricia Seabourne______
Title:_Bacterial Resistance of Serratia marcescens to Hand Sanitizer ______
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.
The purpose of the experiment was to determine the resistance strength of the Bacterium Serratia marcescens to plain Purell hand sanitizer and aloe scented Purell hand sanitizer. Nutrient Agar plates were divided into three sections. Plain Purell was placed in one section, Aloe scented Purell was placed in another section, and distilled water was placed on the third section. After incubation zones of inhibition were measured and surviving bacteria were placed on nutrient broths. This procedure was repeated for three generation. The results indicated that the zone of inhibition in areas where Purell and aloe scented Purel were present, decreased with each successive generation. The zones where distilled water was present remained unchanged.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge.
The paper is good. The purpose of the experiment, and the procedures used are clear and well explained. Even thought the results pertain to only one bacterial species (Serratia marcescens), it is still a very good idea to test the effects of hand sanitizers in that one specie. It will add valuable knowledge to be used in further experiments. One suggestion could be to have used three or more different types of bacteria and to have compared the results between them. Also it could have been interesting to find literature indicating a particular bacterium in which inhibition radii does not change, and try to determine why this does not occur in that particular bacteria. There are a few grammar errors, such as long sentences and absence of commas. There are a few verb errors. Overall, the paper is clear, and easy to understand. The paper does not have any serious weaknesses.
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.
This paper was a final version This paper was a rough draft
Mistake / Explanation1- Opinion / Provide evidence
2- Long Sentences / Break sentences into shorter ones.
3-Verb Tense / Substitute Were for Was
4- Commas / Use commas after words such as and.
5-Word Omission / Write word
Recommendation
This paper should be published as is
This paper should be published with revision
This paper should not be published