THE EFFECTS OF SUCRALOSE AND SUCROSE ONSTAPHYLOCOCCUS AUREUS[O1]
Layla Fijany and Sahar Meshkat
Department of Biological Sciences
Saddleback College
Mission Viejo, CA,[O2]92692
(Indent paragraph)(Provide an introductory sentence in the abstract with insight into what your project is about (ie. what sucrose and sucralose are and what possible inhibitory affects they may have); remember, abstract should be intro, hypothesis, methods, results, discussion/conclusion all in one paragraph).The objective of this study was to analyze and determine the effect of sucrose and sucralose on the growth inhibition of the bacteria, Staphylococcus aureus. Three trials were conducted with nutrient agar plates of bacteria and different concentrations of sucrose and sucralose. Agar plates with sugar concentrations of 5%, 10%, 20%, 30%, 40%, 50%, 80%, 90%, and 100% were prepared and placed for an incubation period of 18-48 hours at 37ºC. No zones of inhibition were visible on any of the plates and there was no significant difference between the types of growth seen. The experimental results indicate that sucrose and sucralose do not inhibit the growth of bacteria. A fourth trial was performed with non-nutrient agar mixed with sucrose and sucralose at varying concentrations (25%, 50%, 75% and 100%) into separate Petri dishes with S. aureus [O3](The name can be abbreviated to reduce redundancy).using a streaking technique. No bacterial growth was observed after an 18-hour incubation period at 37ºC. From these results, it can be concluded that sucrose or sucralose environmentsdo not enable S. aureus to grow; however,but that the nutrient agar provided enough nutrients that theto allow bacterial survivalcould withstand thewith high concentrations of sucrose and sucraloseboth sugars. These results mightalso suggest that S. aureus is able to metabolize sucralose and sucrose and reproduce in an environment of low water activity (high osmotic pressure).in which they are still able to reproduce.
Introduction
Bacteria are classified mostly as unicellular organisms that are able to multiply rapidly under favorable conditions when variables such as temperature, oxygen content, pH, osmotic pressure and nutrients available are ideal in their environment (Chirife et al., 1983). Bacteria, like all other forms of life, require water for growth, and their water requirements are best defined in terms of water activity of the substrate (Chirife et al., 1983). When the aqueous solutions in the environment of the microorganism are concentrated by the addition of a solute, such as sugar (sucrose), this creates a decrease in the water activity level results, which causinginhibition of microorganismto inhibit growth (Chirife et al., 1983).
Bacterial growth may either help or hinder human life. Therefore, it is important to understand the environment in which growth may be controlled or inhibited. Food-borne staphylococcal poisoning, caused by the ingestion of one or more preformed toxins in food contaminated with Staphylococcus aureus, is one of the most prevalent causes of gastroenteritis worldwide (Cole et al., 2002; Anas et al., 2008; Jamshidi et al., 2008). Knowing the precise boundary for the growth/no growth interface of S. aureus and determining the conditions needed for bacterial growth inhibition is necessary for food safety risk assessment (Jamshidi et al., 2008).
Sucrose is veryeffective at inhibiting bacterial growth, as it is routinely used as a natural food preservative and wound remedy(Source?)[O4]. Sugar's success as an antimicrobial agent in wound remedy and food preservation lies primarily in its ability to deprive bacteria of water that is essential for growth. Theexplanationoftheroleof sugar in the treatmentofinfectedwoundsis complex and perhapsimpossibletoreducetoa single mechanism, suchasitsantibacterialaction.The study by Chirife et al.and colleagues (1983) studyhas proposedthat afunctionofsugaristocreateanenvironment of lowwater activity (meaning aor high osmotic pressure),whichinhibitsbacterialgrowth.The study concluded that sugar may play a role in the control of infection by diminishing bacterial virulence in patients with lowered defense mechanisms (Chirife et al., 1983).
The global population is presenting rises inoverweight, obesity, diabetes, hypertension, cardiovasculardiseases, hyperlipidemia and hypercholesterolemia- asituation that has created concern about changes in lifestyleand balanced diet [O5](Azoubel et al., 2009; Astrup et al., 2011). As a result, there is acceleration in [O6]theuse and consumption of light food products,ordiet products, and in the consequentconsumptionofartificial (?) sweeteners. Given that the use of sucralose,one of the newest sweeteners hasbeen gradually increasing, new studies are necessary to attest its affects on bacterial cells and the human body.[O7] Sucralose is poorly absorbed in the intestinal tract, and is almost entirely excreted in unaltered formsthrough the feces (Azoubel et al., 2009).Recent studies have proven anassociation between ingestion of sweeteners andnephrotoxicity, hepatoxicity, orand retardation of placental andfetal development (Azoubel et al., 2009).
Microbial growth models are typically developed when the objective is to understand the responses of microorganisms when part of thein a range of conditions studied permitting growth to occur (Cole et al., 2002). Such models can describe the increase in numbers[O8] with time, the conditions allowing growth or no growth inhibitionto occur, or the chance of growthoccurring. In the past several years, there has beena growing interest in the effects of artificial sweeteners; however, the effects of whether sucralose exhibits similar growth inhibition as sucrose on Staphylococcus aureus[O9]however, are still unclear.
Therefore, it can be concluded that if sucralose exhibits similar properties on bacterial cells as sucrose, then this type of artificial sweetener may be effective at treating wounds and acting as a defense at such sites against invading bacteria and other foreign materials. A better understanding of the effects of sucralose on Staphylococcus may contribute to the development of treatment solutions dealingwithinvolving topical medications. The current study representedfurther attemptsought to better understand the growth inhibition of sucralose and sucrose on S. aureusby focusing on varying concentrations of sweetener solutions in agar plates. We[O10]will[O11]measured the diameter of the zones of inhibition on the agar to further determine if there is a similarity in the antimicrobial properties of sucralose and sucrose.
Materials and Methods
The aim of this in vitro study was to determine the efficacy of artificial sugar, sucralose, and granulated cane sugar, sucrose, against S. aureus.All The sugars used wereconsisted of a commercially available pure granulated white cane sugar (table sugar) and a Ralph’s brand artificial sweetener, made of sucralose. All sugar stock solutions were made with deionized water.
Three trials were performed in this study to examine zones of growth inhibition of S.aureus in a nutrient agar in response to sucrose and sucralose solutions. All measurements were conducted at Saddleback College in Mission Viejo, CA from 2 November to 10 November 2011[O12]. Trial 1[O13] consisted of 5 agar plates of 5%, 10% and 20% solutions of sucrose and sucralose (separately) made with DI water, for a total of 30 experimental plates and an additional 10 control plates made with DI water (no sugar). Trial 2 tested 30%, 40%, and 50% solutions of sucrose and sucralose, as well as a water control group. Trial 3 tested 80%, 90% and 100% solutions of sucrose and sucralose with a water control group. Each trial consisted of the same number of nutrient agar plates and was conducted using the same procedure. The nutrient agar solution was preparedthrough boilingwith 23 g of agar and 1000mL of DI water and was heated to boil untila clear solution was attained. The dry mass of the sucrose and sucralose was first measured, then placed in a 10 mL-graduated cylinder, which was filled up to 10mL with DI water. Percent solutions and agar (liquid) were placed in the autoclave to be sterilized. Forty Petri dishes were filled with agar and, once hardened, 0.3 mL of S. aureus was transferred onto each plate using aseptic technique. Three chads, each containing 5 µL of corresponding solution, wereplaced equidistance from each other in a triangle formation on each agar plate. , with 5 µL of corresponding solution on each chad, that were placed equidistance from each other in a triangle formation. After all the agar plates were completelypreparation, the agar plates theywere placed in an incubator set at 37 ˚C. Trial 1 was incubated for 45 hours; trials 2 and 3 were incubated for 18 hours. After the results of trial 1 were obtained, the principal investigators decided to decrease incubation to see if the zones of inhibition were present in earlier bacterial growth and in order to conduct more trials within a given time period. After the incubation period, zones of inhibition were measured (or lack thereof).
Due to the lack of growth inhibition, a fourth trial was conducted using 3 non-nutrient agar plates of 25%, 50%, 75% and 100% solutions of sucrose and sucralose (separately) for a total of 24 experimental plates. The agar solution was prepared by mixing 2.3 g of non-nutrient agar, 100 mL of DI water,and sucrose or sucralose, ofand 25 g, 50 g, 75 g and 100 gof either sucrose or sucralose. The agar solution was boiled until clear and then placed into the autoclave [O14]for sterilization. S. aureus was added to the plates using a streaking technique, in order to isolate the bacterial colonies. Once the agar plates were prepared, they were incubated for 18 hours at 37 ºC. After 18 hours of incubation, isolated colonies were counted and measured to determine the average size of the colonies present.
Results
There were no zones of inhibition present. Therefore, the mean diameter of the growth inhibition was 0.0 cm. This result was observed for trials 1,2, and 3, which included 5%, 10%, 20%, 30%, 40%, 50%, 80%, 90%,100% sucrose and sucralose solutionsand for the water control as well. [O15]After the fourth trial was complete, no bacterial growth was visible on the non-nutrient agar plates (mixed with the corresponding amount of sugar). As a result, no isolated bacterial colonies were present.
Discussion
The first three trials resulted in no zones of growth inhibition present on any of the nutrient agar plates with the sucrose or sucralose solutions when tested. Staphylococcus aureus displayed the same growth in water and in the two types of sugar solutions. This result may suggest a multitude[O16] of conclusions: thatS. aureus is able to withstand and replicate in environments of low water activity or high osmotic pressure, and still be able to replicate, or that the nutrient present in the agar powder provided a sufficient enough environment that the to allow survival of Staphylococcuswas able to survive in. Totest whether the latter hold[O17] true, we [O18]conducted the fourth trial with a non-nutrient agar to remove the variable of a nutrient enriched environment. The result of the fourth trial suggested that S. aureusis not able to survive in the sugar and DI water agar alone, thus supporting that the nutrients in the agar led to the successful growth of the bacteria and the lack of zones of inhibition.[O19]
Sugar possesses inherent microbial properties, some of which are due to high osmotic pressure and low water activity, which is inhibitory to the growth of the majority of bacteria (Naama, 2009). Water activity is a measure of the consequential effect of the average intermolecular forces between water molecules, being increased when water molecules become oriented on the surface of the solute molecules [O20](Cooper et al., 1999). When numerous molecules are tied up in this way, the water molecules are, on average, less free to act, e.g. to hydrate something, so the ‘activity’ is lower (Cooper et al., 1999). [O21]When applied topically to wounds, osmosis would be expected to draw water from the wound into the sugar, helping to dry the infected tissue and reduce bacterial growth (Dealey et al., 2011). Even when diluted with water, sugar would be likely to retain a water activity sufficiently low enough to inhibit most bacteria.
The ability of topical disaccharides, in the form of sugar, to cleanse infected wounds and facilitate healing has been recognized for many years (Efkors et al., 1999; Naama, 2009; Dealey et al., 2011). Surgeons have applied sugar dressings to contaminated wounds on animals. There is also support that sugar therapy has been used on infected surgical wounding, pressure ulcers, deep tissue infections and for other skin defects that need a healthy granulated bed (Efkors et al., 1999; Naama, 2009). In some developing countries, where modern advanced wound products are unavailable or unaffordable, granulated sugar as a contact dressing for open wounds is seen as the product of choice – its ability to reduce surface contamination and promote granulation is well recognized (Dealey et al., 2011). A study done by Naama (2009) found that S. aureus displayed inhibition of 20 mm and 11 mm at sugar concentrations of 100% and 75%, respectively, using honey solutions in a disc diffusion test. Also, According to this study, no effect was observed at 25% or 50% (Naama, 2009). The high antimicrobial effect of the honey sample in Naama’s 2009 study may be attributable to the presence of glucose oxidase. Glucose oxidase is activated by dilutions[O22] in water, resulting in the production of hydrogen peroxide, which is toxic to bacteria.
The expected results were not obtained because there were no zones of inhibition for any of the concentrations of sucrose or sucralose. These results suggest that the nutrient agar supplied an adequate environment for Staphylococcus, or that this strain of bacteria has a metabolic pathway that utilizes sucrose and sucralose at high concentrations. Although our[O23] intentions of discovering antimicrobial properties of sucralose and sucrose were not achieved, we believe future studies may further determine new treatments pertaining to Staphylococcus inhibition. Future studies regarding the effects of sucralose and sucrose against a variety of bacteria may present new breakthroughs in clinical treatments of infected wounds. The next step of this study would be to focus on the metabolic pathway of S. aureus and how this microorganism processes the uptake of nutrients in its environment and how this affectsfor survival and reproduction of the bacteria.
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Review Form
Department of Biological Sciences
SaddlebackCollege, Mission Viejo, CA92692
Author (s):Layla Fijany and Sahar Meshkat
Title: The Effects of Sucralose and Sucrose on Staphylococcus aureus.
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.
The current experimenters sought to determine the inhibition of bacterial growth in sugar environments with sucralose and sucrose. Agar plates were prepared and streaked with S. aureus and incubated for 1-2 days in a warm environment. Although previous studies revealed sugar to have an inhibiting effect on bacteria, no zones of inhibition were observed in this study. The current study revealed, however, that S. aureus was not able to survive in an environment with sugar and non-nutrient agar alone. The results were explained through either sugar adaptation in this particular bacterial strain, different metabolic pathways, or the nutrient availability in the agar promoting bacterial growth despite sugar presence.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge.
Overall, the paper was good. The introduction, methods, results, and discussion were clear and easy to understand. There are some grammatical errors (as seen above in color) that need to be looked over.Verb tense of some sections (mostly when describing their results in certain sections) were in future tense, and needed to be in the past tense. Sentence structure was adequate, apart from a few run-ons. The discussion is slightly vague in explaining their results, though. My suggestion would be to support the current results (that no inhibition occurred) with any type of study, instead of mainly focusing on studies that support that inhibition usually occurs. Perhaps a study somewhere found no growth inhibition as well. The rest of the discussion is well supported and requires no serious alterations. The abstract needed an introductory sentence, providing some background into their topic; the rest was adequate, consisting of the hypothesis, results, methods, and conclusion. Lastly, some paragraphs addressed the authors in the first-person, which should be corrected into third-person. The paper does not have any serious weaknesses though.
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.