Treatment of Candida Albicans[TH1]: Assessing the Efficacy of Two Popular Home Remedies
Jackie Mitchell, Kim Lehmann, and Karina Gadea
Department of Biological Science
Saddleback College
Mission Viejo, CA 92692
Candida albicans, an opportunistic and polymorphic fungus, can infect superficial mucosal membranes in the mouth and vagina, causing oral thrush and yeast infections. Common home remedies for vaginal yeast infections include apple cider vinegar and theLactobacillus[TH2] acidophilus bacteria found in unpasteurized yogurt. These home remedies were predicted to inhibit C. albicans growth either by inducing apoptosis[TH3] or by adhering to the vaginal epithelium and competing with C. albicans for receptor-binding sites. Potato dextrose agar was prepared at a pH of 4 and a pH of 7 and inoculated with a streak of C. albicans. Candida[TH4]thrives at a neutral pH, and its growth is inhibited as the pH of its environment becomes more acidic. Sterilized chads with deionized water, apple cider vinegar, or Lactobacillus acidophilus were placed on the plates. After incubation, clear zones of inhibition around each sterile chad were measured. Plates set to a pH of 7 and treated with apple cider vinegar were able to significantly inhibit the growth of C. albicans (ANOVA, Bonferroni Correction, p<0.00833).
Introduction
Candida albicans is a naturally occurring fungus in the human mouth and vagina and is the most common human fungal pathogen (Dai et al. 2012). C. albicansis an “opportunistic and polymorphic fungal pathogen[TH5]” and its yeast-hyphal transition is one of its key virulence factors, allowing it to invade our tissues, evade macrophages, and form biofilms (Vediyappan et al., 2013). Overgrowth of C. albicans can cause superficial mucosal infections such as oral thrush in the mouth and throat and yeast infections in the vagina. Instead of using anti-fungal medications, which can produce unwanted side effects, there are many common home remedies used to treat and prevent yeast infections. These include apple cider vinegar and probiotics like Lactobacillus acidophilus,which is commonly found in yogurt (Jeavons, 2002; Pinto et al., 2008). Vaginal pH ranges from 3.8-4.4 under regular conditions and is regulated in part by Lactobacilli bacteria. Lactobacilli help to maintain acidity in the vagina by producing lactic acid.[TH6]; the acidic environment helps to prevent overgrowth of C. albicans. When vaginal conditions change even slightly[TH7], overgrowth can occur. Both vinegar and Lactobacillus work by lowering the vaginal pH down[TH8] to its ideal level to inhibit C. albicans growth. It has been found that after exposure to Lactobacilli bacteria, C. albicans cells died off due to a lack of metabolic activity (Kohler et al., 2012). In another recent study regarding apoptotic stimuli, C. albicans cells induced programmed cell death after being exposed to 60mM acetic acid for 3 hours (Dai et al. 2012). Our study aims to support that Lactobacillus acidophilus and apple cider vinegar are two different affordable and highly effective treatments to reduce C. albicans growth.
Methods and Materials
Researchers used[TH9] Difco Potato Dextrose agar formula obtained in the Saddleback College Biology Lab to fill 40 sterile Petri Dishes. The Difco Potato Dextrose consisted of 4.0g potato starch (from infusions), 20.0 g dextrose, and 15.0g of agar.
During the process of making the agar, once it became transparent, the pH was adjusted by dropwise addition of sodium hydroxide and hydrochloric acid. To ensure a proper pH reading, a Thermo Scientific Orion 815600 pH probe was accurately stabilized and then used to periodically measure the pH of the agar. Three of the flasks were adjusted to a pH of 7.0, while the fourth flask was adjusted to a pH of 4.0. The NaOH and HCl were added drop-wise until goal pH was attained. [TH10]
The agar was autoclavedand[TH11] then allowed to cool to room temperature.After the agar was sufficiently cooled, but not cool enough to solidify, thirty Petri dishes were filled with agar at a pH 0f[TH12] 7, while the ten remaining dishes were filled with agar at a pH of 4. Plates were prepared aseptically in order to prevent growth of unwanted microorganisms on the C. albicans Petri dishes. After pouring, plates were closed and allowed to cool for a period of ten minutes before being stored upside down to prevent condensation. The plates were left in refrigerated storage for five days before being inoculated with C. albicans.
The potato dextrose plates were inoculated to produce a lawn[TH13] of C. albicans. Sterilized chads were immediately placed on the inoculated plates and treated with apple cider vinegar, Lactobacillus acidophilus, or deionized water. All of the plates were inoculated with C. albicans. Of the thirty plates set to a pH of 7, ten were also inoculated with apple cider vinegar to inhibit growth, ten additional plates were inoculated with 10mL of Lactobacillus on sterilized chads to inhibit growth, and the final ten plates were treated with deionized water, to serve as a positive control. The ten plates set to a pH of 4 were then divided into three parts, with each section receiving either apple cider vinegar, Lactobacillus, or deionized water. Each plate at a pH of 4 was inoculated with one sterile chad of each treatment. The plates were then left to incubate for 24 hours at 37ºC in a normal aerobic atmosphere.
After the growth period, the plates were inspected for clear zones surrounding the sterile chads with apoptotic stimuli. The clear zone of inhibition around the sterilized chad was measured; because zones of inhibition were small, zones were measured in reticle units using a microscope and later converted to millimeters. Once all measurements were made, all data were inputted into MS Excel (Microsoft Corporation, Redmond, Washington) so that data analysis could be completed. After the data were compiled, an Analysis of Variance (ANOVA) was completed, followed by a Bonferroni correction to analyze the inhibition of C. albicans growth depending on which, if any, apoptotic stimuli was used.
Results[TH14]
Each zone of inhibition was measured in reticle units, converted to millimeters, and recorded to run a data analysis. Neither of the positive control tests showed any inhibition of C. albicans growth. Plates set to a pH of 7 and treated with deionized water were negative for inhibition (0.00, n=30) and the plates set to a pH of 4 were negative for inhibition as well (0.00, n=10).On average, plates at a pH of 7 that were treated with apple cider vinegar showed a zone of inhibition 0.0525 ± 0.0203 mm (±SEM, n=30), while plates set to a pH of 4 showed average inhibition zones 0.0175 ± 0.00917 mm (±SEM, n=10). Plates treated with Lactobacillus bacteria showed less inhibition of C. albicans at a pH of 7 and slightly more inhibition at a pH of 4. Plates set to pH 7 and treated with Lactobacillus showed average zones of inhibition 0.0183 ± 0.00722 mm (±SEM, n=30), and plates at pH 4 that were treated with Lactobacillus showed zones of inhibition 0.0200 ± 0.0117 mm (±SEM, n=10). An ANOVA, followed by a Bonferroni Correction post test, revealed that plates set to a pH of 7 and treated with apple cider vinegar were significantly different from the plates set to a pH of 7 that were treated with only deionized water. (ANOVA, Bonferroni Correction, p<0.00833). Plates set to a pH of 4 and treated with only deionized water, another positive control, were not shown to be significantly different than the plates set to a pH of 7 and treated with apple cider vinegar due to the pH 4 control test’s small sample size. Although both deionized water control tests showed no inhibition of C. albicans growth, the pH 4 plates had a smaller sample size, thereby[TH15] increasing the likelihood that chance affected the results.
Figure 1: Mean Clear Zone of Inhibition of Candida Albicans growth based on which type of stimuli was used. When treated with deionized water, no zones of inhibition were observed for plates set to a pH of 4 (0.00 mm ± 0.00 SEM, n=10) or a pH of 7 (0.00 mm ± 0.00 SEM, n=30). When treated with apple cider vinegar, plates at a pH of 4 showed average zones of inhibition 0.0175 mm (± 0.00917 SEM, n=10) while plates set to a pH of 7 showed zones an average of 0.0525 mm (± 0.0203 SEM, n=30). Plates treated with Lactobacillus bacteria showed average zones of inhibition 0.0200 mm (± 0.00117 SEM, n=10) on pH 4 plates and zones 0.0183 mm (± 0.00722 SEM, n=30) on pH 7 plates. An ANOVA followed by a Bonferroni correction showed that the only significant difference was between pH 7 plates treated with apple cider vinegar and the plates treated with deionized water.
Discussion
In this study, only one sample group was able to inhibit a significant amount of C. albicans growth. Plates set to a pH of 7 and treated with apple cider vinegar showed the highest amounts of inhibition as compared to the ability of Lactobacilli and deionized water to inhibit growth of C. albicans.When treated with apple cider vinegar, C. albicans cells are exposed to acetic acid, prompting a weak acid stress response. This weak acid stress response activates the Ras-cAMP pathway, sending signals that accelerate programmed cell death in C. albicans (Ramsdale et al., 2008). It was discovered that MNL1, a gene found in C. albicans that was previously believed to have no known function, regulates weak acid stress response. Over time, MNL1 helps C. albicans adapt to weak acid stress responses, but the short-term exposure to apple cider vinegar in our study was able to inhibit C. albicans growth.
Other studies have shown that Lactobacilli can also inhibit growth, but our results did not support this (Jeavons, 2003).Under normal conditions, Lactobacilli are naturally present in the human vagina and help to keep C. albicans in check. Three main mechanisms have been proposed as to how C. albicans growth is regulated by Lactobacilli (Jeavons, 2003). First, Lactobacilli produce lactic acid as a by-product of its glycogen metabolism, helping to maintain the vagina’s ideal pH level of 3.8-4.5 and inhibiting the growth of many pathogenic microbes, including C. albicans. Secondly, Lactobacillus acidophilus produces hydrogen peroxide, further inhibiting microbial growth. Third, and possibly most importantly, Lactobacilli can strongly adhere to the vaginal epithelial cells and compete with pathogens for receptor-binding sites.
In this study, inhibition of growth was expected for Lactobacilli as well as apple cider vinegar, but Lactobacilli was not able to significantly inhibit growth of C. albicans. There are a number of possible explanations as to why Lactobacilli did not sufficiently inhibit growth. In this study, C. albicans plates were only treated once with each stimuli[TH16], whereas individuals treating Candida[TH17] overgrowth would most likely repeat treatments multiple times over the course of a few days. Therefore, we cannot conclude that Lactobacilli would not be able [TH18]to inhibit growth of C. albicans, but this study supports that apple cider vinegar is more able [TH19]to inhibit C. albicans growth.
Literature Cited
Dai B., Wang Y., Li D., Xu Y., Liang R., Zhao L., Cao Y., Jia J., Jiang Y. 2012. Hsp90 Is Involved in Apoptosis of Candida albicans by Regulating the Calcineurin-Caspase Apoptotic Pathway. PLoS ONE 7(9): e45109. doi:10.1371/journal.pone.0045109
Jeavons, H. S. 2003. Prevention and Treatment of Vulvovaginal Candidiasis Using Exogenous Lactobacillus. Journal of Obstetric, Gynecologic and Neonatal Nursing 32(3): 287-296.
Kohler G.A., Assefa S., Reid G. 2012. Probiotic Interference of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 with the Opportunistic Fungal Pathogen Candida albicans. Infectious Diseases in Obstetrics and Gynecology 2012. doi:10.1155/2012/636474
Pinto T., Neves A., Leao M., Jorge A., 2008, Vinegar as an Antimicrobial Agent for Control of Candida spp. In Complete Denture Wearers. Journal of Applied Oral Science 16(6): 365-90.
Ramsdale, M., Selway, L., Stead, D., Walker, J., Yin, Z., Nicholls, S., Crowe, J., Sheils, E., and Brown, A. 2008.MNL1 Regulates Weak Acid-induced Stress Responses of the Fungal Pathogen Candida Albicans.Molecular Biology of the Cell 19: 4393-4403.
Vediyappan, G., Dumontet, V., Pelissier, F., and d’Enfert, C. 2013. Gymnemic Acids Inhibit Hyphal Growth and Virulence in Candida Albicans.PLoS ONE 8(9): 1-11.
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s): Karina Gadea, Jackie Mitchell, Kim Lehmann
Title:Treatment of Candida Albicans: Assessing the Efficacy of Two Popular Home Remedies
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.
**The paper talked about two home remedies to treat Candida albicans (yeast infection). One of the remedies was with apple cider vinegar while the other remedy was Lactobacillis acidophilus, which is found in unpasteurized yogurt. They created a potato dextrose agar at two different pH levels of 4 and 7. They then inoculated the plates with a streak of C. albicans, and put sterile chads with DI water, apple cider vinegar and Lactobacillis acidophilus and incubated them for a few days. Once the incubation period was over, they measured the zones of inhibition on each plate.
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 great. Good organization! Not much to say other than the paper has minor grammatical errors and a few parts were confusing, but can be easily clarified. It would definitely be to your advantage for clarity to the reader to add a data table! Also, stick to one abbreviation for the word Candida albicans to refrain from confusing the reader. Other than those minor errors the paper is good.
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 versionThis paper was a rough draft
Recommendation
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This paper should not be published
[TH1]Needs to be italicized and the “A” in albicans needs to be lower cased
[TH2]I would take out the word “the” and add a comma after the word bacteria
[TH3]Define this word
[TH4]You keep using different terms… stick to one term. Either “C.albicans or Candida to keep away from confusion
[TH5]You already stated this exact comment in the paragraph above
[TH6]Delete this period
[TH7]“When vaginal conditions change, even in the slightest manner, overgrowth can occur” (Or something of this sort… It is a bit confusing)
[TH8]delete this word
[TH9]insert “a”
[TH10]You already stated this a few sentences prior
[TH11]“and WAS then allowed”
[TH12]Change the “0” to an “o” so it says “of” not “0f”
[TH13]Lawn spread?
[TH14]Add a data table to your results section!
[TH15]Sample size, AND thereby
[TH16]Stimulus would be a better word
[TH17]Stick to C. albicans or Candida… don’t use both words
[TH18]Say “ Lactobacilli would be incapable of inhibiting”
[TH19]“apple cider vinegar is able to inhibit”