Abstract

Introduction

Lyme disease is a leading tick-borne multisystemic disease caused by the spirochete Borrelia burgdorferi. The bacterium is transmitted by Ixodes ticks, which could feed on white-footed mice, rodents, deer, and birds [1, 2]. In the United States, there are approximately 300,000 people diagnosed with Lyme disease each year [3]. The frontline treatment for Lyme disease is antibiotics such as doxycycline for adults and amoxicillin for children [4-8]. These antibiotics are found effective in most cases of patients diagnosed with Lyme disease [5-8]. However, according to the Centers for Disease Control (CDC), approximately 10-20% of the Lyme disease patients treated with anti biotics for a recommended 2 to 4 weeks experienced symptoms of fatigue, pain or joint and muscle aches [9]. In some patients, the symptoms even lasted for more than 6 months [9]. This condition was termed as “post-treatment Lyme disease syndrome (PTLDS)” or “chronic Lyme disease” [9].

The mechanism associated with this condition in patients remains unclear. Though not proven, there are a couple of suggested explanations, such as the inability of the immune system to completely clear B. burgdorferi persisters [10], or due to the presence of antigenic debris, which might cause immunological responses [11]. Another possibility of Borrelia evading the host immune clearance after antibiotic treatment is not well understood [12, 13].

Previous in vivo studies on mice, dogs, and nonhuman primates have shown that B. burgdorferi could not be fully eliminated by various antibiotics such as doxycycline, ceftriaxone, and tigecycline. Also, a recent study had demonstrated the presence of Borrelia DNA in mice following 12 months of antibiotic treatment [14]. However, the culturing of viable organisms in Borrelia growth media could not be achieved in these studies [14-17]. A recent study reported the presence of Borrelia DNA from a patient with PTLDS after antibiotic treatment [18]. Prospective clinical studies demonstrated no significant effective antibiotic therapy and failed to show evidence of the continued presence of B. burgdorferi in patients with long-term symptoms [19, 20]. Other trials using prolonged intravenous ceftriaxone treatment only improved fatigue symptoms [21]. In summary, findings suggest that conventional treatments may not completely eliminate Borrelia persisters.

The life style of B. burgdorferi is complex as it exists in different morphological forms like the spirochetes, spheroplast (or L-form), round bodies, and biofilms [7, 13, 22-25]. Several studies show that Borrelia can change into round bodies when conditions become unfavorable such as changes in temperature, high or low pH, starvation, antibiotic exposure, and/or even an attack from the immune system [7, 23-25]. Borrelia in these defensive forms becomes dormant, immobile, and remains in this morphological state until it finds favorable conditions to return to its spirochete form [7, 22, 24, 26]. The most effective hiding place proposed for B. burgdorferi is the recently suggested biofilm form [24]. Bacterial biofilms are organized communities of cells enclosed in a self-produced hydrated polymeric matrix or extracellular polymeric substances (EPS), which is a complex mixture of polysaccharides, lipids proteins, nucleic acids, and other macromolecules [24, 27]. This unique matrix protects the underlying cells from antimicrobial agents [24, 27]. Elimination of pathogenic bacteria in their biofilm form is very challenging since these sessile bacterial cells can endure the host immune responses and are much less susceptible to antibiotics or any other biocides than their individual planktonic counterparts [24, 27]. Biofilm resistance is based upon multiple mechanisms, such as phenotypic changes of cells forming in the biofilm, the expression of efflux pumps, and the presence of persister cells, which resist killing when exposed to antimicrobial agents [28, 29].

Recently, we provided evidence that B. burgdorferi is capable of forming biofilms in vitro [24]. The aggregation of spirochete and round body forms with several different protective layers which makes up the biofilm and extracellular polymeric substances (EPS) is proposed to be a significant factor in antibiotic resistance [24]. It is also reported that the biofilms have higher population of the persister cells, which could lead to the antimicrobial resistance portrayed by these forms [24, 29].

Our previously published results on the in vitro effects of doxycycline on B. burgdorferi showed that different morphological forms have unique sensitivities to antimicrobial agents [7]. A recent study by J. Feng et al. showed that doxycycline was effective in reducing the spirochetes but not the persisters of B. burgdorferi [30, 31]. Studies have also showed that doxycycline and amoxicillin could eliminate the spirochetal form of B. burgdorferi, but the dormant persisters/biofilm-like aggregates/microcolonies were not susceptible to these antibiotics [7, 31]. Therefore, there is an urgent need to find effective agents, which can target/eliminate all the morphological forms of Borrelia.

Natural antimicrobial agents, which have been used for thousands of years, have been shown to be effective against various pathogens [32]. Many in vitro and clinical studies have demonstrated their effectiveness not only against B. burgdorferi but also against many other pathogens [33-38]. Stevia rebaudiana which belongs to the Asteraceae family is typically referred to as honey leaf or sweet leaf, and due to its natural sweetness, it is used as a natural substitute to synthetic sweetener [39-41]. The leaf extract of Stevia possesses many phytochemicals, which include austroinullin, β-carotene, dulcoside, nilacin, rebaudi oxides, riboflavin, steviol, stevioside, and tiamin with known antimicrobial properties against many pathogens [40, 42, 43]. The role of these compounds is mainly to protect the plant from microbial infection and adverse environmental conditions [38-43]. Stevia is also well known in traditional medicine for its use in treatment of many diseases like diabetes, high blood pressure, and weight loss [44, 45]. In a few clinical studies, it is reported that the phytochemical stevioside reduces blood pressure in patients experiencing mild hypertension and reduces blood glucose levels in type 2 diabetic patients [44, 45]. It was also demonstrated that the patients did not encounter any adverse effects from the use of stevioside [44, 45].

Considering the effectiveness of Stevia leaf extract in laboratory and clinical studies, we evaluated the antimicrobial potential of Stevia (whole leaf extracts) against the Lyme disease causing pathogen, B. burgdorferi, in a goal to eliminate all the different morphological forms in vitro. To effectively evaluate the whole Stevia leaf extract, we compared the antimicrobial effect of Stevia with antibiotics (doxycycline, cefoperazone, daptomycin) and their combination, which were recently found effective against Borreliapersisters….

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…Stevia leaf extract is a widely used sugar substitute [39-41, 57]; however, recent studies show that one of the major glycosides, stevioside, could have antimicrobial effect against Bacillus cereus, Bacillus subtilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa [42]. These antimicrobial studies used a high concentration of purified stevioside, and in our study, we achieved similar antimicrobial effect against Borrelia by using a lower concentration of the whole leaf extract. Our data with purified Stevioside did not show any significant antimicrobial effect on Borrelia spirochetes and persisters compared to the whole leaf extracts of Stevia (Fig. 1). This finding suggests that other components within the Stevia whole leaf extract could have antimicrobial activity against B. burgdorferi, which are yet to be identified in future studies. In a good agreement with our findings, Stevia leaf extract has also demonstrated antimicrobial activity against pathogens such as E. coli, S. aureus, Vibrio mimicus, Salmonella typhimurium, S. mutans, Bacillus subtilis, Shigella dysenteriae, and Vibrio cholera [38-42]. The next question is whether Stevia could be safely used as a therapeutic agent. Toxicological studies have shown that Stevia does not have mutagenic, teratogenic, or carcinogenic effects [57], and recent studies demonstrated its safety at high dietary intake levels [57-59]. In a study examining the mutagenicity of Stevioside and Steviol, it was noted that Stevioside at 10 mg/ml did not induce any mutation in S. typhimurium [60]. Apart from these studies, there are two important clinical studies based on the glycoproteins present in Stevia. In a randomized, double-blinded study on Chinese men and women experiencing mild hyper tension, it was reported that the glycoprotein stevioside decreased the systolic and diastolic blood pressure and also improved quality of life without causing any adverse effects compared to the placebo [44]. In another study, the acute effects of stevioside in type 2 diabetic patients were analyzed [45]. Compared to the control group, stevioside reduces postprandial blood glucose levels in type 2 diabetic patients [45]. It was noted that both these studies used an encapsulated powdered form of stevioside and whole leaf extract that had been taken orally [44, 45]. It was also observed that one of the clinical studies used a whole leaf preparation, which contained 91% stevioside, 4% rebaudioside A, and 5% of other derivatives of stevio-side [45]. The outcome from these clinical studies demonstrates that the patients did not encounter any adverse effects from the use of stevioside [44, 45]. Although the safeness of Stevia is widely studied, more in vivo studies are warranted before Stevia could be used as an antimicrobial agent for any infectious diseases.

Our future goal is to further investigate the individual components of whole leaf Stevia extract against B. burgdorferi and to identify the most effective component responsible for its significant antimicrobial effect. In conclusion, the overall antimicrobial effectiveness of Stevia A extract on the different morphological forms of B. burgdorferi was comparable to the combination of certain antibiotics. Although the results of this preliminary study cannot be extrapolated directly to clinical practice, further follow-up studies are necessary which can address the safeness of Stevia and to further identify the most effective component(s) against Borrelia.