Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 4, No. 1, 33-36 2020 DOI: 10.33805/2576-8484.176 © 2020 by the authors © 2020 by the authors History: Received: 29 March 2020; Accepted: 18 May 2020; Published: 25 May 2020 * Correspondence: drmarkcannon@outlook.com Inhibition of Autism Spectrum Disorder Associated Bacteria and C. difficile by Polyols Cannon ML1*, Merchant M1, Kabat W1, Unruh B1, Ramones A1 1Ann and Robert Lurie Children’s Hospital, Northwestern University, Feinberg School of Medicine, Chicago, USA; drmarkcannon@outlook.com (C.M). Abstract: Objectives: To determine the effectiveness of erythritol and xylitol in the inhibition of gut bacteria possibly associated with Autism Spectrum Disorder (ASD) and Clostridium difficile Infection (CDI). Methods: Seven bacterial strains associated with ASD, or with CDI and a control probiotic were tested for polyol inhibitory activity: Clostridium histolyticum, Bacteroides vulgatus, Bifidobacterium longham, and two strains each of Clostridium bolteae and difficile. Each strain was grown in brain heart infusion/sucrose media with polyol concentrations varying from 0% to 15% for erythritol and 0% -30 % for xylitol. Growth of Clostridium histolyticum and Bifidobacterium longham was measured after 24 hours while all other strains were evaluated at 48 hours to permit additional growth. Optical density was measured using a spectrophotometer and the plates were read at 620 nm. Results: All strains had results indicating polyol inhibition of growth. Clostridium histolyticum (Chis), Bifidobacterium longham (Blof), and both Clostridium bolteae (Cbol) strains showed reduced growth with increasing polyol concentration with an inflection point of about 4% for both xylitol and erythritol (complete or near complete inhibition relative to control wells). Bacteroides vulgatus (Bvul) grew very lightly in the BHI/sucrose. This strain has visible growth but very low OD values. Inhibition of growth with increasing polyol concentrations was observed but assessing the polyol inhibition break point was difficult with this strain. Conclusions: Xylitol and erythritol at sufficient concentrations were able to inhibit the growth of bacterial strains that have been associated with the development of Autism Spectrum Disorder in recently published studies. Keywords: Autism spectrum disorder, Polyol, Bacterial strains, Optical density . Abbreviations: ASD-Autism Spectrum Disorder, CDI-Clostridium Difficile Infection, Cbol- Clostridium bolteae. 1. Introduction Polyols have been used for decades as a substitute for sucrose [1-4]. The most commonly used polyols for consum p t ion are sorb itol, mannitol, xylitol, erythritol, maltitol, lactitol and isomalt [5]. Besides having fewer calories than regular sucrose, i.e., table sugar, polyols have other reported health benefits, especially in regards to oral health [6]. Well publicized studies showing the effectiveness of xylitol at reduc ing dental disease have been reported for decades, all with results demonstrating safety and effectiveness [7-9]. The well-publicized “Turk u” and the “Belize” studies reported on the caries reduction by xylitol, with xylitol being more effective than sorbitol [10, 11]. Xylitol chewing gum s, toothpastes, lollipops, candies and mouth rinses are all part of a complete dental oral hygiene program [12]. Erythritol and xylitol are polyols that repeatedly have been demonstrated to possess anti-cariogenic and anti-periodontal disease properties [13]. Polyols (particularly the non-hexitol alditols or “sugar alcohols” erythritol and xylitol) have been found effective in inhibiting the transition to and maturation of biofilms from planktonic cells [14]. Xylitol clearly inhibits the formation of mixed species biofilms, in vitro [15]. Erythritol suppresses the maturation of biofilms and contributed to a healthier oral ecosystem [16]. Polyols can suppress the growth and virulence expression of mixed bacterial biofilms. Erythritol was the most effective polyol in suppressing the growth and organization of dental pathogens. Erythritol also exerted inhibitory effects on several pathways reduced growths through DNA and RNA deple tion , at tenuated extracellular matrix production and alterations of dipeptide acquisition and amino acid metabolism [17]. The bacteria associated with Autism Spectrum Disorder have been reported in the literature, with similar results independent of research institution and locality [18]. Autism Spectrum Disorder (ASD) has been linked to propionic acid producing bacterial species, such as, Clostridia bolteae and Clostridia histolyticum [19-22]. Conversely the presence of Clostridia sporogenes could help protect against ASD by combining propionic acid with indole to produc e 3 - Indole Propionate, a neural protective metabolite, thereby neutralizing the epigenetic effect of propionic acid [23-25]. It has b een theorized that the absence of C. sporogenes in the soil is related to the use of glyphosate, known by the trade name Roundup [18]. Absence of C. sporogenes in the soil and the environment could possibly shift the maternal microbiome, resulting in epigenetic changes in the fetus or infant. Bacteroides vulgatus also has been implicated in ASD as reported in the Frontiers in Microbiology by Coretti, et al. [26]. Clostridia difficile (Cdif) is a gram-positive bacterium that is implicated in antibiotic-associated diarrhea. The relatively recent emergence of a newer hyper-virulent North American strain (NAP1) has been associated with the increase in incidence and severity of C. difficile infections (CDI) over the last decade [27]. Antibiotic overuse remains the leading risk factor for C. difficile infection. Several classes of antibiotics such as penicillins, cephalosporins, fluoroquinolones, and clindamycin have been implicated in causing CDI. Besides antibiotic usage, other risk factors are reported to include advanced age, chemotherapy, use of proton pump inhibitors, c hronic renal disease, chronic liver disease and malnutrition [28, 29]. Treatment options include discontinuing the causative antibiotic and administering either vancomycin or fidaxomicin. Another option is fecal transplantation, the process in which feces from a healthy donor are transplanted into the intestinal tract of a person with the disrupted microbial balance. This protocol has reported an 80% to 90% success rate in reducing the recurrence of C. difficile infections [30]. There remains some opposition to Fecal Transplantation Therapy due to the basic nature of the procedure and potential complications [31]. A simpler, safer and “cleaner” technique would be more appealing to patients and clinicians. 2. Materials and Methods Bacterial isolates and media: C. bolteae and C. histolytica strains were kindly provided by Dr. Emma Allen- Verc o PhD. (Univ ersi ty of Guelph/Canada). B. vulgatis (8482) and B. longum (15707) were obtained from the American Type Culture Collection (ATCCC/Manassas Va .). C. difficile strains 5555 and 5557 were provided by Dr. Larry Kociolek MD (Lurie Children’s Hospital, Chicago, IL). All studies u sed a b as al media of Brain Heart Infusion broth supplemented with 2% sucrose (BHI/Suc). Polyols were prepared separately at high c oncentrat ions in BHI/Suc for assay plate preparations. Xylitol was added to 60% (w/v) and Erythritol was prepared at 30% (w/v) in BHI/Suc. The se p olyol levels were the maximum achievable based on solubility. Final media preparations were sterilized and placed in an anaerobic c hamber for at least 2 hours after preparation to cool and remain in a reduced state. 3. Assay Procedures Assays were prepared in the anaerobic chamber. 96 well plates were employed with each test preparation in triplicate wells by adding 100 mcL of BHI/Suc at 2x concentration to all test wells. Bacterial preparations were made in BHI/Suc adjusted to a Macfarl and standard 34 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 4, No. 1: 33-36, 2020 DOI: 10.33805/2576-8484.176 © 2020 by the authors concentration of 0.5. Final assay inocula of each strain with a further 1:100 fold dilution. 100 mcL of bacterial inocula was added to eac h test well with or without a polyol. Plates were incubated anaerobically for 24 or 48 hours and terminated when bacterial growth reached a easi ly visible level in control wells. Plate were then transferred to a plate spectrophotometer and read at 620 nm wavelength. Mean OD v alues for each well were calculated and OD values vs. polyol concentration were plotted. 4. Results Seven strains were tested for polyol inhibitory activity C. histolyticum, B. vulgatus, C. bolteae (x2), C. difficile (x2), and Bifidobacterium longham. All strains grew to variable bacterial density levels. B. vulgatus had the poorest growth but still had measurable mean OD v alues to suggest polyol activity. Detailed OD values vs. polyol concentration are plotted as follows with relative inhibition inflection points (Figures 1 - 7). Figure 1. Xylitol begins inhibiting C. histolyticum with only a 2% concentration . The probiotic is also inhibited and supplementation may be advised. Figure 2. Erythritol inhibits at around a 2% concentration but inhibits the probiotic more that xylitol. Figure 3. C. bolteae strains inhibited by a very low concentration of xy li tol, b u t less so at a 2% level. Figure 4. Erythritol inhibits the C. bolteae strains at a very low conc entrat ion and in a more linear path than xylitol. 35 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 4, No. 1: 33-36, 2020 DOI: 10.33805/2576-8484.176 © 2020 by the authors Figure 5. Both erythritol and xylitol inhibits B. vulgatis at only a 0.25% concentration. Figure 6. At 2% concentration, xylitol generally inhibit C. difficile strains, although C.diff 5557 is inhibit in a more linear manner and at a lower concentration. 5. Discussion Erythritol inhibits ASD bacteria at a lower concentration than xylitol. Both polyols were capable of significant inh ibition o f the A SD associated bacteria, in addition to the inhibition of antibiotic resistant C. diff strains. Erythritol may inhibit Bacteroide s vulgatus b e tter than xylitol but additional studies with a more optimal media for B. vulgatis need to be performed. Figure 7. Erythritol seems to inhibit C. diff only after reaching a higher concentration. Most dental products contain 20% xylitol for effectiveness. There are no popular dental products with a high erythritol concentration, although some commercial drinks use erythritol as a sweetener. However, Xylitol should be considered as a treatment for C. difficile infection due to its low cost and availability. In addition, xyli tol and erythritol are considered safe food additives with decades of use in the prevention of oral diseases, such as p eriodontal d is ease and dental cavities. Autism spectrum disorders are likely caused by a combination of microbiome, environment, and the epigenetic interac t ion [ 3 2-34] . Recent research shows that more than 50% of children with autism have GI symptoms, food allergies, and maldigestion or malabsorption issues [35]. Propionic acid is used as a food additive and is also a bacterial byproduct. Propionic acid uptake may be related to lack of the b ac terial gluten metabolizers and resultant leaky gut. Elimination of calcium propionate as a bread additive/preservative may be benefi cial in reducing the behaviors associated with ASD [36]. Shifting the oral and gut microbiome with polyols may also be successfu l in reduc ing b ehaviors associated with ASD. More research, large well-designed clinical trials are indicated for protocols illuminating therapies effective with reducing the symptoms of ASD [37]. 6. Conclusion Xylitol and erythritol at sufficient concentrations were able to inhibit the growth of bacterial strains that have been assoc iated with the development of ASD. 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