Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 4, No. 1, 5-8 2020 DOI: 10.33805/2576-8484.173 © 2020 by the authors © 2020 by the authors History: Received: 6 December 2019; Accepted: 30 January 2020; Published: 5 February 2020 * Correspondence: drmarkcannon@outlook.com Inhibition of Rothia Species by Over-the-Counter Products and Bacterial Antagonists Cannon L Mark1,2*, Kabat B1, Yogev R1,2, Jantra L1, Awan A1, Le C1, White K1, Vorachek A1 1Ann and Robert Lurie Children’s Hospital of Chicago; drmarkcannon@outlook.com (C.L.M.). 2Northwestern University Feinberg School of Medicine. Abstract: The interaction between the human host micro biome and over the counter products has recently been investigated, with surprising results. Some over the counter items may negatively affect the health of the host, supporting the concept of the “hygiene hypothesis”, that is, that disease may be actually caused by the lack of beneficial commensal bacteria. Recent reports on the gluten metabolizing genus, Rothia, and a possible association with Celiac Disease beg the question, what happened to the Rothia? In this study inhibitory factors, such as, Over the Counter oral hygiene products and antagonistic bacteria were investigated and, in vitro, significantly inhibited the gluten metabolizing bacteria, possibly affecting human digestion and contributing to gluten sensitivity. Keywords: Gluten oral bacteria, Rothia mucilaginosa, Streptococcus salivariu. 1. Introduction The human body is host to trillions of microorganisms, including bacteria, molds, yeasts, viruses and archaea. In addition, the contribution of the microbiome to human health has become thoroughly established with roles such as educating the immune response, resisting pathogens, and digestion. As a result, the human microbiome project was designed to ascertain the microbial composition of the entire human body. Meanwhile, the oral microbiome has been extensively determined and reported in the literature. The current reported microbiome of the oral cavity region contains 619 taxa, derived from 13 phyla [1-4]. An additional 36,043 gene clones have been sequenced, identifying an additional 434 unique oral taxa that (after further validation) may be added to the database. Amongst the oral strains sequenced to date, two important gluten metabolizing species, Rothia mucilaginosa and Rothia aeria have been identified [4,5]. R. mucilaginosa and R. aeria are of the Rothia genus under the phyla Actinobacteria. R. aeria was named after its isolation from air in the Russian space laboratory Mir and is an oral inhabitant [6,7]. R. mucilaginosa is primarily found in the oral cavity but has been reported in the upper respiratory tract and also the duodenum [8-11]. Interestingly, mucosal damage in celiac disease is mostly found in this area of the gastro-intestinal system [12]. Oral micro-organisms that in vitro degrade dietary proteins may mean that they play an in vivo role in food metabolism. During mastication, ingested food is mixed with stimulated whole saliva and oral micro-organisms. This process accelerates food digestion while the bolus is still churning in the oral cavity [13]. For example, nitrate reducing bacteria have been described as being indispensable in the production of nitric oxide which regulates blood pressure and cardiovascular health and this further emphasize the importance of the oral microbiome in systemic health [14-16]. A favorable and potential source for gluten-degrading enzymes would be the micro-organisms inhabiting the human gastro-intestinal tract. It is well reported that bacteria residing in and on the human body supply the host with numerous functions that are not encoded by the human genome [17]. For instance, bacteria that colonize the large intestine ferment starches that are resistant to mammalian digestive enzymes [18]. In addition, it has been reported that human breast milk contains a number of oligosaccharides that are only digested by gut bacteria, not the breast-feeding child [19,20]. Therefore, recent publications that report gluten-degrading bacteria as natural residents of the oral cavity are not surprising after all [21,22]. This discovery is also very significant, since the oral cavity represents the gateway to the gastro-intestinal system in which gluten is mixed with the oral microorganisms in human saliva. The finding of gluten-degrading oral microbes then begs the questions, what are they susceptible to and what common source may reduce the gluten metabolizers or decrease their effectiveness of gluten processing, leading to gluten “sensitivity”? 2. Objective The purpose of this study was to determine if there is any inhibition of beneficial oral biofilm species such as Rothia aeria, R. mucilaginosa and R. dentocariosa, Streptococcus mutans (pathogen-negative control) and also Lactobacillus reuteri strains (isolated from periobalance Probiotic) by Over The Counter (OTC) oral antimicrobials utilizing in vitro laboratory technique. The secondary objective was to determine the antagonism, if any, of the Rothia genus by Streptococcus species (mutans and salivarius) and known pathogens. Rothia aeria and R. mucilaginosa are reported to be important in the processing of gluten. Inhibition of these beneficial bacteria by OTC products, either directly or indirectly, would increase gluten sensitivity in patients. Beneficial bacteria may be indirectly inhibited by certain antagonistic bacteria that are relatively less sensitive to OTC products. 3. Methods 3.1. Susceptibility Experiment Three colonies of R. aeria, R. dentocariosa, R. mucilaginosa, S. mutans, or Lactobacillus were obtained from isolation plates and grown in Mueller-Hinton media to a McFarland Standard of 0.5. Either Brucella agar plates, Rogosa agar, or Mueller- Hinton agar plates with 5% sheep blood were wholly spread to create a lawn with one cotton swab inoculation of chosen target bacteria. Five cotton discs were evenly distributed on the plate and 10 microliters of full strength OTC reagent was pipetted directly onto each corresponding disc. The plates were evaluated after 30 hours of growth at 36oC. Calipers were used to measure zones of inhibition in millimeters. 3.2. Diffusion Experiment Trypticase Soy Agar (TSA) was autoclaved and cooled to 56 degrees and aliquots of 25 mL were cooled and inoculated with 2 mL of 0.5 McFarland Standard suspensions of target organisms: R. dentocariosa, R. mucilaginosa, Streptococcus salivarius, Escherichia coli or Pseudomonas aeruginosa prior to pouring agar plates. Impregnated plates were then inoculated in punched zones using a disposable 10 microliter loop with 0.5 McFarland Standards of test inhibiting bacteria species: Streptococcus salivarius, Staphylococcus aureus, Vancomycin-resistant Enterococcus, Pseudomonas aeruginosa , Escherichia coli, and R. dentocariosa or R. mucilaginosa. The plates were evaluated after 24 hours of growth at 36oC. Calipers were used to measure zones of inhibition. 6 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 4, No. 1: 5-8, 2020 DOI: 10.33805/2576-8484.173 © 2020 by the authors 4. Results Bacterial growths of all tested bacteria were inhibited by Crest ProHealth™, ACT™, Listerine SmartRinse™, and Chlorhexidine. R. aeria and R. mucilaginosa were also inhibited by Embrace™ varnish Table 1. Table 1. Susceptibility experiment: The effect of over the counter oral hygiene products on oral bacteria. Reagent R. aeria on blood agar R. dentocariosa R. mucilaginosa Perio probiotic (Lactobacillus) S. Mutans on blood agar on blood agar on Brucella on blood agar on Brucella On blood agar On rogosa Spry xylitol mouthwash™ 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Crest prohealth™ 9.9 12.12 11.11 14.16 14.10 15.13 16.13 12.12 ACT fluoride rinse™ 10.10 11.12 14 12.14 16.14 17.15 16.15 13 Listerine smartrinse™ 9.9 10.11 9.9 14.14 9.8 14.12 13.12 11.11 Chlorhexidine (11.6% alcohol) 13.12 18.18 13.12 14.14 11.11 16.15 15.15 15.14 Listerine™ (27% Alcohol) 0.0 0.0 0.0 0.0 0.0 9.9 0.0 0.0 Phosphate buffered saline (PBS) 0 0.0 0 0.0 0 0 0 0 27% alcohol 0.0 0.0 10 0.0 0 10 0 0 Embrace varnish™ (Has xylitol) 8.9 0.0 0.0 12.12 0.0 0.0 0.0 0.0 Spry™ xylitol toothpaste gel 0.0 0.0 0.0 10.12 0.0 0.0 0.0 0.0 50% spry™ xylitol toothpaste gel in PBS - 0.0 - 0.0 - - - - Levoflaxacin (5 micrograms) 30 30 30 36 20 0 0 20 Spry™ Xylitol Toothpaste Gel inhibited R. mucilaginosa, L. reuteri, a probiotic that inhibits many oral and pathogens, was significantly inhibited by OTC oral products, except the xylitol based. Xylitol based oral products did not inhibit the commensal S. salivarius nor the pathogens, S. aureus, E. coli and P. aeruginosa (Table 2). Table 2. Susceptibility experiment: The effect of OTC oral hygiene products on other bacteria of the human flora. S. aureus S. salivarius E.coli P. aeruginosa VRE Spry™ mouthwash 0 0 0 0 0 Embrace™ varnish 0 0 0 0 0 Spry™ xylitol gel diluted in PBS 0 0 0 0* 0 PBS control 0 0 0 0 0 Xylitol based oral products do inhibit many oral pathogens and have been extensively used in dentistry for decades. Growth of P. aeruginosa was inhibited by R. dentocariosa and growth of S. aureus was inhibited by R. mucilaginosa. The zones of inhibition by the gluten metabolizers were demonstrably large. The inhibition of the beneficial gluten metabolizers and probiotic bacteria by OTC oral products may have been the result of fluoride concentration. An alcohol based product, ListerineTM, did not greatly inhibit the gluten metabolizers (Figure 1). Figure 1. Example of Inhibition of pathogen P. aeruginosa by gluten metabolizer, R. dentocariosa. 5. Discussion In vitro results are not always applicable to the clinical situation. Indeed, the complexity of the human oral microbiome would make it difficult to predict a response to any oral intervention with certainty. The results of the present study are o f a pilot nature, a negative finding would mean that there is little need for further investigation. However, in vitro studies are always necessary before progressing into more extensive, time consuming, and financially demanding clinical studies. The mere fact that OTC products, sometimes used ad libitum by patients, contribute to a reduction in beneficial bacteria should be a concern to all health practitioners. Of greater interest should be the extent of inhibition, as the zones of inhibition were quite significant in diameter. The average diameter of inhibition with an OTC product was 13 mm [Range: <6-18 mm] (Figure 2). 7 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 4, No. 1: 5-8, 2020 DOI: 10.33805/2576-8484.173 © 2020 by the authors Note: Rothia aeria is inhibited by: 1. Chlorhexidine, 2. Listerine Smartrinse™. Note: Rothia mucilaginosa inhibition by: 3. ACT fluoride rinse™, 4. Crest Prohealth™. Figure 2. Examples of inhibition plates by OTC products. Note: Rothia dentocariosa inhibition by: 5. Levofloxacin, 6. 27% alcohol. The mode of inhibition should be discovered, as it appears that the fluoride concentration of the OTC products may have been contributory. An alcohol based product was only inhibitory of the probiotic in this study, and not the gluten metabolizers. With dental disease at an increasing rate in developing countries due to the shift to a higher carbohydrate diet, with addition of processed foods containing added sugars, health professionals should be cautioning about the over use of OTC products. The dental caries rate is not decreasing, as would be expected with all the OTC utilization, and dental expenditures are increasing every year. Perhaps the OTC products help with limiting the pathogenic bacteria but only at the expense of also eliminating many beneficial bacteria. This is a no win situation for the population, spending vital resources on products that may indeed create more pathology, such as, gluten sensitivity, and fail to protect from dental caries. The beneficial effect of fluoride for caries protection may be somewhat decreased by the possible inhibition of oral probiotic bacteria by over use of OTC products. Unsupervised use of a daily fluoride mouth rinse by a child could possibly create a gluten sensitivity issue, and due to lack of regulatory oversight, this severe side effect would never be discovered (Table 3). Table 3. Diffusion experiment: Bacterial species inhibition of each other. R. dentocariosa R. mucilaginosa S. salivarius E. coli P. aeruginosa R.mucilaginosa 0.0 0.0 0.0 0.0 0.0 VRE 0.0 0.0 0.0 0.0 0.0 E. coli 0.0 0.0 0.0 0.0 0.0 P.aeruginosa inhibits 0.0 0.0 0.0 0.0 S.Salivarius 0.0 0.0 0.0 0.0 0.0 R.dentocariosa 0.0 0.0 0.0 0.0 0.0 S. aureus 0.0 inhibits 0.0 0.0 0.0 Another very important aspect of this study was the interaction between pathogenic and beneficial bacteria. The interaction, or rather, the inhibition of different bacterial species actually determines the health of the host and as such, is paramount in importance. The results were significant in that growth of Rothia species was inhibited by other bacteria. This suggests that if the oral flora equilibrium is changed by using OTC oral hygiene products, a domino effect can change the entire oral microbiome, which is the gateway to the digestive tract. The gastric microbiome is now recognized as a vital component of the host’s health, both mental and physical. Increased oversight concerning the over uses of anti-microbial, food preservatives that are also anti-microbial, and OTC products that inhibit commensal bacteria, is essential. Required testing of OTC products and better population education into the importance of the holobiome should be a health priority. The connection between the increase in chronic diseases and the significant shift reported in the modern human microbiome should be further investigated. 6. Conclusion Rothia and Lactobacillus species may be decreased in quantity by the overuse of oral antimicrobials. OTC products may alter the oral microbiome creating a situation less conducive for the survival of essential beneficial bacteria. The use of OTC products may decrease the enzymatic degradation of gluten containing foods by Rothia bacteria. This can possibly result in gluten sensitivity, thereby increasing the clinical prevalence of celiac disease. Further studies are required before any clinical implications may be concluded, but oral antimicrobials should be used only when necessary. References [1] J. G. Camp, M. Kanther, I. Semova, and J. F. Rawls, "Patterns and scales in gastrointestinal microbial ecology," Gastroenterology, vol. 136, no. 6, pp. 1989-2002, 2009. https://doi.org/10.1053/j.gastro.2009.02.075 [2] R. E. Ley, D. A. Peterson, and J. I. Gordon, "Ecological and evolutionary forces shaping microbial diversity in the human intestine," Cell, vol. 124, no. 4, pp. 837-848, 2006. https://doi.org/10.1016/j.cell.2006.02.017 [3] P. J. Turnbaugh, R. E. Ley, M. Hamady, C. M. Fraser-Liggett, R. Knight, and J. I. Gordon, "The human microbiome project," Nature, vol. 449, no. 7164, pp. 804-810, 2007. [4] T. Chen, W.-H. Yu, J. Izard, O. V. Baranova, A. Lakshmanan, and F. E. Dewhirst, "The human Oral microbiome database: A web accessible resource for investigating oral microbe taxonomic and genomic information," Database: The Journal of Biological Databases and Curation, vol. 2010, p. baq013, 2010. https://doi.org/10.1053/j.gastro.2009.02.075 https://doi.org/10.1016/j.cell.2006.02.017 8 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 4, No. 1: 5-8, 2020 DOI: 10.33805/2576-8484.173 © 2020 by the authors [5] F. E. Dewhirst et al., "The Human Oral Microbiome," Journal of Bacteriology, vol. 192, no. 19, pp. 5002-5017, 2010. [6] G. Wei, M. Zamkhcharfi, F. Dewhirst, D. Schuppan, and F. Oppenheim, "Isolation and characterization of gluten degrading bacteria from the oral cavity," Gasteroenterol, vol. 140, pp. S-641, 2011. https://doi.org/10.1016/s0016- 5085(11)62656-5 [7] Y. Li et al., "Rothia aeria sp. nov., Rhodococcus baikonurensis sp. nov. and Arthrobacter russicus sp. nov., isolated from air in the Russian space laboratory Mir," International Journal of Systematic and Evolutionary Microbiology, vol. 54, no. 3, pp. 827-835, 2004. https://doi.org/10.1099/ijs.0.02828-0 [8] C. Kazor et al., "Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients," Journal of Clinical Microbiology, vol. 41, no. 2, pp. 558-563, 2003. https://doi.org/10.1128/jcm.41.2.558-563.2003 [9] M. Collins, R. Hutson, V. Båverud, and E. Falsen, "Characterization of a Rothia-like organism from a mouse: Description of Rothia nasimurium sp. nov. and reclassification of Stomatococcus mucilaginosus as Rothia mucilaginosa comb. nov," International Journal of Systematic and Evolutionary Microbiology, vol. 50, no. 3, pp. 1247- 1251, 2000. https://doi.org/10.1099/00207713-50-3-1247 [10] E. Zaura, B. Keijser, S. Huse, and W. Crielaard, "Defining the healthy" core microbiome" of oral microbial communities," BMC Microbiology, vol. 9, no. 9, p. 259, 2009. https://doi.org/10.1186/1471-2180-9-259 [11] G. Ou et al., "Proximal small intestinal microbiota and identification of rod-shaped bacteria associated with childhood celiac disease," Official Journal of the American College of Gastroenterology| ACG, vol. 104, no. 12, pp. 3058-3067, 2009. https://doi.org/10.1038/ajg.2009.524 [12] M. Bonamico et al., "Patchy villous atrophy of the duodenum in childhood celiac disease," Journal of pediatric Gastroenterology and Nutrition, vol. 38, no. 2, pp. 204-207, 2004. https://doi.org/10.1097/00005176-200402000- 00019 [13] K. Kodukula et al., "Gut microbiota and salivary diagnostics: The mouth is salivating to tell us something," BioResearch Open Access, vol. 6, no. 1, pp. 123-132, 2017. https://doi.org/10.1089/biores.2017.0020 [14] E. R. Hyde et al., "Metagenomic analysis of nitrate-reducing bacteria in the oral cavity: Implications for nitric oxide homeostasis," PLoS One, vol. 9, no. 3, p. e88645, 2014. https://doi.org/10.1371/journal.pone.0088645 [15] J. E. Koopman, M. J. Buijs, B. W. Brandt, B. J. Keijser, W. Crielaard, and E. Zaura, "Nitrate and the origin of saliva influence composition and short chain fatty acid production of oral microcosms," Microbial Ecology, vol. 72, pp. 479- 492, 2016. https://doi.org/10.1007/s00248-016-0775-z [16] V. Kapil, S. M. Haydar, V. Pearl, J. O. Lundberg, E. Weitzberg, and A. Ahluwalia, "Physiological role for nitrate- reducing oral bacteria in blood pressure control," Free Radical Biology and Medicine, vol. 55, pp. 93-100, 2013. https://doi.org/10.1016/j.freeradbiomed.2012.11.013 [17] S. R. Gill et al., "Metagenomic analysis of the human distal gut microbiome," Science, vol. 312, no. 5778, pp. 1355- 1359, 2006. [18] L. V. Hooper, T. Midtvedt, and J. I. Gordon, "How host-microbial interactions shape the nutrient environment of the mammalian intestine," Annual Review of Nutrition, vol. 22, no. 1, pp. 283-307, 2002. [19] N. Kirmiz, R. C. Robinson, I. M. Shah, D. Barile, and D. A. Mills, "Milk glycans and their interaction with the infant- gut microbiota," Annual Review of Food Science and Technology, vol. 9, pp. 429-450, 2018. https://doi.org/10.1146/annurev-food-030216-030207 [20] S. Moossavi et al., "Integrated analysis of human milk microbiota with oligosaccharides and fatty acids in the CHILD cohort," Frontiers in Nutrition, vol. 6, p. 58, 2019. https://doi.org/10.3389/fnut.2019.00058 [21] E. J. Helmerhorst, M. Zamakhchari, D. Schuppan, and F. G. Oppenheim, "Discovery of a novel and rich source of gluten-degrading microbial enzymes in the oral cavity," PloS One, vol. 5, no. 10, p. e13264, 2010. https://doi.org/10.1371/journal.pone.0013264 [22] N. Tian et al., "Salivary gluten degradation and oral microbial profiles in healthy individuals and celiac disease patients," Applied and Environmental Microbiology, vol. 83, no. 6, pp. e03330-16, 2017. https://doi.org/10.1128/aem.03330-16 https://doi.org/10.1016/s0016-5085(11)62656-5 https://doi.org/10.1016/s0016-5085(11)62656-5 https://doi.org/10.1099/ijs.0.02828-0 https://doi.org/10.1128/jcm.41.2.558-563.2003 https://doi.org/10.1099/00207713-50-3-1247 https://doi.org/10.1186/1471-2180-9-259 https://doi.org/10.1038/ajg.2009.524 https://doi.org/10.1097/00005176-200402000-00019 https://doi.org/10.1097/00005176-200402000-00019 https://doi.org/10.1089/biores.2017.0020 https://doi.org/10.1371/journal.pone.0088645 https://doi.org/10.1007/s00248-016-0775-z https://doi.org/10.1016/j.freeradbiomed.2012.11.013 https://doi.org/10.1146/annurev-food-030216-030207 https://doi.org/10.3389/fnut.2019.00058 https://doi.org/10.1371/journal.pone.0013264 https://doi.org/10.1128/aem.03330-16