ACTA SCIENTIFIC MICROBIOLOGY (ISSN: 2581-3226) Volume 2 Issue 6 June 2019 Research Article
The Effect of Sewak on Oral Microbiota Composition
Yosra Modafer1*, Xiaofei Wang1 and Mohammad Kamese2 1Department of Biological Sciences, Tennessee State University, USA 2Department of Respiratory Therapy, Tennessee State University, USA *Corresponding Author: Yosra Modafer, Department of Biological Science, Tennessee State University, Nashville, USA. Received: April 24, 2019; Published: May 07, 2019 DOI:
10.31080/ASMI.2019.02.0225 Abstract Considerable data by many experimental studies have shown that the Salvadora persica plant and its chewing sticks (sewak)
display beneficial effects for oral hygiene. The aim of this study was to evaluate the potential effects of sewak on salivary microbiome obtained from six healthy volunteer participants. Samples were processed and DNA was isolated using QlAamp kit. The variable V3 profiles using 16s metagenomics sequencing approach. Surveys and mouthwash samples with and without the use of sewak were
and V4 regions of the 16S rRNA gene were sequenced. Each sample was sequenced to a depth of 380,000 reads in the paired-end 27 species level taxa decreased and those of 16 taxa increased (P < 0.05). Streptococcus fryi and Streptococcus vestibularis were two 2x300 read format. Taxonomic identification and statistical analysis revealed that after using the sewak treatment, the proportions of
bacterial species were not changed among tested samples between the two conditions. There were also differences in the propor streptococcus species that were significantly lowered after sewak treatment in comparison to the control samples (P < 0.05). Other tion of different taxa between samples without sewak treatment. It is possible that sewak use may reduce oral bacteria in a global, - proportional manner, such that the microbiome approach could not detect such changes since the method is designed to measure composition changes, not absolute changes. Keywords: Sewak; Chewing Stick; Miswak; Oral Microbiome; Bacteria; Salvadora persica
Introduction bial shift toward a disease ce range of some oral bacterial species could be considered as a [4]. Significant increase in the abundan-
The history of oral microbiota study began in 1683 when An- oral bacterial abundances with increased risk cell cancer . For his mouth using his handmade microscope. It has been a long jour sign for some serious diseases. Several studies have linked specific thony van Leeuwenhoek decided to examine the film growing in example, Streptococcus anginosus is linked the carcinogenesis of ney from these simple experiments to the comprehensive study of [5] - , while the oral micro the human oral microbiota. Oral microbiota is considered the most head and neck squamous cell carcinoma [6] - dual's body and includes an unknown number of archaea, fungi, biota in head and neck squamous cell carcinoma patients is distin- complex and most significant microbial community in the indivi- bacteria, dental hygiene, includes using a toothbrush, toothpaste viruses, and bacteria. To date, there are only about 750 different guishable from non-cancer patients [7]. To control disease-causing and mouthwash, must be practiced every single day . Interes Prevotella, tingly, the earliest common method of dental hygiene is the use of Streptococcus, Leptotrichia, Actinomyces, Eikenella, Peptostrepto- [2,8] - bacterial species identified, including those of genera a wood stick called sewak that was used 7000 years ago and is still coccus, Veillonella, Fusobacterium, Porphromonas, Haemophilis, used by some people . Sewak is an Arabic word that refers to a Treponema, Nisseria, Eubacteria, Lactobacterium, Capnocytophaga, Salvadora Staphylococcus, and Propionibacterium . Stability of the oral [9] persica used as a natural toothbrush for obtaining good oral hygie tooth-cleaning stick, made from the branches or twigs of [1-3] - microbiome composition is essential to prevent dysbiosis-a micro-
Citation: Yosra Modafer., et al. “The Effect of Sewak on Oral Microbiota Composition”. Acta Scientific Microbiology
2.6 (2019): 08-13. The Effect of Sewak on Oral Microbiota Composition
09 ne and as an oral remedy for religious and social purposes. S. per- sica plant is grown initially in Rajasthan (India), Nepal, and Malay one week apart in the morning of Friday, as a control before any two-week period. The first two mouthwash samples were collected eating or drinking. After the second sample collection, participants - Mauritania . Other names of sewak are “arak” or “miswak” in were asked to use sewak for one week besides their regular brush sia, Pakistan, Iran, Iraq, Saudi Arabia, Sudan, Ethiopia, Egypt and [9] Then, each participant was asked to collect the third mouthwash Arabic, “koyoji” in Japanese, “qesam” in Hebrew, “qisa” in Aramaic, routine, at least five times a day or frequently whenever possible. by the Greek and Roman empires, and then by ancient Egyptians sample early in the morning on Friday. All eighteen samples were and “mastic” in Latin. It was first used by Babylonians, followed centrifuged, vortexed; then, the buccal cells were suspended and and Muslims. Moreover, the mechanical cleansing, anti-plaque, an- to regular use of sewak in some previous studies. The inhibitory ti-caries, anti-bacterial, and anti-decay actions have been linked DNAstored extraction in the 80°C and freezer amplification until use. action of sewak extract against certain bacterial species that were growing on Petri dishes has been extensively studied . The The QlAamp Mini kit (Qiagen) was used for genomic DNA isola se studies were often focused on a few microbial species. No study tion from the eighteen samples, following the manufacturer’s pro [10-12] - - reported so far has examined the effect of sewak use on the com - prehensive composition of oral microbiota. Thus, this study aims cleaning solution steps were applied, and ATL buffer was added - tocol. Briefly, the bead beating lysis was done in ATL buffer. Three to comprehensively analyze oral microbiota composition, using to the cleansing number 3 solution and samples were incubated young healthy individuals, before and after the use of sewak. Samples were washed twice with AW1 and AW2. Finally, pellets 16S rRNA gene sequencing, in oral fluids samples collected from at 55 °C for 1 hour with proteinase-K (Sigma-Aldrich, 100 ug/ul).
Materials and Methods the purity and concentration of each extracted DNA were assessed were resuspended in DNase-RNase-free water (Sigma-Aldrich) and Project approval, participant enrollment and survey using a Nano Drop spectrophotometer (NanoDrop Technologies, The research was approved by the Institutional Review Board at Tennessee State University (IRB# FWA00007692). Ten healthy, Willmington, DE). The amplification of extracted DNA was done 04). randomly selected participants were recruited for this study. Writ using the NEXTflex 16S V1-V3 amplicon sequencing kit (196-4202- ten consent was obtained from each of the volunteer participants. - 16S rRNA gene sequencing Of this group, one was excluded, and three were lost to follow up. The six healthy individuals who continued in this study were from both genders aged between 24 and 34 with no symptoms of oral each mouthwash sample were done using the 16S metagenomics Phylogenetic classifications analysis of the oral microbiota in diseases, as claimed themselves. A survey questionnaire contai- sequencing library approach (on the Illumina platform, by Omega neral information that needed 10 minutes to complete was given V4 regions using a primer pair that creates a single amplicon of ning questions of health history, food habits, oral health and ge- Bioservices). The protocol includes the amplification of the V3 and to volunteers before samples collection. Written permission forms approximately ~460 bp. A full complement of Nextera XT indices were signed from all subjects after providing information about this study and details about sample collection and use of sewak. was used for library preparation. For sequencing on MiSeq, pai- red-end 300‐bp reads and MiSeq v3 reagents were used. For taxo- Sewak brand and instructions of use nomic classification, kingdom, phylum, class, order, family, genus Sewak sticks, Alfalah brand, labeled with the instruction of use and species, the Metagenomics Workflow was performed using the were shipped from Saudi Arabia to the USA. Participants were Greengenes database (http://greengenes.lbl.gov/) showing genus and species level classification in a graphical format. The Illumina requested to chew sewak tip repeatedly until the fibers appeared protocol with a benchtop sequencing system, primary analysis, and 24 hours). Then, they were asked to apply the cleaning action di like a toothbrush (the fibers were trimmed by participants every secondary analysis using BaseSpace (cloud‐based software) has rected away from the gingival margin of the teeth. - been applied to provide a comprehensive workflow for 16S rRNA amplicon sequencing. Collection criteria and processing of samples Statistical analysis
Each participant was asked to provide 3 mouthwash samples, each about 10 ml, using Scope mouthwash (original brand) in a Recorded data were statistically analyzed using a paired t-test, ANOVA and Chi-square test.
Citation: Yosra Modafer., et al. “The Effect of Sewak on Oral Microbiota Composition”. Acta Scientific Microbiology
2.6 (2019): 08-13. The Effect of Sewak on Oral Microbiota Composition
10 Results and Discussion cies of this family correspond with the healthier oral cavity . All previous studies of sewak used traditional methods to test published literature that shows that the relatively higher frequen- its inhibition activity against some selected bacterial species. In [13] contrast, this study used 16s rRNA gene sequencing to obtain a munity structure changes of oral microbiota in saliva samples comprehensive profile and comparisons of the diversity and com-
(before and after the use of sewak) of six healthy, non-smoker par- provided two samples along with his or her oral hygiene routine ticipants (aged ≥24 years, in Nashville, TN, USA). Each participant and one sample after one week of sewak treatment.
6,620,906 reads in total, 6,620,630 reads were bacteria, while 235 Around 384,344 reads per sample were obtained. Among the and 41 reads were viruses and Archaea respectively (Figure 1).
Bacterial reads were classified based on the Greengenes database dom, phylum, class, order, family, genus, and species). (http://greengenes.lbl.gov/) at different taxonomic levels (King-
Figure 2:
samplesThe distributioncontrol, and of3= the after major treatment phyla found samples. in the 18 mouthwash samples, 1=first control samples, 2=second control
Figure 1: Reads distribution in saliva samples.
Figure 3: The distribution of major families (abundance > 1%) families, 644 genera, and 1,643 species in total. The mean propor 2=second control samples, and 3= after treatment samples. We were able to detect 28 phyla, 58 classes, 110 orders, 236 tions of bacterial phyla and families among samples from all par found the 18 mouthwash samples, 1=first control samples, - ticipants was shown in Figures 2 and 3. Phyla Firmicutes had most - abundant reads, followed by Bacteroidetes, Protobacteria, and Acti- The proportions of oral bacteria species were compared betwe en samples collected before and after sewak treatment. Differences nobacteria (see Figure 2). About 50% of reads belonged to Firmicu- - tes in which Streptococcaceae was found most abundant at family applied to compare each “taxa” of bacteria between the control and level in all samples (see Figure 3), which agrees with previously were found at several taxonomic levels when paired-end t test was
Citation: Yosra Modafer., et al. “The Effect of Sewak on Oral Microbiota Composition”. Acta Scientific Microbiology
2.6 (2019): 08-13. The Effect of Sewak on Oral Microbiota Composition
11 the treatment (P < 0.05). At the species level, the proportions of markedly (P < 0.05). This bacterium is abundant in the rumen of 27 “taxa” were reduced with the sewak treatment; whereas those ruminants, playing an important role in breakdown of plant poly saccharides . The proportion of bacteria in genus Leptotrichia - proportions of the majority of bacterial species level taxa were un of 16 taxa were increased (P < 0.05, see Table 1, 2). However, the [17] changed (Data not shown). abundant in the human oral cavity. Five species of Leptotrichia are - was also decreased about 6-fold. These bacteria were moderately known from human source . They may be associated dental ca Several streptococcus species (i. e., S. vestibularis, S. fryi, S, mi- ries . Rothia dentocariossa is also moderately abundant. After lleri, S. anginosus [18] - sewak treatment for one week, the proportion of R. dentocariosa [19] ) were reduced significantly after sewak treat- S. ment (P˂0.05, Treatment
Citation: Yosra Modafer., et al. “The Effect of Sewak on Oral Microbiota Composition”. Acta Scientific Microbiology
2.6 (2019): 08-13. The Effect of Sewak on Oral Microbiota Composition
12 In contrast, the proportions of reads of Haemophilus parainflu- opportunistic pathogen . Neisseria mucosa was very abundant enzae, Neisseria mucosa, Snowella rosea, Selenomonas noxia, Geme- N. mucosa rarely [22] lla cunicula, Gemella sanguinis and Gemella haemolysans increased causes disease . Gemella sanguinis and Gemella haemolysans but increase by 160% after one-week sewak use. H. parainfluenzae were abundant in mouthwash samples in which their proportions [23] G. haemolysans is reco after one-week sewak use (P˂0.05, Table 2). gnized to cause disease occasionally . The rest of the increased was one of the very abundant oral bacteria. The proportion of H. were significantly increase after sewak use. - parainfluenzae was increased by 125% after one-week of sewak [24] use. This is often non-pathogenic, but is increasingly recognized as species after one-week sewak use were in low abundance (Table 2). Control Treatment Order Family Genus Species P mean mean Pasteurellales Pasteurellaceae Haemophilus parainfluenzae 6370 0.04 Neisseriales Neisseriaceae Neisseria mucosa 4725 12251 0.005 14387 Gemellales Gemellaceae Gemella sanguinis 2226 0.03 Gemellales Gemellaceae Gemella haemolysans 5436 0.02 3839 Flavobacteriales Flavobacteriaceae Capnocytophaga leadbetteri 127 290 0.02 1816 Clostridiales Peptococcaceae Desulfotomaculum indicum 102 124 0.007 Clostridiales Veillonellaceae Selenomonas noxia 111 0.033 Halanaerobiales Halanaerobiaceae Halanaerobium alcaliphilum 23 55 0.011 58 Chrysiogenales Chrysiogenaceae Desulfurispirillum alkaliphilum 23 55 0.03 Bacillales Planococcaceae Lysinibacillus parviboronicapiens 26 0.03 Burkholderiales Oxalobacteraceae Oxalobacter vibrioformis 13 22 0.04 18 Lactobacillales Lactobacillaceae Lactobacillus johnsonii 6 121 0.03 Burkholderiales Burkholderiaceae Burkholderia ubonensis 5 12 0.005 Chroococcales Gomphosphaeriaceae Snowella rosea 5 19 0.02 Alteromonadales Alteromonadaceae Marinobacter arcticus 4 0.01 Clostridiales Veillonellaceae Selenomonas infelix 3 16 0.0003 8 Table 2: Bacteria increased after sewak use.
sewak. Firmicutes was found to be most abundant taxon in all sam
Statistical analysis in this study shows substantial inter-indi- - the structure of the human microbiota within one site varies even cantly lower after sewak treatment were S. fryi and S. vestibularis vidual differences which confirms previous studies claiming that ples. Two streptococcus species, belonging to Firmicutes, signifi- in the same individual and variation can be detected in the heal (P < 0.05). Some bacterial species were increased after sewak, and thy people . When comparing sample 1 and sample 2, we the majority were unchanged. The microbiome approach could not - also found differences at the similar extent which suggest that the detect overall abundancy changes since the method is designed to [25,26] effect of sewak s relatively small. This could result from the short measure composition changes, not absolute changes. A greater nu period of treatment. Moreover, the number of subjects in this study mber of samples are needed to minimize the possible effect of in - - associated with sewak antimicrobial activity might become appa is relatively small, and inter-individual differences of microbiota ter-individual variability. Taking all limitations into consideration, rent when larger numbers of subjects are included. ce our understanding of sewak antimicrobial activity. - 16s r RNA metagenomics library sequencing technique will advan- Conclusion Acknowledgments
In this study, the use of 16s rRNA sequencing approach, taxo- This work is supported in part by Meharry (MMC)-Vanderbilt of oral microbiota composition in saliva samples after the use of authors are thankful to the Saudi Arabian Cultural Mission (SACM) nomic classification, and statistical analysis determined changes (VICC)- Tennessee State University (TSU) Cancer Partnership. The and Jazan University (JazanU).
Citation: Yosra Modafer., et al. “The Effect of Sewak on Oral Microbiota Composition”. Acta Scientific Microbiology
2.6 (2019): 08-13. The Effect of Sewak on Oral Microbiota Composition
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Citation: Yosra Modafer., et al. “The Effect of Sewak on Oral Microbiota Composition”. Acta Scientific Microbiology
2.6 (2019): 08-13.