E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668
ORIGINAL RESEARCH ARTICLE
*1Garba S.,1Sulaiman M. T., 1Muhammad S., 1Sani A. and 1Mohammed R. B.
1Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences Ahmadu Bello University, Zaria, Kaduna, Nigeria
Salvadora persica L., commonly referred to as Miswak, is a chewing stick commonly used to maintain dental hygiene. The aim was to determine the antibacterial effectiveness of the methanol crude extract of S. persica against gram-positive bacteria obtained from orally infected individuals who were registered at the University Health Clinic of Ahmadu Bello University. A total of twenty (20) samples were collected from patients who were diagnosed with oral infections. These samples underwent traditional biochemical and microbiological testing. The antibiotic susceptibility test was conducted using the Kirby-Bauer disc diffusion method, and the findings were interpreted following the CLSI standards. The extraction of Salvadora persica L. was carried out using cold maceration with methanol. The crude extract was evaluated using the agar well diffusion and broth dilution techniques. Phytochemical screens were conducted using established procedures. The findings revealed that Staphylococcus aureus had the highest prevalence rate of 60%, whereas Streptococcus sp. had a prevalence rate of 10%. A high level of resistance was detected for amoxicillin (100%), chloramphenicol (85%), ciprofloxacin (60%), ceftriaxone (50%), and gentamycin (47.5%). The cold maceration extraction process yielded in % crude extract yield of 11.59%. The phytochemical elements were saponin, tannin, alkaloid, flavonoid, phenol, heart glycoside, carbohydrate, steroid, and terpenoid. An area of inhibition measuring 18-22mm and a minimum inhibitory concentration (MIC) of 25mg/ml were employed to detect a significant degree of activity. The methanolic extract of Salvadora persica L. has shown significant therapeutic efficacy against gram-positive bacteria recovered from orally infected patients.
Key terms: Gram-positive bacteria, infection of the oropharynx, antibiotic
Oral infections and dental lesions are important global health issues that impact individuals globally. Maintaining good hygiene is the most effective way to prevent periodontal disease and dental infections. A variety of equipment and substances are accessible to uphold and safeguard dental and oral well-being, such as toothbrushes, mouthwash, and toothpaste (Kheddouma et al., 2021). Oral health conditions have been widespread since ancient times, and their frequency has notably risen due to the heightened consumption of processed sugar (Bifulco et al., 2016). W.D. Miller was the first to propose the presence of oral microorganisms in the human oral cavity that can convert carbohydrates into acid, leading to the formation of tooth decay. In 1949, J.K. Clarke successfully isolated the initial strain of Streptococcus mutans (Ferdpus et al., 2019).
Different studies have linked several species to oral infections (Ferdpus et al., 2019). These species
include Streptococcus mutans, Prevotella intermedia, Bacteroides
forsythus, ampylobacter rectus, Actinobacillus actinomycetemcomitans,
Eikenella corrodens, Porphyromonas gingivalis, Eikenella corrodens,
Eubacterium species, Fusobacterium nucleatum, Fusobacterium nucleatum,
Streptococcus salivarius, Streptococcus sanguinis, Streptococcus mitis,
Eubacterium species and oral spirochetes like Treponema
denticola.
Tooth infections, such as dental caries and periodontal disorders, occur
when pathogenic bacteria multiply and spread in the oral cavity. Without
treatment, these infections can cause significant discomfort, agony, and
even problems that affect the entire body. An abscessed tooth, also
known as a periapical abscess, occurs when an infected tooth develops a
cavity filled with pus in its root (Rasouli et
al., 2014). This disorder can potentially spread to the
adjacent bone and teeth. As a result of the emergence of antibiotic
resistance in microorganisms, scientists have been motivated to explore
new antibacterial chemicals from various sources, such as medicinal
plants. Kheddoumaet al. (2021) have
shown that many plant extracts have substantial therapeutic efficacy
against infectious pathogens. Using natural plant extracts for mouthwash
and oral hygiene has gained considerable attention in the last twenty
years. Salvadora persica, also called Miswak, Arak, or
toothbrush tree, is an extensively utilized chewing tool within the
Muslim community. The World Health Organisation (WHO) has endorsed using
the fibrous branches of S. persica for oral hygiene, given that
they have been found to protect against specific oral pathogens(Rasouli et al., 2014).
The Salvadora Stem Collection and Identification involved acquiring Salvadora persica stems from Ahsan Islamic chemist Gwammaja Kano specifically to use them as chewing sticks. The samples were initially gathered and treated in Pakistan by M SHAN and subsequently recognized in the herbarium of the Department of Botany, Ahmadu Bello University, Zaria, by DrNamadiSunusi, with the specific identification number: ABU026607.
The stick was rinsed with distilled water, divided into small segments, and left to air-dry at room temperature for two weeks. It was then diminished in size into smaller components. The methanol extract was obtained by soaking 500g of fragmented Salvadora persica in 2.5L of pure methanol for 72 hours. After filtration, the filtrate was evaporated using a vacuum evaporator at a temperature of 40°C. The extract was preserved in a hygienic plastic container and refrigerated until needed. Before testing, the miswak extract was reconstituted in distilled water to achieve a final 200mg/ml concentration. Subsequently, the same method was used to generate a series of dilutions ranging from 200mg/mL to 6.25mg/mL.
Evaluation of phytochemical constituents such as cardiac glycosides, steroids, flavonoids, tannins, alkaloids, anthraquinones, saponins, etc. The results were determined using conventional techniques outlined by (Musa, 2005; Sofowora, 2008; Evans, 2009).
Twenty-three (23) samples were collected from oral-infected patients who visited the Ahmadu Bello University Medical Center's Dental Clinic using antiseptic swab sticks. The samples were conveyed to the Department of Pharmaceutical Microbiology microbiology laboratory, where they were inoculated into a sterile nutrient broth and incubated at a temperature of 37°C for 24 hours.
The isolation procedure involved pouring freshly made nutrient agar into a sterile petri plate and allowing it to harden. Afterward, the inoculating loop was sterilized, and a loopful of the 24-hour cultured nutrient broth was evenly streaked over the nutrient agar. The plate was incubated at 37°C for 24 hours (Supriya, 2023).
Fresh blood agar plates were prepared. A single colony of 24-hour agar culture was picked and streaked onto the sterile blood agar plate using a sterilized loop, which was then incubated at 37°C for 24 hours.
These were identified using microbiological standard protocols, microscopic examination of cell shape and structure (Gram's Staining), and biochemical assays, such as the slide and tube reaction test for catalase and coagulase, as well as growth on mannitol salt agar (Cheesebrough, 2010).
Modified Kirby-Bauer disc agar diffusion tests were conducted on each of the previously identified isolates using the Clinical Laboratory Standard Institute suggested technique. The experiment utilized a disc that included the following antibiotics: ciprofloxacin (5μg), gentamicin (10μg), chloramphenicol (30μg), amoxicillin (30µg), and ceftriaxone (30µg) (Oxoid Ltd. Basingstoke, London).
The antibacterial activity of Salvadora Stem extracts was assessed using the Agar well Diffusion Method. The antimicrobial efficacy of the extract derived from Salvadora persica was assessed using the cup plate method on Mueller Hinton agar. The agar plate was pre-saturated with the isolated Staphylococcus aureus. A total of six perforations were made on the agar plate using a sterilized cork borer. Afterward, a small amount of liquefied agar was placed into the well to seal the bottom. Five drilled holes with a diameter of 6mm were utilized to introduce the Salvadora persica extract at five separate concentrations: 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml, and 6.25mg/ml. A conventional antibiotic, Ciprofloxacin 5µg/ml, was placed into the sixth opening to serve as a control. The plate was left unattended for one hour to ensure proper diffusion of the pre-diffusion period into the matrix. Afterward, it was placed in an incubator at 37°C for 24 hours. The zone of inhibition was identified following the identical process conducted using the isolated Streptococcus as the inundated organism.
The data obtained in this investigation was displayed as mean values and proportions in the relevant tables and figures.
S. aureus was determined to be the most common pathogen, with a prevalence rate of 60%, followed by Streptococcus species at 10%, as depicted in Figure 1. Amoxicillin demonstrated complete resistance (100%), while chloramphenicol exhibited a resistance rate of 85%. Ciprofloxacin showed a resistance rate of 60%, ceftriaxone had a resistance rate of 50%, and gentamycin had a resistance rate of 47.5%. These resistance rates are depicted in Figure 2. The cold maceration extraction resulted in a crude extract yield of 11.59%. The following phytochemical compounds were identified: saponin, tannin, alkaloid, flavonoid, phenol, cardiac glycoside, carbohydrate, steroid, and terpenoid (see Table 1). The 18-22mm Zone of Inhibition (Figure 3) and 25mg/ml Minimum Inhibitory Concentration (MIC) (Figure 4) showed significant effectiveness.
Figure 1: Distribution of Gram-positive
F
igure 2:
Antibiotic resistant profile of Gram-positive
isolates.
Table 1: Preliminary Phytochemical Screening of Salvadora persica extract
| Phytochemicals | Salvadora persica MeOH |
| Carbohydrate | + |
Flavonoids Steroids/ Terpenoids Saponins Cardiac glycoside Phenols Tannins Alkaloids |
+ + + + + + + + |
Figure 3: In vitro antibacterial activity of Salvadora persica extract Gram-positive
Figure 4: MIC OF Salvadora persica Extract against of Gram-positive
Gram-positive bacteria such as Streptococcus spp, S. aureus, Bacillus spp, Enterococcus spp, and Actinobacillus are commonly found in the oral microbiota. This study found that S. aureus was the most common gram-positive bacteria, followed by Streptococcus species. This agrees with the study carried out in Minna, Northern Nigeria, as reported by Daniyan et al. in 2011. Also, India (Umesheppa et al., 2021) and Pakistan (Jamil et al., 2020) reported the same. On the contrary, studies in southern Nigeria (Enitan et al., 2021; Philips et al., 2021) and Cameroon (Bissong et al., 2014) revealed that Streptococcus spp were the most predominant gram-positive bacteria isolated. Others indicated Bacillus spp (Sakthivel et al. 2016) and Enterococcus spp (Zawadzki et al. 2016). However, the differences in organisms can be ascribed to various factors, such as the sample site and size, the duration of the investigation, the severity of the infections, and the geographical location in which the research was conducted. High resistance observed in this study with amoxicillin, chloramphenicol, and ciprofloxacin was also reported in other studies carried out in Nigeria (Daniyan et al., 2011; Philips et al., 2021). Furthermore, this is similar to the findings in Ghana (Donkar et al., 2020), India (Batabyel et al., 2012), and Korea (Kim et al., 2015). However, this is also dissimilar to the results of previous studies conducted in Ogun, Nigeria (Enitan et al., 2021), Pakistan (Jamil et al., 2020), and Poland (Garbacz et al., 2021), which showed that the isolates were susceptible to the same antibiotics. The high prevalence of antibiotic resistance can be ascribed to the selective pressure caused by frequent improper usage, excessive administration of antibiotics, and insufficient sanitary conditions in hospitals (Abdulaziz et al., 2022). In addition, the phytochemical contents discovered in this study were similar to those found by Adigun et al. (2023) and in ethanol and aqueous extracts (Abdallah et al., 2015). However, the therapeutic and physiological qualities of the plants are ultimately dictated by the presence of these phytochemicals. The antibacterial susceptibility test revealed activity against the isolates at different concentrations. These findings align with the results of previous studies conducted by Al-Ayed et al. in 2016 and Adigun et al. in 2023, which reported efficacy against multidrug-resistant S. aureus and Streptococcus. Adigun et al. (2023) reported high activity against S. aureus, with a similar minimum inhibitory concentration (MIC) of 25 mg/ml as seen in our study, compared to Streptococcus. Kumar et al. (2016) reported the efficacy of the same extract against Bacillus subtillis. In addition, other researchers (El-sherbiny et al., 2023) have reported that the petroleum ether extract of the same plant exhibits antibiofilm action and is effective against beta-lactam-resistant Streptococcus.
The methanolic extract of Salvadora persica L. has shown significant efficacy against gram-positive oral pathogens, making it a promising option for treating oral infections and promoting oral hygiene.
The extract should undergo further examination, including HPLC-MS and column chromatography fractionation, to discover active chemicals and isolate their constituents.
The authors express their profound gratitude for the invaluable technical support and assistance rendered by the Pharmaceutical Microbiology, Pharmacognosy, and Drug Development faculty members at the Faculty of Pharmaceutical Sciences A.B.U Zaria and Ahmadu Bello University Medical Centre.
The authors confirm that they do not have any conflicts of interest.
Abdallah, Emad & Al-Harbi, Khalid. (2015). Phytochemical screening and antibacterial activity of crude aqueous and ethanol extracts of Salvadora persica L. Stem (Miswak) from Saudi Arabia. The Journal of Phytopharmacology. 4. 243-247. [Crossref]
AbdulAziz Z. A., Onaolapo J. A., Ibrahim Y. K. E., Olayinka B. O., Abdulaziz M. M. (2022). Prevalence and Antimicrobial Resistance Profile of Methicillin Resistant Staphylococcus aureus isolates from Wound Infections in Zaria, Nigeria. Journal Of Current Biomedical Research, 2: 475-489. [Crossref]
AdigunT, Muhammed M. Funmilayo A., Alli A.,Fagboro O., Oluwatosin, O.,Akinsehinwa S.,Oyebola A, (2023). Antimicrobial Evaluation of Salvadora persica Methanolic Stem Extract and In vivo Modelling of its Identified Constituting Compounds Revealed Antibacterial Effect of 2-[(5Iodosalicylidene)hydrazino]-4-morpholino-6-(1-pyrrolidinyl)-1,3,5-triazine in the Treatment of Dental Plague. [Crossref]
Al-Ayed MS, Asaad AM, Qureshi MA, Attia HG, AlMarrani AH. (2016). Antibacterial Activity of Salvadora persica L. (Miswak) Extracts against Multidrug Resistant Bacterial Clinical Isolates. Evid Based Complement Alternat Med:7083964. [Crossref]
Batabyal B. Shibendu B. Sukanta C. Priti D. And Navonil S. (2012). Prevalence and drug sensitivity pattern of staphylococcus aureus in post-operative surgical oral & maxillofacial infections. International journal of life science and pharma research. 2 (4), 150-158
Bifulco, M., Amato, M., Gangemi, G., Marasco, M., Caggiano, M., Amato, A., Psanti, S. (2016). Dental care and dentistry practice in the Medieval Medical School of Salerno, 221,87-89. [Crossref]
Bissong M. E. A., fon, P. N., kamga, F. H. L. & Akenji1, T. N. (2014). Microbiological profile of oral infections in diabetic Patients and non-diabetic controls in the southwest, Cameroon. African journal of clinical and experimental microbiology. 15(3): 138-143. [Crossref]
Cheesebrough, M. (2010). District Laboratory Practice in Tropical Countries. Second edition update, Part 2: Cambridge University Press, 137-188, 395-396.
Daniyan, S.Y., &Abalaka, M. (2011). Prevalence and Susceptibility Pattern of Bacterial Isolates of Dental Caries in a Secondary Health Care Institution, Nigeria. shiraz e medical journal, 12, 135-139.
Donkor ES, Kotey FC. Methicillin-Resistant Staphylococcus aureus in the Oral Cavity: Implications for Antibiotic Prophylaxis and Surveillance. Infect Dis (Auckl). 2020. 14;13:1178633720976581. [Crossref]
Elsherbiny, Gamal &Gazelly, Ahmed & Sharaf, Mohamd&Moghannem, Saad & E, Shehata & K. A. Ismail, Mahmoud & El-Hawary, Ahmad. (2022). Exploitation of the Antibacterial, Antibiofilm and Antioxidant Activities of Salvadora Persica (Miswak) Extract. Journal of Bioresources and Bioproducts. 8. 10.1016. [Crossref]
Enitan, Comfort & Enitan, Samson &Adeolu, Oluremi&Okeleke, Ochei&Akele, Richard &Usiobeigbe, Osahon& Emmanuel, Ileoma&Tajudeen, Rukayah& Samson, Seyi. (2020). Assessment of Oral Bacterial Profile and Antibiogram of Patients Attending Dental Clinic of a Private Tertiary Hospital in Ogun State, Nigeria. 5. 11-23. [Crossref]
Evans, W. C. (2009).„„Trease and Evans pharmacognosy‟‟, 16th edition, W.B.Saunders Ltd., London, Pp. 10 – 11
Ferdpus, M. J., Ahmed, T. (2019). Microbiological study of oral cavity of patients seeking dental treatments in Dhaka Metropolis, Bangladesh. Iran Journal of Medical Microbiology, 13(4),284-293. [Crossref]
Garbacz K, Kwapisz E, Piechowicz L, Wierzbowska M. Staphylococcus aureus Isolated from the Oral Cavity: Phage Susceptibility in Relation to Antibiotic Resistance. Antibiotics. 2021; 10(11):1329. [Crossref]
Jamil, Johar& Zaman, Khuzaifa& Iftikhar, Ali & Ullah, Saad & Khan, Ahmad & Wazir, Kalsoom& Ullah, Noor & Ullah, Zakir. (2019). Prevalence and antimicrobial susceptibility profile of MRSA and VRSA strains isolated from out -door patients of Swabi region, Pakistan. International Journal of Biosciences (IJB).
Johar Jamil, Khuzaifa Zaman, Iftikhar Ali, Saad Ullah, Faheem Ahmad Khan, Kalsoom*, Noor Ullah, Zakir Ullah (2019). Prevalence and antimicrobial susceptibility profile of MRSA and VRSA strains isolated from outdoor patients of Swabi region, Pakistan. International Journal of Biosciences, Vol. 14, No. 1, p. 156-161, 2019. [Crossref]
Kheddouma, A., Cherraben, Y. (2021). In vitro antimicrobial activity of Salvadora persica and Juglans regia extracts against microbial strains from oral cavity. Biocatalysis and Agricultural Biotechnology, 33(10),2003. [Crossref]
Kim GY, Lee CH. Antimicrobial susceptibility and pathogenic genes of Staphylococcus aureus isolated from the oral cavity of patients with periodontitis. J Periodontal Implant Sci. 2015. 45(6):223-8. [Crossref]
Kumar, Sanjay &, Navneet & Gautam, Shiv & Kumar, Vinod. (2016). Preliminary Phytochemical Screening and Antimicrobial Activity of Salvadora persica Linn. Extracts against Oral Pathogens. Fungal Genomics & Biology. 7. 1-4. [Crossref]
Philips, Orhue& Timothy, Omoregie&Charlse, Idehen&Osamuyimen, Iserhienrhien. (2021). The Incidence of Bacterial Isolates Among Dental Caries Patients Attending Clinic AtIrrua Specialist Teaching Hospital, Irrua, Nigeria. Sumerianz Journal of Medical and Healthcare. 166-171. [Crossref]
Rasouli GAR, Rezaei A, Mohseni SSH, Yaghoobee S, Khorsand A, Kadkhoda Z, MoosaviJazi M, Rokn Amir R. (2014). Inhibitory activity of Salvadora persica extracts against oral bacterial strains associated with periodontitis: An in-vitro study. Journal of Oral Biology and Craniofacial Research, 4(1),19-23. [Crossref]
Sakthivel M., Ayyasamy P. M, Arvind Prasanth D. (2016). Identification and antimicrobial susceptibility testing of pathogenicMicro-organism from dental patients. Asian Journal Pharmacy Clinical Research, 9 (2),226-230. [Crossref]
Sofowora, A.(2008).Medicinal Plants and Traditional Medicine in Africa. Spectrum Books Limited, 3rd Edition. Ibadan, Nigeria, Pp.200-202.
Supriya N. (2023). Biology Reader; Isolation of bacteria.
Temidayo Olamide Adigun, Maikarfi Muhammed, AdisaMofoluwaso Funmilayo, AbdulhameedOluwatomi Alli, FagboroOluwayemisi Olufunke, Oyebamiji Samson Oluwatosin, Akinsehinwa Samuel Ayodeji, Aina Kehinde Oyebola 2023. Antimicrobial Evaluation of Salvadora persica Methanolic Stem Extract and In vivo Modelling of its Identified Constituting Compounds Revealed Antibacterial Effect of 2-[(5-Iodosalicylidene) hydrazino]-4-morpholino-6-(1-pyrrolidinyl)-1,3,5-triazine in the Treatment of Dental Plague. BioRxiv.546444;
Umeshappa H, Shetty A, Kavatagi K, Vivek GK, Vaibhav N, Mohammed I. Microbiological profile of aerobic and anaerobic bacteria and its clinical significance in antibiotic sensitivity of odontogenic space infection: A prospective study of 5 years. Natl J Maxillofac Surg. 2021 Sep-Dec;12(3):372-379. [Crossref]
Zawadzki PJ, Perkowski K, Padzik M, Mierzwińska-Nastalska E, Szaflik JP, Conn DB, Chomicz L. Examination of Oral Microbiota Diversity in Adults and Older Adults as an Approach to Prevent Spread of Risk Factors for Human Infections. Biomed Res Int. 2017; 2017: 8106491. [Crossref]