Advances in Microbiology, 2024, 14, 163-174 https://www.scirp.org/journal/aim ISSN Online: 2165-3410 ISSN Print: 2165-3402 DOI: 10.4236/aim.2024.143012 Mar. 13, 2024 163 Advances in Microbiology Antimicrobial Activities of Extracts of Macrosphyra longistyla against Gram-Positive Oral Biofilm-Formers from School Children in Southwestern Nigeria and Toxicity Studies Using Brine Shrimps Chukwuemeka Emmanuel Nwankwo1,2*, Onikepe Folarin1,2, Adeleke Osho1 1Department of Biological Sciences, Faculty of Natural Sciences, Redeemer’s University, Ede, Nigeria 2African Centre of Excellence for Genomics of Infectious Diseases (ACEGID), Redeemer’s University, Ede, Nigeria Abstract The world will benefit from more effective antimicrobial agents against oral conditions arising from the actions of biofilm forming bacteria. Also, infor- mation is lacking on the oral biofilm-forming bacterial diversity in South- western Nigeria. In this study, we isolate and characterize oral biofilm pro- ducing bacteria in the oral cavities of schoolchildren in Southwestern Nigeria. We also investigate the antimicrobial properties of Macrosphyra longistyla extracts against the biofilm-formers and the toxicity of potent extracts. Sam- ples were obtained from 109 schoolchildren aged 4 - 14 years from Lagos, Oyo and Osun States. Agar well diffusion technique was used in the antimi- crobial susceptibility testing. Toxicity testing was done using brine shrimps (Artemia salina). Biofilm-formers in this study are Klebsiella sp., Streptococ- cus sp., Staphylococcus sp., and Micrococcus sp. Ethanol leaf extracts had the highest activity against all biofilm-producing bacteria. Ethanol stem bark ex- tract, which elicited activity against Klebsiella only, was found to be less toxic than the ethanol leaf extract. Staphylococcus showed >10 mm susceptibility to the ethanol and aqueous extracts of Macrosphyra longistyla. Streptococcus and Micrococcus were susceptible to the antimicrobial actions of the ethanol- ic leaf extracts. Although the ethanol extracts of the leaves had lower mini- mum inhibitory concentrations than the ethanol extracts of the stem bark, toxicity studies showed ethanol extracts of the stem-bark to be more toxic than the ethanol extracts of the leaves. In conclusion, ethanolic extracts of Macrosphyra longistyla show potential as sources of antimicrobials against gram-positive, oral biofilm-forming bacteria. How to cite this paper: Nwankwo, C.E., Folarin, O. and Osho, A. (2024) Antimicrobi- al Activities of Extracts of Macrosphyra lon- gistyla against Gram-Positive Oral Bio- film-Formers from School Children in Southwestern Nigeria and Toxicity Studies Using Brine Shrimps. Advances in Micro- biology, 14, 163-174. https://doi.org/10.4236/aim.2024.143012 Received: February 2, 2024 Accepted: March 10, 2024 Published: March 13, 2024 Copyright © 2024 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access https://www.scirp.org/journal/aim https://doi.org/10.4236/aim.2024.143012 https://www.scirp.org/ https://doi.org/10.4236/aim.2024.143012 http://creativecommons.org/licenses/by/4.0/ C. E. Nwankwo et al. DOI: 10.4236/aim.2024.143012 164 Advances in Microbiology Keywords Biofilms, Plant Extracts, Toxicity, Bacteria, Susceptibility, Antimicrobial 1. Introduction The presence and growth of microorganisms in the oral cavity contributes to the pathogenesis of diseases in the mouth and in internal organs. Oral diseases caused by biofilms include caries and periodontal disease. Biofilms are defined as aggregation of cells of microorganisms embedded in an extracellular matrix composed of single or multispecies organisms [1] [2] [3]. These conditions arise due to prolonged exposure to causal microorganisms in individuals with poor oral healthcare as children or adults [4] [5]. The microorganisms in the oral cav- ity can get disseminated to internal organs of the body. Internal diseases origi- nating from the activities of oral biofilm-producing microorganisms include ga- strointestinal diseases cardiac issues, and some rare forms cancers. Due to the need to prevent infectious diseases, and to identify causal agents of certain dis- eases, the detection of biofilm-producing bacteria can serve as a way to identify markers of underlying diseases in the host. One of the ways biofilm-producing bacteria cause harm in hosts is by the disruption of the gut microflora by the in- gestion of infectious agents [6] [7]. Sources of related diseases that arise include microorganisms spread via the fecal-oral axis. This explanation seems to throw light on the importance and strategic nature of the mouth, as an axis for the flow of both harmful and unharmful or commensal bacteria introduced into the hu- man body [8] [9]. This is one of the reasons why caries has been termed the most contagious disease. There are also investigations into the link between oral-associated biofilm-producing bacteria and diseases of the brain, by the no- tion that such microorganisms can cross the brain-barrier. The disruption of the gut microbiome by dento-gingival microorganisms also provides a link to an under- standing of the link between oral-associated microorganisms and stress-mediated illnesses [10]. When microorganisms produce biofilms, they are shielded from the action of antimicrobial agents [11]. They also adopt mechanisms that accelerate destruc- tion of cells of the mucous membranes of the mouth. In periodontal diseases, there is possibility, in severe cases, where gums and surrounding tissues and bones are affected [12] [13]. Various associations exist among bacterial species involved in oral biofilm formation or progression. They may be antagonistic to- wards each other by competing for nutrients or by feeding on another. They may also be commensal and they may co-cause multi-bacterial infections. There are numerous biofilm-producing bacteria in the oral cavity. The prevalence of mul- ti-drug resistant biofilm-producing bacteria with or without pathogenicity isl- ands is an emerging study in the biological sciences [14]. The need therefore arises to address the problems and effects of oral biofilm-producing bacteria. https://doi.org/10.4236/aim.2024.143012 C. E. Nwankwo et al. DOI: 10.4236/aim.2024.143012 165 Advances in Microbiology The first step in the control of these pathogens would be to isolate and charac- terize the microorganisms responsible for this situation. In the microbiology la- boratory, this would traditionally involve obtain oral samples (plaque, saliva or epithelial swabs) and incubation including the growth of the microorganisms on agars fortified with indicators that detect the presence of biofilm-producing bac- teria. Subsequent steps will involve antimicrobial activity with therapeutic agents which are known or which are novel [15]. Removal of biofilms is by brushing regularly and the use of chlorohexidine [16]. It is important to control the presence of oral biofilms, its progression, and pathogenesis due to microorganisms present in it. This underlies the need to find alternatives to the current aid measures. Oral care will benefit from the cre- ation of any chemical that can prevent caries formation, or the dissolution of oral biofilms. In severe cases, there might be the need to control the biofilms by mechanical removal by a dentist. In order to control the activities of oral bio- film-producing bacteria, there is the race to find novel antimicrobials which can prevent the growth of these bacteria or aid in their removal. Scientists have tried a wide variety of plants and synthetic chemicals for this purpose [17]. It is im- portant to note that in folk medicine, the use of Macrosphira longistyla as mouth cleaning device (chewing stick) is common. It is also common to use herbs along with alcohol in the morning to clean the mouth. However, due to hardness of sticks and the possibility of its destruction of gums, it is pertinent to obtain ex- tracts of promising natural products in order to concentrate and characterize them in the race to create novel oral care products with less toxicity. Macros- phyra longistyla is common in African folk medicine for use as chewing stick. However, little is documented about its activities against microorganisms, oral microorganisms and oral biofilm-forming bacteria. In our yet-to-be-published preliminary study, we characterized components of the plant and determined its preliminary antimicrobial activity. However, this study characterizes microbio- logically, stemming from the fact that it has been found to be effective, its activi- ty against oral biofilm-producing bacteria. We identify some of the bacteria re- sponsible for biofilm-formation in schoolchildren in southwestern Nigeria in order to uncover the strains unique to this environment. Therefore, this study will support ongoing research on bacterial diversity in Southwestern Nigeria and a further investigation into the antimicrobial and toxicity research in the race to discover novel antimicrobials. 2. Material and Method 2.1. Sample Collection and Tests for Biofilm Production Ethical approvals were obtained from Redeemer’s University Directorate of Re- search Innovation and Partnerships (DRIPs, Redeemer’s University’s institu- tional review board), Local Government Education Authority of Lagos State (LGEA), State Universal Education Board in Oyo state (SUBEB), Health Re- search and Ethics Committee, Ministry of Health, Osun State. Samples were col- https://doi.org/10.4236/aim.2024.143012 C. E. Nwankwo et al. DOI: 10.4236/aim.2024.143012 166 Advances in Microbiology lected after informed consent from parents or guardians. Gargled water samples from the participants (109 healthy children aged 4 - 14 years old) from 4 differ- ent schools in Lagos, Oyo and Osun states in southwestern Nigeria were ob- tained. Subjects gargled potable drinking water and released salivary exudates into sample bottles, ensuring asepsis. This method of sample collection for the purpose of biofilm research is supported by the study by [18]. This is an effective means to achieve our aim. The bottles were then labelled and transported to the laboratory where samples were enumerated and pure isolates were tested for biofilm production. The pour plate method was used to enumerate and obtain pure colonies for identification and characterization. This involved the inocula- tion of 1ml of gargled water sample onto sterile petri-dish, to which molten nu- trient agar (at 45˚C) was added. This petri-dish was then swirled clockwise and anti-clockwise to ensure uniform distribution. The petri dish containing sample was then allowed to set and labelled. Incubation at 37˚C for 18 - 24 h took place and enumeration was done. Pure isolates were then obtained and their 6-hour embryos were stored by streaking in slant method on nutrient agar in bijou bot- tles, which was corked, labelled and stored at −6˚C for use in subsequent tests. Tests were performed on pure isolates only. Inoculation of each individual rep- resentative bacterium on brain-heart infusion agar with Congo-red dye was the method used to determine biofilm production. Black coloration after 18 - 24 h incubation meant biofilm production. 2.2. Identification Biofilm Positive Bacteria Biofilm producing bacteria were subjected to morphological and biochemical identification tests. Tests included Gram-staining, gas production, sugar fer- mentation. Sugar fermentation tests were performed with bromocresol agar with yellow pigment production as positive results and bubble for gas production (Table 1). Tests were maltose, sucrose, glucose, lactose, mannitol and citrate [19]. Biochemical tests included hydrogen sulphide, Indole, Methyl-red, Voges Proskauer, urea, motility, oxidase, and catalase tests. Incubation was at 37˚C for 24 hours [20]. Table 1. Sugar fermentation of biofilm-producing isolates. Sample code Sugar Fermentation Test Maltose Sucrose Glucose Lactose Mannitol Citrate Gas 124_C a+ + + + + + + 124_A + + + + b− cN/A + V_3 + + + + − N/A + 33_B + + + + + + + VI_3 + − − − + − − a+ positive reaction. b− negative reaction. cN/A not applicable. https://doi.org/10.4236/aim.2024.143012 C. E. Nwankwo et al. DOI: 10.4236/aim.2024.143012 167 Advances in Microbiology 2.3. Obtaining Plant Extracts Fresh leaves and stem bark of Macrosphyra longistyla (Dc.) Hiern (Family: Ru- biaceae; LUH: 9431) were identified by Professor Ernest Durugbo and deposited in the Herbarium of University of Lagos. Plant parts were air-dried at room temperature for three weeks after which they were ground to powder using a homogenizer. Ethanol and aqueous extracts were obtained by introducing 250 g of plant parts in 250 ml solvent. Extracts were then placed into rotary evaporator to reduce liquid content. The extracts were then dried overnight in an oven and weighed to ascertain the yield of each extract. 2.4. Antimicrobial Susceptibility and Minimum Inhibitory Concentration (MIC) Agar well diffusion technique was used to determine susceptibility of the bacte- ria to the plant extracts and zones of inhibition were recorded. 18 H bacterial isolates were inoculated into petri dishes containing Mueller Hinton agar using sterile swab sticks. Wells were created using 6mm cork-borers and 100 μL of each extract was dispensed into each well using a micropipette and incubated at 37˚C for 24 h. Inspection was done and zones of inhibition produced by each extract were compared to determine the ones that MIC would be performed on. Minimum inhibitory concentration (MIC) was determined on the susceptible bacteria with zones of inhibition of 15 mm and above by measuring their optical density (OD) at 600 nm and by visual inspection using 96-well microtiter plate. The MIC was given as the minimum concentration of plant extract that was able to inhibit visible growth of the bacteria. Toxicity testing was performed on ex- tracts with higher antimicrobial activity (15 mm and above). 2.5. Toxicity Testing Artemia salina were hatched in artificial sea water with aeration and an electric lamp above the side of the tank for 24 hours to obtain nauplii. 10 nauplii were then aspirated by pipetting and introduced into each test tube containing serial dilutions (1, 10, 100, and 1000 PPM) of the plant extracts and incubated. Nauplii survivors were counted at 6 hours, 18 hours and 24 hours [21]. Mortality rate was calculated and the data were subjected to statistical analyses to determine the Lethal Concentration (LC50). LC50 was determined using the Quest Graph™ LC50 Calculator [22]. 3. Results and Discussion 3.1. Identification of Biofilm-Producing Isolates Ethanol extracts of the leaves and stem barks of Macrosphyra longistyla show higher activity against these biofilm-producing bacteria than aqueous extracts of same plant. On a broader scale, ethanol extract of the leaves show activity against a higher number of bacteria isolated in this study than ethanol extracts of the stem bark. This seems to justify the combination of this herb with tinctures of https://doi.org/10.4236/aim.2024.143012 C. E. Nwankwo et al. DOI: 10.4236/aim.2024.143012 168 Advances in Microbiology alcohol. Staphylococcus showed susceptibility to all extracts used in this study, suggesting that in control of this microorganism, the use of this plant can be a good option. Other microorganisms in this study showed varying degrees of susceptibility to the plant extracts. Biofilm-producing bacteria isolated in this study were identified biochemically and found out to be Klebsiella, Streptococcus, Staphylococcus and Micrococcus (Table 2). These bacteria are commonly isolated from studies on the microbi- ome of the oral cavity [23] [24] [25]. Recently, Klebsiella has been a reason of public health concern, not just in Nigeria, but globally. Multi-drug resistant strains of Klebsiella have been isolated in nosocomial infections. Hence, it is important to mitigate its actions using novel approaches [26] [27] [28] [29] [30]. Even though gram positive and gram-negative bacteria exist in different associa- tions and have complex relationships in biofilm modes in inhibiting the action of antimicrobials [31], all biofilm-producing isolates were gram-positive. The presence of Streptococcus in the oral cavities of schoolchildren in two different states, supports its abundance in studies on the oral cavities of pupils from this region. The presence of Streptococcus in oral biofilms presents a concern in its treatment and complications arising from the actions of this bacterium present a global health threat [32] [33] [34]. Staphylococcus is commonly isolated as a contaminant in nosocomial and environmental samples [35] [36] [37]. However, its biofilms are a contribution to antimicrobial resistance due to the acquisition of virulence genes and their existence in pathogenicity islands [38] [39] [40]. Drug resistant strains of Micrococcus have been identified in hospital and oral samples in Nigeria by [41] and [42]. 3.2. Antimicrobial Susceptibility of the Biofilm-Formers to the Plant Extracts All biofilm-positive isolates were susceptible to the antimicrobial action of etha- nol extracts of Macrosphyra longistyla leaves with zones of inhibition greater than 15 mm (Table 3). However, Klebsiella was especially susceptible to the ac- tion of ethanol stem bark extract. This effect led to the selection of the isolates for minimum inhibitory concentration (MIC) determination. Our results agree with other similar studies, with the plant exhibiting results similar to the action of standard antibiotics [43] [44]. The lowest MIC was exhibited by 124_C while the highest was exhibited by VI_3 (Table 4). Low MIC values indicate to the re- searcher that such compound shows great potential in drug design as it will be required in low concentration to inhibit the growth of potentially pathogenic bacteria. Antimicrobial research will benefit from further studies on the effect of the extracts on isolates with zones of inhibition which were medium but were not characterized to check their MIC. 3.3. Toxicity of Active Plant Parts High LC50 values indicate is less toxicity, especially than compounds with lower https://doi.org/10.4236/aim.2024.143012 C. E. Nwankwo et al. DOI: 10.4236/aim.2024.143012 169 Advances in Microbiology LC50 values. Data on proper scientific studies on the use of this plant in oral care is lacking. This research will therefore add to the limited existing knowledge of this plant. Toxicity assay showed the stem bark of the ethanol extract, with lower LC50 value, to be more toxic than the leaf extract (Table 5). Also, the leaf extract showed higher antimicrobial potential than the stem. If this plant’s extract should be used at all in oral care, the active compounds must be isolated from the leaf. From the data presented, therefore, it is therefore recommended the stem not be used for oral care due to its toxic nature. Table 2. Identification of biofilm-producing isolates. Sample code Phenotypic Test Performed Gram H2S Indole dMR eVP Urea Oxidase Catalase Probable organism 124_C a+ + + + + + + + Klebsiella sp. 124_A + + cN/A N/A b− − N/A − Streptococcus V_3 + + N/A N/A - − N/A − Streptococcus 33_B + - − + + + − + Staphylococcus VI_3 + + − − + N/A + + Micrococcus a+ positive reaction. b− negative reaction. cN/A not applicable. dMR Methyl red. eVP Vogues Proskauer. Table 3. Antimicrobial susceptibility results of ethanol and aqueous extracts of Macrosphyra longistyla. Sample code Extract Ethanol Leaf (mm) Aqueous Leaf (mm) Ethanol Stem-bark (mm) Aqueous Stem-bark (mm) 124_C 15.7 ± 0.54 - 15.6 ± 0.8 - 124_A 15.7 ± 1.97 7.1 ± 0.68 - 12.7 ± 0.12 V_3 15.4 ± 1.00 9.1 ± 0.35 - 6.5 ± 0.09 33_B 15.6 ± 0.10 11.4 ± 0.67 13.5 ± 0.27 10.5 ± 0.57 VI_3 15.8 ± 0.35 10.7 ± 0.58 11.1 ± 0.2 - Table 4. Minimum Inhibitory Concentration (MIC) of the highly-active plant extracts. Sample code MIC Ethanol Leaf (µg/ml) Ethanol Stem Bark (µg/ml) 124_C 2.5 0.1563 124_A 2.5 N/A V_3 2.5 N/A 33_B 2.5 N/A VI_3 >2.5 N/A N/A not applicable. https://doi.org/10.4236/aim.2024.143012 C. E. Nwankwo et al. DOI: 10.4236/aim.2024.143012 170 Advances in Microbiology Table 5. Toxicity of the active plant extracts. dMSL Conc. Ethanol Leaf (µg/mL) Effect of extract on Brine shrimps T1 (6 hrs) T2 (18 hrs) T3 (24 hrs) No. of Nauplii Survivors % Mortality aLC50 (µg/mL) 1 10 10 9 29 96.6 10 8 8 5 21 70 100 10 9 9 28 93.3 1000 8 8 6 22 73.3 422.05 MSL bConc. Ethanol Stem Bark (cppm) 1 9 9 9 27 90 10 10 10 10 30 100 100 9 9 5 23 76.6 1000 8 4 2 14 47 47.23 Control-Distilled Water (ppm) 1 10 10 9 29 96 10 10 9 9 28 93 100 10 10 10 29 96 1000 10 10 9 30 100 498.58 aLC50: lethal concentration. bConc. Concentration. cPpm: Parts per million. dMSL: Macrosphyra longistyla. 4. Conclusion Due to the burden and the problems associated with oral biofilms, results from this study become important. Biofilm producing bacteria identified in this study shows diversity in morphological and biochemical characteristics. They vary in their antimicrobial susceptibility pattern to extracts from Macrosphyra longisty- la. The extracts show in-vitro promise in mitigating the effects of these oral bio- film-producing bacteria. It is suggested that the leaves be concentrated and used in drug design. Acknowledgements We would like to acknowledge the directorate of the African Centre of Excel- lence for Genomics of Infectious Diseases (ACEGID) and the department of Bi- ochemistry, Redeemer’s University for technical support. Conflicts of Interest The authors declare no conflict of interest. https://doi.org/10.4236/aim.2024.143012 C. E. Nwankwo et al. DOI: 10.4236/aim.2024.143012 171 Advances in Microbiology References [1] Buzalaf, M.A.R., Ortiz, A.D.C., Carvalho, T.S., Fideles, S.O.M., Araujo, T.T., Mo- raes, S.M. and Reis, F.N. 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AIMS Microbiology, 8, Article No. 239. https://doi.org/10.3934/microbiol.2022019 https://doi.org/10.4236/aim.2024.143012 https://doi.org/10.32604/biocell.2023.024635 https://doi.org/10.1016/j.heliyon.2023.e16253 https://doi.org/10.1007/978-3-031-24408-7_3 https://doi.org/10.3390/microorganisms10061239 https://doi.org/10.3390/microorganisms10091783 https://doi.org/10.1016/j.scib.2023.09.040 https://doi.org/10.1016/j.jormas.2022.07.018 https://doi.org/10.3934/microbiol.2022019 Antimicrobial Activities of Extracts of Macrosphyra longistyla against Gram-Positive Oral Biofilm-Formers from School Children in Southwestern Nigeria and Toxicity Studies Using Brine Shrimps Abstract Keywords 1. Introduction 2. Material and Method 2.1. Sample Collection and Tests for Biofilm Production 2.2. Identification Biofilm Positive Bacteria 2.3. Obtaining Plant Extracts 2.4. Antimicrobial Susceptibility and Minimum Inhibitory Concentration (MIC) 2.5. Toxicity Testing 3. Results and Discussion 3.1. Identification of Biofilm-Producing Isolates 3.2. Antimicrobial Susceptibility of the Biofilm-Formers to the Plant Extracts 3.3. Toxicity of Active Plant Parts 4. Conclusion Acknowledgements Conflicts of Interest References