Phenotypic Analysis, Molecular Characterization, and Antibiogram of Caries-Causing Bacteria Isolated from Dental Patients

Dental caries is a biofilm-mediated, sugar-driven, multifactorial, dynamic disease that results in the phasic demineralization and remineralization of dental hard tissues. Despite scientific advances in cariology, dental caries remains a severe global concern. The aim of this study was to determine the optimization of microbial and molecular techniques for the detection of cariogenic pathogens in dental caries patients, the prevalence of cariogenic bacteria on the basis of socioeconomic, climatological, and hygienic factors, and in vitro evaluation of the antimicrobial activity of selected synthetic antibiotics and herbal extracts. In this study, oral samples were collected from 900 patients for bacterial strain screening on a biochemical and molecular basis. Plant extracts, such as ginger, garlic, neem, tulsi, amla, and aloe vera, were used to check the antimicrobial activity against the isolated strains. Synthetic antimicrobial agents, such as penicillin, amoxicillin, erythromycin, clindamycin, metronidazole, doxycycline, ceftazidime, levofloxacin, and ciprofloxacin, were also used to access the antimicrobial activity. Among 900 patients, 63% were males and 37% were females, patients aged between 36 and 58 (45.7%) years were prone to disease, and the most common symptom was toothache (61%). For oral diseases, 21% used herbs, 36% used antibiotics, and 48% were self-medicated, owing to sweets consumption (60.66%) and fizzy drinks and fast food (51.56%). Staphylococcus mutans (29.11%) and Streptococcus sobrinus (28.11%) were found as the most abundant strains. Seven bacterial strains were successfully screened and predicted to be closely related to genera S. sobrinus, S. mutans, Actinomyces naeslundii, Lactobacillus acidophilus, Eubacterium nodatum, Propionibacterium acidifaciens, and Treponema Pallidum. Among plant extracts, the maximum zone of inhibition was recorded by ginger (22.36 mm) and amla (20.01 mm), while among synthetic antibiotics, ciprofloxacin and levofloxacin were most effective against all microbes. This study concluded that phyto extracts of ginger and amla were considered suitable alternatives to synthetic antibiotics to treat dental diseases.


Introduction
The oral cavity (the first part of the alimentary canal) is responsible for three primary functions: digestion, communication, and respiration. The structure of the oral cavity is critical in speech, perception of taste, and the first stages of digestion [1]. The oral cavity The intricate ecology of microorganisms in the human mouth is made up of hundreds of bacterial species. There is a lot of information out there on the role of particular species in pathogenesis; however, the system-level processes that lead to illness are still a mystery [25]. Based on studies, from the complex oral microbial flora some bacterial stains (Streptococcus sobrinus, Staphylococcus mutans, Actinomyces naeslundii, Lactobacillus acidophilus, Eubacterium nodatum, Propionibacterium acidifaciens, and Treponema pallidum) were selected due to least study present in the selected area of study.

Bacterial Isolation and Identification
Plaque sample (0.1 mL) was streaked on sterile 5% blood agar and Mitis Salivarius Bacitracin (MSB) Agar, incubated at 37 • C for 24 h and 48 h. Pure cultures were produced by sub-culturing a single colony on fresh blood agar and MSB agar and cultivating the resultant cultures. A pure colony was suspended in saline and a loopful of this suspension was streaked on blood agar, incubated at 37 • C for 24 h [26]. Selective colonies were picked for Gram staining after observing colony morphology and were evaluated for the biochemical reaction using a commercially available rapid bacterial detection kit (API-Biomeurix France) [27,28].

Nucleic Acid Extraction and Estimation and PCR Amplification
Bacterial nucleic acid was extracted and estimated using the standard protocols of Wright et al. (2017) [32] and Wilson (2001) [33] and User Manual Nano-Drop 2000, respectively. Specific primers were designed for each isolate based on the previously published literature (Table S1). The bands of specific lengths were visualized by gel electrophoresis.
PCR (50 µL) contained 10 ng of DNA, 1 µL of PCR buffer,1.5 U of Taq polymerase, 0.2 µL of each primer (Table S1), 200 lM of each dNTPs, and 1.75 mM of MgCl 2 . The DNA amplification conditions were initial denaturation at 95 • C for 5 min, 35 cycles of 95 • C for 1 min, 55 • C for 40 s, 72 • C for 1 min 40 s, and final extension at 72 • C for 10 min. The PCR products were analyzed on 1% agarose gel electrophoresis.

Determination of Zone of Inhibition by Herbal Extracts and Synthetic Agents
Agar well diffusion technique [72] was used to measure the antibacterial activity of the plant extract tested against each isolated bacterial strain. The freshly isolated colony of bacteria was suspended in sterile saline to achieve a turbidity of 0.5 McFarland standard after being isolated overnight, and 0.1 mL of this suspension was applied to the Muller Hinton agar. Each medicinal extract (aqueous, methanolic, and ethanolic) was added to wells (8 mm in diameter) at concentrations of 25 µg/mL, 50 µg/mL, 75 µg/mL, and 100 µg/mL and incubated at 37 • C for 24 h. The zone of inhibition was determined in millimeters (mm). Triplicates of each extract were taken. Selected antibiotics, such as Penicillin and Amoxicillin 25 µg/mL each; Erythromycin and Clindamycin 5 µg/mL each; Metronidazole, Ceftazidime, Levofloxacin, and Ciprofloxacin 30 µg/mL each; and Doxycycline 5 µg/mL, were also used with the plant extracts to measure the antibacterial activity.

Statistical Analysis
Graph Pad Prism 8, Statistica version 8.1, and Microsoft Excel software (2018) were used to analyze the data statistically. Results were displayed in mean ± standard deviation of triplicate values for each test. The confidence level was set at 95% for the three-way ANOVA and values were considered significant when p < 0.05.

Usage of Medicine Other Than Prescription
Among all study participants, 32.22% (N = 290) did not ever self-medicate to treat dental problems, while a large number (48%, N = 432) self-medicated when they had any oral infection. Additionally, 11.67% (N = 105) sometimes self-medicated, while only 8.11% (N = 73) rarely used medicine as a treatment for dental problems on their own ( Figure 2).

Usage of Medicine Other Than Prescription
Among all study participants, 32.22% (N = 290) did not ever self-medicate to treat dental problems, while a large number (48%, N = 432) self-medicated when they had any oral infection. Additionally, 11.67% (N = 105) sometimes self-medicated, while only 8.11% (N = 73) rarely used medicine as a treatment for dental problems on their own ( Figure 2).

Usage of Medicine Other Than Prescription
Among all study participants, 32.22% (N = 290) did not ever self-medicate to treat dental problems, while a large number (48%, N = 432) self-medicated when they had any oral infection. Additionally, 11.67% (N = 105) sometimes self-medicated, while only 8.11% (N = 73) rarely used medicine as a treatment for dental problems on their own ( Figure 2).

Comparison between Herbal Treatment and Suggested Dentist Treatment Plan
Herbal medicine as a traditional and local treatment is used for various diseases. The a itudes towards the best treatment for dental caries were tried to be determined by the participants. Figure 7 shows the detailed a itude of participants while comparing herbal medicine with prescribed medicine.

Comparison between Herbal Treatment and Suggested Dentist Treatment Plan
Herbal medicine as a traditional and local treatment is used for various diseases. The attitudes towards the best treatment for dental caries were tried to be determined by the participants. Figure 7 shows the detailed attitude of participants while comparing herbal medicine with prescribed medicine.

Selection of Bacterial Strains
Based on the microscopic and macroscopic analysis, such as colony morphology, Gram staining, biochemical, and physiological tests, seven bacterial strains, DKF 001, DKF 002, DKF 003, DKF 004, DKF 005, DKF 006, and DKF 007, were isolated and predicted to be closely related to the genera S. sobrinus, S. mutans, A. naeslundii, L. acidophilus, E. nodatum, P. acidifaciens, and T. Pallidum, respectively. Furthermore, gene sequencing of the strains is required to confirm the genera and exactly find out the species belonging to these genera.

Isolation of Bacterial Strains, Gram Staining, and Morphological Characteristics
The growth pa ern of bacterial isolates on MSB, blood agar, BHI agar, and SB-20M agar was observed for each strain ( Figure 8; Table 3). The Gram-staining pa ern, including color, shape, and size, was observed for each strain.

Selection of Bacterial Strains
Based on the microscopic and macroscopic analysis, such as colony morphology, Gram staining, biochemical, and physiological tests, seven bacterial strains, DKF 001, DKF 002, DKF 003, DKF 004, DKF 005, DKF 006, and DKF 007, were isolated and predicted to be closely related to the genera S. sobrinus, S. mutans, A. naeslundii, L. acidophilus, E. nodatum, P. acidifaciens, and T. Pallidum, respectively. Furthermore, gene sequencing of the strains is required to confirm the genera and exactly find out the species belonging to these genera.

Isolation of Bacterial Strains, Gram Staining, and Morphological Characteristics
The growth pattern of bacterial isolates on MSB, blood agar, BHI agar, and SB-20M agar was observed for each strain ( Figure 8; Table 3). The Gram-staining pattern, including color, shape, and size, was observed for each strain. spore-forming, and non-motile. DKF 001 showed purple-colored colonies, spherical shape, appear in pairs or chains, and were non-spore-forming and non-motile. The culture conditions depicted that this strain was anaerobic. DKF 002 retained a purple stain and appeared as cocci, arranged in pairs or short chains. DKF 003 were rod-shaped. DKF 004 were rod-shaped coccobacilli, having clubbed ends with branching. DKF 005 were rod-shaped with branches. The growth conditions depicted it as an anaerobe. DKF 006 were approximately 2.6 µm long and 0.75 µm wide and exhibited pigment-producing ability. DKF007 were Gram-negative spirochete, microaerophilic, thin, corkscrew-shaped, 6-20 µm long, 0.1-0.2 µm wide, and tightly coiled.

Biochemical Identification
Enzyme Activity Analysis DKF 003 showed highly positive results for esculin, urease, starch, and OPNG hydrolysis analysis. It was able to reduce nitrate, and hydrogen sulfide gas was produced and positive for phenylalanine dehydrogenase. The enzymatic activity of other isolates is discussed in Table 4.

Biochemical Identification Enzyme Activity Analysis
DKF 003 showed highly positive results for esculin, urease, starch, and OPNG hydrolysis analysis. It was able to reduce nitrate, and hydrogen sulfide gas was produced and positive for phenylalanine dehydrogenase. The enzymatic activity of other isolates is discussed in Table 4. Table 4. Enzyme activity analysis of isolates from oral microbial flora. NEA, no enzymatic activity; ONPG, O-nitrophenyl-beta-D-galactopyranoside; MR, methyl red; VP, Voges-proskauer; PAD, phenylalanine deaminase.

Physiological Analysis of Isolated Strains
All isolated strains showed positive growth under 2% and 5% NaCl concentrations (except DKF 001 and DKF 007 for 5%), while DKF 005 exhibited positive growth at all concentrations (Table 6). The catalase-negative test indicated that DKF 001 and DKF 002 were streptococci and were able to grow on Mitiis-Salivarius (MS) agar. These strains' growth on SB-20M media differentiated them as S. mutans and S. sobrinus, as reported by Saravia et al. [73].

Physical Properties of Extracts
The results of plant extracts' physical features, such as color, odor, and consistency, are given in Table 7. The percentage of plant extracts in aqueous, methanol, and ethanol solvents was described in Table 8. The maximum yield of garlic was seen in methanol (34%). In ethanol, the yield was found to be 32.93% and in water, it has only a 26.07% yield. Methanolic extract (62.93%) > ethanolic extract (43.6%) > aqueous extract (56.23%) were the yields of ginger. In Neem, the trend of yield was ethanolic extract (40.87%) > methanolic extract (35.47%) > aqueous extract (32.47%). Tulsi, amla, and aloe vera showed maximum yield in aqueous media (47.33%, 43.14%, and 51.27%, respectively).

Phytochemical Analysis of Extracts
The preliminary phytochemical screening revealed the presence of alkaloids and saponins in all plant extracts. Carbohydrates, tannins, flavonoids, cardiac glycosides, fats and fixed oils, proteins, and amino acids were also found in different extracts of the desired plants; details of phytochemicals have been given in Table 9.

Alkaloids Detection
Methanolic and ethanolic extracts of garlic have alkaloids, while in ginger, only ethanolic extract showed the presence of alkaloids. Both aqueous and methanolic extracts of neem showed the presence of alkaloids. Alkaloids were found in the ethanolic extract of tulsi and in the aqueous extracts of aloe vera.

Detection of Carbohydrates, Proteins, and Amino Acids
All the extracts of herbal plants, except the aqueous extract of neem, aqueous and methanolic extracts of tulsi, methanolic and ethanolic extracts of amla, and methanolic extract of aloe vera, showed the presence of carbohydrates. Only the aqueous extract of garlic and amla, ethanolic extract of ginger, amla, and aloe vera, and methanolic and ethanolic extracts of neem and tulsi showed the presence of proteins. The aqueous extracts of garlic, ginger, neem, and amla, methanolic extract of neem, tulsi, and aloe vera, and ethanolic extract of tulsi showed the presence of amino acids.

Detection of Saponins, Tannins, and Flavonoids
The aqueous extract of ginger, methanolic extract of neem, and ethanolic extracts of tulsi, amla, and aloe vera showed negative results during the saponins detection in the extracts of herbal plants. Tannins were also not present in the aqueous extract of garlic, ginger, and neem, methanolic extracts of amla and tulsi, and ethanolic extracts of garlic and aloe vera. Flavonoids were present in most of the extracts, except the methanolic extract of garlic, neem, tulsi, and amla; among the aqueous extracts, flavonoids were absent in ginger, amla, and aloe vera. All ethanolic extracts had tannins.

Detection of Cardiac Glycosides, Fats, and Fixed Oils
All the garlic extracts were positively detected for cardiac glycosides, while only the methanolic extract of ginger had cardiac glycosides. In neem and tulsi, except the aqueous extract, the others have cardiac glycosides. In amla, only ethanolic extract showed positivity to the cardiac glycosides. Aqueous and ethanolic extracts of aloe vera also showed a positive response during testing. Fats and fixed oils were also not found in the methanolic extract of garlic, aqueous extract of ginger, aqueous and methanolic extracts of neem, and aqueous extracts of amla and aloe vera.

Antibacterial Activity by Synthetic Antibiotics
Ciprofloxacin and levofloxacin were most effective against all microbes. Erythromycin had least activity against S. sobrinus (14.31 mm) and S. mutans (15.03 mm). Against A. naeslundii, ciprofloxacin showed the maximum inhibition (29.8 mm), while amoxicillin (25 mg/mL) showed the minimum inhibition (9.1 mm). Against L. acidophilus and E. nodatum, amoxicillin exhibited inhibition activity of 12.3 mm and 15.4 mm, respectively. Penicillin was found to be least effective against P. acidifaciens (7.3 mm). Against T. pallidum, penicillin and amoxicillin indicated the lowest activity (7.8 mm) (

Statistical Analysis
ANOVA was conducted for the antibacterial activity of herbal extracts against S. sobrinus (Table S2), S. mutans (Table S3), A. naeslundii (Table S4), L. acidophilus (Table S5), E. nodatum (Table S6), P. acidifaciens (Table S7), and T. pallidum (Table S8). Tables S2-S8 indicate that the values are statistically significant when comparing the strength of extract × plant, type of extracts × strength of extract, and type of extracts × plant. All results are statistically significant with p < 0.05.

Discussion
Dental caries and associated disorders are the most frequent diseases to be found in people worldwide [74]. The incidence of these illnesses is growing as a result of the shift in eating habits among individuals. Dental caries is a multifactorial illness that affects both the teeth and the gums [75]. The condition is impacted by a variety of variables, such as age, gender, food, microbiota in the mouth, salivary flow, tooth shape, and genetic tendency. In India, the prevalence of dental caries is estimated to be between 60% and 84% [76]; in Pakistan, the prevalence is estimated to be 56% [77].
Several experiments on oral microorganisms and their products have gained acceptance in the prediction of dental caries. These studies focused on Lactobacilli count and S. mutans, which are linked to dental caries. Although dietary carbohydrates are important etiological and predisposing variables for dental caries [78], the link between Lactobacilli count and the Snyder test positivity was determined by Snyder and Clarke [79]. They claimed that when Lactobacilli count exceeded 10,000 L/cc in saliva, 33.4% of samples tested positive in 24 h and 90% in 48 h.
A study conducted in Karachi, Pakistan reported that the majority of children between the ages of 9 and 18 were suffering from caries, with >40% of those suffering from the condition going untreated. The results of this study are not in line with our study; more individuals in the middle-aged group were affected as per our study results [80].
According to our study results, males are found to have more prevalence of caries as compared to females. The high prevalence in males may be due to different habitual patterns like smoking, more intake of sweets, and others [81,82]. A significant relationship was found between oral hygiene and dental caries; to maintain oral hygiene, it is necessary to adopt daily brushing and avoid sugary foods (refined) [83].
Dental caries is a serious public health concern in many areas of the globe, and the mechanical removal of oral biofilms is still the first line of defense against the development of caries and periodontal disease. Antibiotics are used to combat caries, but due to antibiotic resistance, the administration of antibiotics will not be sufficient to entirely block demineralization [84].
Bacterial antibiotics are frequently utilized in the treatment of dental caries and other dental-related disorders, both therapeutically and prophylactically [85]. Dental surgeons commonly prescribe antibiotics because they are concerned that the oral cavity, which ordinarily contains a large number of microorganisms as part of the natural flora and which might cause infections in their patients, would get infected [86].
Diet has an important impact on the development of dental caries and the degradation of enamel. Dental caries is a complex illness that emerges from interactions among a susceptible host, caries-related microorganisms, and cariogenic foods [87]. Organic acids that develop in the dental plaque are responsible for the demineralization of the tooth enamel and dentine, which is caused by anaerobic microbes metabolizing carbohydrates from the diet [88].
Acidogenic and cariogenic properties are present in soft drinks due to the presence of both acids and sugars, which may result in tooth caries and enamel loss [89]. According to the findings of many research studies, consuming soft drinks is associated with an increased risk of dental caries and erosion. Children aged 2-10 years who drank a high volume of carbonated soft drinks also had a considerably greater prevalence of dental caries than children who had a high volume of juice, milk, and water in their diet [90].
The biochemical analysis including sugar fermentation and enzyme activity was in agreement with the data reported by Soumya and Nampoothiri [91]. DKF 007 after the Gram staining was predicted to be the species belonging to Treponema. However, the spirochetes are difficult to culture on media and repeated culturing is required to obtain colonies. This strain was unable to grow on media for biochemical analysis. The difficulty in dealing with this organism in the microbiology lab has already been reported [92]. The findings were consistent with earlier research. Since different commercial and in-house systems have varied substrate specificities, buffering capacities, and therefore sensitivities, it might be challenging to interpret previously published results on enzyme reactions and fermentation studies. It is critical to understand the roles that these Gram-positive rods play in oral and non-oral environments and diseases, even if they are difficult to identify down to the species level.
According to the literature, S. mutans is more common in the oral cavity than S. sobrinus [93]. S. mutans has been implicated as one of the most important etiological agents for dental caries. Literature studies have found a positive relationship between caries experience and the presence or degree of S. mutans in saliva or plaque [94]. The incidence of caries is higher when S. mutans and S. sobrinus are present in the same oral microbiota. Okada et al. demonstrated that preschool children who have both S. mutans and S. sobrinus in their plaque samples have a higher incidence of dental caries than those who only have S. mutans [95]. It has also been demonstrated that S. sobrinus, in contrast to S. mutans, has not been discovered in any healthy control patients, and it may be a more effective caries-causing agent than S. mutans. Based on the current data, it is possible that the true incidence of S. sobrinus is higher than what has been believed [96].
The presence of significant levels of ureolytic activity in human dental plaque has been linked to the maintenance of plaque pH homeostasis and plaque ecology and the development of dental caries, calculus, and periodontal disease. Although the organisms responsible for this activity have not been identified with certainty, the molecular aspects of ureolysis in dental plaque have not been studied in depth [97]. While several dental plaque organisms have demonstrated ureolytic activity when isolated, Actinomyces strains are the most abundant. They can be found in high concentrations in both supragingival and subgingival plaque, indicating that they have the potential to be significant contributors to total plaque ureolysis. Thus, specific primers for urease gene ureC were designed to amplify the DNA of A. naeslundii [98].
Various techniques are used to extract phytochemicals. Extraction utilizes the selected solvents to separate medicinally active components. The conventional extraction procedure was used to retrieve required fraction and remove undesired material using solvent [99]. These items include alkaloids, glycosides, terpenoids, flavonoids, and lignones, among others. Phytochemical extraction must be thorough, efficient, simple, fast, and affordable. Soxhlet extraction and plant tissue homogenization are two procedures that have been developed throughout time [100,101].
Chemicals found in plants that are not nutritional but have protective or diseasepreventing qualities are referred to as phytochemicals [102]. As a result of the antimicrobial activity of several phytochemicals generated by plants, these plants may be investigated and employed in the creation of novel antimicrobial medications. The phytochemical characterization of plant material is significant since it is related to the therapeutic effects of the plant material in question [103]. It is self-evident that various plant species would have a variety of chemical compounds of varying strength. However, these variations might include distinct types or even the same variety produced in a different place or harvested at a different time, depending on the circumstances. Different plant components, including the leaves, bark, seeds, roots, flowers, and pods, might contain a variety of active ingredients that vary from one another [104].
Kavya et al. conducted a preliminary phytochemical investigation of Abrus pulchellus and Abrus precatorius plant extracts and discovered the presence of flavonoids, alkaloids, and saponins in both plant extracts [105]. Abrus precatorius seeds were subjected to a phytochemical screening procedure, which indicated the presence of alkaloids, tannins, and flavonoids but not anthraquinones or glycosides [106]. A phytochemical screening of the methanol extract of Piper betle L. leaves was carried out, which revealed the presence of flavonoids, tannins, sterols, and phenols in the leaves of Piper betle L. Through the methanol extract, various compounds, such as flavonoids, tannins, steroids, alkaloids, and glycosides, as well as carbohydrates, proteins, phenols, flavonoids, and alkaloids were discovered [107].
In herbal medicine, 20% of plants are used to cure a variety of disorders, including diseases caused by pathogenic bacteria and fungus [108]. Wolde et al. found that garlic extracts have a high range of antibacterial activity against both Gram-negative and Grampositive bacteria. The garlic extracts were also found effective against antibiotic-resistant bacteria and their toxic products. This effect was because of garlic compounds. Especially, allicin affects the growth of bacteria by partially inhibiting their DNA and protein synthesis and primarily inhibiting RNA synthesis as the main target [109]. According to Kshirsagar et al., garlic extract showed antimicrobial activity against S. mutans and L. acidophilus. During their study, they found that 18 mm to 24 mm of inhibition was observed against the above-mentioned bacteria [110]. The antibacterial, antifungal, and antiviral qualities of garlic (Allium sativum) make it a valuable addition to any kitchen. Garlic extracts were shown to suppress the development of harmful bacteria in aqueous, ethanol, and chloroform solutions, however, with different degrees of sensitivity to the extracts [111,112].
Garlic extract was shown to be effective against Gram-positive bacteria that are diseasecausing, especially S. mutans. As the concentration of garlic extract increases, the width of the non-growth zone increased as well. The bacteria L. acidophilus was responsive to varied doses of garlic extract when cultured on blood agar medium, in a similar fashion. Once again, the width of the inhibitory zone grew in proportion to the rise in the concentration of garlic extract [113]. Bacillus subtilis, S. aureus, Escherichia coli, and Salmonella typhi, were used against the garlic extracts, and the zones of inhibition were calculated to be 29 mm, 26 mm, 46 mm, 31 mm, and 25 mm in diameter, respectively. While using streptomycin, the inhibitory zone was determined to be 35 mm, 33 mm, 29 mm, and 31 mm in width, respectively [114]. With diameter zones of inhibition ranging from 4.40 cm to 3.80 cm, garlic extract had the greatest antibacterial effect against skin pathogenic bacteria S. aureus, followed by S. epidermidis with diameter zones of inhibition ranging from 4.13 cm to 3.57 cm, and finally Strep. pyogenes with diameter zones of inhibition ranging from 3.40 cm to 2.67 cm. P. aeruginosa was found to have the smallest inhibitory zones, which were 2.32 cm-1.55 cm in diameter [115]. A 31 mm inhibitory zone has been observed for both fresh local and imported garlic extracts when used against methicillin-resistant S. aureus (MRSA). S. aureus was the most susceptible bacterium to garlic extract, with a 26 mm diameter zone of inhibition, followed by S. enteritidis, while B. cereus was found to be the most resistant bacteria to garlic extract [116]. Because of its broad range of antibacterial action, garlic extract has the potential to be used in the creation of broad-spectrum antibiotics [117].
A similar pattern was seen when comparing the zone of inhibition (9 mm) of garlic extract to that of antibiotic (ciprofloxacin) when 50% of the garlic extract was used against Pseudomonas aeruginosa. However, when tested against Pseudomonas aeruginosa, garlic extract and antibiotics were shown to be ineffective in 20% of cases [118]. The antibacterial activity of the alcoholic extract of garlic against S. aureus was determined to be 9 mm for the concentration of 10 mg/mL and 23 mm for the concentration of 100 mg/mL. The concentrations of 10-20 mg/mL were relatively inactive in preventing the development of S. aureus, the concentrations of 40-60 mg/mL were moderately inactive, and the concentrations of 80-100 mg/mL were very effective in preventing the growth of S. aureus [119].
According to the literature, the ethanolic extract of ginger has shown significant activity against P. aeruginosa and B. subtilis with zone of inhibition ranging from 70.4 mm at 25 mg/mL to 23 mm at 100 mg/mL, and the MIC ranges from 6.25 mg/mL to 12 mg/mL against B. subtilis and C. albicans. At low doses, the activity of the aqueous tract was very low; however, at higher concentrations, a significant amount of activity was found [120]. In addition to Escherichia coli, Staphylococcus aureus, S. epidermidis, Klebsiella pneumoniae, Salmonella typhi, S. typhimurium, Pseudomonas aeruginosa, Proteus sp., Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Streptococcus faecalis, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus sp., ginger shows antibacterial properties against various other Gram-positive and the Gram-negative bacteria [121]. The antimicrobial effectiveness of fresh, natural, and commercial dried ginger extracts has been evaluated against seven local clinical bacterial isolates using the agar disc diffusion technique. The results demonstrated that the chloroform and diethyl ether extracts of ginger, with the exception of P. aeruginosa and E. coli, displayed a more substantial inhibitory zone of the pathogens examined [122].
Neem extracts are high in antibacterial components, making them potentially effective in the control of certain foodborne pathogens and other spoilage organisms. Neem leaf extracts have antibacterial characteristics, and the extract exhibited considerably higher zones of inhibition than 3% sodium hypochlorite, indicating that the extract has antimicrobial capabilities [123].
Bacterial strains were evaluated based on the width of the growth inhibition zone achieved by using concentrations of 20 mg/mL and 40 mg/mL Ocimum methanol extract, respectively. By using this procedure, the extracts can diffuse more effectively into the medium, increasing interaction with the organisms. The antibacterial activity of tulsi extracts was tested against four pathogenic species: Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, and Enterococcus faecalis. The results showed that the extracts were effective against all four pathogenic organisms. The findings revealed that the Ocimum extracts at final concentrations of 40 mg/mL were effective. In contrast, the final concentration of 40 mg/mL methanol extract of the antioxidant was higher in the liver than in the muscle systems when exposed to oxidative stress [124].
Variya et al. indicated that both the aqueous and methanol extracts of amla were effective against a variety of pathogenic organisms, including Klebsiella pneumoniae, E. cloacae, and E. coli [125]. The antibacterial activity of amla has been seen to vary when tested against Gram-positive and Gram-negative pathogenic bacterial species, including Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, which were successfully treated using silver nanoparticles created by green synthesis using the fruits and leaves of amla [126].
Ramanuj et al. observed the bacteriostatic effect of Emblica officinalis seed fractions against Acinetobacter baumannii [127]. Amla's antiplasmodial effect has been studied in vivo and in vitro. Both chloroquine-resistant and chloroquine-sensitive isolates of P. falciparum were found to be resistant to amla leaves extract in ethyl acetate. The antimalarial effect was shown against both chloroquine-sensitive and chloroquine-resistant plasmodium species [128]. The ethanolic extract, chloroform, acetone, and aqueous portions of amla fruits exhibited promising antimicrobial activity against a variety of bacterial species, including Escherichia coli, Proteus sp., Salmonella paratyphi, Pseudomonas sp., Staphylococcus aureus, Klebsiella sp., Bacillus sp., and Salmonella [128]. A potential inhibitory effect against Candida galbrata and Cryptococcus neoformans was seen in the ethanolic fraction of amla; however, there was no cytotoxic activity observed in the vero cell line [125]. It was discovered that the chloroform soluble fraction of the amla methanol extract exhibited considerable and promising antibacterial activity, as well as significant cytotoxicity, when tested against a variety of pathogenic Gram-positive and Gram-negative bacteria [129].
Aloe vera plant extracts possess antimicrobial properties that kill or inhibit the development of microorganisms (including bacteria (antibacterial activity)), fungi (antifungal activity), and viruses (antiviral activity). Fruit rot is a significant factor impacting the postharvest quality of fresh food after it has been harvested. A number of previous research have shown that the application of aloe vera gel as an edible coating has beneficial effects on the prevention of fruit deterioration and microbiological spoiling [130,131]. Aloe vera gel has an inhibiting impact on the growth of mycelium (Penicillium digitatum and Aspergillus niger) [132]. The rate of mycelium development increased with the concentration of gel used. Aloe vera gel at a concentration of 500 mL/l was shown to suppress the growth of P. digitatum and A. niger at 100% and 64%, respectively [133].
Dental caries is a multifactorial human disease found all over the world. It is considered one of the main problems of public health. During the last two decades, dental caries remains a severe global concern. There is a significant correlation between having poor dental health and an increased likelihood of developing systemic diseases. The results of our study are in line with the previous literature, where our study results are supported by the previous studies; herbal plants have been used against the varieties of microbes. Bacterial antibiotics are frequently utilized in the treatment of dental caries and other dental-related disorders, both therapeutically and prophylactically. Further research studies on the topic are needed and necessary to verify the results obtained in this study [85].

Conclusions
The results obtained during this study showed that selected herbal plants have antimicrobial activities against the oral microbes obtained from patients with dental caries. The current research study concluded that the disease prevalence was higher in males as compared with females, more prevalent in middle-aged patients, and toothache was the most common symptom of dental caries. Seven cariogenic bacterial strains were isolated, among which S. mutans and S. sobrinus were found as the most abundant bacterial strains. Among synthetic antibiotics, ciprofloxacin and levofloxacin were the most effective against all isolated cariogenic microorganisms. Comparatively, among plant extract antimicrobial activity, ginger and amla exhibited the maximum antibiotic activity against all isolated strains, making them the most suitable alternatives to synthetic antibiotics for treating dental diseases. Plant extract antimicrobial activity against cariogenic bacterial strains is the most preferable choice in the future to avoid upcoming bacterial resistance to antibiotics.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms11081952/s1, Table S1: Primers used in PCR amplification of conserved region in 16S rDNA gene for bacterial strains; Table S2: ANOVA for antibacterial activity of herbal extracts for S. sobrinus; Table S3: ANOVA for antibacterial activity of herbal extracts for S. mutans; Table S4: ANOVA for antibacterial activity of herbal extracts for A. naeslundii; Table S5: ANOVA for antibacterial activity of herbal extracts for L. acidophilus; Table S6: ANOVA for antibacterial activity of herbal extracts for E. nodatum; Table S7: ANOVA for antibacterial activity of herbal extracts for P. acidifaciens; Table S8: ANOVA for antibacterial activity of herbal extracts for T. pallidum. References [134][135][136][137][138] are cited in the supplementary materials.