Susceptibility of Porphyromonas gingivalis and Streptococcus mutans to Antibacterial Effect from Mammea americana

The development of periodontal disease and dental caries is influenced by several factors, such as microorganisms of bacterial biofilm or commensal bacteria in the mouth. These microorganisms trigger inflammatory and immune responses in the host. Currently, medicinal plants are treatment options for these oral diseases. Mammea americana extracts have reported antimicrobial effects against several microorganisms. Nevertheless, this effect is unknown against oral bacteria. Therefore, the aim of this study was to evaluate the antibacterial effect of M. americana extract against Porphyromonas gingivalis and Streptococcus mutans. For this, an experimental study was conducted. Ethanolic extract was obtained from seeds of M. americana (one oil phase and one ethanolic phase). The strains of Porphyromonas gingivalis ATCC 33277 and Streptococcus mutans ATCC 25175 were exposed to this extract to evaluate its antibacterial effect. Antibacterial activity was observed with the two phases of M. americana extract on P. gingivalis and S. mutans with lower MICs (minimum inhibitory concentration). Also, bactericidal and bacteriostatic activity was detected against S. mutans, depending on the concentration of the extract, while on M. americana extract presented only bacteriostatic activity against P. gingivalis. These findings provide important and promising information allowing for further exploration in the future.


Introduction
Oral diseases are a worldwide public health problem. Many epidemiological studies report that diseases such as dental caries and periodontal disease are the most prevalent oral disorders of humanity [1][2][3]. These conditions are caused by poor oral hygiene and biofilm forming bacteria residing in the mouth, able to communicate with each other through mechanisms of Quorum sensing [4]. Biofilms are complex structures, where different bacterial species are arranged to form a superorganism with advanced properties unlike planktonic bacteria. Dental or bacterial plaque is a type of biofilm on the tooth surface that plays an important role in the development of these oral conditions [5,6]. Streptococcus mutans colonize the tooth surface and initiate biofilm formation by their ability to synthesize extracellular polysaccharides from sucrose [7]. The further accumulation of biofilm around the supra-and subgingival region leads to a shift in its microbial composition from Streptococcus spp., Actinomyces spp., and Porphyromonas gingivalis [8,9]. Therefore, these microorganisms are considered to be the major etiological agents involved in dental caries and periodontal disease [3].
Clinical practice seeks to prevent the occurrences of these oral conditions or apply minimally invasive treatments, avoiding in most cases surgical interventions [10,11]. Therefore, antimicrobial or antibacterial agents against these oral pathogens could play an important role in the prevention and treatment of dental caries and periodontal disease, principally those that can inhibit or reduce the growth of these microorganisms, inhibit biofilm formation, influence the adhesion of bacteria to surfaces, and reduce the clinic symptoms [12,13]. Many of the currently available oral antimicrobials can change oral microbiota and have adverse side effects such as diarrhea, vomiting, and teeth staining. Therefore, it is necessary to find safe and effective therapeutic agents for everyone. Traditional herbal medicines are considered as 2 Advances in Pharmacological Sciences a good source of therapeutic alternatives, depending on their properties [14,15]. Antimicrobial agents isolated from plants represent a huge and poorly exploited source of drugs with great therapeutic potential. Some of these compounds are effective in the treatment of infectious diseases, while having the advantage of causing few side effects which are often associated with synthetic antimicrobials [16].
Mammea americana is an evergreen tree of the family Calophyllaceae, native of the Antilles, and introduced into central and northern South America, although it is currently cultivated in other tropical and humid areas. It is commonly known as mammee, mammee apple, mamey, mamey apple, Santo Domingo apricot, South American apricot, mamey de cartagena de indias, or mamey de santo domingo. Its fleshy fruit is edible and frequently consumed; they have a yellowreddish and aromatic pulp and are round or slightly irregular, with a thick brown rind. Their diameter ranges from 10 to 20 cm. Small fruit contain a single seed, while larger ones might have up to four. The seeds are brown, rough, oval, and around 6 cm long [17].
Oral lore has referred the use of M. americana as a natural therapeutic alternative. However, the medicinal properties of the leaves, fruits, and seeds have not been widely elucidated. Several studies have reported some medicinal properties, such as anticonvulsant, antipyretic, antimalarial, anthelmintic, and digestive tonic, as well as a remedy for parasitic skin diseases [18]. It also provides inhibitory activity against Mycobacterium tuberculosis [19] and molluscicidal properties against Biomphalaria glabrata [20]. Furthermore, antitumor activity has been reported, as well as some coumarin, phloroglucinol derivatives [21,22], xanthones, and benzophenones [23]. Currently, there are few studies reporting the M. americana antimicrobial activity, specifically for oral bacteria. Therefore, the aim of this study was to identify the mamey's antibacterial activity against pathogenic bacteria involved in dental caries and periodontal disease.

Extract Preparation.
From the ripe fruit of M. americana, we extracted the seeds. These were air dried at 25 ∘ C for 3 weeks and ground in a seed grinder into a fine powder. The powdered materials (1313 g) were soaked in ethanol (50% w/v) at room temperature for 72 h in dark conditions and then were filtered. The total extract was dried, and solvent was evaporated in a rotary evaporator (Laborota 4001, Heidolph) under reduced pressure at 50 ∘ C. We obtained two phases from the total extract: one hydroalcoholic phase called "ethanolic phase" and one "oily phase, " 90 g and 109 g, respectively. Both phases were further dried at room temperature; before evaluating the antibacterial activity, each phase was subjected to a solubility test in 2% ethanol, 2% methanol, 1% DMSO (dimethyl sulfoxide), and combinations from these solvents. We observed that 1% DMSO was the best solvent for both phases. In addition, 1% DMSO did not exert any damage on the bacterial strains.
From a few microbial colonies of P. gingivalis and S. mutans bacterial cultures, inocula and bacterial growth curves were performed for each strain. Isolated colonies were suspended in their corresponding culture media and turbidity of the inoculum was adjusted to reach 0.5 on the McFarland scale (optical density between 0.08 and 0.10) using a microplate reader (Multiscan EX, Thermo Scientific) at 620 nm, which is equivalent to 1-2 × 10 8 CFU/mL. S. mutans reached its stationary phase for approximately 13 h, whereas P. gingivalis required 22 h of incubation. These preliminary tests allowed establishing ideal experimental conditions. 2.5. Antibacterial Activity Assay. Antibacterial activity was determined by the microdilution technique with 96-well microplates. Using this technique, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were obtained for the ethanolic phase and oily phase against the microorganisms under study. All assays were performed in triplicate. (MIC). The ethanolic and oily phases were serially diluted, ranging from 500 g/mL to 0.06 g/mL in the 96-well plate * * with bacterial suspension (5 × 10 5 CFU/mL); gentamicin (16 g/mL) was used as negative control for bacterial growth, and a broth solution was used as control sterility. Elsewhere, microbial growth was indicated as changes in optical density from bacterial inoculum (positive control). The 96-well plates were incubated at 37 ∘ C under anaerobic conditions, for 13 h (S. mutans) and 22 h (P. gingivalis). The MIC was defined as the lowest concentration that inhibited microbial growth.

Determination of the Minimum Bactericidal Concentration (MBC).
The MBC was determined by adding 10 L of the suspensions from the wells, which did not show any growth during MIC assays in petri dishes with corresponding agar to each bacterium. These petri dishes were incubated at 37 ∘ C under anaerobic conditions for 5 days (P. gingivalis) and for 48 h (S. mutans). After this incubation period, bacterial colonies were observed. The ethanolic and oily phases were designated as bacteriostatic (those in which bacterial colonies grew in petri dishes) or bactericide (those in which bacterial colonies did not grow in petri dishes).

Statistical Analysis.
The data were analyzed using Graph-Pad Prism v5 and compared by nonparametric Kruskal-Wallis and multiple-comparison tests as the Dunnett's test; this was applied for comparison between each treatment concentration and the respective control. The chosen level of significance for all statistical tests was < 0.05.

Antibacterial Activity.
The two phases of M. americana extract proved antibacterial activity against S. mutans and P. gingivalis strains. These bacteria were significantly sensitive to the extract from 500 g/mL, from a mean optical density (OD) of 0.93 for the inoculums of P. gigivalis to 0.033 ( = 0.005) and 0.022 ( = 0.005) for wells with bacteria exposed to the oily and ethanolic phases, respectively. These results are similar and do not differ from those of gentamicin OD: 0.034, ( = 0.32); therefore, mamey extract inhibits bacterial growth by 96% (Figure 1(a)). S. mutans was susceptible to mamey and inhibits bacterial growth by approximately 31.5%, from a mean OD of 0.170 for the inoculums of S. mutans to 0.053 ( = 0.013) for oily phase and 0.055 ( = 0.013) for ethanolic phase (Figure 1(b)). This antibacterial activity was interpreted as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The oily and ethanolic phases showed very promising data on Gram-positive bacteria, which was reflected by their MICs against S. mutans (MIC: 15.62 g/mL and 62.5 g/mL, resp.), comparing broth solution at all tested concentrations (Figures 2(a) and 2(b)). While the two phases did not show prominent results against P. gingivalis, their MICs showed antibacterial activity; the MIC of the oily phase was 250 g/mL and 500 g/mL for the ethanolic phase (Figures 3(a) and 3(b)).
MBC was determined from 500 g/mL of each phase. The oily phase showed bactericidal property against S. mutans from 500 g/mL to 125 g/mL, and concentrations <125 g/mL behaved as bacteriostatic. While the ethanolic phase showed bactericidal activity from 500 g/mL to 250 g/mL, lower concentrations were bacteriostatic. M. americana extract did not show bactericidal activity against P. gingivalis. Both oily and ethanolic phases only showed bacteriostatic behavior even at 500 g/mL (Table 1).

Phytochemical
Screening. The phytochemical screening of the oily and ethanolic phases showed the presence of phenolic compounds, tannins, and coumarins ( Table 2). These metabolites possibly contribute to the antibacterial activity of M. americana extract.

Discussion
Oral bacteria have been amply tested for antimicrobial susceptibility to various plant extracts and natural substances. According to our knowledge, this is the first report on the antibacterial effect of M. americana extract against bacteria representative of oral diseases such as dental caries and periodontal disease such as S. mutans and P. gingivalis [8,9]. M. americana, commonly known as "mamey, " is a tree widely used for its medicinal properties and its fruit is sought in communities of the Antilles and central and northern South America. Morris and Pagán. were the first to report some properties of mamey, including antimicrobial and toxic properties of fruit seeds of this tree [25]; other parts of mamey, such as leaves, stems, and fruit, have also shown medicinal properties. Frame et al. evaluated the antituberculous effect against Mycobacterium smegmatis from 50 extracts of plants belonging to tropical flora in Puerto Rico. The Mammea americana extract showed the best antituberculous effect with low concentration (50 g) unlike 49 remaining extracts whose concentrations are close at 500 g, this showed promising properties for M. americana [19]. We also observe similar promising results of fruit seeds of mamey against oral bacteria. For

Porphyromonas gingivalis
Oily It showed bacteriostatic activity at all concentrations. Ethanolic It showed bacteriostatic activity at all concentrations. showed antibacterial activity in one of two bacterial strains, but only the leaves extract of M. americana inhibited bacterial growth of the two strains tested [30]. We also obtained similar results, considering that this property was assessed on S. mutans and P. gingivalis.
It remains to elucidate the active compounds and cytotoxic effect of M. americana seeds extract. However, some metabolites present in mamey have reported antimicrobial activity for other medicinal plants. The major metabolite of mamey reported in other studies is coumarin [31], including Mammea africana, another species of the Guttiferae family; coumarins of this species exhibit significant antimicrobial activity against S. aureus [32]. Other metabolites found in mamey are the phenolic compounds, the antimicrobial capacity of these, acting by the alteration of the permeability of the cell membrane that could result in the uncoupling of oxidative phosphorylation, inhibition of active transport, and loss of pool metabolites due to cytoplasmic membrane from bacteria [33,34]. These could be two possible explanations for the antibacterial activity of the M. americana seeds extract reported in our study.
In conclusion, we may point out that the Mammea americana extract showed antibacterial activity against Porphyromonas gingivalis ATCC 33277 and Streptococcus mutans ATCC 25175, and the MIC-MBC obtained in this work allows us to hypothesize that M. americana presents promising effects against oral bacteria which need to be further studied.

Ethical Approval
This study was approved by the Institutional Ethics Committee of the Universidad de Cartagena, according to resolution no. 62 of 28-02-2013.