In Vitro analysis of the Antibacterial action of the Extract of Costus Spiralis (Costaceae) on Enterococcus Faecalis

— Enterococcus faecalis is an important cause of endodontic and nosocomial infections, making the search for new antimicrobial drugs important. To analyze in vitro the antibacterial action of Costus spiralis extract on E. faecalis. The antimicrobial sensitivity test was performed using the well agar diffusion method. E. faecalis strains (NEWProv-0012) were revitalized in BHI broth and incubated at 37 ° C for 18 h. Subsequently, the microbial suspension was adjusted to a concentration of 5x105 CFU mL-1 and sown on Mueller Hinton agar, after which 6 mm wells were made. The crude alcoholic extract paste of C. spiralis was used as a positive control 2.5% sodium hypochlorite and as a negative control 96% ethanol and BHI broth. The halos of minimal inhibitory inhibition (MIC) using the crude extract paste and solutions prepared with BHI broth in different concentrations. The viability and microbial quantification of E. faecalis in culture with MIC were determined by serial dilution after 24, 48 and 72 h. Results: The crude extract of C. spiralis has an effective action in inhibiting the growth of E. faecalis. MIC was observed at a concentration of 25% of the extract in solution and promoted the inhibition of E. faecalis growth by 50% after 24 h and 98% in 48 h of incubation. Conclusion: The C. spiralis extract has antibacterial action and can be a therapeutic alternative for infections caused by E. faecalis. The formulations with concentrations of the crude extract of caninha do-brejo from 25% analyzed in this study showed action against E. faecalis. Previous research using plant extract of Costus spiralis shows antibacterial activity in vitro against different gram-negative bacteria Pseudomonas Shigella sonnei, Salmonella sp., Vibrio cholerae and against gram positive bacilli, In none of these studies, the species E. faecalis was included among the strains analyzed, not allowing the comparison of results with the present study.


INTRODUCTION
Enterococcus are characterized as gram-positive cocci arranged in a single form, in pairs or in short chains. They are facultative anaerobic microorganisms, producers of lactic acid and negative catalase (IKE, 2017). It has the ability to form biofilm, which can be found in the gastrointestinal tract, oral cavity and vagina ( In view of these considerations, it is necessary to investigate and develop new alternative antimicrobial options to those already used, and it is relevant to verify the effectiveness of medicinal plants found in our territory. Therefore, the present study sought to analyze the antibacterial action and the determination of the Minimum Inhibitory Concentration (MIC) in vitro of the crude ethanolic extract of the Costus spiralis plant on Enterococcus faecalis.

II. MATERIALS AND METHODS
The leaves and branches of caninha-do-brejo (Costus spiralis [Jacq.] Roscoe) were collected, following the herborization techniques of Fidalgo&Bononi (1984), at the Sítio Novo farm, municipality of Divinópolis do Tocantins, in the western region of state of Tocantins, Brazil, between the geographical coordinates 9 ° 54'32 "south latitude and 49 ° 09'27" west longitude. The species was identified by the botanist responsible for the herbarium of the Federal University of Tocantins campus Porto Nacional.
The exsiccates are incorporated into the collection of the referred herbarium, under registration number 12098 HTO. The leaves were washed with water and dried, then crushed and subjected to maceration with 96% ethanol for five days. After that period the macerate was filtered on filter paper and the ethanolic solution was concentrated on a rotary evaporator, with temperature controlled at 40 ° C, to recover the solvent and obtain the crude extract, with 6.44% yield.
After the microorganisms were inoculated in a plate of nonselective nutrient medium (Nutrient agar, KASVI) by the depletion technique and they were again incubated at 37 ° C ± 2 ° C for 24 h. In order to carry out the tests, the microbial concentration was adjusted to the concentration of 5x105 CFU / mL of BHI broth, controlled by reading the turbidity in a spectrophotometer with a wavelength of 600 nm and later counting of plate colonies by performing serial dilutions. in 0.95% saline solution.
To determine the antibacterial action on plates, Mueller Hinton agar (MH, KASVI) was prepared, previously melted, sterilized and cooled to 45-50 ° C, then distributed in 150 mm diameter Petri dishes until reaching a thickness of approximately 4 mm. After obtaining solid consistency of the MH agar, sterile swabs were used to collect microorganisms in the bacterial suspension (≈ 5x105 CFU / mL) and the surface seeding technique was performed in three directions on each plate, paying attention to uniform distribution, avoiding the growth of isolated colonies. Then, 6 mm diameter wells were made, with four perforations in each plate.
The MIC determination was performed by diluting the crude extract in brain heart infusion broth (BHI, KASVI), to prepare solutions in different concentrations: 100%, 50%, 25%, 12.5%, 6.25%, 3, 1%, 1.5% and 0.7%, to determine the MIC. As a positive control, 2.5% sodium hypochlorite was used and as a negative control, 96% ethanol and BHI broth. The inhibition halos were measured with the aid of a caliper, the results being expressed in millimeters. MIC was considered to be the lowest concentration of the extract solution that enabled the formation of an inhibition halo. The wells were completely filled with sufficient quantities of crude extract or controls and incubated at 37 ° C ± 2 ° C.
After MIC determination, the microorganisms (≈ 5x105 CFU mL-1) were grown in a bacteriological incubator at 37 ° C ± 2 ° C, in the presence or absence of the crude extract of C. spiralis at 25%. For the analysis of viability and microbial All experiments were carried out in triplicates. To determine the antimicrobial action of the C. spiralis extract, the final result was the arithmetic mean of the inhibition halos or the number of CFU / mL obtained from triplicates of three experiments carried out consecutively.

III. RESULTS AND DISCUSSION
The results obtained show that the vegetable preparation of the leaves of Costus spiralis has potential antimicrobial activity against the tested strain of Enterococcus faecalis. The crude extract was able to inhibit microbial growth after 24 h of incubation, with inhibition halos with an average diameter of 13 mm (Figure 1), which were maintained even after 30 days of culture.  The determination of the Minimum Inhibitory Concentration (MIC) was performed with the extract of C. spiralis in paste and with solutions prepared in concentrations 100%; 50%; 25%; 12.5%; 6.2% 3.1%, 1.5% and 0.7%, (Table 1). Growth inhibition was directly proportional to the concentration of the extract, with MIC being fixed in the dilution of the crude extract in 25% solution in BHI medium, with the formation of an inhibition halo of 7 mm in diameter. Crude extract in solution 100%

Fig.2: Growth curve of in vitro E. faecalis in the presence and absence of Crude Extract of C. spiralis
The rate of inhibition of microbial growth in the presence of the crude extract of C. spiralis at MIC 25% was compared with microbial growth free of any limiting factor. The extract at MIC 25% was able to inhibit microbial growth by 54% in 24 h, 98% after 48 h and in 72 h it inhibited 100% ( Figure 3).

IV. FINAL CONSIDERATIONS
It was found in in vitro studies that Costus spiralis leaf extracts have antimicrobial activity against Enterococcus faecalis from concentrations of 25% crude extract. The evidence from the present study indicates a relevant contribution of C. spiralis as a raw material for new arsenals of antimicrobial substances that will combat the feared infections by E. faecalis. The results encourage the development of new studies to prove its effectiveness, isolation of active ingredients, synergism with other substances and clinical applicability, enabling its use as an auxiliary substance in future treatments.