Cyathea gigantea (Cyatheaceae) as an antimicrobial agent against multidrug resistant organisms

Background Rapid emergence of multidrug resistant (MDR) organisms in hospital and community settings often result into treatment failure, thus leading the clinicians with fewer treatment options. Cyathea gigantea, an ethnomedicinally important fern used in cuts and wound infections. So, if this medicinal plant is used in treating the MDR infections then it might bring certain relief in future treatment options. Methods Antibacterial activity of C. gigantea against MDR bacteria was assed using well diffusion and broth microdilution methods to determine the diameters of growth inhibition zones, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Synergistic activity was also determined with the conventional antibiotics by disc diffusion method followed by FIC index of each of the tested antibiotic was calculated. The active extract was then subjected to fractionation by column chromatography and antibacterial activity was done with each of the collected fractions. Results Crude extract of C. gigantea was found to be active against all the tested organisms. The MIC was 200 μg/ml against Gram-positive i.e., Staphylococcus aureus ATCC 25923 and 400 μg/ml against Gram-negative i.e., Escherichia coli ATCC 25922 and Pseudomonas aeruginosa PAO1, while the MBC was 400 μg/ml in case of Gram-positive and 800 μg/ml for Gram-negative. The synergistic activity revealed that the plant extract increased the antibacterial property of the studied antibiotics and the FIC index showed that significant synergistic activity was shown by ciprofloxacin followed by tetracycline, ampicillin and oxacillin. Antibacterial activity with the fractionated extract showed that the FR II, FR III and FR IV were active against both Gram-positive and Gram-negative bacteria, whereas FR I, FR V and FR VI did not show antibacterial property against any of the tested bacteria. Conclusions Extracts of C. gigantea was found active against both selected Gram-positive and Gram-negative organisms and thus offers the scientific basis for the traditional use of the fern. The present study also provides the basis for future study to validate the possible use against multidrug resistant organisms.


Background
The worldwide emergence of multidrug resistant bacteria is the primary cause of various treatment failure and thus leading the clinicians with fewer treatment options. Natural products of plant origin are sources of various secondary metabolites and thus known to possess potential antimicrobial properties. In particular, the drugs derived from natural products are important in the treatment of life-threatening conditions [1]. Hence, if medicinal plants are used in treating the MDR infections, they might bring certain relief in future treatment options.
The fern possesses an important role in folklore medicine although neglected in modern days. The family Cyatheaceae includes the worlds' tallest tree ferns. C. gigantea is an ethno-medicinal important fern and a native of Southern Assam [2].Paste of apical soft portion of caudex is applied on cuts and wounds which prevents microbial growth and inhibits abscess formation. The plant is also effective in immediate blood clotting [3][4][5]. Hentriacontane, âsitostanone, diploterol, sitosterol, hop-29-ol and oleanolic acid were isolated from the hexanesoluble fraction of the tree fern, C. gigantea [6]. The phytochemical study showed the presence of steroid, flavonoid and saponin and TLC profiling of plant extracts in different solvent system confirms the presence of diverse group of phytochemicals [7]. Therefore, in this study an attempt has been taken to explore the antibacterial property of the fern C. gigantea available in Southern part of Assam, India.

Plant material
Fresh plant material (fronds) of C. gigantea was collected from Southern Assam. The identification of the fern was done by Miss Kathakali Nath using standard manual [8,9] and was formally confirmed by Dr. M. K. Bhattacharya. It was also matched with the available herbarium of the species at Central National Herbarium of Botanical Survey of India, Calcutta. A voucher specimen of the fern has been deposited in the Karimganj College Herbarium, Karimganj, Assam bearing deposition number M. 1213.

Extraction and fractionation
With tap and subsequently by sterile water the fresh fronds were cleaned, which were then shad-dried and pounded to powder. For extraction 50 g of powdered plant material was added to three solvents (500 ml) of increasing polarity (ethyl acetate, methanol and water) using soxlet evaporator [10]. Boiling method was adopted for preparation of water extract.

Micro-organisms
The strains (KKN) were isolated from Silchar Medical College and Hospital Silchar, India from the patients who were admitted or visited the clinics of the hospital. The samples were collected on recommendation of the clinician for routine culture and sensitivity in the Department of Microbiology of Silchar Medical College and Hospital. The investigators have collected the isolates from the above mentioned Department and characterized for ESBL producing E. coli (KKN5 and KKN 6) and MBL producing P. aeruginosa (KKN1 and KKN2) production using PCR based assay and sequencing. The same was done for characterization of MRSA (KKN3, KKN4, KKN10, KKN11, and KKN12) strains as well. E. coli ATCC 25922 and S. aureus ATCC 25923 and P. aeruginosa PAO1 were used as controls.
Antibacterial activity of extracts and fractions from C. gigantea Determination of diameters of growth inhibition zones Agar well diffusion method was used for determining the antibacterial activity of different extracts. Aided with sterile swab, each bacterial culture (10 5 cfu/ml) was spread on Mueller Hinton Agar plates. Followed by wells (5 mm diameter) were punched in the agar medium where 20 μl of extract was added along which same volume of dimethyl sulphoxide (DMSO) served as negative control and antibiotic discs of ampicillin (10 μg) and ciprofloxacin (5 μg) were used as positive control. The plates were then incubated and the zone of inhibition was measured in mm [12]. The test was carried out in triplicate.
The different fractions obtained were allowed to stand at room temperature till the solvent evaporated. The dried fractions were dissolved in di-methyl sulphoxide (DMSO) and used for screening the antibacterial activity against the reference strains of E. coli ATCC 25922, P. aeruginosa PAO1 and S. aureus ATCC 25923 by well diffusion method [11].

Determination of minimum inhibitory concentrations (MIC) and determination of minimum bactericidal concentrations (MBC)
Broth dilution method was performed for determination of MIC. Test tubes containing culture media along with different dilutions of extract i.e., 25-800 μg/ml dissolved in dimethyl sulphoxide (DMSO) was used. To each of the test tubes bacterial cultures (10 5 cfu/ml) were added. Positive control containing growth media with each Ethyl acetate extract 10.67 ± 0.577 12.33 ± 0.577 11.17 ± 0.288 11.33 ± 0.577 12.67 ± 0.577 13.83 ± 0.763 11.5 ± 1.32 10.5 ± 0.500 12.67 ± 0.577 11.33 ± 1.52 9.67 ± 1.52 10.67 ± 0.763 The test was done in triplicate. Diameter of the zone of inhibitions (mm) is given here as mean ± standard deviation organisms and negative control containing only extract and media was used [13]. The test was carried out in triplicate.
For determination of MBC, test tubes from MIC study where no visible growth observed were subsequently sub-cultured (100 μl) into agar medium [14]. The test was carried out in triplicate.

Association of extracts with conventional antibiotics
Bacterial culture of 10 5 cfu/ml were seeded on Mueller Hinton agar plates to which antibiotic disc viz. ampicillin (2 μg), ciprofloxacin (5 μg), erythromycin (15 μg), oxacillin (1 μg) and tetracyclin (30 μg) were placed. To each of the disc 6 μl of extract (1x MIC) was added. The plates were then incubated [15] and the results were interpreted as per CLSI guidelines 2015 [16]. The test was carried out in triplicate.
The FIC index of each antibiotic combined with extract was calculated using eq. 3.
The sum of FIC A and FIC B determines FIC index (eq. 3) The effects of combination results into synergy, indifference, antagonism and additive and is defined as ∑FIC ≤ 0.5, ∑FIC ≤ 4, ∑FIC > 4 and 0.5 ∑FIC ≤ 1 respectively Gas chromatography mass spectroscopy Gas chromatography mass spectroscopy was done at AIRF, Jawaharlal Nehru University, Delhi and was carried out with GCMS-QP2010 Plus, Shimadzu, Kyoto, Japan. The type of column used was RXi-5 Sil MS (30 m X 0.25 mm i.d. X 0.25 μm film thickness) whose initial temperature was 60°C and the carrier gas used was helium. The compounds were identified using two libraries i.e., NIST14.lib and WILEY8.lib (Additional file 1).

Statistical analysis
All the values were expressed as mean ± standard deviation of three replicates. Statistical significance was determined by p-value < 0.05.

Results
The ethyl acetate, methanol and water extracts of C. gigantea were studied for their antibacterial activities against MDR bacteria. Of the three extracts, ethyl acetate extract was found to possess antibacterial activity especially against E. coli, P. aeruginosa, S. aureus as well as against reference strains, E. coli ATCC 25922 and S. aureus ATCC 25923. The diameters of inhibition zones varied from 11 to 13 mm and from 8 to 12 mm against Gram-negative and Gram-positive bacteria, respectively (Table 1).
Since the ethyl acetate extract of C. gigantea was found to possess significant antibacterial property so, the ethyl acetate extract was fractionated by column chromatography. The elution was done using petroleum ether, ethyl acetate and methanol in different ratios and six fractions were collected. A varying degree of antibacterial activity was observed with the FR II, FR III and FR IV fractions of ethyl acetate extract against all the tested organisms. Highest zone of inhibition was shown by FR IV fraction followed by FR II and FR III fraction in case of Gram-positive i.e., S. aureus, whereas in case of Gram-negative a consistent increase was observed from FR II to FR IV which was in accordance with increase in polarity of the solvent. However, FR I, FR V and FR VI did not show any antibacterial property.
The MBC values (Table 3) were found to be two-fold greater than the MIC values on the corresponding microorganisms.
The combination of the ethyl acetate extract of C. gigantea with conventional antibiotics: ampicillin, ciprofloxacin, erythromycin, oxacillin and tetracycline against methicillin resistant S. aureus (Fig. 1) and reference strain, S. aureus ATCC 25923, revealed that the antibacterial activity of all studied antibiotics increased in the presence of the plant extract. The FIC index of each antibiotic combined with extract was calculated using eq. 3 ( Table 3). The analysis of results revealed synergistic effects with ciprofloxacin, tetracycline, ampicillin and oxacillin ( Table 4).

Discussion
The spread of drug-resistant microorganisms is a serious threat to successful therapy of microbial diseases. Therefore there is an urgent need to search new compounds characterized by diverse chemical structures and mechanisms of  The test was done in triplicate. Diameter of the zone of inhibitions is given here as mean ± standard deviation  [25] action. Plants are rich in a wide variety of secondary metabolites such as tannins, terpenoids, alkaloids and flavonoids which have been found in vitro to possess antimicrobial properties. C. gigantea, a tree fern belonging to the family Cyatheaceae, possesses immense and diverse ethnomedicinal importance [3][4][5]. Preliminary phytochemical analysis of different extract of the fern was carried out which have brought foreword the presence of various bioactive secondary metabolites viz., steroids, alkaloids, phenolic groups, cardiac glycosides, flavonoids, saponins, tannins and terpenoids [26]. This presence of bioactive secondary metabolites has investigated to study the plant for its various bioactivity. In this study the antibacterial activity of C. gigantea is evaluated. Ethyl acetate extract was recorded to be the active extract against all the tested organisms and this might be due to the presence of various phytochemicals. The antimicrobial property also may be due to the presence of coumarin, which is an antimicrobial agent naturally present in plant [27]. In addition some other isolated compounds viz., 2methylbutane-1,4-diol, 3-(1-ethoxyethoxy) may also impart to the antibacterial property of the plant [26].
The fractionated crude ethyl acetate extract also showed potential antibacterial activity against both Gram-positive and Gram-negative. Of the six fractions, FR II, FR III and FR IV exhibited antibacterial property while FR I, FR V and FR VI did not show any activity. The fractions (FR II, FR III and FR IV) showed significant variations in their inhibition zones against the studied organisms.
The MIC was found to be 200 μg/ml against Grampositive and 400 μg/ml against Gram-negative bacteria. The efficacy of the extract is determined by its MIC value. Extracts having MIC value lesser than 1000 μg of crude extracts or 100 μg of isolated compounds is considered to be effective concentration. In the study the MIC value varied from 200 to 400 μg/ ml against the tested bacteria indicating the fern to be an effective antibacterial agent. The higher MIC against Gram-negative i.e., E. coli ATCC 25922 and P. aeruginosa PAO1 in comparison to S. aureus ATCC 25923 may be due to the presence of outer membrane in their cell wall [28].
The differences in MBC and MIC suggests that the extract possesses a selective antibacterial property. The extract was found to show good bactericidal activity against the tested organisms as the ratio of MBC/MIC was equals to 2 [29].
The present study also focused on the combined effect of ethyl acetate extract and antibiotics. The results revealed the synergistic and additive interactions between the phytochemicals present in the ethyl acetate extract and antibiotics, which are cell wall inhibitors (ampicillin, oxacillin), protein synthesis inhibitor (erythromycin, tetracycline) and DNA synthesis inhibitor (ciprofloxacin). The FIC index showed that significant synergistic activity was shown by ciprofloxacin followed by tetracycline, ampicillin and oxacillin.

Conclusion
Extracts of C. gigantea showed activity against both Gram-positive and Gram-negative organisms and thus offers the scientific basis for the traditional use of the fern. The study also highlighted that FR IV of ethyl acetate extract of C. gigantea showed the highest antibacterial activity which is due to the presence of various active phytochemicals with high peak area shown in GCMS. The present study also provides the basis for future study to validate the possible use against multidrug resistant organisms.