The possibility of using Sansevieria aethiopica ( Thunb ) leaf extracts in combinations with gentamicin for the treatment of oral enterococcal infections

Sansevieria aethiopica (Thunb) has been reported to be used for the treatment of oral infections in Eastern Cape of South Africa. Based on ethnobotanical survey, the plant was selected for the possible synergistic effects of its acetone, ethanol and methanolic extracts with gentamicin on the planktonic and sessile cells of Enterococcus faecalis ATCC 29212 and Enterococcus faecalis KZN. In vitro interactions between the plant extracts and gentamicin were studied using checkerboard macrodilution method and anti-biofilm activity of the iso-effective combinations was determined by semi-quantitative adherence assay. Acetone extract of S. aethiopica has the highest inhibitory activity. The minimum concentration of gentamicin that inhibited the two isolates was the same (0.016 mg/ml). Different isoeffective points were observed with fractional inhibitory concentration indexes ranged between 0.375 and 1.9313. Out of the iso-effective points observed only four were synergistic while one was addictive. The maximum biofilm reduction was observed when the two antibacterial agents were combined. We therefore suggest that the extracts of the plant at the test concentrations can be used in combination with gentamicin for oral hygiene.


INTRODUCTION
The prevalence of dental caries in school aged children is up to 90% and the majority of adults are also affected (Petersen et al., 2005) and chronic diseases like diabetes and HIV have been reported to enhance oral infections (Petersen, 2003).Oral infection is caused by different bacteria out of which Enterococcus faecalis is emerging.E. faecalis has been mentioned with increased frequency with regard to oral with post-treatment diseases.Enterococci are Gram positive coccus bacterium and a part of the normal flora in the oral cavity and gastrointestinal tract.E. faecalis has been reported as the most common bacterium recovered from different oral infections with failed treatment and persistence (Rams etal., 1992;Colombo et al., 2002;Rocas et al., 2004).The pathogen survives in situations where conventional therapy has eliminated other bacteria; this attribute has resulted in the emergence of drug resistance strain of the bacterium (Sundqvist et al., 1998).
E. faecalis accounts for around 80% of all infections caused by enterococci (Sahm, 2000).Enterococci pose increasing problems in medicine due to an increased resistance to various antibiotics.It account for 10% of the dental infections among healthy individuals (Smyth et al., 1987) and higher rate of 60% was observed among longterm patients (Murray, 1990).
The genus Enterococcus has been implicated in 60% of dental infections among school children and 75% of patients with endodontic infection (Gold et al., 1975).Sedgley et al. (2004) reported the presence of E. faecalis in 29% of oral rinse samples, 55% of tongue dorsum and 22% of gingival sulcus samples from 41 endodontic subjects.The genus is well known for biofilm formation *Corresponding author.E-mail: aafolayan@ufh.ac.za.Fax: +27-866-282-295.
which serves as an important reservoir of cells that can repopulate colonized sites hence lead to persistency (Costerton et al., 2011;Li and Yu-mei, 2011).Biofilms are responsible for persistent pathogens on infection sites (Costerone et al., 1999;Anderson et al., 2004;Li and Yumei, 2011).
The use of plant for oral hygiene is age long.The Babylonians recorded the use of chewing sticks in 7000 BC and its use ultimately spread throughout the Greek and Roman Empires (Almas and al-Lafi, 1995).It has been documented that medicinal plants confer considerable antibacterial activity against various bacterial pathogens including those responsible for dental infections (Jonathan et al., 2000).
Sansevieria aethiopica (Thunb) is a medicinal plant belonging to the family Asparagaceae.It is a perennial shrub with tough, semi-succulent and erected leaves (Van Wyk et al., 2000).The hard, mottled leaves arise from thick fleshy rhizomes and like other members of the genus, it flourishes under low light conditions.S. aethiopica is called isikholokotho, isikwendle or isitokotoko in South Africa where it has been long recognized as a remedy for the treatment of dental infections, otitis, ulcers among others (Watt and Breyer-Brandwijk, 1962;Hutchings et al., 1996;Newton, 2001;Von Koenen, 2001).
The aim of this study was to investigate the in vitro interactions between extracts of S. aethiopica and gentamicin and the activities of the resultant iso-effective combinations against biofilm of E. faecalis ATCC 29212 and E. faecalis KZN.

Plant material
Plant material was collected in March 2012 from a single population of S. aethiopica growing around Alice Township in Nkokobe Municipality, Eastern Cape Province (32°47' S, 26°50' E and altitude 589 m).The species was authenticated by Prof. Donald S. Grierson, University of Fort Hare, Alice, South Africa and a voucher specimen (DavMed, 2012/2) was prepared and deposited in the Giffen Herbarium of the University of Fort Hare, Alice, South Africa for future reference.Plant sample was dried in the oven at the temperature (40°C).
The dried leaves of the plant sample were pulverized.Powdered plant material (40 g each) was separately extracted in acetone, ethanol and methanol for 48 h on an orbital shaker (Stuart Scientific, Manchester, UK).The extracts were filtered through Whatman No. 1 filter paper.The extracts were evaporated to dryness under reduced pressure at 40°C using a rotary evaporator (Laborota 4000-efficient, Heldolph, Germany).Individual crude extracts was diluted using 50% dimethylsulphoxide to give 50 mg/mL stock solution (Taylor et al., 1995).This was then diluted to the required concentrations for the bioassay.

Test organisms
Two strains of E. faecalis (E.faecalis KZN and E. faecalis ATCC 29212) used in this study were obtained from the Department of Biochemistry and Microbiology, University of Fort Hare, Alice, South Africa.Each strain was maintained on Nutrient Agar (Biolab, South Africa) plates.The grown cultures were used for preparation of bacterial suspensions in sterile distilled water with densities adjusted to 0.5 McFarland standard.

Antimicrobial susceptibility tests (AST)
The checkerboard method was used to examine the effects of combinations of S. aethiopica extract and gentamicin against strains of E. faecalis.Twofold serial dilutions of crude extract of S. aethiopica and gentamicin were added to make up to final concentrations of 0.0488 to 50.0mg/ml and 1.0 to 64.0 μl/l, respectively in Mueller Hinton broth.The tubes were incubated for 24 h at 37°C.The tube with least concentration and with no sign of growth was taken for MIC of each of the antibacterials and the iso-effective combinations.Fractional inhibitory concentration (FIC) index, which represents the sum of the FICs of each drug tested, where the FIC is determined for each drug by dividing the MIC of each drug when used in combination by the MIC of each drug when used alone.
Where, MICExt alone and MICGen alone were respectively the MICs of extract and gentamicin when acting alone, and CExt comb and CGen comb are concentrations of extract and gentamicin at the iso-effective combinations respectively.The interpretation of FICI was made as follows: synergistic (<0.5), additive (0.5 to 1.0), indifferent (>1), or antagonistic (>4.0) according to White et al. (1996).

Biofilm eradication test
Mueller Hinton broth (5 ml) was prepared in the test tubes and inoculated with then test organism.The tubes were incubated at 37°C for 6 h after which the MICs of the S. aethiopica extracts and the isoeffective combinations were added to separate tubes except the control.The tubes were further incubated aerobically for 24 h at 35°C.After the incubation, the contents of tubes were discarded and the tubes washed three times with sterile distilled water.The remaining attached bacteria were fixed with methanol after 15 min of which the tubes were emptied, air dried and stained with 10 ml of 1.0% crystal violet for 5 min.Excess stain was rinsed off with water.After the plates were air dried, the dye bound to the adherent cells was extracted with 10 ml of 33% (v/v) glacial acetic acid (Merck, Darmstadt, Germany) per tube.The optical density of each tube was measured at 570 nm using UV-3000 PC spectrophotometer (Optima Scientific).The percentage reduction in biofilm was calculated as %Biofilm reduction= (AC-AT)/AC) x100 Where, AC = absorbance of the control, AT = absorbance of the test

RESULTS AND DISCUSSION
In this study, S. aethiopica leaf extracts in combination with gentamicin were screened against the E. faecalis strains planktonic and sessile cells.S. aethiopica alone and in combinations with gentamicin produced good inhibitory activity on the isolates.The result of MIC study is represented in Table 1.Against E. faecalis KZN, methanolic and ethanolic extracts had lower inhibitory activity, compared to acetone extract.The MICs of ethanolic extract of S. aethiopica were 0.3906 and 0.1953 mg/ml against E. faecalis KZN and E. faecalis ATCC 29212 respectively.The minimum concentration of genta-  micin that inhibited the two isolates was the same (0.016 mg/ml).Our report is similar to that of Van Wyk and Gericke (2002) and Aliero et al. (2008) that reported that Sansevieria spp.are very rich in antimicrobial compounds.The plant has also reported to inhibits bacteria associated with otitis (Hutchings et al., 1996).Philip et al. (2011) reported antibacterial activity of closer member of Genus; Sansevieria roxburghiana on common pathogens.They also reported, in agreement with our results, that the methanolic is more active on E. faecalis than acetone extract of the plant.The S. aethiopica extracts could be good alternatives against enterococcal infections.Enterococcal oral infections are developing resistance against antibiotics and different researchers have suggested alternates to antibiotic treatment of the infections (Rocas et al., 2004;Stuart et al., 2006;Zehnder and Guggenheim, 2009).The FIC index is the most frequently used method to determine the interaction between antifungal drugs.The interpretation is based on the value got as synergistic interaction of anti-bacterial which has FIC that is less than one.This means that the action is better when in combination than they are separately (Isenberg, 1992).FIC results were provided to allow re-evaluation of the results and also indicate whether the degree of interaction (Eliopoulos and Eliopoulos, 1988).Though bulk of the thirty six iso-effective points observed were indifferent, only four showed synergistic interactions and one was addictive, none was antagonistic as shown in Table 2.The FIC indexes calculated for different isoeffectives concentrations ranged between 0.375 and 1.9313.The interaction between the combinations of S. aethiopica extracts and gentamicin against two strains of E. faecalis were predominantly indifference.FICI values greater than 1 were considered as indifference and as the value decreases the interaction tends towards synergism.The FIC index is based on the assumption that neither the S. aethiopica extract nor gentamicin would interact with itself, therefore the effect of a selfdrug combination will always be additive, with an FIC index of 1.An FIC index lower or higher than 1 indicates synergy or antagonism, respectively, because less or more of either of the extracts of gentamicin would be required in order to produce the same effect as either alone.
As shown in Tables 3 and 4, at the MIC of ethanolic extract, there was reduction in biofilm formed by the two test organisms.The maximum biofilm reduction was observed when two antibacterial agents were combined as shown in Table 5.The inhibitory effect of the extracts (alone) on the biofilm of the two enterococci was noticed to be more pronounced than the gentamicin except for the methanolic extract on the biofilm of E. faecalis ATCC 29212.The combinations of extracts and gentamicin achieved inhibition of cells in suspension and eradication of the biofilm of the test bacteria.The recognition that most oral infections developed from a biofilm explains why some dental infections like periodontitis have been reported to be very difficult to prevent and treat (Kishen et al., 2004;Rocas et al., 2004;Arias-Moliz et al., 2009).All the S. aethiopica extracts were effective at reducing the E. faecalis biofilms at concentrations that will inhibit the planktonic cells of the pathogens.Gentamicin, like other aminoglycoside antibiotics, affects Gram positive bacteria by production of transferase enzyme and impairment of permeability of cell membrane (Simjee et al., 1999; Katzung , 2006).It also impaired 30S ribosomal subunit and inhibits protein synthesis by causing misreading of mRNA and complete inhibition of protein synthesis at low and high levels, respectively (Kaye, 2004;Poole, 2005).Medicinal plants have different sites of actions due to the different bioactive compounds (Cushman et al., 1991;Qin et al., 2010).The actions of these antimicrobial must have complemented each other to achieve this activity.The variation in the activities of the combinations may be due to different bioactive compounds present in the S. aethiopica extracts as suggested by Gatsing et al. (2010).The S. aethiopica extracts reduced the pre-formed biofilm at varying degrees, though there was no total eradication at the MICs (of both the extracts and gentamicin) and also at the iso-effective concentrations.This is in agreement with the work of George et al. (2005), Arias-Moliz et al., (2009, 2010) and Nett et al., (2008) that reported that higher concentrations than MIC are needed to clear already established biofilm due to the protection offered by the glycoprotein.Pathogens within a biofilm environment behave very differently from freefloating bacteria (Lima et al., 2001;Kishen et al., 2008;Arias-Moliz et al., 2010;Balaei-Gajan et al., 2010).Protective extra-cellular slime matrix makes bacteria extremely resistant to antibiotics, antimicrobial agents and host defense mechanisms (Colombo et al., 2002;Rocas et al., 2004;Rolland et al., 2006;Daneshmehr et al., 2008).
The combinations of the agents were able to break the barrier created by the biofilm.This is necessary before dental pathogen embedded in the biofilm could be eradicated or reduced.The efficacies of the extracts of S. aethiopica (singly and in combination with gentamicin) justifies its usage in oral hygiene and also suggests it as an important candidate for the formulation of paste or tincture for oral hygiene and treatment of enterococcal infections.However, isolation and characterization of the active ingredients in this plant together with their mechanisms of actions on pathogens are still open for further investigations.

Table 1 .
Combined activity of extracts of S. aethiopica and gentamicin on planktonic cells of E. faecalis strains.

Table 2 .
Numbers of the iso-effective combinations and interactions of extracts of S. aethiopica and gentamicin on planktonic cells of E. faecalis strains.

Table 3 .
Effect of ethanolic extracts of S. aethiopica in combination with gentamicin on biofilm of E. faecalis strains.

Table 4 .
Effect of methanolic extracts of S. aethiopica in combination with gentamicin on biofilm of E. faecalis strains.

Table 5 .
Effect of acetone extracts of S. aethiopica in combination with gentamicin on biofilm of E. faecalis strains