Bactericidal activity of acetone extract of Alpinia galanga on multidrug resistant clinical isolates of Enterococcus faecalis

Introduction: The objective of the study was to evaluate the inhibitory activity of five traditional Indian medicinal herbs against multidrug resistant clinical isolates of E. faecalis. Methods: Sixty five E. faecalisderived from clinical specimens were tested for their resistance pattern to 7 antibiotics by disc diffusion method. Extracts from 5 medicinal plant parts were obtained and tested for inhibitory activity against multi drug resistant clinical E. faecalisisolates. The minimum bactericidal concentration (MBC) for the active plant extract exhibiting highest activity towards the susceptible isolateswas determined by macro broth dilution method followed by sub culturing on agar plate. Results: All the clinically derived E. faecalis isolates were resistant to two or more antibiotics. Acetone extract of Alpiniagalanga inhibited 67.69% of E. faecalis and concentration of <5.0mg/ml was found to exert bactericidal activity against multi drug resistant isolates. Conclusion: Acetone extract of A. galanga exhibit bactericidal activity against multi-drug resistant clinical isolates of E. faecalis and hence could be potential phytotherapeutic candidate.


Introduction
India is a land of rich heritage for employing medicinal plants in treating illness among its diverse indigenous cultures. Several of these folklore medicines, employing herbs and medicinal plants are validated through scientific research and made accessible to the global population. Therapy through plant products benefits its users by providing generalised immunostimulatory and immunomodulatory effects apart from abating the side effects of antibiotics and monetary expenditure for cure [1,2].
Enterococci are one among the leading nosocomial pathogens commonly associated with urinary tract infections, blood stream infections and catheter related infections. Of more than a dozen species in the genus, Enterococcus faecalis is involved in 70 -80% of clinical infections [3]. Infections by multidrug resistant E. faecalis encompassing even the last line drug of choice are emerging around the globe, accounting for increase in morbidity and mortality rates [4,5].

Plant extracts employed for study:
The plants were chosen based on i) their documented use as antimicrobials in literature and ii) traditional use of these plants among the regional folklore against infections [8]. The plants employed in the study (Table  1) were harvested in Cuddalore district and taxonomically identified.Crude extracts from the plants were extracted with solvents mentioned in Table 1 as per the method described by Eloff [9]. The extracts were stored in the form of dried powder at -20 ºC until it was used for further biological evaluation.

Determination of inhibitory activity of the plant extracts by disk diffusion test:
The plant extracts were tested for their inhibitory activity against the 25 multidrug resistant E. faecalis isolates derived from clinical specimens. A single colony from overnight growth on blood agar plates were inoculated in Brain heart infusion broth and incubated at 37 ºC for 4 h.
This bacterial suspension was adjusted to 0.5 Mc Farland Standard and used as inoculum to obtain a lawn culture on Mueller Hinton agar plates (Sisco Research Laboratories Pvt. Ltd., India). Discs (6 mm in diameter) obtained from Whatmann No.1 filter paper was soaked with the plant extracts reconstituted with 20% DMSO (to obtain a concentration of 5mg/ml) and placed on the inoculated plates. Suitable controls were included. Following incubation at 37 ºC for overnight period, the plates were evaluated for the presence of zone of inhibition around the discs.The extracts were considered to exhibit anti-E. faecalis activity if a inhibition zone >10mm was observed was found around the extract impregnated disc.

Determination of the minimum bactericidal concentration (MBC) of A. galanga against E. faecalis isolates:
Macrobroth dilution test was employed to determine the minimum inhibitory concentration (MIC) of A. galangaon susceptible multidrug resistant E. faecalis isolates according to the protocol of CLSI, 2012 [7]. Extract obtained from A. galanga was diluted with Mueller Hinton broth in test tubes to obtain a final concentration of 2.0, 2.5,3.0, 3.5, 4.0, 4.5mg/ml. E. faecalis adjusted to 1.5 x 10 8 CFU/ml was used as inoculum at a quantity of 1ml on the tubes containing A.galanga extract. Mueller Hinton broth containing E. faecalis suspension alone and A. galanga extract without the inoculum were included as controls. The minimum concentration of A. galanga extract which inhibited the visible growth of E. faecalis was considered as MIC value. The MBC of the extract was determined by spot inoculating 10 μl of the suspension from macrobroth dilution tubes lacking the growth of E. faecalis, on Mueller Hinton agar plates. Following incubation at 37 ºC for overnight period the minimum concentration of the extract that produced no visible colonies was recorded as the MBC value for the strain tested.

Results
Isolation of E. faecalis from clinical specimens: Sixty five isolates of E. faecaliswas isolated from urine, pus, blood and ascitic fluid at the isolation rate of 44%, 28%, 16% and 12% respectively.
Antibiotic resistance pattern of clinical E. faecalis isolates: Antimicrobial resistance pattern for E.faecalis isolates showed 72% of isolates to be high-level gentamicin resistant and 68% of isolates demonstrated resistance to 2 antibiotics -erythromycin and ciprofloxacin. None of the isolates were found vancomycin resistant ( fig. 1).
The clinicalisolates of E. faecalis were found to be resistant to a combination of 2 or more antibiotics ( fig.  2). Combined resistance to three antibiotics was the frequent observation and was seen among 35% of isolates. However, none of the isolates were found resistant to all the 7 antibiotics used in the study.  .3). Since, A. galanga exhibited inhibitory activity onthe majority of the E.faecalis isolates used in our study, this extract and the strains inhibited by the extract were chosen for further studies.

Minimum bactericidal concentration (MBC) of A.
Galangal extract for the test isolates: The 44E. faecalis isolates which were inhibited by A. galanga extractas determined by disc diffusion test was further studied for the MBC value of A. galanga extract required to inhibit them.A concentration of 3.0 mg/ml of A. galanga extract was found to be the MBC value for a majority of E. faecalis isolates (36.3%). MBC value for more than 50% of our isolates was ≤3.0 mg/ml (table 2).

Discussion
In our study, E.faecalis was commonly isolated from urine, followed by pus and blood samples. The predominance of this pathogen in urinary tract infections and pyogenic infections are reported widely [10,11]. The 1 st line drug of choice for treating enterococcal infections is with a synergistic combination of beta lactam and aminoglycoside antibiotics [12]. In our study, 42% and 27% of isolates exhibited resistance to beta lactam class of antibioticspenicillin and ampicillin respectively. Further, highlevel resistance to aminoglycosides-gentamicin and streptomycin was noted with 72% and 46% of isolates respectively. With the prevalence of higher resistance rates towards the synergistic antibiotic combinations and the occurrence of multidrug resistance among our clinical isolates, the treatment options for infections demands the role of 2 nd and last line drug of choice which are also noted for their adverse effects [10,13].
The isolation of multidrug resistant E. faecalis from clinical specimens in our geographical locality makes the situation imperative to find an alternate treatment option. The phytochemicals of the extracts among the 5 plants selected in the study have proven antibacterial activity against common Gram positive and Gram negativepathogens [8, 14,15]. In our study, the extracts from the five selected plants exhibited inhibitoryactivity on 13.84% -67.69% of E. faecalis isolates with A.galangademonstrating inhibition on a majority of isolates. A.galanga is widely cultivated medicinal herb in India and the traditional medicinal systems have used the rhizome for diuretic, expectorant, carminative purposes and in treating tubercular glands, bronchitis, kidney and heart diseases [16]. The chief chemical constituent of rhizome is flavonoid which exerts antibacterial activity through inhibition of nucleic acid synthesis, inhibition of cytoplasmic membrane function, inhibition of energy metabolism, inhibition of the attachment and biofilm formation, inhibition of the porin on the cell membrane, alteration of the membrane permeability and attenuation of the pathogenicity [17,18].
The inhibitory activity of M.fragranssucceeds A.galanga in our study, and was active on 47.69% of E. faecalis. Methanol extracts of M.fragrans was earlier reported to have strong antibacterial activity against multi-drug resistant S.typhi [19]. Extracts from A. graveolens,B. serrate and H. indicus inhibited 9%, 41.53% and 18.46% of our isolates. Literatures citing the inhibitory activity of these medicinal plants against several Gram positive, Gram negative bacterial pathogens, yeast and fungi are recorded earlier [20 -22].
The acetone extract of A.galangaemployed in our study proved to exhibit inhibitory activity on E.faecalis. However previous studies indicate ether, ethyl acetate, aqueous extract and essential oil of A.galangafor significant antibacterial activity against S.aureus and S.pyogenes [23]. In our study, the MBC of acetone extract of A.galanga for the clinical isolates of E.faecalis ranged between 2.0 -4.5 mg/ml, and the concentration of 3.0 mg/ml of the extract inhibited 36.3% of isolates.
This concentrationwas found to be lesser when compared tothe study of Okonogi et al. wherein 8mg/ml was the MBC of essential oil of A.galanga against S.aureusATCC 25923 [24]. In another study involving human pathogens, an active component 1'-acetoxy chavicol acetate from the ethyl acetate extract of A.galanga demonstrated antibacterial activity against P.acne with the MIC and MBC values of 156 and 312 μg/ml [25]. A study by Warit et al. showed the extract of A. galanga and its major component S-enantiomer of acetoxy chavicol acetate exhibited anti-tubercular activity on clinical isolates at 2.0 μg/mL [26].

Conclusion
As an attempt to elude from the repercussion of antibiotic usagefor pathogenswhich exhibit multiple drug resistance, an extensive search is made towards herbal therapy. The study documents the inhibitory activity of acetone extract of A.galanga to be superior compared with other medicinal plants for their anti-E. faecalis activity. Further, bactericidal activity was demonstrated at concentration of <5.0mg/ml against all the multidrug resistant isolates used in our study. This substantiates the efficacy of A.galanga as an alternative therapeutic option to mu