Antibacterial Activity of Plants Extracts against Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococcus faecalis

Antibacterial Activity of Plants Extracts against Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococcus faecalis Prashant Agarwal1*, Neeraj Agarwal1, Ritika Gupta1, Meenu Gupta2 and Bindu Sharma3 1Department of Biotechnology, Meerut Institute of Engineering and Technology, Meerut (MIET), 250005, India 2Department of Biotechnology Indian Institute of Technology, Roorkee (IITR), 247667, India 3Department of Biotechnology, Devanagri Post-Graduate College, Meerut, 250002, India


Introduction
The emergence and spread of multidrug resistant (MDR) bacterial pathogens have substantially threatened the current antibacterial therapy [1]. Even though pharmacological industries have produced a number of new antibiotics in the last three decades; resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents [2]. It has been estimated that between 60-90% of the populations of developing countries use traditional and botanical medicines almost exclusively and consider them to be a normal part of primary healthcare [3]. The most problematic bacteria include, but are not limited to, extended-spectrumβ-lactamaseproducing Escherichia coli (ESBL-EC) and Klebsiella pneumoniae (ESBL-KP), carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) [4,5]. Thus, Infectious Diseases Society of America has recognized MRSA, VRE, ESBL-EP, ESBL-KP and Pseudomonas aeruginosa as notorious pathogens among the six major pathogens to which therapies with effective newer antimicrobials are urgently required [1,5]. Incidences of epidemics due to drug resistant microorganisms are now a common global problem posing enormous public health concerns [6]. The global emergence of multi drug resistant bacterial strains is increasingly limiting the effectiveness of current drugs and significantly causing treatment failure of infections [7]. Antimicrobial drug resistance is also of economic concern with impact on doctors, patients, health-care administrators, pharmaceutical companies and the public [8]. The non-availability and high cost of new generation antibiotics with limited effective span have resulted in increase in morbidity and mortality [9]. Therefore, there is a need to look for substances from other sources with proven antimicrobial activity. Consequently, this has led to the search for more effective antimicrobial agents among materials of plant origin, with the aim of discovering potentially useful active ingredients that can serve as source and template for the synthesis of new antimicrobial drugs [10,11]. A vast number of medicinal plants have been recognized as valuable resources of natural antimicrobial compounds [12]. Medicinal plant extracts offer considerable potential for the development of new agents effective against infections currently difficult to treat [6]. A wide range of phytochemicals present in plants are known to inhibit bacterial pathogens [13][14][15]. Many plant species have been used by the indigenous people of India as traditional medicines, including as treatments for infectious diseases. Successful determination of such biologically active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. Organic solvents such as ethanol, acetone, and methanol are often used to extract bioactive compounds [16]. Ethanol, however, is the most commonly used organic solvent by herbal medicine manufacturers because the finished products can be safely used internally by consumers of herbal extracts [17]. The aim of this study is to investigate the antibacterial properties of these extracts by exploring the activities against a collection of clinical isolates of MRSA and VRE.

Plant material
Four plant samples were collected on the basis of traditional medicinal history of India from garden, local markets were studied. All the plant materials were further identified in the Department of Botany, D. N PG College Meerut, UP, India. Table 1 shows the botanical name, family, parts used and ethno-medicinal use of the plants under this study.

Preparation of plant extracts
The plant extracts were prepared with some modifications in the guidelines of [18]. Plant material was freeze-dried and milled to a coarse powder to extract sequentially with hot water and 80% ethanol. Each solvent was replaced three times with fresh solvents and was allowed to remain in contact with the plant materials for 48 hrs. The concentrated extract was centrifuged at 10,000 × g for 20 min at 20°C. The supernatant was recovered, filter sterilized evaporated to dryness in vacuum and stored at -70°C.

Microbial strains
The MRSA strains used in this study were clinical isolates from patients presenting with symptoms of S. aureus-associated diseases. The isolates were identified as S. aureus according to colonial and microscopic morphology, positive catalysis, hemolysis and coagulase production. Strains of Enterococcus faecalis were isolated from patients suffering from nosocomial infections and identified on the basis of glucose fermentation without gas production, catalysis reaction, gamma hemolysis, reduction of litmus milk. Standard strains of Enterococcus faecalis (EFS) and Staphylococcus aureus (SAS) were done in the Department of Zoology, D. N PG College Meerut, UP, India. Standard strains and clinical isolates of MRSA and VRE.

Assay for antibacterial activity
After identification on selective medium all bacteria were grown on Nutrient Agar (Hi-Media M001A) and in Nutrient Broth (Hi-Media M002) at 37°C. Antimicrobial activity of the plant extracts was determined by the agar well diffusion method (Holder et al 1994), with modifications. 200 μL of overnight NB culture were added to 15 ml of molten MUELLER-HINTON Agar (Hi-Media M-173), mixed well, poured into a sterile PETRI dish and allowed to set. A sterile cork-borer (5 mm diameter) was used to make wells in the set agar. 25 μL of plant extract, diluted 1:200 in sterile water, were added to triplicate wells and the plates were incubated overnight at 37°C. Antibacterial activity was recorded as a zone of growth inhibition of greater than 5 mm around the well. All S. aureus isolates were tested for methicillin resistance. The disk diffusion method outlined by the National Committee for Clinical Laboratory Standards (NCCLS) was used with a 1 µg oxacillin disk (Oxoid). Zone sizes were read after incubation at 35°C for 24 h. Isolates with zone sizes 10 mm were considered methicillin resistant.

Results and Discussion
The plant extracts were prepared in different concentrations ranging from 0.2 mg/ml to 2.0 mg/ml using four different plants, the best results were obtained by enlisted concentration of plants-Ageratum conyzoides (Family-Asteraceae) 0.42 mg/ml and 1.52 mg/ ml, Phayllanthus emblica (Family-Phyllanthacea) 0.75 mg/ml and 0.85 mg/ml, Camellia sinensis (Family-Theaceae) 0.85mg/ml and 0.70 mg/ ml and Mentha longifolia (Family-Lamiaceae) 1.32 mg/ml and 0.55 mg/ml for MRSA and VRE, respectively. As the bioactivity of plant extracts depends on the water and ethanol concentration used in the extraction process. Since ethanol is safe to ingest thereby it is most commonly used organic solvent to extract plants extracts which is used as potential therapeutic agent by human society. These extracts were tested for antimicrobial activity against gram positive bacteria Methicillin resistant Staphylococcus aureus (MRSA) isolated from burnt patients and gram positive bacteria Vancomycin Enterococcus faecalis (VRE) isolated from patients suffering from nosocomial infections. These micro-organisms are responsible for causing serious infection in the human body thus need to be treated. The antimicrobial activity was studied using zone of inhibition and zone >5mm was considered as positive result as shown in Table 2 and Figure 1 for MRSA and Table  3 and Pneumonia [19], bacteriocide, antidysenteric [20], fever, rheumatism, headache and colic [21,22] Phyllanthus emblica Phyllanthaceae Amla Arial Part Antimicrobial [23], antioxidant [24,25], anti-inflammatory [26], analgesic and antipyretic [27,28], Adaptogenic [29], hepatoprotective [30].
can serve as potential therapeutic agents however, the application of any compounds to medicine will require safety and toxicity issues to be addressed.

Conflict of Interests
The authors have no conflict of interests.

Plant Species
Antibacterial activity against* *Activity of plants extracts was determined from agar well diffusion assay. Numbers indicates zone of inhibition of test cultures around the wells. Each number was the average of quadruplets Zone of inhibition >5mm was considered as positive result. A negative symbol indicates no inhibition. Numbers in the square brackets indicates incomplete inhibition (hampered growth but not complete abolition of growth) was observed within the specified zone. Concentrations of extracts were 1.52, 0.85, 0.70 and 0.55 mg/ml for Ageratum conyzoides, Phyllanthus emblica, Camellia sinensis, Mentha longifolia, respectively, Penicillin G was used as a reference control drug Table 3: Activity of plant extracts against VRE isolates.