Asian Pac J Trop Med 2014; 7(Suppl 1): S435-S441

S435

Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm

Document heading

doi: 10.1016/S1995-7645(14)60271-6

B actericidal

activity of selected medicinal plants against multidrug resistant bacterial strains from clinical isolates Biswajit Chakraborty, Anupam Nath, Himadri Saikia, Mahuya Sengupta* Department of Biotechnology, Assam University, Silchar, Assam 788011, India

ARTICLE INFO

ABSTRACT

Article history: Received 22 Jul 2014 Received in revised form 29 Aug 2014 Accepted 8 Sep 2014 Available online 17 Sep 2014

Objective: To investigate the antibacterial effect of Curcuma longa (C. longa), Zingiber officinale (Z. officinale) and Tinospora cordifolia (T. cordifolia) against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Bacillus subtilis and Proteus mirabilis of clinical origin. Methods: The antimicrobial efficacy of said medicinal plants and establishment of multidrug resistant character of these bacteria were carried out using disc diffusion, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods. Results: The results of MIC and MBC showed that these clinical bacterial isolates were phenotypically multidrug resistant against standard antibiotics (>500 µg/mL). Compared to standard antibiotics, C. longa, Z. officinale and T. cordifolia were more effective in killing these microbes as evident from MIC and MBC values (5 to 125 µg/mL). Moreover, C. longa had highest antibacterial efficacy compared to Z. officinale and T. cordifolia. Conclusions: The result thus obtained suggests that bioactive principles of these plants can be used particularly against these multidrug resistant bacteria of clinical origin.

Keywords: Bactericidal Medicinal plants Clinical origin MIC and MBC

1. Introduction Antibiotic resistance is now considered a major public health issue with reports of an increasing trend of resistance in clinical isolates[1]. Recently the acceptance of traditional medicine as an alternative form of health care and the development of microbial resistance to the available antibiotics have reaffirmed the need to

*Corresponding author: Dr. Mahuya Sengupta, Assistant Professor, Department of Biotechnology, Assam University, Silchar, Assam 788011, India. E-mail: [email protected] Foundation Project: Supported by Senior Research Fellowship (SRF) Extended fellowship grant under Council of Scientific and Industrial Research (CSIR), Govt. of India (Grant No: 9/747 (0010)/2012-EMR1).

probe the antimicrobial activity of medicinal plants[2]. Curcuma longa L . ( C. longa ) , popularly known as turmeric, has long been used as a spice in S outheast Asia. It has also been used in Oriental folk medicines to treat infectious diseases ( e.g. sinusitis, cough ) , cholecystitis and cholangitis and used as a therapy for hepatic disorders, rheumatism and anorexia[3]. Previous works have shown that C. longa inhibited the growth of activity of some bacteria and fungi[4,5]. C. longa has been reported to show antibacterial activities against methicillin-resistant Staphylococcus aureus (S. aureus) and lower the minimum inhibitory concentrations (MICs) of β-lactams[6]. The water extract of C. longa inhibits the growth of Helicobacter pylori[7]. In a recent report, it has been observed that very low doses of plant extract were

S436

Biswajit Chakraborty et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S435-S441

effective against multidrug resistant clinical isolates[8]. However, little is known about the antimicrobial effects of C. longa on multidrug resistant strains. Zingiber officinale Rosc. (Z. officinale) is a slender, perennial rhizomatous herb. A broad range of biological activities have been attributed to Z. officinale and these include insecticidal[9], antibacterial[10,11], antiprotozoal[12], antioxidant [13] , antiemetic [14,15] , antirhinovirus [16] , immunomodulatory[17], and antihepatotoxic[18]. The herb Tinospora cordifolia ( M enispermaceae ) ( T. cordifolia ) belongs to a group of medicinal plant that grows in the tropical and subtropical regions of I ndia. T he herb is extensively used in the Indian System of Medicine; the extract of different parts of the herb has found wide use in variety of diseases. It is known for its antidiabetic[19], hepatoprotective [20] , and immunomodulatory [21,22] . R ecently, N arayanan et al. reported the antibacterial activity of various medicinal plants including T. cordifolia against multidrug resistant uropathogens[23]. C ompared to other two plants, very few reporting has been observed for T. cordifolia as antimicrobial agents. M oreover, very little is known about the comparative evaluation of antibacterial properties among these three plants against clinical bacterial isolates. So, the present study was intended to make a comparative evaluation of antibacterial properties of C. longa, Z. officinale and T. cordifolia against S. aureus, Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis) and Proteus mirabilis (P. mirabilis) of clinical origin. 2. Materials and methods 2.1. Collection of plant materials and microbes C. longa, Z. officinale and T. cordifolia were selected from agricultural land neighboring A ssam U niversity, S ilchar, A ssam, I ndia. R hizome of C. longa and Z. officinale were then collected, whereas in case of T. cordifolia, the stem parts were collected. Collection of plant material was independent of season. All the plants sample specimens were maintained in the U niversity H erbarium, D epartment of L ife S cience, AUS ( V oucher No- TC 11.21A; CL 11.22, ZO 11.23). The microbial strains viz, S. aureus (SMCSA-324), P. aeruginosa (SMCPA-544), K. pneumoniae (SMCKP-121), E. coli (SMCEC-422), B. subtilis (SMCBS-111) and P. mirabilis (SMCPM-301) were collected

from Microbiology Department of Silchar Medical College, Assam, India. 2.2. Plant extracts preparation for antimicrobial studies Four portions (100 g) of each T. cordifolia (stem parts), C. longa (rhizome parts) and Z. officinale (rhizome parts) were soaked separately in 1 000 mL of acetone (Merck), ethanol ( M erck ) , methanol ( M erck ) and distilled water for 72 h at room temperature. For water extraction, 100 g of each powdered plant sample was boiled in 1 000 mL of hot water for 60 min. T he filtration of the extracted solution was done twice at first with W hatman N o. 1 paper (Merck) and after that the filtrate was again filtered with mixed cellulose ester membrane filter (0.30 µm pore size ) ( M erck ) . F ollowing filtration of the extracts, the evaporation was done under reduced pressure[24]. From these extracted compounds, different concentrations were prepared for antibacterial studies (Table 1). Table 1 Yield of plant extract using different solvents (g). Plant

T. cordifolia C. longa Z. officinale

Methanol

Ethanol

Acetone

Water

0.800

0.845

0.956

0.782

1.000 0.800

1.250 1.000

1.500 0.600

0.980 0.800

2.3. Disc diffusion method The paper disc diffusion method was used to determine

antibacterial activity, which is based on the method described previously[25] . W hatman paper N o. 1 filter paper was used to make sterile discs in order to screen for the antibacterial activities of C. longa, Z. officinale and T. cordifolia. Filter paper was punctured to the shape of commercial antibiotic disc and discs were autoclaved at 121 °C for 5 min. The sterile filter papers were then dipped into the solubilized extract solution at different concentrations. The suspension of bacteria culture was prepared according to the MacFarland standard 0.5 and layered onto the Mueller Hinton agar plates to produce the bacteria field. A series of sterile punctured filter papers containing the extract were placed on the bacteria field by a sterile forceps. D istilled water, ethanol, methanol and acetone were used as negative control while the commercial antibiotic discs viz, ampicillin, clindamycin, penicillin G, erythromycin, chloramphenicol and vancomycin were used as a positive. F inally, the plate was incubated at 37 °C and the zone of inhibition

S437

Biswajit Chakraborty et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S435-S441

was observed after 24-48 h.

2.5. Statistical analysis

2.4. Determination of MICs and minimum bactericidal concentration (MBC)

O ne way ANOVA was carried out to compare the difference between mean (n=3) MIC and MBC values for each plant extract against each resistant microbe.

S ensitivity of bacteria to different T. cordifolia

extracts can be measured by using a tube dilution technique, which determines the MBC . T hese tests were done to determine the lowest concentration of T. cordifolia extract, where it can show the bactericidal or bacteriostatic effect. Each tube contained an inoculum density of 5伊105 CFU/mL of each of the test organisms. All organisms were grown in Mueller Hinton broth. Then, the suspension of all the six bacterial culture was added into tubes containing diluted sample of T. cordifolia extract ( 1 - 100 µ g/ml ) . T he dilution of the samples was done with Mueller Hinton broth. Finally, the tubes containing diluted sample of T. cordifolia and bacteria was then incubated overnight at 37 °C with constant shaking on the shaker. The growth of the microorganisms was determined by turbidity. Clear tubes indicated absence of bacterial growth. For every experiment, a sterility check (methanol, ethanol, acetone and medium ) , negative control (methanol, ethanol, acetone, medium and inoculums) and positive control ( methanol, ethanol, acetone, medium, inoculum and different standard antibiotics individually) were included. The MIC of the alkaloids was the lowest concentration in the medium that completely inhibited the visible growth. T he solvent value was deducted accordingly to get the final results of activity. The MBCs were determined by inoculating the surfaces of Muller Hinton agar plates with 25 µL of samples taken from the clear tubes. A fter the bacterial suspensions had been fully absorbed into the agar, the plates were further incubated at 35 °C for 20 h and were examined for growth in daylight. The MBC 50% and 90% were defined as the concentration of drug that resulted 50% and 90% killing of the bacterium relative to the concentration of bacterium that was present in test wells at 0 h. The ATCC strains of S. aureus, P. aeruginosa, K. pneumoniae, E. coli, B. subtilis and P. mirabilis were purchased from HiMedia company and used as reference strains (data not shown). After the completion of experimental work, the used microbial strains, media and plastic wares were sterilized and discarded as per the guideline of institutional biosafety committee.

3. Results To determine the antimicrobial activity of C. longa, Z.

officinale and T. cordifolia plant crude extract against six clinical isolates, zone of inhibition, MIC and MBC were measured. All the experiments were done in triplicate and results are expressed as mean依standard error mean. The comparative studies of different extracts against each bacterial strain were statistically analyzed by One way ANOVA. The antimicrobial study was carried out against B. subtilis, K. pneumoniae, P. mirabilis, S. aureus, P. aeruginosa and E. coli of clinical origin. A ll of these strains were found to be phenotypically resistant to multiple antibiotics and the resistant property of these selected microbial strains was established from lack of zone of inhibition as well as from the MIC, MBC values of multiple standard antibiotics (Table 2). Table 2 A ntibacterial activity of six standard antibiotics ( 30 µg ) against microbial strains of clinical origin. Bacteria

E. coli P. aeruginosa K. pneumonaie S. aureus P. mirabilis B. subtilis

CL R R S R R S

PE R R R R R S

CH R R R R R S

ER R R R R S R

AM R S R R S R

VA R R S R R R

R : R esistant; S : S ensitive; CL : C lindamycin; PE : P enicillin G ; CH : C hloramphenicol; ER : E rythromycin; AM : A mpicillin; VA : V ancomycin. R esistant: less than 15 mm of inhibiting zone and MBC>0.5 mg/mL; Sensitive: greater than 15 mm of inhibiting zone and MIC