Bactericidal activity of selected medicinal plants against multidrug resistant bacterial strains from clinical isolates

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

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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

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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

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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<0.1 mg/mL.

3.1. Antibacterial effects of C. longa against six clinical isolates The MIC and MBC 90% values of different fractions of C.

longa against B. subtilis, K. pneumoniae and S. aureus ( T able 3 ) showed that the aqueous extract had highest antibacterial activity. In addition, the result of zone of inhibition confirmed that the aqueous extract of C. longa was more effective against these three strains than other extracts ( P< 0 . 000 1 ; T able 4 ) . T he value of MIC [ ( 5 . 92 依 0.04) µg/mL] and MBC 90% [(19.57依0.24) µg/mL] of different

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Table 3 MIC and MBC (mean依SEM) of different extracts of C. longa against six microbial strains of clinical origin (µg/mL). Microbial strains

MIC

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

Methanol extract 20.25依0.12

26.83依0.14

12.43依0.11

27.83依0.14

33.9依0.08

12.68依0.08 9.33依0.14

12.62依0.06

: P<0.000 1.

MBC 90%

6.62依0.16

12.13依0.11

****

MBC 50%

30.17依0.36

MIC

Ethanol extract MBC 50%

11.64依0.11 30.42依0.18 42.50依0.41

41.77依0.12

12.12依0.12 34.38依0.18 44.62依0.16 6.52依0.11

13.8依0.13 18.97依0.12

26.07依0.05

33.83依0.07

12.44依0.09 26.75依0.31 41.83依0.14

23.67依0.27

43.67依0.27

12.09依0.08 24.93依0.05 42.60依0.25

18.60依0.08

28.83依0.07

10.67依0.27 22.87依0.06 32.73依0.10

fractions of C. longa against P. mirabilis (Table 3) showed that the ethanol extract had highest antibacterial activity as compared to other extracts which was confirmed from zone of inhibition (Table 4) against P. mirabilis of clinical origin [(20.67依0.59) mm] (P<0.000 1). The value of MIC [(7.27依0.05) µg/mL] and MBC 90% [(24.67依0.15) µg/mL] of different fractions of C. longa against P. aeruginosa ( T able 3 ) showed that the acetone extract had highest antibacterial activity as compared to other extracts. The result of zone of inhibition [ ( 17 . 17 依 0 . 49 ) mm] ( T able 4 ) also confirmed that (P<0.000 1). The value of MIC [(7.58依 0.07) µg/mL] and MBC 90% [(21.73依0.14) µg/mL] of different fractions of C. longa against E. coli (Table 3) showed that the acetone extract had highest antibacterial. In addition, the result of zone of inhibition (Table 4) against E. coli of clinical origin also showed that the acetone extract of C. longa was more effective than other extracts (P<0.000 1). The MIC and MBC values of different extracts of C. longa against different bacterial strains ranged from 5 to 55 mg/ mL. Table 4 Zone of inhibition (mm) against six microbial strains of clinical origin at (200-300 CFU) different extracts of C. longa at 30 g/disc. Microbial strains Methanol extract Ethanol extract Aqueous extract Acetone extract

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

: P<0.001,

***

21.17依0.44

15.17依0.6

13.67依0.88

21.50依0.62

16.00依0.94

20.67依0.59

16.50依0.41

19.17依0.36

15.67依0.54

18.17依0.49 10.33依0.27

12.17依0.49

10.40依0.33

: P<0.000 1.

****

MBC 90%

10.83依0.36

15.00依0.47 13.83依0.14 13.17依0.36 11.67依0.27

10.33依0.17

****

11.17依0.36

11.33依0.54

****

***

12.33依0.72

MIC

Aqueous extract MBC 50%

MBC 90%

6.50依0.08 16.33依0.27 20.83依0.14

MIC

Acetone extract MBC 50%

24.75依0.12

7.60依0.07 14.13依0.47

21.77依0.12

6.80依0.13 13.70依0.09 21.39依0.16

14.72依0.08 40.57依0.28

6.77依0.15 13.77依0.12 21.65依0.10

14.43依0.08 29.82依0.08

12.73依0.05 25.00依0.09 44.47依0.20

7.58依0.07 14.53依0.24

5.92依0.04 10.37依0.19 19.57依0.24 14.08依0.18 28.87依0.05 48.10依0.45

MBC 90%

7.43依0.05 13.67依0.14

7.27依0.05 14.80依0.09

****

52.85依0.37

**** ****

49.78依0.01

****

24.67依0.15

****

21.73依0.14

****

3.2. Antibacterial effects of Z. officinale against six clinical isolates From the MIC [(8.17依0.07) µg/mL] and MBC [(30.00依0.24) µg/mL] values of different extracts of Z. officinale against

B. subtilis, the methanol extract had highest antibacterial activity as compared to other extracts (Table 5), where the acetone extract [(14.67依0.67) mm] at 30 µg showed highest inhibiting zone against this strain (Table 6). The aqueous extract of Z. officinale showed highest activity compared to other extracts, against three Gram positive bacteria viz. K. pneumoniae, P. mirabilis and S. aureus as was evident from the results of MIC, MBC and zone of inhibition (Tables 5 and 6). After comparing the antibacterial activity (MIC, MBC and zone of inhibition) of different extracts of Z. officinale against two Gram negative bacteria (P. aeruginosa and E. coli), it was found out that acetone extract had highest activity ( T ables 5 and 6 ) . T he MIC and MBC values of different extracts of Z. officinale range between 8 to 95 µ g/m L . T his result suggests that polar fractions of Z. officinale have higher antibacterial activity against Gram positive bacteria where less polar fraction of Z. officinale is more active against Gram negative bacteria. C. longa also showed the same trends of antimicrobial results as Z. officinale against these six clinical isolates. Table 6 Zone of inhibition (mm) against six microbial strains of clinical origin at (200300 CFU) different extracts of Z. officinale at 30 µg/disc. Bacterial strains Methanol extract Alcohol extract Acetone extract Aqueous extract 11.33依0.54 10.67依0.67 14.67依0.67 12.00依0.00 B. subtilis K. pneumoniae 12.33依0.27 12.33依0.33 12.33依0.17 12.67依0.41 P. mirabilis 11.67依0.54 11.17依0.44 10.67依0.44 12.13依0.24 S. aureus 10.33依0.27 12.17依0.44 12.50依0.50 10.73依0.50 P. aeruginosa 13.67依0.72 11.67依0.67 11.67依0.44 11.43依0.23 E. coli 11.33依0.27 10.67依0.33 12.67依0.33 10.57依0.35

***

17.17依0.49

****

17.50依0.62

****

Table 5

MIC and MBC (mean依SEM) of different extract of Z. officinale against six microbial strains of clinical origin (µg/mL). Microbial strains

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

: P<0.000 1.

****

MIC

Methanol extract

8.17依0.07

MBC 50%

MBC 90%

25.25依0.35 30.00依0.24

MIC

Ethanol extract MBC 50%

MBC 90%

MBC 90%

MIC

Acetone extract MBC 50%

45.57依0.24

10.50依0.24

29.70依0.05

23.50依0.24 60.83依0.36 81.25依0.20

19.25依0.20 46.67依0.27

65.67依0.54

27.90依0.08

73.67依0.54

18.27依0.22 51.33依0.27 64.50依0.36

20.32依0.18 58.52依0.24 67.50依0.24

26.33依0.98 60.67依0.54 86.50依0.62

MBC 50%

11.50依0.41 31.17依0.14

17.63依0.26 52.30依0.13 66.4依0.70

16.13依0.11 41.87依0.11 44.67依0.27

Aqueous extract

10.67依0.27 28.60依0.31 36.8依0.16

16.40依0.17 45.57依0.24 61.83依0.14 20.50依0.24 52.33依0.27 74.33依0.27

MIC

18.90依0.08 47.30依0.25 52.83依0.36 34.00依0.94 84.33依0.27 94.17依0.49

15.80依0.13 42.67依0.27 13.13依0.11 39.33依0.27 21.33依0.54 44.77依0.12 20.17依0.49 64.33依0.27

53.60依0.25 45.67依0.72 55.87依0.38 78.20依0.34

21.85依0.06 22.80依0.30 11.53依0.38 13.33依0.36

MBC 90%

40.08依0.07

****

52.73依0.35

77.85依0.06

61.90依0.08

75.93依0.48

35.93依0.43

37.50依0.85

****

94.23依0.51

**** ****

40.28依0.42 50.50依0.62

**** ****

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

3.3. Antibacterial effects of T. cordifolia against six clinical isolates U nlike the antimicrobial results of C. longa and Z.

officinale against these selected clinical isolates, entire extract of T. cordifolia did not show the antibacterial effect. The aqueous extract of T. cordifolia only showed the antibacterial effect (Tables 7 and 8) against these microbial strains and also higher MIC and MBC values as compared to C. longa and Z. officinale. The MIC and MBC values of aqueous extract against six clinical isolates ranged from 25 to 125 µg/mL.

Table 7 MIC and MBC (µg/mL) of aqueous extract of T. cordifolia against six microbial strains of clinical origin (200-300 CFU). Microbial strains

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

MIC

MBC 50%

MBC 90%

27.83依3.34

66.67依0.27

80.50依0.41

10.97依0.45

34.67依0.54

40.33依0.27

10.17依0.36 7.50依0.41

37.07依0.48 43.50依0.26

31.50依0.24 21.50依0.62

86.83依0.14

100.27依0.73

36.67依0.27 27.00依0.47 96.93依0.47

124.57依0.71

Table 8 Zone of inhibition (mm) by aqueous extract of T. cordifolia at 30 µg/disc against six microbial strains of clinical origin (200-300 CFU). B. subtilis 29.50依0.24

K. pneumoniae 23.33依0.54

P. mirabilis 31.00依0.47

S. aureus

23.50依0.62

P. aeruginosa 15.93依0.52

E. coli

19.17依0.36

4. Discussion Emergences of antibiotic resistance can develop from

the selection of intrinsic resistance microbes or it can develop from environmental factor. R esistance is by definiting an in vitro phenomenon. The mechanisms of acquired resistance fall into one of the five categories, and these are 1) Enzymatic modification or destruction of the antibiotic, 2) Reduced antibiotic uptake into the bacterium, 3 ) I ncreased efflux of antibiotic from the bacterium, 4) Alteration or production of a new target site and 5) Over expression of the drug target[26]. In hospital associated microbial pathogen, the existence of drug resistant microbes has been increasing very rapidly. This situation generally develops by the misuse of standard antibiotics or selection of intrinsic resistant pathogen and subsequently transferring this character to other microbes via conjugation, transformation or transduction. Moreover, depending on the environment, microbes can also acquire resistance in response to particular antibiotic during

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nosocomial infection as a result of phenocopy. So, it is very important to measure the response of a particular microbial pathogen to a particular antibiotic in clinical isolates. R ecent accumulation suggests that medicinal plants have been used against resistant microbial pathogen of clinical isolates and compared to single compounds, the plant extract has more profound pharmacological activity due to their multiple mechanism of action or due to synergistic interaction between the different compounds in a mixture[27]. In the present investigation, B. subtilis, K. pneumoniae, P. aeruginosa, S. aureus, P. mirabilis and E. coli of clinical origin were found to be phenotypically multidrug resistant and the resistance character of these microbes were established from the MIC, MBC (90%) values compared to standard antibiotics. The presence of resistant disposition of these six microbial strains might have arisen through nosocomial infection. The present study also reports that B. subtilis, a Gram positive bacteria which can cause severe disease in immucompromised patients, is phenotypically resistant against erythromycin, ampicillin and vancomycin. Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections and it inhibits the enzyme transpeptidase thus blocking the final stage of cell wall synthesis. E rythromycin is a macrolide antibiotic that interferes with aminoacyl translocation by binding with the 50S subunit of 70S ribosome rRNA complex, preventing the transfer of the t RNA bound at the A site of the rRNA complex to the P site of the r RNA complex and thus preventing protein synthesis. Vancomycin prevents incorporation of N-acetylmuramic acid and N-acetylglucosamine peptide subunits into the peptidoglycan matrix, which forms the major structural component of Gram-positive cell walls. This study showed that B. subtilis was resistant to these standard antibiotics thus establishing the fact that this microorganism overcame the entire mechanism of action exerted by these antibiotics. H owever, the crude extracts of C. longa, Z. officinale and T. cordifolia were found to be effective against this phenotypically resistant B. subtilis, as was evident from MIC, MBC values. T he comparative antibacterial studies among the different crude extracts of C. longa and Z. officinale individually, suggested that the aqueous and methanolic extracts of C. longa and Z. officinale respectively, had the highest effectiveness against this resistant B. subtilis. The antibacterial efficacy of C. longa and Z. officinale against B. subtilis were established to be

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equal in range. However, only the aqueous extract of T. cordifolia showed antibacterial activity against B. subtilis. The possible mechanism of showing antibacterial activity by these three plants was either the compounds of these plants altering the modified form of targeted biomolecules or the mixture of compounds present in selected plants acted on multiple targets in this particular microbe. In addition to erythromycin, ampicillin and vancomycin, our study also reported that S. aureus of clinical origin, which can cause range of diseases starting from mild skin infection to severe toxic shock syndrome, was phenotypically resistant against clindamycin, penicillin, and chloramphenicol. C lindamycin is a lincosamide antibiotic having bacteriostatic effect. It is an inhibitor of bacterial protein synthesis that acts by impairing ribosomal translocation, in a way similar to macrolides. It does so by binding to the 50S rRNA of the large bacterial ribosome subunit. P enicillin is an antibiotic of β-lactam group. Bacteria constantly remodel their peptidoglycan cell walls, simultaneously building and breaking down portions of the cell wall as they grow and divide. β-Lactam antibiotics work by inhibiting the formation of peptidoglycan crosslinks in the bacterial cell wall. R esistance to these antibiotics by S. aureus creates a threat against the treatment of this bacteria associated diseases. However, the crude extract of C. longa, Z. officinale and T. cordifolia showed antibacterial activity against this antibiotic resistant S. aureus strain probably by overcoming or bypassing this entire resistance mechanism present in it. The trend of results showed by these plants against this S. aureus strain was similar to the result as obtained against B. subtilis. T he antibacterial activity of C. longa, Z. officinale and T. cordifolia against P. mirabilis, P. aeruginosa, K. pneumoniae and E. coli was also studied and it was found that this four Gram negative strains were resistant to almost all selected six standard antibiotics. But the acetone extract (less polar) of C. longa was exceedingly effective against G ram negative P. aeruginosa and E. coli. T he acetone fraction of Z. officinale also showed highest activity against these two G ram negative strains as compared to other extracts. However, the activity of Z. officinale was observed to be subordinate as compared to C. longa. Comparative antibacterial studies among different extracts of C. longa and Z. officinale against other two selected Gram negative bacteria viz, K. pneumoniae and P. mirabilis showed contrasting results i.e. aqueous fraction (polar compounds)

had the highest activity. In case of T. cordifolia, only the aqueous extract showed the antibacterial activity against all these selected six microbial strains but at thrice more concentration than C. longa and Z. officinale. Thus, it can be suggested that probably the relevant extracts of C. longa and Z. officinale penetrate the envelope’s outer membrane of these two Gram negative bacteria while the aqueous fraction of T. cordifolia follows an analogous kind of mechanism. Another promising rationalization of showing antibacterial activity against six phenotypically resistant strains by all these three plants was that probably it was the mixture of compounds present in the respective plant extracts that overcame the resistance mechanism by interacting with these microbes probable via multiple pathways and targets. This study showed the efficacy of C. longa, Z. officinale and T. cordifolia as antimicrobial agents against phenotypically resistant bacteria of clinical origin. Comparative antibacterial evaluation among these plants carried out in this study also showed that C. longa was more effective in killing these selected bacteria of clinical origin. Thus, the study not only explored the emergence of multidrug resistant bacteria in clinical isolates but also explored the fact that medicinal plants still have great reservoir of new antimicrobial agents, which are further needed to be explored in more details. Conflict of interest statement The authors declare that there is no competing interest among the authors. Acknowledgements W e gratefully acknowledge the D epartment of

Microbiology, Silchar Medical College, Silchar, Assam,

I ndia and their associated staffs for supplying these selected microbial strains. We also acknowledge CSIR for giving the SRF Extended Fellowship to Dr. Biswajit Chakraborty and also to Assam University authority for giving permission to carry out this research work. This paper was 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).

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

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