Antimicrobial Activity of Extracts against MRSA

 

Ms. Sharma. Shalini1*, Dr. Upadhayay U. M2, Dr. Mistry Sunil3

1PhD Scholar, R.K. University, Rajkot

1Assistant Professor, Babaria Institute of Pharmacy, Varnama, Gujarat.

2Principal, Sigma Institute of Pharmacy, Bakrol, Vadodara.

3Associate Professor, Malhotra College of Pharmacy, Bhopal, (M.P)

*Corresponding Author E-mail: shalineesharma29@gmail.com

 

ABSTRACT:

MRSA  is the common cause of diabetic foot infections. By the market survey, 26 drugs were found to be present in  various antidandruff  herbal preparations and were selected for the present study. Aqueous extract of these drugs were screened for antibacterial activity on  MRSA .During this research this was  observed that  out of 26 extracts 11 showed more consistent and prominent antimicrobial activity  in terms of zone of inhibition (ZOI).The study  on MRSA  was surprising because MRSA  is resistant to many antibiotics. From the above finding ,it can be concluded that  the selected medical plants have  great potential as antimicrobial agent, and could  be employed in antimicrobial herbal formulation further for fungal  and bacterial infections of the skin, nails and hair caused by dermatophytes, Candida  albicans  and  Malassezia furfur. MIC of extracts  showed prominent  response was done.

 

KEYWORDS: MRSA, ZOI, Antimicrobial, Diabetic foot, Extracts.

 


 

INTRODUCTION:

MRSA (Methicillin resistant staphylococcus aureus) is responsible for worldwide infections in infants, children and adults. Diabetic foot infection is also caused as a result of MRSA. There are certain complications in treating the infectious diseases due to the presence of drug resistant bacteria and fungal pathogens.1  In recent years, antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world.

 

Medicinal plants contain many types of naturally occurring anti microbial compounds that can be effectively used against microbial infections.

 

Alkaloids, flavones (flavonoids, flavonols, Quinones), essential oils, lectins, polypeptides, phenolics, polyphenols, tannins and terpenoids are the major groups of naturally occurring antimicrobial agents that can be extracted from medicinal plants.2In the present study 26 Indian medicinal plants were selected and, their aqueous extracts were screened against multi drug resistant bacteria, the major causative pathogen for diabetic foot infections.

 

The selection of plant was on the basis of literature review and market survey, traditional drugs used in herbal antidandruff  preparations as  anti microbial agent  were selected for study on MRSA. However most of these extracts were not previously screened against multi-drug resistant  pathogen .The substances that can either inhibit the growth of pathogens or kill them and have no or least toxicity to host cells are considered candidates for developing new antimicrobial drugs.

 

MATERIAL AND METHODS:

The invitro antibacterial studies were carried out by agar cup-plate method. Gentamicin and Amikacin were used as standards. Each well in the plate was loaded with 300 µ / ml of extracts and standards. Antibacterial activity was done at Department of Microbiology, R.D. Gardi Medical College, Ujjain (MP). 

 

Material

Plant extracts – All 26 extracts were obtained from Amsar Private Limited , Indore (M.P.)

Microorganism Used - MRSA (ATCC No.2267)

 

Preparation of Inoculum  

Suspension of organism was prepared as per McFarland standard (0.05%) 24 hours old culture (for bacteria) Suspension of organism was made in the sterile isotonic solution of sodium chloride (0.9% w/v) and turbidity was adjusted such that it contains approximately 1x103 Cells/ ml. It was obtained by adjusting the optical density of bacteria suspension to that of a solution of 0.05 ml of 1.175% of Barium Chloride and 9.95 ml of 1% sulphuric acid. 3

 

Preparation of culture media

A.  Nutrient agar media (NA)

Direction – Suspend 28.0 gms in 1000 ml distilled water. Heat to boiling to dissolve the media completely. Sterilize by autoclaving at 15 lbs. pressure (121o C.) for 15 minutes.

 

Table I  Standard Formula

S.No.

Ingredients

Gms/ltr

1

Peptic digest of animal tissue

5.00

2

NaCl

5.00

3

Beef extract

1.50

4

Yeast extract

1.50

5

Agar

15.00

 

PH at 25oC – 7.4 + 0.2

Uses – A general culture media which may be used as an enrichment medium by incorporating 10 % blood or other biological fluids.

 

B.  Muller Hinton Agar (MHA)

Direction – Suspend 38.0 gms in 1000 ml distilled water. Heat to boiling to dissolve the media completely. Sterilize by autoclaving at 15 lbs. pressure (121o C.) for 15 minutes. Mix well before pouring.

 

Table II  Standard Formula

S.No.

Ingredients

Gms/ltr

1

Beef infusion

300.00

2

Casein acid hydrolysate

17.50

3

Starch

1.50

4

Agar

17.00

PH at 25oC – 7.3 + 0.2

Uses – For cultivation of Neisseria and for determination of susceptibility of microorganism and antimicrobial agents.

 

Preparation of seeded media4 100 ml of sterile molten agar media was seeded by organism (about 6 ml according to McFarland standard). In semi hot condition (40o C) was poured aseptically in sterile Petri plate and allowed to solidify at RT (room temperature).

 

Anti bacterial activity

Evaluation of activity was carried out by agar well method. Antibacterial activity was measured in terms of zone of inhibition (ZOI) and minimum inhibitory concentration.5

 

Agar well method

The medium was prepared by dehydrated media but dissolving in distilled water and subjected to sterilization in autoclave at 121o C for 15 minutes. The petri plates were washed thoroughly and sterilized in hot air over at 160o C for two hours. 30 ml of sterile molten agar media was seeded by organism (about 2 ml according to McFarland. Standard) in semi hot condition was poured aseptically in sterile Petri plates and allowed to solidify at RT.

 

Bores of 6 mm diameter were made on medium using sterile borer and extracts were added to respective bores. Amikacin 30 mg and Gentamicin 10 mg were taken as standards. The petri plates seeded with organism containing extracts and the standard were kept in room temperature for one hour to facilitate the diffusion of extracts and the standards into the media. After diffusion the plates were incubated at 37oC (+1) for 24 hours incubation and zone of inhibition was observed.

 

Determination of Minimum Inhibitory concentrations (MICs)

MIC assay was performed using the crude extracts with concentration of 10 to 500 μl/ml. After incubation at 370C for 24 hrs, the dilution showing highest microbial growth inhibition was recorded as Minimum Inhibitory Concentration (MIC) respectively for each pathogen. Standard used were Amikacin 30 Mg, Gentamicin 10 mg for antibacterial activity and zone size observed was more than 24 mm.

 

TABLE   III  Assessment of Anti bacterial activity in terms of zone of inhibition (ZOI)

Bacterial strains

Extracts                     

Herbal extracts  (300µ/ml)

Zone of inhibition (mm)

 1

 2

 3

 4

 5

 6

 7

 8

9

10

11

12

13

MRSA

 -

14

 24

14

19

 16

 14

  -

 -

 -

 -

 -

14

Standard

  All zones were more than 24 mm

* 1.Glycerrhiza, 2.Bakuchi, 3.Bahera, 4.Turmeric, 5.Redsandal,6. Gulab, 7.Neem bark, 8.Bhrami, 9.Amahaldi, 10.Amla, 11.Ashwagandha, 12.Reeta, 13.Shikakai.* Standard used   Amikacin 30 Mg, Gentamicin 10 mg

 

 TABLE IV Assessment of Anti bacterial activity in terms of zone of inhibition (ZOI)

 Bacterial   strains

Extracts                      

Herbal extracts  (300µ/ml)

Zone of inhibition (mm)

14

15

16

17

18

19

20

21

22

23

24

25

26

MRSA

 -

 -

14

  -

18

  -

  -

  -

  -

 19

28

 -

  -

Standard

  All zones were more than 24 mm

14. Neel, 15.Bhringraj, 16.Jasund, 17.Shatavari, 18.Lemon, 19.Guduchi, 20.Sarsaparilla,21. Meethi, 22.Neem leaf 23.Heena, 24.Harir, 25.Jatamansi, 26.Kumari* Standard used   Amikacin 30 Mg, Gentamicin 10 mg

 

 

Table V Assessment of anti bacterial activity in terms of minimum inhibitory   concentration (MIC)

Bacterial

Strain

Conc.

µ/ml

Diameter of zone in (mm)

 

 

 

MRSA

 

Extracts

1

2

3

4

5

6

7

8

 9

10

11

 

10

-

-

-

-

-

-

-

-

-

-

-

50

-

-

-

-

-

-

-

-

-

-

-

100

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

200

-

18

-

-

-

-

--

-

-

-

--

300

14

24

14

19

16

14

14

16

18

19

 

400

16

29

15

22

18

15

18

17

19

19

 

500

17

30

15

26

19

16

22

21

20

20

 

*1.Bakuchi, 2.Bahera, 3.Turmeric, 4.Red Sandal, 5.Gulab, 6.Neem bark, 7.Shikakai, 8.Jasund, 9.Lemon, 10.Heena, 11.Harir

 


Assessment of Anti bacterial activity of extracts on MRSA in terms of ZOI  

     

Fig 1: ZOI Glycerrhiza,2.Bacuchi,3.Bahera, 4.Turmeric, 5.Redsandal,6. Gulab7.Neem bark,8.Bhrami,9.Amahaldi, 10.Amla, 11.Ashwagandha, 12.Reeta, 13.Shikakai, 14.Neel,15.Bhringraj,16.Jasund,17.Shatavari, 18.Lemon, 19.Guduchi, 20.Sarsaparilla,21.Meethi, 22. Neem, 23.Heena, 24.Harir, 25.Jatamansi, 26.Kumari

 

 


RESULTS:

Based on antimicrobial susceptibilities of MRSA, there is a need to search an antimicrobial material in the natural sources Total 26 extracts (aqueous). Screened for potential antibacterial activity against MRSA. ZOI were observed in Bakuchi, Bahera, Turmeric, Red Sandal, Gulab, Neem Bark, Shikakai, Jasund, Lemon, Heena, Harir.

 

Out of these 11 extracts, Red Sandal, Harir and Shikakai provided more consistent and prominent antimicrobial activity as compared to other extracts (Table III and IV).Susceptibity of MRSA to different antibiotics and plant extracts has been documented in literature, .it was surprising to note ZOI of  these extracts. Inhibition provided by Harir appeared to be highly active. (Fig .1). The extracts with promising antimicrobial activity (with zone of inhibition > 10 mm) was subjected for the MIC(minimum inhibitory concentration) assay to find out the lowest concentration of extract that inhibits the growth of the test organisms The data obtained through MIC (Table V) revealed variability in the inhibitory concentrations of each extracts for given bacteria.

 

DISCUSSION:

Our study was designed to look at antimicrobial agents present in various marketed herbal antidandruff preparations. From the above study, it can be concluded that the selected medicinal plants have great potential as antimicrobial agent against MRSA.

 

MRSA is the major cause for so many skin infections, including diabetic foot infections which can be controlled or cured by the further investigation done on these extracts. Extracts individually or in the combinations with antibiotics. This comprehensive in vitro study will provide valuable scientific information towards the development of more effective herbal preparation for superficial infections with no synthetic base, less or no interaction, more stable with less or no preservatives.

 

ACKNOWLEDGEMENTS:

The author wish to thank the Director , Microbiology, RDGD, Medical College, Ujjain, (M.P.), India for providing all facilities for this work.We also thank  Mrs.Shah (HOD, Microbiology, RDGD) and Imran (Microbiologist, RDGD, Ujjain) their tremendous support and guidance and to Amsar Private Limited, Indore, (M.P) for providing plant extracts used in this study.

 

REFERENCES

1.       Ellile J.C.G. Diabetic foot infections. Diabeted Care, volume 19, Number 6,June 1996.

2.       Samy RP, et al.  Therapeutic potentials of plants as anti-microbials for drug  discovery. Evidence-Based Complementary  and Alternative Medicine.7(3); 2010:283-294.

3.       Jarald E.et al. Anti microbial activity of leaves of Lantana camara and Calotropis procera,   Indian  Drugs ,45 (9);2008

4.       Roopashri TS.et al, Anti bacterial Activity of anti psoriatic herbs: Cassia tora, Momordica charantia and Calendula officinalis International  Journal  of Applied Research in Natural Products Vol. I (3);2008

5.       Praveen D., Sharmishtha P., Phytochemical Screening and Antimicrobial Activity of Some Plants Against Multi-drug Resistant Bacteria from Clinical Isolates. Indian Journal of  Pharmaceutical  Science,74(5);2012: 443–450.

 

 

 

Received on 15.12.2016          Modified on 26.12.2016

Accepted on 31.12.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(12):2283-2286

DOI: 10.5958/0974-360X.2016.00460.1