Wageningen Academic P u b l i s h e r s
Beneficial Microbes, September 2011; 2(3): 53-61
Antibacterial activity of selected medicinal plants against multiple antibiotic resistant uropathogens: a study from Kolli Hills, Tamil Nadu, India A.S. Narayanan1, S.S.S. Raja2, K. Ponmurugan1, S.C. Kandekar3, K. Natarajaseenivasan4, A. Maripandi5 and Q.A. Mandeel6 1K.S. Rangasamy College of Arts and Science, PG and Research Department of Microbiology, KSR Kalvi Nagar, Thokkavadi
Post, Tiruchengode 637 215, Tamil Nadu, India; 2Bharathidasan University College, Department of Microbiology, Perambalur, Tiruchirapalli, Tamil Nadu, India; 3KSR Institute of Dental Science & Research, KSR Kalvi Nagar, Thokkavadi Post, Tiruchengode 637 215, Tamil Nadu, India; 4Bharathidasan University, Department of Microbiology, Tiruchirapalli 620 024, Tamil Nadu, India; 5King Saud University, School of Biological Sciences, P.O. Box 2454, Riyadh 11451, Saudi Arabia; 6University of Bahrain, Department of Biology, P.O. Box 32038, Sakhir Campus, Bahrain;
[email protected] Received: 4 August 2010 / Accepted: 26 August 2011 © 2011 Wageningen Academic Publishers
Abstract The increasing incidence of antibiotic resistance among bacterial pathogens necessitates medicinal plants as an alternate therapy in restricting the resistant infectious organisms. In this primitive study, the antibiotic resistance of organisms isolated from urinary tract infected patients was evaluated using the National Committee for Clinical Laboratory Standards (NCCLS) method and Multiple Antibiotic Resistance (MAR) index values, and the MAR values was also calculated for plant extracts. The 10 common medicinal plants collected from Kolli hills, Namakkal, south India were extracted using the chloroform, methanol, acetone, ethanol and saponification procedure. The efficacy of the extracts on the uropathogens was tested by agar disc diffusion method in order to analyse the inhibitory activity of plant extract on the organisms. Azadiracta indica A. Juss., Tinospora cordifolia (Wild.) and Euphorbia hirta Linn. exhibited high inhibitory activity against most of the 11 tested organisms followed by Cassia javanica Linn. and Phyllanthus niruri Linn. The maximum zone size of 46.3 mm was exhibited by methanol extract of P. niruri Linn. against Pseudomonas aeruginosa. Asparagus racemosus Willd. and Eupatorium triplinerve Vahl had the least activity against resistant pathogens. Saponified lipids of most of the plants exhibited maximum antibacterial activity. Among the tested organisms, P. aeruginosa and Staphylococcus epidermidis were the most susceptible and Serratia marcescens, Enterobacter cloaceae, Citrobacter koseri, and Citrobacter freundii were the least inhibited by most of the extracts of medicinal plants. It is concluded that revised antibiotic policies and more importantly the development of herbal medicine as an alternative may be incorporated in urological practice. Keywords: antibacterial activity, multiple antibiotic resistance, uropathogens, Azadiracta indica, Tinospora cordifolia, Euphorbia hirta
1. Introduction Urinary tract infections (UTI) are the most common form of bacterial infection affecting people throughout their life (Barnett and Stephen, 1997; Foxman, 2002). It is one of the common reasons for adults to seek medical help and is one of the most frequently occurring nosocomial infections (Gastmeier et al., 1998). The treatment of UTI
has become more complicated because of the appearance of pathogens with increasing resistance to antimicrobial agents (Murakami et al., 1995). Such antibiotic resistant organisms are difficult to eliminate during the course of an infection and are lethal to the human population. In addition, antibiotics in these cases are sometimes associated with adverse effects on the host, which include hypersensitivity, depletion of beneficial gut and mucosal microorganism,
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2011.0033
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A.S. Narayanan et al.
immunosuppressant and allergic reactions (Idose et al., 1968). Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases in general and of UTI in particular. During the last decade, the use of traditional medicine has expanded globally and is gaining popularity as an alternative medicine. It has continued to be used not only for primary health care of the poor in developing countries, but also in countries where conventional medicine is predominant in the national health care system (Lanfranco, 1999). According to the World Health Organization (WHO), herbal medicines serve the health needs of about 80% of the world’s population, especially for millions of people in the vast rural areas of developing countries (WHO, 2001). Hence, pharmacognostic investigations of plants are carried out to find novel drugs or templates for the development of new therapeutic agents. In spite of intensive investigation, only 5-10% of more than 250,000 species of higher plants have been chemically investigated (Nahrstedt, 1996). There is an increasing need for the documentation and development of alternative anti-pathogenic substances. Accordingly, the antimicrobial properties of certain Indian medicinal plants were reported based on folklore information (Ashmad et al., 1998; Dayal and Purohit, 1971; Hook and Thomas, 1995; Mehmood et al., 1999; Reddy, 1995; Suresh et al., 1995) and a few attempts were made to study inhibitory activity against certain pathogenic bacteria and fungi (Taylor et al., 1995). Ayyanar and Ignacimuthu (2005), Duraipandiyan et al. (2006), Natarajan et al. (1999), Rajan et al. (2002) and Sandhya et al. (2006) reported the practice of green pharmacy by the population in Tamil Nadu state of India. Considering the frequent emergence of antibiotic resistant strains and the immense potential of traditional medicines of plant origin, this work has been designed to study the efficacy of 10 medicinal plants of the Kolli hills of Namakkal, south India, against 11 multiple antibiotic resistant bacterial strains isolated from infected persons. The medicinal plants were selected based on folkloric medicine used by traditional healers dwelling at the foot of the Kolli hills. The significance of the present study lies in the fact that this is the first of its kind in evaluating the efficiency of indigenous plants species against multiple antibiotic resistant bacterial strains causing nosocomial UTI with significant Multiple Antibiotic Resistant (MAR) values.
2. Materials and methods Study area The Kolli hills, a part of Eastern ghat situated in the district of Namakkal, Tamil Nadu, south India lies between 11°10’ to 11°30’N and 78°15’ to 78°30’. It has an area of 482 km2 and stretches 29 km from north to south and 19 km from east to west. The highest point in the Kolli hills is 1,380 54
meter above sea level, but the general level of the upper surface of the hill is not more than 1000 m (average rainfall 942 mm and temperature 27 °C during study period). The bioresource, especially the flora, is largely unexplored for its identity and biological functions.
Plant collection The medicinal plants included in the Table 1 were collected fresh from the Kolli hills during the period of June 2006 – March 2007 and maintained in the KSR medicinal garden in college premises. The plants were identified and authenticated by Prof. P. Ponmurugan (PG Department of Biotechnology, KSR College of Technology, Tiruchengode, Namakkal, India) and preserved in our college herbarium. The identifications were based on the descriptions by Chatterjee et al. (1997). The leaves, stems and roots of medicinal plants were washed in running tap water to clean the adhering particles, sterilised with 0.1% mercuric chloride for 1 min and washed again with distilled water. Then the plants were subjected to the antibacterial activity tests.
Bacterial isolates and their multiple antibiotic resistance index Three Gram positive and eight Gram negative organisms included in this study are indicated in Table 2. The pathogenic organisms were isolated from urine specimen of persons suffering from UTI and identified by standard methods (Koneman et al., 1997). The cultures were maintained in the department laboratory. The antibiotic susceptibility patterns of the test organisms were performed using standard procedure (NCCLS, 2003) and their MAR index was calculated by the ratio of the number of ineffective antibiotics to the total number of antibiotics tested (Krumperman, 1983) against different numbers of isolates. The MAR index value was also calculated for the herbal extracts. The various concentrations of antibiotics as per the practical applications for in vitro studies have been employed (Lee et al., 2009; Samie et al., 2005). The antibiotics (Himedia, Mumbai, India) used in the study were: ampicillin (10 µg), amoxicillin (30 µg), amikacin (30 µg), cephalothin (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), ceftriaxone (30 µg), clindamycin (2 µg), co-trimoxazole (1.25 µg), erythromycin (15 µg), gentamycin (10 µg), kanamycin (30 µg), methicillin (5 µg), novobiocin (30 µg), nalidixic acid (30 µg), norfloxacin (10 µg), ofloxacin (5 µg), penicillin G (10 units), rifampicin (5 µg), streptomycin (10 µg), tetracyclin (30 µg) and vancomycin (30 µg). The reference strains used for the study were Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Staphylococcus epidermis ATCC 14990, Klebsiella pneumoniae ATCC 12658, Serratia marcescens ATCC 274, Proteus mirabilis ATCC 35491, Citrobacter freundii ATCC 8090 and Enterobacter cloacae ATCC 10699. Beneficial Microbes 2(3)
Antibacterial activity of medicinal plants against uropathogens
Table 1. Antibacterial activity of the crude medicinal plant extracts (40 µg/disc) against antibiotic resistant uropathogens. Plant name (local name)
Parts used
Solvent3 Bacterial species1,2 (mean inhibition zone in mm)
Phyllanthus fraternus Webster (Keezhanelli)
leaves
Asparagus racemosus Willd. (Thaneervittan kizhangu)
roots
e m a c s e m a c s e m a c s e m a c s e m a c s e m a c s e m a c s e m a c s e m a c s e m a c s
Eupatorium triplinerve Vahl leaves (Ayapana)
Leucas aspera Spreng. (Thumbai)
leaves
Glycosmis pentaphyllia Ritz leaves (Kuttivila)
Azadirachta indica A. Juss. leaves (Vembu)
Tinospora cordifolia Wild. (Shindilakodi)
stem
Euphorbia hirta Linn. (Amman pacharisi)
leaves
Phyllanthus niruri Linn. (Keezhanelli)
leaves
Cassia javanica Linn. (Konrai)
seeds
Se
Stf
Sa
Pa
Kp
Ecl
25.1 25.4 20.2 18.1 18.2 20.1 25.1 20.3 16.2 18.1 17.1 22 17 7 8 12 28 7 7 7 11 32 11 14 12 24 18 19.1 15 16.1 11.1
20.1 22 15.6 12.1 18.1 20.1 18.1 7 7 7 8 22 8 5 10 14 19 10 13 20 20.4 15 -
20.4 18.1 15.2 14.4 15.1 15.1 13.2 16 7 14 7 20 7 8 12 15 24 14 18 18 23 14.2 15.3 19.1 17 9.2 11 14.5
32.4 46.3 19 40.1 20.7 35.1 40.3 25 30 18.1 13.2 5 7 9 24 9 7 13 8 19 15 13 14 14 18.1 22.2 16 23.1 19.5 14.4 18.3
12.1 20 5 8 18 22 10 7 11 11 22 13 12 12 16 21.9 21.1 16.3 9.4 13
15.5 13.2 7 10 9 12 32 7 8 13 9 27 17 17 14 17 15.1 24.1 -
Pm 20.1 20.2 18.1 8 12 7 8 23 8 11 12 22 14 16 15 19 20.3 -
Sm
Cf
Ck
Ec
15.6 15.7 -
10.8 9.3 9 9 10 10 12.2 -
8.1 8.2 9.6 9 10 7 9 10 11 11 8 7.2
9.3 8.1 7 10 11 9 8 9.3
14.3 17.4 17.3 18 17 18 13.2 21.1
1
Bacterial species: Staphylococcus epidermidis (Se), Streptococcus faecalis (Stf), Staphylococcus aureus (Sa), Pseudomonas aeruginosa (Pa), Klebsiella pneumoniae (Kp), Escherichia coli (Ecl), Proteus mirabilis (Pm), Serratia marcescens (Sm), Citrobacter freundii (Cf), Citrobacter koseri (Ck), Enterobacter cloacae(Ec). 2 Zone of inhibition includes the diameter of the disc (6 mm); - no zone formation. 3 Solvents used: E = ethanol; M = methanol; A = acetone; C = chloroform; S = saponified lipid.
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A.S. Narayanan et al. Table 2. Multiple antibiotic resistance index values of antibiotic resistant organisms and number of isolates used in this study. Organism
Number of isolates
MAR value1
Number of ineffective Name of ineffective antibiotics2 antibiotics
Staphylococcus epidermidis Streptococcus faecalis Staphylococcus aureus Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli Proteus mirabilis Serratia marcescens Citrobacter freundii Citrobacter koseri Enterobacter cloacae
9 9 9 9 8 10 2 1 1 1 1
0.2 0.41 0.64 0.32 0.36 0.5 0.32 0.24 0.64 0.73 0.91
5 9 14 7 8 11 7 6 14 16 20
S, T, Nv, C, Ch Na, E, Va, T, Of, Nx, Ci, C, Ak S, T, Nv, C, Ch, M, A, Ac, Na, Nx, P, Ci, G, Ch A, Ac, Ci, G, Of, P, C A, Ac, Ci, G, Of, P, C, Cd A, Ac, Ci, G, Of, P, C, Cd, Ak, Ch, T A, Ac, Ci, G, Of, P, C A, Ac, Ci, G, Of, P, S, T, C, Ch, M, A, Ac, Cf, Ak, Of, P, Ci, G, Ch S, T, C, Ch, M, A, Ac, Cf, Ak, Of, P, Ci, G, Ch, R, Va S, T, Nv, C, Ch, M, A, Ac, Na, Nx, P, Ci, G, Ch, K, Cf, Co, Va, E, G
1 A multiple
antibiotic resistance (MAR) value near to 1 indicates the antibiotics are ineffective. Antibiotic abbreviations: ampicillin (A), amoxicillin (Ac), amikacin (Ak), cephalothin (Ch), chloramphenicol (C), ciprofloxacin (Cf), ceftriaxone (Ci), clindamycin (Cd), co-trimoxazole (Co), erythromycin (E), gentamycin (G), kanamycin (K), methicillin (M), novobiocin (Nv), nalidixic acid (Na), norfloxacin (Nx), ofloxacin (Of), penicillin G (P), rifampicin (R), streptomycin (S-10 µg), tetracycline (T), vancomycin (Va). 2
Preparation of extract and discs Fresh matured leaves, stems, seeds and roots of plant specimens were dried in shade and powdered by a mechanical mortar separately. Powdered plant materials (10 g) were used for extraction of the bioactive compounds in Soxhlet extractor with organic solvents chloroform, methanol, acetone or ethanol. After concentrating the extract at 50 °C for 6 h it was stored at 4 °C until further use. For the extraction of saponified lipid content of medicinal plants, a modified saponification process as applied to plants was employed (Padmini et al., 1986). For preparing the plant extract disc, 1 ml of concentrated extract was incorporated into the pre-weighed watch glass and evaporated by heat at 50-60 °C for 1 hour and weighed again. Thus, the weight of the bioactive component in 1 ml was calculated and hence different concentrations were loaded onto sterile discs (Himedia, Mumbai, India) to reach 40 μg. For the present study, herbal extract discs were prepared at concentrations of 10, 20, 30 (data not shown) and 40 μg.
Determination of antimicrobial activity of solvent extracted medicinal plants Two sets of Mueller Hinton agar (HiMedia) plates were prepared and seeded with 16 h broth cultures of different pathogenic organisms. Before making the lawn culture with the inoculum, the turbidity was adjusted to 0.5% of McFarland solution (1-2×107 cfu/ml). Seeded inoculum was treated with different concentrations of herbal extract 56
discs. The plates were incubated at 37 °C for 24 h. The experiment was repeated twice and the average of the zone of inhibition was recorded.
3. Results and discussion The MAR index values of the test organisms are reported in Table 2 and the MAR index value for herbal extracts are reported in Table 3. The MAR value is a ratio of the number of effective antibiotics to that of ineffective antibiotics tested against the different number of isolates. The antibacterial activities and minimum inhibitory concentration values of different solvent extract of 10 common medicinal plants of Kolli hills are given in Tables 1 and 4. Azadirachta indica, Tinospora cordifolia and Euphorbia hirta exhibited a high degree of inhibitory activity against most of the 11 tested organisms followed by Cassia javanica and Phyllanthus niruri. Saponified lipids of these plants exhibited maximum activity. Among the pathogens, P. aeruginosa and S. epidermidis were the most susceptible followed by S. aureus and Enterococcus faecalis, E. cloaceae, S. marcescens, Citrobacter koseri and C. freundii were the least inhibited by most of the extracts of medicinal plants. Due to the over-usage of antibiotics (Sydney et al., 1980), mutation and environmental stress the antibiotic resistant organisms have become a major challenge in hospital acquired infections. This often causes difficulties for the treatment of UTI patients. Because of this drug resistance, the search for new antibiotics continues unabated. In this connection, plants continue to be a rich source of therapeutic drugs. The active ingredients of many drugs are found in plants, or Beneficial Microbes 2(3)
Antibacterial activity of medicinal plants against uropathogens
Table 3. Multiple antibiotic resistance index values of herbal plant extracts on antibiotic resistant organisms used in this study. Name of the organism
Staphylococcus epidermidis Streptococcus faecalis Staphylococcus aureus Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli Proteus mirabilis Serratia marcescens Citrobacter freundii Citrobacter koseri Enterobacter cloacae
No. of extracts used
50 50 50 50 50 50 50 50 50 50 50
No. of extracts not inhibiting the growth
MAR value1
19 28 22 19 30 32 33 40 43 38 42
0.38 0.56 0.44 0.38 0.60 0.64 0.66 0.80 0.86 0.76 0.84
1 A multiple
antibiotic resistance (MAR) value near to 1 indicates that the extracts are ineffective.
are produced as secondary metabolites and are increasingly popular among village communities because there is real improvement of disease conditions after herbal treatment and harmful side effects and the high cost of other forms of treatment are very much reduced. Methicillin resistant S. epidermidis (MRSE) in urinary isolates is responsible for complicated UTI (Sakumoto et al., 1996). In our study, S. epidermidis exhibited a MAR value of 0.2 but P. niruri, Eupatorium triplinerve, A. indica, T. cordifolia, E. hirta and P. niruri exhibited inhibitory activity over the organism. The maximum inhibitory zone of 32 and 28 mm was exhibited by saponified lipid of T. cordifolia and A. indica respectively. E. faecalis susceptibilities to erythromycin and minocycline have undergone significant changes; the former decreased significantly in the last 5 years (P
