Int.J.Curr.Microbiol.App.Sci (2013) 2(12): 411-417 ISSN: 2319-7706 Volume 2 Number 12 (2013) pp. 411-417 http://www.ijcmas.com Original Research Article Inhibitory effect of Anisomeles indica Linn. against multidrug resistant urinary tract pathogens M.N.Vivek1, Yashoda Kambar1, S.Pallavi1, T.R.Prashith Kekuda1*and T.N.Ravi Kumar2 1P.G. Department of Studies and Research in Microbiology, Sahyadri Science College campus, Kuvempu University, Shivamogga-577203, Karnataka, India 2P.G. Department of Microbiology, K.M.C, Manipal University, Manipal, Karnataka, India *Corresponding author A B S T R A C T Urinary tract infections are the infections of urinary tract and are the common K e y w o r d s infections in both community and hospital settings. The aim of the present study was to investigate inhibitory effect of methanolic extract of Anisomeles indica Linn. (Lamiaceae) leaf against antibiotic resistant urinary tract pathogens. Agar Anisomeles well diffusion assay was performed to screen the antibacterial effect of leaf extract indica; against five isolates viz., Staphylococcus aureus, Enterococcus faecalis, Urinary tract Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Gram infection; positive bacteria have shown higher susceptibility to leaf extract than Gram antibiotic negative bacteria. S. aureus and E. coli were inhibited to high extent among Gram resistance; positive and Gram negative bacteria respectively. K. pneumoniae was inhibited to Agar well least extent. The plant can be a potential source for the development of agents diffusion. active against urinary tract pathogens. Further studies are to be carried out to isolate active principles from the crude leaf extract and to determine their inhibitory activity against urinary tract pathogens. Introduction The discovery of antibiotics remains one antibiotic resistance. Staphylococcus of the significant events in the field of aureus, Pseudomonas aeruginosa, chemotherapy. Antibiotics have Mycobacterium tuberculosis, revolutionized the field of medicine and Enterococcus faecalis, coliforms such as subsequent use of antibiotics saved Escherichia coli, are among the most countless individuals from infection by important antibiotic resistant pathogenic microorganisms. However, microorganisms which have developed microbial strains developing resistance are resistance against a wide range of continuously appearing because of wide antibiotics. These antibiotic resistant spread use which appears to be the major pathogens make the treatment of infections selective force for development of difficult. This alarming situation triggers 411 Int.J.Curr.Microbiol.App.Sci (2013) 2(12): 411-417 the search for and development of activity of leaf extract of A. indica against antimicrobial agents from other sources. antibiotic resistant pathogens of urinary From ancient time, plants have been used tract infection. all over the world as drugs and remedies for treatment of various kinds of ailments. Materials and Methods The chemicals (phytochemicals) present in these plants even serve as prototype for the Collection and extraction of plant development of more effective and less material toxic drugs (Cooke, 1976; Niemi et al., 1983; Carmeli et al., 1999; Cowan et al., The plant material was collected at college 1999; Sharma et al., 2009; Demain and campus during September 2013. The Sanchez, 2009; Davies and Davies, 2010; leaves were separated, washed well in Onanuga and Awhowho, 2012; Kekuda et order to remove extraneous matter and al., 2012; Fernandes and Dhanashree, dried under shade. The dried leaves were 2013; Kekuda et al., 2013). powdered mechanically and a known quantity of powdered leaf material (10g) Anisomeles indica Linn. is an was extracted using methanol (HiMedia, ethnomedicinally important aromatic plant Mumbai) in Soxhlet apparatus. After and belongs to the family Lamiaceae. The extraction, the solvent extract was filtered plant is commonly called Catmint and is through Whatman No. 1 filter paper, used for the treatment of various kinds of concentrated in vacuum under reduced ailments in various parts of the world pressure and dried in the desiccator (Batish et al., 2007; Alagesaboopathi, (Kekuda et al., 2012). 2009; Kunwar et al., 2010; Sutha et al., 2010). The crude extracts, essential oils Test bacteria and purified compounds from various parts of the plant (such as roots, leaves and The antibacterial efficacy of leaf extract of flowers) have shown to exhibit several A. indica was tested against two Gram bioactivities such as antimicrobial positive bacteria viz., Staphylococcus (Yadava and Barsainya, 1998; Usher et al., aureus, Enterococcus faecalis and three 2010; Rao et al., 2012; Lien et al., 2013; Gram negative bacteria viz., Escherichia Kundu et al., 2013), antioxidant (Huang et coli, Pseudomonas aeruginosa and al., 2012; Kundu et al., 2013), attenuation Klebsiella pneumoniae isolated previously of inflammation (Lien et al., 2013), from urinary tract infections. The isolates analgesic (Dharmasiri et al., 2003), anti- were resistant to antibiotics and Table 1 inflammatory (Rao et al., 2009), antiviral shows the name of antibiotics against (Alam et al., 2000), antiplatelet which the isolates are resistant. aggregation activity (Chen et al., 2008) and others. The root and leaf powder of Antibacterial activity of extract A. indica when applied as mulch significantly reduced the emergence and We employed Agar well diffusion assay to growth of weeds of wheat crop similar to determine the potential of leaf extract to herbicide, without any negative effect on inhibit urinary tract pathogens. In brief, 24 the wheat growth and yield (Batish et al., hour old Nutrient broth (HiMedia, 2007). The present study was conducted Mumbai) cultures of test bacteria were with an aim to determine inhibitory swabbed aseptically on sterile Nutrient 412 Int.J.Curr.Microbiol.App.Sci (2013) 2(12): 411-417 Table.1 UTI isolates and the antibiotics against which the isolates are resistant Isolates Antibiotic Ampicillin, Norfloxacin, Amoxicillin, Cefuroxime, Cotrimazole, E. coli Cefazolin, Aztreonam, Cefpirome, Imipenem Ampicillin, Norfloxacin, Amoxicillin, Cefuroxime, Cotrimazole, K. pneumoniae Cefazolin, Aztreonam, Cefoperazone, Imipenem P. aeruginosa Gentamycin, Amikacin, Ceftazidime, Ciprofloxacin, Tobramycin S. aureus Ampicillin, Gentamycin, Norfloxacin, Penicillin E. faecalis Ampicillin, Gentamycin, Norfloxacin, Penicillin agar (HiMedia, Mumbai) using sterile Urinary tract is an important system which cotton swabs. With the help of a sterile collect, store and release urine. It include cork borer, wells of 6mm diameter were kidneys, ureters, bladder and urethra. punched in the inoculated plates. 100 l of Urinary Tract Infections (UTIs) refers leaf extract (20mg/ml of dimethyl infections that are caused by sulfoxide [DMSO]), reference antibiotic microorganisms anywhere in the urinary (Chloramphenicol, 1mg/ml of sterile tract. These UTIs are one among the distilled water) and DMSO (25%) were common infections in both community and transferred into respectively labelled wells. hospital settings. UTIs been reported in The plates were incubated for 24 hours at people of all age groups in both sexes and 37oC. The zones of inhibition formed are more common in females than in around the wells were measured using a males. It can be classified as symptomatic ruler (Kekuda et al., 2012). or asymptomatic; complicated or uncomplicated. It can also be classified Statistical analysis based on the infection site (bladder [cystitis], kidney [pyelonephritis], or urine The experiment was done in triplicates. [bacteriuria]. These infections form a The result is taken as Mean±Standard serious health problem and affect millions deviation (SD). of people globally each year. UTIs represent the leading cause of Gram- Result and Discussion negative bacteraemia and are the most common hospital-acquired infections Table 2 shows the result of antibacterial (Okonko et al., 2010; Beyene and activity of leaf extract of A. indica against Tsegaye, 2011; Humayun and Iqbal, clinical isolates of UTI. A dose dependent 2012). inhibition of test bacteria was observed. Overall, the extract was found to inhibit A number of bacteria are implicated in Gram positive bacteria to high extent causing UTIs. Escherichia coli, Klebsiella when compared to Gram negative bacteria. pneumoniae, Enterobacter sp., Among bacteria, highest and least Pseudomonas aeruginosa, Proteus sp., susceptibility was observed in case of S. Enterococcus faecalis, Staphylococci and aureus and K. pneumoniae respectively. E. Streptococci common UTI causing coli was inhibited to higher extent among bacterial agents. Community acquired Gram negative bacteria. DMSO was not UTIs is caused by bacteria such as E. coli, found to inhibit any of the clinical isolates. K. pneumoniae, P. mirabilis, 413 Int.J.Curr.Microbiol.App.Sci (2013) 2(12): 411-417 Table.2 Inhibitory activity of extract against clinical isolates of UTI Zone of inhibition in cm (Mean±SD) Isolates Extract 20mg/ml Extract 10mg/ml DMSO E. coli 1.8±0.2 1.5±0.2 0.0±0.0 P. aeruginosa 1.2±0.0 0.9±0.2 0.0±0.0 K. pneumoniae 1.0±0.1 0.0±0.0 0.0±0.0 E. faecalis 1.8±0.2 1.5±0.0 0.0±0.0 S. aureus 2.3±0.1 1.8±0.3 0.0±0.0 S.saprophyticus or E. faecalis whereas the to possess inhibitory effect against urinary hospital acquired UTIs are associated with tract pathogens. In the present study, we bacteria such as E. coli, P. aeruginosa, evaluated the efficacy of leaf extract of A. Proteus sp, Enterobacter sp.,