International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 3(9), 57-64, September (2014) Int. Res. J. Biological Sci. International Science Congress Association 57 Potent Inhibition of swarming growth of Uropathogenic Proteus Bacteria by Ethanolic extracts of Emblica officinalis fruit and Tamarindus indica barkMamunur Roshid, Abdul Wadud and Aktar Uzzaman Chouduri* Department of Pharmacy, University of Rajshahi, Rajshahi-6205, BANGLADESHAvailable online at: www.isca.in, www.isca.me Received 29th March 2014, revised 3rd June 2014, accepted 28th July 2014Abstract Medicinal plants have been the principal source for most of the drugs. Phytochemicals having antimicrobial properties can be of great significance in therapeutic treatments. Several pathogenic features of uropathogenic Proteus species isolated from municipal water have earlier been studied. Strong resistance to cephalosporin was found in those strains. This study aimed to evaluate the role of medicinal plants to interfere with the growth and virulence of those strains as an alternative therapy of UTI. Six strains with high pathogenicity were undertaken to a test of bactericidal activity by normal human serum (NHS). Then NHS resistant strains were targeted to combat them by phytochemicals. Five medicinal plants, Emblica officinalis, Asparagus racemosus, Azadirachta indica, Abroma augusta, and Tamarindus indica, were selected for the purpose. Four strains named 11(Pv), 91(Pm), 91(Pm), 66(Pp) were resistant to NHS being 91(Pm) the strongest. Ethanol extracts of A. augusta had no anti-Proteus activity and that of A. indica, A. racemosus showed noticeable inhibition of Proteus cells. However, very low concentration of extracts of E. officinalis (400µg/ml) and T. indica (25µg/ml) completely inhibited the swarming although they had essentially no effect on cell proliferation. Ethanol extracts of E. officinalis and T. indica are potent inhibitor of Proteus swarming. Edible fruit (E. officinalis) known as Indian gooseberry/Amla can be used as food supplement to cure UTI and to control bacterial pathogens having swarming abilities. Keywords:Alcoholic extract, Emblica officinalis, swarming inhibitor, Tamarindus indica. Introduction Herbal care or conventional systems of medicine have been using from ancient times. Medicinal plants especially herbs have been the original source for most of the drugs. Now-a-days about 70% of the world population is depending on medicinal herbs. Medicinal plants contain so many chemical compounds which are the major source of therapeutic agents to cure human diseases. For a long period of time, medicinal plants have been the valuable source of natural products for maintaining human health, especially in the last decade, with more intensive studies for natural therapies. The use of phytochemicals for pharmaceutical purposes has been gradually increased. According to World Health Organization the medicinal plants could be the best source to obtain a variety of drugs. Approximately 80% people in developed countries use traditional medicine, which has compounds derived from medicinal plants. Therefore, such plants should be investigated to better understand their properties, safety and efficiency. The use of plant extracts and phytochemicals, both with known antimicrobial properties, can be of great significance in therapeutic treatments. In the last few years, a number of studies have been conducted in different countries to prove such efficiency3-7Many plants have been used because of their antimicrobial traits, which are due to compounds synthesized in the secondary metabolism of the plant.The antimicrobial properties of medicinal plants have been investigated by many investigators worldwide, especially in Indian region. Thirty one medicinal plant species have been reported by traditional healers as being used for UTIs, including leucorrhea, frequent or infrequent urination, cloudy urination, and burning sensations during urination in Bangladesh. The Proteus pathogens are thought to be the principal cause of UTI, CAUTI and wound infections. We isolated pathogenic Proteus bacteria from municipal tap water that were multidrug resistant especially to cephalosporin,10 and several pathogenic features of those isolates have already been reported11-12. The increasing evidence of antibiotic resistance in bacterial pathogens necessitates medicinal plants as an alternative therapy in restricting the antibiotic resistant infectious organisms. We selected here five medicinal plants having potential antimicrobial properties that are traditionally used as folk medicine for urological disorder. Leaves of Abroma augusta Linn (Family: Malvaceae) has been widely investigated and its antibacterial potentials have been reported by researchers13-14. Emblica officinalis Gaertn (Family: Phyllanthaceae) is commonly known as Indian gooseberry or amla. The alcoholic extract of E. officinalis exhibited strong and broad spectrum antibacterial activity against various pathogenic bacteria and numerous biological activities has also been reported15-17. This tree grown or cultivated in subtropical and tropical parts of China, India, Indonesia and the Malay Peninsula has been used for anti-inflammatory and antipyretic treatments of rural populations in these areas18. The root extract of Asparagus International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(9), 57-64, September (2014) Int. Res. J. Biological Sci. International Science Congress Association 58 racemosus (Family: Asparagaceae) which is locally known as shotomuli showed antibacterial activity against resistant uropathogens isolated from patients having UTI19. The alcoholic extract of Azadirachta indica leaf (Family: Meliaceae) which is locally named neem showed potential antimicrobial activities including Proteus mirabilis20. Nwodo et al. 2011 found the significant antimicrobial activities in aqueous and alcoholic extract of Tamarindus indica bark (Family: Leguminosae)21. Therefore, this study aimed to investigate the role of ethanolic fraction of test medicinal plants to interfere with the growth and virulence of cephalosporin resistant uropathogenic Proteusbacteria isolated from municipal supplied water in our previous study. Material and Methods Bacterial strains: Six Proteus strains belonging four species i.e. P. vulgaris (hereafter termed as Pv), P. mirabilis (Pm), P. hauseri (Ph), and P. penneri (Pp) designated as 11(Pv), 66Pp), 66Ph), 66Pp), 91Pm) and 91Pm) isolated from municipal tap water (Rajshahi City, Bangladesh) in our previous studyhave been used. Those strains were multidrug resistant to broad spectrum antibiotics and possessed several pathogenic features including swarming motility, urease production, extracellular proteases, biofilm formation as reported earlier10-12. Strains stored at 40ºC in Luria-Bertani (LB) broth supplemented with 12% (v/v) glycerol were freshly grown at 37ºC to carry out this study. Growth media and culture conditions: Nutrient agar media (1% yeast extract, 0.5% peptone, 0.5% NaCl, 1.5% agar) purchased from Difco was used for antibacterial activity assay of the extracts. The strains were incubated at 37°C for overnight as described elsewhere9-10. Cells were cultured in nutrient broth media with mild shaking on a water bath (Advantec Lab-Thermo Shaker, TS-20, Toyo Kaisha, Ltd) at 37°C to test the growth rate of bacterial cell. Swarming motility test: In 10ml of LB broth medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) Proteus strains were grown overnight at 37ºC with shaking (200 rpm). Then 5l of fresh cell culture was spotted at the center of LB agar plates (LB medium containing 1.5% agar) previously dried to remove water drops from the surface of the agar medium as described in other reports22 and incubated at 37ºC for 24 hrs unless it is mentioned otherwise. Then the diameters of swarming zones were measured in millimeter at three different directions. Plant material: Plant parts were collected from the medicinal plant garden, Department of Pharmacy, University of Rajshahi and around Rajshahi City area, Bangladesh on Nov 2013, and duly identified by a plant taxonomist Mr. Arshed Alom, Department of Botany, University of Rajshahi, Bangladesh where a specimen voucher (75/05.07.2008) was recorded in the department herbarium for future reference. Plant parts i.e. fruit of Emblica officinalis Gaertn (Family: Phyllanthaceae, local name: Amla/Amloki), root of Asparagus racemosus (Family: Asparagaceae, local name: Shotomuli), leaf of Azadirachta indica (Family: Meliaceae, local name: Neem), bark of Tamarindus indica (Family: Leguminosae, local name: Tentul), leaf of Abroma augusta Linn (Family: Malvaceae, local name: Ulotcombol) were air-dried under shade. Once dried, the plant material was ground, extracted by maceration for 48-72 hours with ethanol, filtered (Paper Whatman No. 3) and the solvent was vacuum evaporated in a Soxhlet apparatus (Rotary Evaporator, RE 300, Bibby Sterilin Ltd, UK). Then solutions were evaporated to dryness and further dilutions were made in the same solvent to obtain the required extract concentrations for the different assays. Table-1 Anti-Proteus activity of ethanolic extracts of five medicinal plants Proteusstrain (spp) Disk potency (500 µg/disk) Cotrimoxazole (5 µg/disk) A. indica A. augusta A. racemosus T. indica E. officinalis 11(Pv) 17 ± 1.14 14 ± 1.12 7 ± 0.07 12 ± 0.22 34 ± 0.84 66 2 Ph) 18 ± 0.38 15 ± 0.13 7 ± 0.03 11 ± 0.98 28 ± 1.21 91 1 Pm) 12 ± 0.19 13 ± 0.09 6 ± 0.08 8 ± 0.88 30 ± 0.22 91 2 Pm) 15 ± 0.51 14 ± 0.22 7 ± 0.54 9 ± 0.78 30 ± 1.11 Disk potency (1 mg/disk) A. indica A. augusta A. racemosus T. indica E. officinalis 11(Pv) 18 ± 0.28 16 ± 0.82 7 ± 0.76 16 ± 0.87 34 ± 1.26 66 2 Ph) 15 ± 0.98 14 ± 0.88 8 ± 0.22 13 ± 0.34 28 ± 1.09 91 1 Pm) 15 ± 0.78 15 ± 0.25 8 ± 0.43 11 ± 1.09 30 ± 0.24 91 2 Pm) 17 ± 1.21 14 ± 0.99 8 ± 0.34 11 ± 0.76 30 ± 0.98 Sign ( ) indicates no zone of inhibition detected. A. indica leaf and A. racemosus root showed notable zone of inhibition but T. indica bark and E. officinalis fruit showed either no or very low zone of inhibition compared to that of reference International Research Journal of Biological Sciences ____________________ Vol. 3(9), 57-64, September (2014) International Science Congress Association Test for antibacterial activity: Each of plant extracts was tested for its anti-Proteus activity following disk diffusion method on nutrient agar media23-25 . The crude extracts were separately dissolved in 1 ml of ethanol and the filter paper discs (6 mm diameter) were impregnated with known amounts of test substances and prepared in various potencies, 25 mg/disc. Discs were placed on pre- seeded bacter of Proteus strain by sterilized forceps. Plates were then kept overnight at 4°C to allow maximum diffusion of components of disc. The plates were then incubated at 37°C for 18 hrs. Then the diameter (in millimeter) of zone of inhibition f extract against tested microorganisms was noted. Reference standard disc of cotrimoxazole (5g, Hi- media, India) was used as positive control and blank disc impregnated with solvent followed by drying off was used as negative control. All tests wer e performed in triplicate and the antibacterial activities were calculated by measuring the diameter of zone of inhibition. Inhibition of swarming motility: Effects of plant extracts on swarming motility of isolates were assessed as described in other report26 . Briefly, an overnight bacterial culture (5 inoculated centrally onto the surface of dry LB agar plates made with or without extracts at various concentrations which were then incubated at 37ºC for 24 hours. The perimetric distance of swarmin g motility was assayed by measuring the fronts of swarming areas in three different directions. Normal human serum (NHS) preparation from five healthy university students was collected in a test tube at university health care center. Af ter collection, the blood was allowed to clot leaving undisturbed at room temperature. The blood clot was removed by centrifugation at 6000 rpm for 20 minutes in a refrigerated centrifuge. The resulting supernatant was designated as serum that apportioned stored at –20°C. Heat inactivation , i.e. the destruction of complement activity was achieved by incubating the serum for 1 h at 56°C before use in bactericidal activity Bactericidal activity of NHS: 50l of standardized (~10 CFU/ml) fresh bacterial cell suspension adjusted with physiological saline (0.9% NaCl) was immediately mixed with equal volume of NHS and incubated at 37ºC for 3 hours while samples were pooled at 0, 0.5, 1, 2 and 3 h of incubation to test the bactericida l activity of NHS against multidrug resistant Proteus strains. Then the samples plated on nutrient agar medium were subjected to the colony counting process by serial dilution method. The colony count of pooled samples was scored as percent of bactericidal activity regarding the value at 0 h as 100%. Those strains were regarded as resistant to bactericidal activity of NHS whose survival in the serum was 50% after 3 h of incubation22, 28-29. Data analysis: For data processing, the software Microsoft Excel 2 007 was used. Results of triplicate experiments were averaged, and means ± standard deviations were calculated. International Research Journal of Biological Sciences ____________________ ____________________________ International Science Congress Association Each of plant extracts was activity following disk diffusion . The crude extracts were separately dissolved in 1 ml of ethanol and the filter paper discs (6 mm diameter) were impregnated with known amounts of test substances and prepared in various potencies, 25g to 1 seeded bacter ial culture plate strain by sterilized forceps. Plates were then kept overnight at 4°C to allow maximum diffusion of components of disc. The plates were then incubated at 37°C for 18 hrs. Then the diameter (in millimeter) of zone of inhibition f or each extract against tested microorganisms was noted. Reference media, India) was used as positive control and blank disc impregnated with solvent followed by drying off was used as negative control. All tests e performed in triplicate and the antibacterial activities were calculated by measuring the diameter of zone of inhibition. Effects of plant extracts on swarming motility of isolates were assessed as described in . Briefly, an overnight bacterial culture (5l) was inoculated centrally onto the surface of dry LB agar plates made with or without extracts at various concentrations which were then incubated at 37ºC for 24 hours. The perimetric distance of g motility was assayed by measuring the fronts of swarming areas in three different directions. Normal human serum (NHS) preparation :The whole blood from five healthy university students was collected in a test tube ter collection, the blood was leaving undisturbed at room temperature. The blood clot was removed by centrifugation at 6000 rpm for 20 minutes in a refrigerated centrifuge. The resulting supernatant was designated as serum that apportioned into 0.5 ml aliquots, , i.e. the destruction of complement activity was achieved by incubating the serum for 1 h at 56°C before use in bactericidal activity 27. l of standardized (~10 9 CFU/ml) fresh bacterial cell suspension adjusted with physiological saline (0.9% NaCl) was immediately mixed with equal volume of NHS and incubated at 37ºC for 3 hours while samples were pooled at 0, 0.5, 1, 2 and 3 h of incubation to test l activity of NHS against multidrug resistant strains. Then the samples plated on nutrient agar medium were subjected to the colony counting process by serial dilution method. The colony count of pooled samples was activity regarding the value at 0 h as 100%. Those strains were regarded as resistant to bactericidal activity of NHS whose survival in the serum was For data processing, the software Microsoft 007 was used. Results of triplicate experiments were averaged, and means ± standard deviations were calculated. Figure Bactericidal activity of NHS to The slope of each linear line indicating the rate of bactericidal activity of NHS to the respective strain are 0.164 (91 (66 ), 0.302 (11), 0.322 (91 Strains whose 50% cells can survive up to 3 hrs of incubation were regarded as NHS resistant Results and Discussion Bactericidal activity of NHS against Proteus strains: Proteus isolates were selected from eleven others based on their species and the extent of pathogenicity depending on few pathogenic factors, i.e. swarming motility, antibiotic resistance, extracell ular proteases, biofilm forming abilities. Two strains, 91Pm) and 66Ph ), had strong swarming abilities as reported earlier11. Strain 91Pm ) also showed strong proteolytic activity measured by well diffusion assay on casein agar plate 11(Pv), 91Pm), 66Pp ) were biofilm former on PVC strip as well as catheter strip12 . These strains were subjected to a bactericidal activity assay with NHS as described in materials and methods section. The ability of NHS to kill the strains was differ ent in each isolate. The decline of the number of survived cells with time which indicated the bactericidal activity of NHS was found to be rapid in the strains 66 66Pp ) (figure 1) whereas 50% bacterial cells of four other strains, 11(Pv), 91Pm), 91 2 in the presence of NHS up to 3 h of incubation at 37°C. The result indicated that these four strains were resistant to NHS. Based on the rate of bactericidal activity of NHS, calculated from the slope of linear plo 91Pm) followed by 66 Pp supports our previous conclusion best proteolytic activity on casein agar plate and 66 the second highest scorer. Therefore, we assumed that the secretion of extracellular proteases especially metalloprotease ZapA was high enough to combat against NHS for its survival. ____________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 59 Figure -1 Bactericidal activity of NHS to Proteus isolates The slope of each linear line indicating the rate of bactericidal to the respective strain are 0.164 (91 ), 0.227 ), 0.302 (11), 0.322 (91 ), 0.720 (66) and 0.866 (66), Strains whose 50% cells can survive up to 3 hrs of incubation were regarded as NHS resistant Results and Discussion Bactericidal activity of NHS against Proteus strains: Six isolates were selected from eleven others based on their species and the extent of pathogenicity depending on few pathogenic factors, i.e. swarming motility, antibiotic resistance, ular proteases, biofilm forming abilities. Two strains, ), had strong swarming abilities as reported ) also showed strong proteolytic activity measured by well diffusion assay on casein agar plate 11. Strains ) were biofilm former on PVC strip as . These strains were subjected to a bactericidal activity assay with NHS as described in materials and methods section. The ability of NHS to kill the Proteus ent in each isolate. The decline of the number of survived cells with time which indicated the bactericidal activity of NHS was found to be rapid in the strains 66 Ph) and ) (figure 1) whereas 50% bacterial cells of four other 2 Pm), 66Pp) were able to survive in the presence of NHS up to 3 h of incubation at 37°C. The result indicated that these four strains were resistant to NHS. Based on the rate of bactericidal activity of NHS, calculated from the slope of linear plo t, the most resistant strain was Pp ), 11(Pv) and 91Pm). This result supports our previous conclusion 11 where 91Pm) showed the best proteolytic activity on casein agar plate and 66 Pp) was the second highest scorer. Therefore, we assumed that the secretion of extracellular proteases especially metalloprotease ZapA was high enough to combat against NHS for its survival.