ANTIBACTERIAL, ANTI-SWARMING POTENTIAL OF ETHANOL EXTRACTS OF PHYSALIS MINIMA L. WHOLE PLANT AND URENA LOBATA L. ROOT ON CEPHALOSPORIN RESISTANT PROTEUS SPECIES

Abstract

This study aimed to search medicinal plants with high antibacterial potentials in order to manage antibiotic resistant uropathogens. Twelve specimens of nine medicinal plants which are available locally were analyzed for their anti-infective properties against resistant uropathogens using disc diffusion method. Remarkable antibacterial activities of ethanol extract of Physalis minima whole plant followed by Azadirachta indica leaf, Asparagus racemosus root, Phyllanthus emblica fruit, Urena lobata root and Tamarindus indica bark were found against eleven test bacteria and eleven resistant Proteus isolates. Physalis minima extract showed the highest zone of inhibition but it had no anti-swarming effect. Interestingly complete inhibition of swarming was found by Urena lobata root extract at 500 μg/ml concentration although its antibacterial activity was very low or nil. Thus, the mixture of two extracts would be a powerful anti-infective agent to combat UTI and/or wound infection caused by resistant Proteus bacteria. The extracts could be further analyzed for the drug development.

Headnote

ABSTRACT

Swarming of Proteus bacteria has been implicated in pathogenesis. In previous study, eleven Proteus strains isolated from municipal water were found to be resistant to cephalosporins and four isolates, 11(Pv), 66^sub 1^(Pp), 91^sub 1^(Pm), and 91^sub 2^(Pm), were resistant to normal human serum. The increasing evidence of antibiotic resistance necessitates medicinal plants to develop alternative strategies of treatment. This study aimed to search medicinal plants with high antibacterial potentials in order to manage antibiotic resistant uropathogens. Twelve specimens of nine medicinal plants which are available locally were analyzed for their anti-infective properties against resistant uropathogens using disc diffusion method. Remarkable antibacterial activities of ethanol extract of Physalis minima whole plant followed by Azadirachta indica leaf, Asparagus racemosus root, Phyllanthus emblica fruit, Urena lobata root and Tamarindus indica bark were found against eleven test bacteria and eleven resistant Proteus isolates. Physalis minima extract showed the highest zone of inhibition but it had no anti-swarming effect. Interestingly complete inhibition of swarming was found by Urena lobata root extract at 500 µg/ml concentration although its antibacterial activity was very low or nil. Thus, the mixture of two extracts would be a powerful anti-infective agent to combat UTI and/or wound infection caused by resistant Proteus bacteria. The extracts could be further analyzed for the drug development.

KEY WORDS: Urena lobata L., Physalis minima L., antibacterial and anti-swarming activities, cephalosporin resistant Proteus bacteria.

ABBREVIATIONS: UTI-Urinary tract infection, CAUTI-Catheter associated urinary tract infection, ESBL- Extended spectrum β-lactamase, NHS- Normal human serum.

INTRODUCTION

Thirty-one species of medicinal plants were reported by traditional healers as being used for UTIs, including leucorrhea, frequent or infrequent urination, cloudy urination, and burning sensations during urination (Hossan et al., 2010). The major parts (flower, bark, root, leaves) of one of these medicinal plants, Urena lobata Linn are used as folk medicine for UTIs (Nandwani et al., 2008; Hossan et al., 2010). U. lobata Linn (common name Ceasar weed) is native to China but it is available in many tropical countries including Bangladesh, India, South America, Africa, Australia, and the United States. Especially U. lobata roots have been shown to bear a broad-spectrum antibacterial activity against Gram-positive and Gram-negative microorganisms (Mazumder et al., 2001).

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 (Wadud and Chouduri, 2013) that were multi-antibiotic resistant especially to cephalosporins (Chouduri and Wadud, 2013) and several pathogenic features of those isolates have already been reported (Chouduri et al., 2014; Chouduri and Wadud, 2014). Although the pharmacological industries have produced a number of new antibiotics in the last four decades, resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents (Cohen, 1992). The increasing evidence of antibiotic resistance among bacterial pathogens necessitates medicinal plants as an alternate therapy in restricting the resistant infectious organisms. Previously it had been reported that recently the extensive use of cephalosporins for the treatment of infectious diseases allows pathogens to be resistant to the antibiotics of cephalosporin group. Therefore, an urgent need is to search new antibiotic or an alternate therapy of infectious diseases. This study aimed to manage the emergence of antibiotic resistance by phytochemicals of selective medicinal plants. To serve the purpose here nine medicinal plants (Table 1) having potential antimicrobial properties have been selected that are traditionally used as folk medicine for urological disorders.

Nwodo et al. (2011) found the significant antimicrobial activities in aqueous and alcoholic extract of Tamarindus indica bark. Fruit of Phyllanthus emblica Gaertn is commonly known as Indian gooseberry or amla. The alcoholic extract of Phyllanthus emblica exhibited strong and broad spectrum antibacterial activity against various pathogenic bacteria and numerous biological activities has also been reported (Ahmad et al., 1998; Khan, 2009; Khosla and Sharma, 2012). The root extract of Asparagus racemosus showed antibacterial activity against resistant uropathogens isolated from patients having UTI (Narayanan et al., 2011). The alcoholic extract of Azadirachta indica leaf showed potential antimicrobial activities including Proteus mirabilis (Yasmeen et al., 2012). Leaves of Abroma augusta Linn has been widely investigated and its antibacterial potentials have been reported by researchers (Saikot et al., 2012; Zulfiker et al., 2013). The extract of Mimosa pudica Linn root is an alternative wound healing agent widely used as folk medicine in Indian subcontinent for the treatment of vaginal and uterine complications. It is very useful in diarrhea, amoebic dysentery, bleeding piles and urinary infections (Joseph et al., 2013). The ethanol extract of Coccinia grandis leaves exhibited antimicrobial activity against biofilm and ESBL producing uropathogenic Escherichia coli strains UPEC- 17 and -82 (Poovendran et al., 2011).

The general acceptance of traditional medicine for health care and the development of microbial resistance to several available antibiotics have led researchers to investigate the activity of medicinal plants against infectious diseases (Low et al., 2002; Yarnell, 2002). Therefore, the aim of this study was to evaluate the role of ethanolic fractions of the medicinal plants to interfere with the growth and virulence of multi-antibiotic, especially cephalosporin resistant uropathogenic Proteus bacteria isolated in our previous study.

MATERIALS AND METHODS

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. Twelve specimens of nine medicinal plants enlisted in table 1 were air-dried under shade. A representative image of two plants and plant parts has been shown in figure 1. Once dried, the plant material was ground, extracted by maceration for more than 72 hrs 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

Bacterial strains

From our laboratory stock five Gram positive bacteria, Staphylococcus aureus, Streptococcus agalactiae, Bacillus cereus, Bacillus megaterium, Bacillus subtilis, and six Gram negative bacteria, Pseudomonas aeruginosa, Shigella flexneri, Shigella dysenteriae, Escherichia coli, Shigella sonnei, Agrobacterium species, were used for antibacterial activity assay of the plant extracts. Eleven Proteus strains of four species: P. vulgaris (hereafter termed as Pv), P. mirabilis (Pm), P. hauseri (Ph), and P. penneri (Pp) named as 11(Pv), 661(Pp), 662(Ph), 663(Pp), 664(Pp), 665(Pp), 666(Pp), 667(Pp), 668(Pp), 911(Pm) and 912(Pm) isolated from municipal tap water (Rajshahi City, Bangladesh) in our previous study (Wadud and Chouduri, 2013) have 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 earlier (Chouduri and Wadud, 2013; Chouduri et al., 2013; Chouduri and Wadud, 2014). Strains stored at -40°C in Luria-Bertani (LB) broth supplemented with 12% (v/v) glycerol were freshly grown at 37°C to carry out this study.

Growth media and culture conditions

Nutrient agar media purchased from Difco, USA was used for antibacterial activity assay of the plant extracts. The bacterial strains were incubated at 37°C for overnight as described elsewhere (Nesa et al., 2013; Chouduri and Wadud, 2013). Fresh cell culture in nutrient broth media prepared on water bath (Advantec Lab-Thermo Shaker, TS-20, Toyo Kaisha Ltd) with mild shaking at 37°C was used to test the swarming motility of Proteus strain.

Test for antibacterial activity

Plant extracts were tested for antibacterial and specifically anti-Proteus activity using disk diffusion method on nutrient agar media as reported elsewhere (Dash et al., 2005; Parvin et al., 2014). The 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 disc with various potencies, 25 µg to 1 mg/disc. Discs were placed on pre-seeded bacterial culture plates and then kept at low temperature (4°C) overnight to allow maximum diffusion of the components. The plates were then allowed to incubate at 37°C for 18 hrs. Then the diameter (in millimeter) of zone of inhibition for each extract against tested microorganisms was noted. Reference standard discs of cefixime (5 µg), ceftazidime (30 µg), kanamycin (30 µg) (Hi-media, India) were used as positive control and blank disc as negative control.

Swarming motility test

Proteus strains were grown overnight in 10 ml of LB broth medium (1% Tryptone, 0.5% Yeast extract, and 0.5% NaCl) at 37°C with shaking (200 rpm). Then 5 µl 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 reports (Kwil et al., 2013) and incubated at 37°C for 24 hrs unless it is mentioned otherwise. Then the mean diameters of swarming zones measured in millimeter at three different directions were used for analysis.

Inhibition of swarming motility

The effects of plant extracts on swarming motility of Proteus strains were assessed as described in other report (Liaw et al., 2000; Roshid et al., 2014). Briefly, an overnight bacterial culture (5 µl) was inoculated centrally onto the surface of dry LB agar plates prepared with extracts at various concentrations which were then incubated at 37°C for 24 hrs. The perimetric distance of swarming motility was assayed by measuring the fronts of swarming areas in three different directions.

Data analysis

For data processing, the software MicrosoftExcel 2007 was used. Results of triplicate experiments were averaged, and means ± standard deviations were calculated.

RESULTS

Antibacterial activities of plant extracts

The ethanol extracts of the plant specimens were tested for their antibacterial activities on five Gram-positive and six Gram-negative bacteria from our laboratory stock. The extracts named Ti-b, Pe-f, Pm-w, Ar-r, Ul-r, and Ai-l showed remarkable antibacterial activities with a wide zone of inhibition whereas Ul-l, Ul-f, Ul-b, Cg-w, and Mp-r were inactive in antibacterial activities (Table 2). However, the antibacterial potentials of the test extracts based on their zone of inhibition were evaluated where Pm-w was the best one showing 15-22 mm clear zone on culture plate followed by Ai-l (18-20 mm), Ar-r (10-19 mm), Pe-f (10- 16 mm), Ul-r (8-14 mm) and Ti-b (9-13 mm). The antibacterial potentials of the extracts Pmw and Ai-l against three Gram-positive bacteria, S. agalactiae, B. megaterium, B. subtilis, and two Gram-negative bacteria, P. aeruginosa, S. flexneri, were very close and comparable to that of reference antibiotic kanamycin (Table 2).

Screening of plant extracts for their abilities to inhibit Proteus

Next our efforts aimed to search medicinal plants to combat these strong cephalosporin resistant Proteus isolates to control and manage UTI caused by these bacteria. To do so, twelve specimens of nine medicinal plants as enlisted in table-1 were selected based on their reported information. The extracts exhibiting high antibacterial activities on several Gram-positive and Gram-negative bacteria were used to test whether they have any inhibitory effect on multi-antibiotic resistant Proteus strains isolated in our previous study (Wadud and Chouduri, 2013). The extract Pm-w showed remarkable zone of inhibition of Proteus strains (22 mm) whereas no clear zone of inhibition was observed for reference antibiotic cefixime (Figure 2). A representative image has been shown in figure 2. The extract Ul-r showed clear zone of inhibition of Proteus strains but relatively higher inhibition was found for the strain 11(Pv). Then the extracts were screened for their effects on swarming motility of the test strains since swarming is one of the crucial pathogenic factors of Proteus bacteria.

Effects of plant extracts on swarming motility of Proteus strain

Eleven Proteus isolates found to be strongly resistant to cephalosporin by disc diffusion method as reported earlier (Chouduri and Wadud, 2014) were subjected to a bactericidal activity assay by NHS where four isolates 11(Pv), 661(Pp), 911(Pm), and 912(Pm) were found to be resistant to NHS (unpublished data). Isolates 912(Pm), 911(Pm) and 662(Ph) were strong swarmer on LB agar media (Chouduri et al., 2014), therefore, these isolates were undertaken to a test of swarming in the presence of various concentrations of plant extracts especially U. lobata extracts (Figure 3) since major parts of this plant are used as folk medicine for UTI (Nandwani et al., 2008; Hossan et al., 2010). No noticeable effects of the extracts except Ul-r were found on the swarming motilities of the test strains (Figure 3). The extract Ai-l accelerated the swarming of Proteus isolates about 2 fold. However, interestingly complete inhibition of swarming was found by the extract Ul-r at 500 µg/ml concentration (Figure 3) although its antibacterial activity was nil or very low by disc diffusion method (Figure 2, Table 3). The lag phase of swarming continued up to 4 hrs of incubation and the basal swarming starts after 4 hrs of incubation in the presence of the extracts. The zigzag pattern of swarming curves was a consequence of swarming-plus-consolidation cycle of the strains. However, anti-swarming effect of U. lobata root extract can be of interest to develop phytomedicine for the management and control of UTI and/or wound infection caused by antibiotic resistant Proteus bacteria. Moreover, the extract of Physalis minima had no anti-swarming effect although its antibacterial activity was stronger than that of others.

Kinetics of U. lobata extract-induced swarming motility

Three Proteus isolates exhibiting enhanced swarming motility were assessed for their abilities to swarm onto the LB agar plate in the presence of U. lobata extracts. The velocities of swarming (mm/h) of the test strains in the presence of U. lobata root extract were found to be zero after the lag phase (4 hrs) (Table 4) and the swarming velocities for other extracts were instantaneously accelerated just after the lag phase (4 hrs) that were the maximal velocities up to 7 hrs of incubation period. The similar duration of lag phase (4 hrs) of clinical isolates of P. mirabilis was reported by Rauprich et al. (Rauprich et al., 1996). The rapid onset of swarming after lag phase is possibly due to the formation of elevated number of flagella on bacteria or multinucleation in the first generation of swarmer cells. The cycle time of swarming-plusconsolidation phase found by Rauprich et al., (1996) is about 3.5 hrs at 37°C on 1.5% agar plate. In this study, the high swarming velocities after lag phase at 4-5 hrs and the subsequent gradual decline of the velocity up to 7 hrs indicated the first cycle of swarming-plusconsolidation phase and the following high velocities at 7-8 hrs indicated the second cycle of swarming-plus-consolidation phase resembling the findings of Rauprich et al. (1996). However, only the U. lobata root extract showed significant inhibition of swarming of the test strains and no noticeable effects of other test extracts on swarming were observed.

DISCUSSION

Among the most common infections UTI is affecting humans and represent a serious health problem for millions of people each year. Proteus is an important opportunistic uropathogen, frequently isolated from catheterized patients or individuals with structural abnormalities of the urinary tract (Khalid et al., 2013; Hoban et al., 2012; Alves et al., 2014) although it does not commonly cause UTI in the normal host. UTI is commonly managed with antibiotic therapy but the increasing evidence of antibiotic resistance is restricting the therapeutic option. Thus the acceptance of traditional medicine as an alternative form of health care and the development of microbial resistance to the available antibiotics have led researchers to investigate the antimicrobial activity of herbal extracts.

The World Health Organization reported that about 80% of the world's population depends primarily on traditional medicine that mainly involves the use of plant extracts (Low et al., 2002). The screening of plant extracts and plant products has shown that medicinal plants represent a potential source of new antiinfective agents. For instance, cranberry has long been of interest for its beneficial effects in preventing UTI (Ahuja et al., 1998; Howell et al., 1998; Howell and Foxman 2002; McCall et al., 2013). Plants containing flavonoids, terpenoids, steroids, phenolic compounds and alkaloids have been reported to have antimicrobial activity. Three compounds (kaempferol, quercetin, tiliroside) isolated from ethyl acetate fraction of U. lobata leaf showed strong antimicrobial activities against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, Bacillus polyxyma and Candida albicans (Adewale et al., 2007). But in this study, the ethanol extract of U. lobata leaf showed no antibacterial activities against test bacterial pathogens including Proteus. In contrast, U. lobata root had a significant antiswarming effect on Proteus isolates although its antibacterial activity was very low. It has been reported that U. lobata root has no significant toxic effects on serum total proteins, albumin and globulins (Omonkhua and Onoagbe, 2011). Therefore, U. lobata root can be used as a source of alternative anti-infective agent for the treatment of UTI and wound infection caused by antibiotic resistant bacteria.

The chloroform extract of P. minima exhibited remarkable cytotoxic activities on NCI-H23 (human lung adenocarcinoma) cell line at dose- and time-dependent manners (Leong et al., 2011). The strong antibacterial activity of ethanol extract of P. minima leaf has been reported (Gavimath et al. 2012). In this study, we found strong inhibition of antibiotic resistant Proteus isolates by the treatment of ethanol extract of P. minima whole plant. Thus, P. minima can also be the plant of interest for the treatment and control of antibiotic resistant uropathogens.

Howell et al., (1998) determined that proanthocyanidins isolated from the cranberry fruit inhibit P-fimbrial adhesion in vitro, and thus may be the compounds responsible for the beneficial effect on UTI prevention (Howell et al., 1998). The urine of humans who consumed cranberry juice cocktail also exhibited antiadhesion activity (Howell and Foxman, 2002), which suggests that a certain level of absorption occurred and that bioactive proanthocyanidins and/or their metabolites have been excreted in the urine to inhibit adhesion. The bactericidal activities of anacardic acid and totarol (a diterpene extracted from the totara tree) on methicillin resistant strains of S. aureus and the synergistic effect of these compounds associated with methicillin have been reported (Muroi and Kubo, 1996). Therefore, more studies pertaining to the use of plants as therapeutic agents should be emphasized, especially those related to the control of antibiotic resistant microbes.

CONCLUSION

The ethanol extract of Physalis minima whole plant showed strong antibacterial activities against cephalosporin resistant uropathogen Proteus and the extract of Urena lobata root showed strong anti-swarming effect on Proteus. Therefore, a mixture of two extracts would be a powerful anti-infective agent to combat UTI caused by antibiotic resistant Proteus. This study could offer scientific basis for the in-depth evaluation of ethanol extract of P. minima whole plant and U. lobata root. The phytochemical(s) in P. minima and U. lobata extracts having the potential antibacterial activities and antiswarming effect are remain to be identified and are required to go through the toxicity analyses before they can be safely applied.

ACKNOWLEDGEMENTS

Authors wish to thank the Department of Pharmacy, University of Rajshahi, Bangladesh for providing laboratory facilities to carry out the entire experiments. We thank the Ministry of Science and Technology, Government of the People's Republic of Bangladesh for the NST fellowship provided to author MR to carry out the research.

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Cite this article:

Mamunur Roshid, Aktar Uzzaman Chouduri (2014), Antibacterial, anti-swarming potential of ethanol extracts of Physalis minima L. whole plant and Urena lobata L. root on cephalosporin resistant Proteus species, Global J Res. Med. Plants & Indigen. Med., Volume 3(5): 184-195

Source of Support: National Science and Technology (NST) Fellowship, Government of Bangladesh

Conflict of Interest: None Declared

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