Antibacterial activity of seed extracts of Callistemon lanceolatus DC on uropathogenic bacteria

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ScienceDirect Journal of Acute Medicine 4 (2014) 6e12 www.e-jacme.com

Original Research

Antibacterial activity of seed extracts of Callistemon lanceolatus DC on uropathogenic bacteria Kumara Shanthamma Kavitha, Sreedharamurthy Satish* Herbal Drug Technological Laboratory, Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India Received 8 November 2013; accepted 23 December 2013 Available online 1 April 2014

Abstract Background: Urinary tract infections are a major public health problem in developing countries. The spread of microbial drug resistance has become a global threat, challenging public health, and can result in increased illness and even lead to death in the chronic stages of infection. There is a desire to identify and exploit newer, ecologically friendly antimicrobial agents from natural sources to overcome this problem. Purpose: This work was carried out to investigate the antibacterial potential of Callistemon lanceolatus DC seed extracts against 11 isolates of uropathogenic bacteria. Methods: Bacteria isolated from urine samples were collected from the Krishna Rajendra Hospital, Mysore, India and were subjected to biochemical tests followed by standard procedures for bacterial identification. The uropathogens were then tested for susceptibility to antibiotics using an assay following the KirbyeBauer method. Phytochemical analyses of seed extracts of C. lanceolatus DC were carried out to identify the various phytocomponents. Antibacterial activity against 11 uropathogenic isolates using petroleum ether, chloroform, ethyl acetate, and methanol seed extracts of C. lanceolatus were carried out following the KirbyeBauer disc diffusion method. Results: The identities of the bacteria were confirmed by the biochemical tests. Antibiogram assays showed the sensitivity of the pathogens towards the test antibiotics. The phytochemical analyses showed the presence of carbohydrates, saponins, and phytosterols in all the test solvent extracts; alkaloids, tannins, and amino acids were absent from all the test solvent extracts. Tests to determine the antibacterial activity of seed extracts of C. lanceolatus against 11 isolates of bacteria showed significant activity against Staphylococcus aureus in petroleum ether and chloroform extracts; the ethyl acetate and methanol extracts showed significant activity against Proteus mirabilis. All the test solvent extracts showed the least activity against Acinetobacter baumannii. Petroleum ether, ethyl acetate, and methanol extracts also showed the least activity against Citrobacter freundii; only ethyl acetate showed moderate activity against Enterococcus faecalis. The methanol extract showed moderate activity against Escherichia coli. Ethyl acetate and methanol extracts showed a moderate zone of inhibition against Klebsiella pneumoniae. Conclusion: This study shows that C. lanceolatus has an inhibitory effect on uropathogenic bacteria. The antibacterial activity of this plant could be exploited as a therapeutic agent for further investigation of the bioactive compounds responsible for antibacterial activity. Copyright Ó 2014, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved.

Keywords: antibacterial activity; antibiogram assay; C. lanceolatus; phytochemical analysis; uropathogenic bacteria

1. Introduction A urinary tract infection (UTI) occurs when microorganisms reside and multiply within the urinary tract and cause an * Corresponding author. Herbal Drug Technological Laboratory, Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore 570 006, India. E-mail address: [email protected] (S. Satish).

infection. About 95% of the microorganisms responsible for such infections are bacteria, although fungi and viruses have also been found to cause UTIs. Among the bacteria, Escherichia coli is the predominant pathogen, causing about 80% of all cases of UTI, followed by other species of bacteria such as Staphylococcus, Klebsiella, Enterobacter, Proteus, and Enterococcus.1 Guidelines vary, but typically a pure culture of between 104 CFUs/mL and 106 CFUs/mL of urine is indicative of a UTI.2 Common symptoms of UTIs include leukorrhea,

http://dx.doi.org/10.1016/j.jacme.2013.12.001 2211-5587/Copyright Ó 2014, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved.

K.S. Kavitha, S. Satish / Journal of Acute Medicine 4 (2014) 6e12

frequent or infrequent urination, cloudy urine, and a burning sensation during urination. Some studies have reported that women are more prone to UTIs than men.3,4 There are several approaches to the treatment and management of UTIs. Antimicrobial treatment may be administered in a regimen depending on the clinical situation. Such treatment may be administered temporally either as a prophylactic or as a therapeutic approach to treat the cause of infection. Antibiotics such as penicillin, sulfanilamide, nitrofurantoin, and cephalexin have been used for the treatment of UTIs.5 The usual treatment for UTIs is a short course of antimicrobial drugs such as a 3-day regimen of trimethorpimesulfamethoxazole.6 Historically, plants have served as a natural reservoir of bioactive components with different functional groups in their structure; their antimicrobial activity is attributed to multiple mechanisms contributing to human health and wellbeing.7 Antimicrobial drugs derived from plants have recently received more attention because of the increasing prevalence of antibiotic-resistant bacteria, the escalating costs of antibiotic treatment, and unsatisfactory therapeutic alternatives for recurrent UTIs. This has triggered an interest in searching for new antimicrobial agents with diverse chemical structures and novel mechanisms of action to treat new and re-emerging infectious diseases.8 Callistemon lanceolatus DC (Myrtaceae) is an evergreen aromatic ornamental shrub. The plant is known to possess antibacterial, antifungal, antioxidant, antidiabetic, antinociceptive, and anti-inflammatory activity.9,10 However, there has been no previous report of antibacterial activity against uropathogenic bacteria using C. lanceolatus seed extracts. The aim of the work reported here was to determine the antibacterial sensitivity of different solvent seed extracts of Clanceolatus against isolates of various bacteria responsible for UTIs. 2. Materials and methods 2.1. Collection of plant material Fresh disease-free seeds of C. lanceolatus DC were collected from Mysore, Karnataka, India. The seeds were washed thoroughly two to three times with running tap water and once with sterile distilled water. The seeds were then shade-dried on sterile blotters. A voucher specimen of this plant material was deposited in the herbarium of the Department of Studies in Botany, University of Mysore, Mysore, India.

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2.3. Preliminary phytochemical screening Chemical tests were carried out on the petroleum ether, chloroform, ethyl acetate, methanol, and also aqueous extracts of C. lanceolatus using standard procedures to identify the phytochemical constituents.12e14 2.4. Bacterial strains 2.4.1. Clinical isolates Acinetobacter baumannii, Citrobacter freundii, Enterobacter aerogenes, Enterococcus faecalis, E. coli, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens, and S. aureus were recovered from urine samples obtained from the Krishna Rajendra Hospital, Mysore, Karnataka, India. The isolates obtained were identified on the basis of colony morphology and biochemical tests.15e17 2.4.2. Preparation of inoculum Stock cultures were maintained at 4
C on slants of nutrient agar. A loopful of stock cultures were transferred to a fresh sterile nutrient broth tubes and incubated for 24 hours at 37
C without agitation. A 0.2-mL volume of culture was inoculated into 5 mL of nutrient broth and incubated until it reached a turbidity equal to a standard 0.5 McFarland solution. This is equivalent to 106e108 CFU/mL measured spectrophotometrically at 600 nm.18 2.5. Antibiogram assay for uropathogenic bacteria The antibacterial sensitivity and resistance patterns of uropathogenic bacteria causing UTIs were detected using the KirbyeBauer disc diffusion method.19 An aliquot of 100 mL (0.5 McFarland equivalent) of the test bacterial inoculums from an approximately 18e24-hour broth culture was spread onto the surface of MuellereHinton agar media. Standard antibiotic discs (Hi-Media, Mumbai) were placed onto the culture lawn. The antibiotics used in this experiment were amoxicillineclavulanic acid (30 mg), norfloxacin (10 mg), gentamicin (10 mg), kanamycin (30 mg), trimethoprim (5 mg), methicillin (5 mg). The plates were then incubated at 37
C for 18e24 hours. Interpretation based on a comparison of the inhibition zones with published criteria for zone diameters were measured using Clinical and Laboratory Standards Institute (CLSI) guidelines.

2.2. Soxhlet extraction 2.6. Antibacterial activity of extracts Dried seeds of C. lanceolatus were powdered using a Waring blender. Fifty grams of the powder were filled in the thimble and extracted successively with petroleum ether, chloroform, ethyl acetate, and methanol for 48 hours using a Soxhlet extractor (Borosil, Mumbai). The extracts were concentrated under reduced pressure using a rotary vacuum evaporator. The extracts were stored in sterile amber bottles until further use.11

The antibacterial activity of seed extracts of C. lanceolatus against uropathogenic bacteria was determined using a modified KirbyeBauer disc diffusion method. A 100-mL aliquot of bacterial suspension was swabbed uniformly onto MuellereHinton agar plates. Volumes of 50 mL (100 mg/mL concentration) of the petroleum ether, chloroform, ethyl acetate, and methanol extracts were then loaded onto sterile 6-mm

þ e

Nitrogen metabolism

Oxygen metabolism

Hydrogen sulfide production Bile esculin IMViC Indole Methyl red Voges proseukar Citrate

VI

VII

VIII IX X

Glucose Lactose Sucrose Mannitol Nitrate reduction Urea hydrolysis Oxidase Catalase

þ ¼ present; e ¼ absent; A ¼ acid production; AG ¼ acid and gas production; B ¼ bacilli; C ¼ cocci; C/B ¼ cocco-bacilli; IMViC ¼ indole, methyl red, Voges-Proskauer, and citrate.

þ e

Negative B þ þ A e A A þ e e þ e e e e þ þ Negative B þ þ e e e A þ e þ þ e e e e e þ Negative B þ e AG e e e þ þ þ þ e þ e þ þ þ Negative B e e AG A A A þ þ e þ e e e e þ þ Negative B þ e AG AG A A þ e e þ þ e e e þ þ Positive C e e A A A A e e e þ e e e Gram stain Cell morphology Motility Pigment Carbohydrate metabolism I II III IV V

Sample number Biochemical test

The cell morphology and results of the biochemical tests carried out on the bacteria isolated from urine samples are shown in Table 1. The biochemical tests for carbohydrate, nitrogen, and oxygen metabolism, the hydrogen sulfide and bile esculin tests, and the IMViC (indole, methyl red, VogesProskauer, and citrate) test allowed the identification of the characteristic features of the bacteria. Table 2 gives the results of the antibiogram pattern assay for the UTI isolates studied. The results (diameter of inhibition zone in millimeters) were interpreted according to the CLSI guidelines and categorized into susceptible, intermediate, and resistant to the individual antibiotics. The antibiotics were chosen based on the resistance of bacteria causing UTIs. The results of the assay showed that A. baumannii was resistant to trimethoprim and intermediate gentamicin. C. freundii exhibited resistance towards standard antibiotics such as amoxicillineclavulanic acid, norfloxacin, and trimethoprim and was sensitive to gentamicin and kanamycin. E. aerogenes showed resistance to amoxicillineclavulanic acid, kanamycin, norfloxacin, and trimethoprim and was sensitive to gentamicin. E. faecalis was resistant to norfloxacin. E. coli was resistant to amoxicillineclavulanic acid, kanamycin, and norfloxacin, showed an intermediate zone of inhibition to gentamicin, and was sensitive to trimethoprim. K. pneumoniae exhibited an intermediate zone of inhibition to gentamicin and was resistant to all other test antibiotics. Methicillin-resistant S. aureus showed resistance to all test antibiotics except gentamicin. S. aureus was resistant to kanamycin, norfloxacin, and trimethoprim, but sensitive to amoxicillineclavulanic acid, gentamicin, and methicillin. P. mirabilis was sensitive to amoxicillineclavulanic acid, gentamicin, and norfloxacin, but was intermediate to kanamycin and resistant to trimethoprim. P. aeruginosa exhibited resistant to kanamycin and was sensitive to gentamicin and norfloxacin. S. marcescens was

Table 1 Biochemical tests on recovered bacterial isolates of urine samples from Krishna Rajendra Hospital, Mysore, India.

3. Results

Negative B þ e A A A A þ e e þ e e e e þ þ

Uropathogenic bacteria

Minimal inhibition concentration was defined as the lowest concentration of extract required to inhibit the visible growth of the test bacteria as indicated by 2,3,5-triphenyl tetrazolium chloride using a microtitre enzyme-linked immunosorbent assay plate.20 The 96 wells were filled with MullereHinton broth media containing different test solvent extracts against uropathogenic bacteria. Antibacterial activity was detected by adding 0.5% 2,3,5-triphenyl tetrazolium chloride aqueous solution (Merck., Mumbai); dead cells are not stained by this solution.

Negative B þ e A A e A þ þ e þ e e e þ e þ

2.7. Micro-broth dilution assay

Negative C/B e e A A e e e e e þ e e e e e þ

discs and the impregnated discs were placed on the inoculated plates. The plates were allowed to stand at 4
C for 2 hours prior to incubation. The plates were then placed in an incubator at 37
C for 24 hours. The inhibition zones formed around the disc were measured in millimeters. The assays were performed in triplicate and the results are given as mean  standard deviation values.19

Positive C e þ A A A A þ e e þ e e e

K.S. Kavitha, S. Satish / Journal of Acute Medicine 4 (2014) 6e12 Acinetobacter Citrobacter Enterobacter Enterococcus Escherichia Klebsiella Proteus Pseudomonas Serratia Staphylococcus baumannii freundii aerogenes faecalis coli pneumoniae mirabilis aeruginosa marcescens aureus

8

S S R S R R R S S ND S S ND S R ND S ND S S I ND S R R S R R R R R I R ND R R R I R ND R S ND ND ND ND R ND I ¼ intermediate; ND ¼ not determined; R ¼ resistant; S ¼ sensitive.

R S S ND R R Amoxycillin Gentamicin Kanamycin Methicillin Norfloxacin Trimethoprim 1 2 3 4 5 6

ND I ND ND ND R

R S R ND R R

Methicillin resistant Staphylococcus aureus Klebsiella pneumoniae Enterobacter aerogenes Citrobacter freundii Acinetobacter baumannii

Antibiotic Sample number

Table 2 Antibiogram activity using standard antibiotics against uropathogenic bacteria.

Enterococcus faecalis

Escherichia coli

Uropathogenic bacteria

Proteus mirabilis

Pseudomonas aeruginosa

Serratia marcescens

Staphylococcus aureus

K.S. Kavitha, S. Satish / Journal of Acute Medicine 4 (2014) 6e12

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resistant to only amoxicillineclavulanic acid and was sensitive to the other test antibiotics. Table 3 shows the presence and absence of phytochemicals in the seed extracts of C. lanceolatus. Carbohydrates, phytosterols, and saponins were present in all test solvent extracts (petroleum ether, chloroform, ethyl acetate, methanol, and aqueous solvent extracts), whereas alkaloids, amino acids, and tannins in absent from all test solvent extracts. Flavonoids and proteins were present in all test solvent extracts except for petroleum ether. Glycosides and phenolic compounds were present in the ethyl acetate, methanol, and aqueous extracts. The antibacterial activity of the four different solvent seed extracts (petroleum ether, chloroform, ethyl acetate, methanol) were tested against 11 isolates of uropathogenic bacteria (Table 4). The zone of inhibition were interpreted on the basis of the CLSI guidelines. All the test solvent extracts showed the least activity against A. baumannii and methicillin-resistant S. aureus; no inhibitory activity was observed against E. aerogenes, P. aeruginosa, or S. marcescens. C. freundii showed the least activity in petroleum ether, ethyl acetate, and methanol extracts and no activity in the chloroform extract. Only the ethyl acetate extract showed moderate activity against E. faecalis; no zone of inhibition was observed in the other test solvent extracts. The petroleum ether and ethyl acetate extracts showed the least activity and the methanol extract showed moderate activity against E. coli, but no antibacterial activity in the chloroform extract. The ethyl acetate and methanol extracts exhibited moderate activity against K. pneumoniae, whereas the petroleum ether and chloroform extracts did not show any zone of inhibition. Significant antibacterial activity was observed with ethyl acetate and methanol extracts against P. mirabilis, but only moderate activity was observed in the petroleum ether and chloroform extracts. Significant activity against S. aureus was observed in the petroleum ether and chloroform extracts, but only moderate activity in the ethyl acetate and methanol extracts. The petroleum ether extract showed the lowest minimal inhibition concentration of about 5 mg/mL against all test bacteria except P. mirabilis and S. aureus, which showed 2.5 mg/mL and 0.625 mg/mL, respectively. Similarly, the chloroform extract showed an MIC of 5 mg/mL against A. baumannii and methicillin-resistant S. aureus, whereas the MIC was 2.5 mg/mL and 0.156 mg/mL against P. mirabilis and S. aureus, respectively. The ethyl acetate and methanol extracts showed the lowest MIC, ranging from 10 mg/mL to 1.25 mg/ mL, against the test uropathogenic bacteria. 4. Discussion Several approaches have been taken towards the treatment and management of UTIs. Among these, the use of botanical agents has suggested a new way of preventing and managing infections. Advances in phytochemistry and in the identification of bioactive compounds from plants which are effective against disease have renewed interest in the treatment of UTIs by herbal medicines. One study reported that a Vaccinium macrocarpon (cranberry) juice cocktail or placebo beverage intake of about

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Table 3 Phytoconstituent analysis of seed extracts of Callistemon lanceolatus DC. Sl. No.

I

II

III

IV

V

VI VII

Chemical test

Alkaloids Dragendorff’s test Mayer’s test Wagner’s test Carbohydrates and glycosides Barford’s test Benedict’s test Fehling’s test Molisch’s test LibermanneBurchard’s test Ligal’s test Modified Borntrager’s test Flavonoids Alkali and acid test Shinoda test Proteins and amino acids Biuret test Millon’s test Xanthoprotein test Ninhydrin test Phytosterols LibermanneBurchard’s test Terpenoids 2,4-DNPH test Anthraquinones Borntrager’s test Steroids Salkowski’s test Saponins Foam test Tannins and phenolic compounds Vanillin hydrochloric acid Iron (III) chloride test

Solvent extract of Callistemon lanceolatus DC Petroleum ether

Chloroform

Ethyl acetate

Methanol

Aqueous

  

  

  

  

  

þ þ þ þ þ  þ

þ þ þ þ þ  þ

þ þ þ þ þ þ þ

þ þ þ þ þ þ þ

þ þ þ þ þ þ þ

þ 

þ þ

þ þ

þ þ

þ þ

 þ þ 

þ þ þ 

þ þ þ 

þ þ þ 

þ þ þ 

þ þ

þ þ

þ þ

þ þ

þ þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

 

 

 þ

 þ

 þ

þ ¼ present;  ¼absent.

300 mL daily for 6 months resulted in the prevention of UTIs in elderly women, although a dose of about 15 mL/kg had no effect in children with neurogenic bladder dysfunction. An intake of cranberry juice diluted with about 15 mL of water twice daily for 4 weeks resulted in decreased bacteriuria.22 Similarly, a mixed cranberryelingonberry juice concentrate with no added sugar, Lactobacillus rhamnosus, or no intervention of about 50 mL (7.5 g cranberrye1.7 g lingonberry) daily for 6 months, or 100 mL Lactobacillus drink 5 days per week was reported to prevent UTIs in college-aged women.21 The administration of Arctostaphylos uva ursi with Taraxacum officinalis UVA-E standardized extracts of uva ursi and dandelion (Taraxacum officinalis) (oral dosage of about 3 tablets 3 times a day for 1 month followed for 1 year) had the effect to prevent UTIs in women.21 In vivo studies of the oral administration of berberine sulfate, an alkaloid compound found in many plants at a concentration of 200 mg/mL for 18 hours resulted in the inhibition of fimbrial synthesis (antiadhesion) and direct growth inhibition of E. coli and P. aeruginosa. An animal study at a concentration of 200 mg/kg (1 dose) resulted in the prevention of cyclophosphamide-induced hemorrhagic cystitis in rats.21,22

The results of the work reported in this study indicate that C. lanceolatus seed extracts show significant antibacterial activity against S. aureus and P. mirabilis and moderate activity against E. faecalis, E. coli, and K. pneumoniae, with least activity against A. baumannii and C. freundii. Methanol and acetone extracts of the leaves of C. rigidus have previously been shown to have antibacterial activity against uropathogenic bacteria such as S. aureus and E. coli.23 These workers also reported the isolation of alkaloids from the same plant which showed significant anti-staphylococcal activity in a UTI isolate.24 Another study using methanol extracts of the leaves of C. lanceolatus showed the presence of carbohydrates, phenolic compounds, saponins, alkaloids, oils and fats, flavonoids, phytosterols, and tannins, which also exhibited antibacterial activity against S. aureus.25 Many reports have also been published about the biological properties of C. lanceolatus; however, these reports are based on the leaf components and essential oils.9,10,26e30 To the best of our knowledge, this is the first report of a phytochemical analysis of C. lanceolatus and determination of the antibacterial activity against uropathogenic bacteria of seed extracts of C. lanceolatus. Further separation and characterization of

0.57

0.57

0.57

0.57

0.00

0.00

0.00

0.00 0.57

0.00 0.57

0.00

0.00

0.00  (ND) 0.00  (ND) 0.00  (ND) 0.00  (ND) 0.00  (ND) 0.00  (ND) 0.00  (ND) 0.00  (ND)

0.00

23.66  (0.625) 28.33  (0.156) 14.66  (5) 18.66  (2.5)

Conflicts of interest

 0.00

 0.57

 0.57

Acknowledgments The authors are thankful to the University Grant Commission (UGC)eRGNFs, Government of India, for providing financial support and to the Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore, India.

0.00  0.00 (e) 0.00  0.00 (e) 15.33  0.57 (2.5) 18.00  0.00 (2.5)

10.33 (5) 12.33 (5) 14.33 (5) 12.00 (5)

 0.57

All contributing authors declare no conflicts of interest.

References

0.00

0.00

0.00

Ethyl acetate Methanol

Data are given as mean  SD of triplicate analyses. p < 0.05. MIC ¼ minimal inhibitory concentration; ND ¼ not done; e ¼ not significant.

13.66  0.57 (5) 0.00  0.00 (e) 10.00  0.00 (5) 14.33  0.57 (5) 0.00  0.00 (e) 0.00  0.00 (e) 14.33  0.57 (5) 0.00  0.00 (e) 0.00

0.00  (ND) 0.00  (ND) 0.00  (ND) 0.00  (ND) 13.66  0.57 (5) 0.00  0.00 (e) 12.00  0.00 (5) 12.66  0.57 (5) 12.66  0.57 (5) 11.33  0.57 (5) 8.00  0.00 (10) 8.00  0.00 (10) Petroleum ether Chloroform

11

the various bioactive compounds in C. lanceolatus and their assessment against the same test panel of multidrug-resistant uropathogens would help in determining novel compounds which could be developed into drugs. In conclusion, the conventional use of antibiotics in the prevention and treatment of UTIs has contributed to the development of antibiotic-resistant uropathogens. The results obtained in the work reported here might be considered sufficient to further isolate and identify the bioactive compounds responsible for the antibacterial activity against uropathogenic bacteria of C. lanceolatus and to evaluate its pharmaceutical significance. The use of these alternative strategies in the control of UTIs could circumvent the risk of bacteriuria and microbial drug resistance induced by the overuse of antibiotics.

15.33  (2.5) 17.00  (2.5) 20.33  (1.25) 21.66  (1.25)

0.57

0.00

Serratia marcescens Pseudomonas aeruginosa Proteus mirabilis Methicillin resistant Staphylococcus aureus Klebsiella pneumoniae Escherichia coli Enterococcus faecalis Citrobacter freundii Acinetobacter baumannii

Enterobacter aerogenes

Isolates of uropathogenic bacteria (zone of inhibition in mm) (MIC in mg/mL) Solvent extract

Table 4 Antibacterial activity measured as zone of inhibition at 50 mL (100 mg/mL) of solvent seed extracts of Callistemon lanceolatus DC against uropathogenic bacteria.

Staphylococcus aureus

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