Phytochemical analysis and antimicrobial study of Fernandoa adenophylla against multidrug resistant urinary tract infection pathogens

In the present study various solvent extracts from the leaves of Fernandoa adenophylla were screened for their phytochemical and antimicrobial potentials. Preliminary phytochemical screening of leaves extracts showed the presence of alkaloids, glycosides, flavonoids, terpenoids, tannins, steroids, reducing sugars and anthracenes. Fourier transform infrared spectroscopic studies showed the presence of different functional groups for compounds like alcohol, aldehydes and ketones, saturated and unsaturated hydrocarbons, amides, amines, carboxylic acids, esters, and ethers. All the extracts showed significant antibacterial activities when tested against eight multidrug resistant bacterial strains isolated from urinary tract infection patients. It was concluded that the leaves extracts of F. adenophylla have several vital phytochemical constituents and significant antibacterial activities against multidrug resistant bacterial strains causing urinary tract infections. Article Info Received: 13 September 2015 Accepted: 9 January 2016 Available Online: 27 January 2016 DOI: 10.3329/bjp.v11i1.24974 Cite this article: Rahim A, Hussain MM, Altaf IUK. Phytochemical analysis and antimicrobial study of Fernandoa adenophylla against multidrug resistant urinary tract infection pathogens. Bangladesh J Pharmacol. 2016; 11: 240-47. Phytochemical analysis and antimicrobial study of Fernandoa adenophylla against multidrug resistant urinary tract infection pathogens Abdur Rahim1, Muhammad Medrar Hussain2 and Ihsan Ullah Khan Altaf1 Department of Pharmacy and Microbiology, Abasyn University, Peshawar, Pakistan. This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor Leaves were collected from the gardens of the University of Peshawar and identified by the Department of Botany, University of Peshawar. The leaves were shade dried and grinded to coarse powder (Sood and Sharma, 2010) using a crusher. Powdered leaves (150 g) were macerated in five different solvents (ethanol, methanol, ethyl acetate, n-hexane and purified water) (Cseke et al., 2006) for 24 hours at room temperature. The supernatant liquids were filtered and collected. The crude extracts were concentrated using rotary evaporator and the solvents were recollected. The concentrated extracts were dried at 60 ± 5°C using water bath. All the extracts were then preserved in separate labeled glass vials and stored at room temperature for further processes. Phytochemical investigation of extracts Qualitative tests were performed for each extract using standard protocols (Kayani et al., 2007; Ayoola et al., 2008) for the presence of different phytochemicals i.e. alkaloids, flavonoids, reducing sugars, terpenoids, saponins, tannins, steroids, glycoside and anthracene. Fourier transform infrared spectroscopy Fourier transformed infrared (FTIR) spectroscopy was carried out using IR Prestige-21 FTIR (Shimadzu, Japan) for the identification and confirmation of different compounds functional groups present in extracts in dried form from 400-4000 cm-1. Isolation and identification of bacterial strains Urine samples from 150 UTI patients were collected from different tertiary care units of Peshawar, Pakistan. These samples were used for isolation of bacterial strains which were identified by specific morphological and biochemical characteristics in the laboratory of the Department of Microbiology, Abasyn University Peshawar. The pathogens isolated were: Escherichia coli, Klebsiella pneumoniae, Citrobacter, Enterobacter, Pseudomonas aeruginosa, Acinetobacter, Providencia, and Methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial susceptibility testing for MDR UTI pathogens Before evaluating the antimicrobial activity of plant extracts, all the isolated pathogenic microorganisms were tested for MDR profile. The antimicrobial susceptibility was carried out by disc diffusion method using Muller Hinton agar (MHA) as a medium (Ushimaru et al., 2007). The sensitivity of the isolated bacterial strains was tested against 10 commonly used antibiotics in triplicate. All the media plates were incubated for 24 hours at 37°C. Antibacterial activity of plant extracts Agar well diffusion method (Obeidat et al., 2012; Janovska et al., 2003) was applied for evaluation of antimicrobial activity of the leaves extracts of F. adenophylla. Each extract (10 mg) was dissolved in 1 mL of dimethyl sulfoxide to get a concentration of 10 mg/mL. On each sterile Muller Hinton agar plat, 100 μL of standard inoculum (0.5 MacFarland turbidity standards, 10-6 CFU/mL) of each test bacterial strain was spread with the help of sterilized cotton swabs while sterile borer was used for wells preparation. An amount of 100 μL of each extract i.e. ethanolic extract (EE), methanolic extract (ME), ethylacetate extract (EAE), n-hexane extract (nHE) and water extract (WE) was added through micropipette under aseptic conditions into specifically marked wells and then incubated at 37 ± 2° C for 18 hours. Zone of inhibition (ZI) was then measured to the closest point in millimeters (mm) (Bobbarala et al., 2009). The test was performed in triplicate and results were presented as mean ± standard deviation.


Introduction
The global emergence of multidrug resistant (MDR) bacteria is increasingly limiting the effectiveness of current drugs and significantly causing treatment failures (Hancock, 2005).One of the most common infectious diseases is urinary tract infection (UTI) (Sampson and Gravett, 1999) which has a high tendency of recurrence (Milan, 2006).Most of the studies have shown that UTI pathogens are resistant to the commonly prescribed antibiotics (Mussa-Aisien and Ibadin, 2003).Although many new antibiotics have been introduced, bacterial resistance is also continuously increasing (Hussain et al., 2014), which diverted the researchers towards the development of novel drugs from plant sources, having antimicrobial potentials (Maiyo et al., 2010).
Fernandoa adenophylla, an essential medicinal plant belongs to the family Bignoniaceae consisting of woody plants with approximately 860 species and 82 genera mainly growing in Africa, Central and South America (Lohmann, 2004).It is distributed in South and Southeast Asian regions i.e.Burma, Pakistan, the Andaman Islands, East Bengal and Assam (Olmstead et al., 2009).The leaves and seeds of F. adenophylla are also used as an antimicrobial agent.It is used for the treatment of urinary tract infections as well as anti-diarrheal and anti -diabetic agents (Muhammad et al., 2012).As a folk medicine, it is employed for the treatment of amenorrhoea, night emission, premature ejaculation and skin diseases.It also has antifungal and antiseptic activities (Rahmatullah et al., 2010).
Therefore, the present research work has been designed to investigate the phytochemical constituents and antimicrobial activities of less investigated F. adenophylla against MDR bacterial strains causing UTIs.
Leaves were collected from the gardens of the University of Peshawar and identified by the Department of Botany, University of Peshawar.The leaves were shade dried and grinded to coarse powder (Sood and Sharma, 2010) using a crusher.Powdered leaves (150 g) were macerated in five different solvents (ethanol, methanol, ethyl acetate, n-hexane and purified water) (Cseke et al., 2006) for 24 hours at room temperature.The supernatant liquids were filtered and collected.The crude extracts were concentrated using rotary evaporator and the solvents were recollected.The concentrated extracts were dried at 60 ± 5°C using water bath.All the extracts were then preserved in separate labeled glass vials and stored at room temperature for further processes.

Fourier transform infrared spectroscopy
Fourier transformed infrared (FTIR) spectroscopy was carried out using IR Prestige-21 FTIR (Shimadzu, Japan) for the identification and confirmation of different compounds functional groups present in extracts in dried form from 400-4000 cm -1 .

Isolation and identification of bacterial strains
Urine samples from 150 UTI patients were collected from different tertiary care units of Peshawar, Pakistan.These samples were used for isolation of bacterial strains which were identified by specific morphological and biochemical characteristics in the laboratory of the Department of Microbiology, Abasyn University Peshawar.The pathogens isolated were: Escherichia coli, Klebsiella pneumoniae, Citrobacter, Enterobacter, Pseudomonas aeruginosa, Acinetobacter, Providencia, and Methicillin-resistant Staphylococcus aureus (MRSA).

Antimicrobial susceptibility testing for MDR UTI pathogens
Before evaluating the antimicrobial activity of plant extracts, all the isolated pathogenic microorganisms were tested for MDR profile.The antimicrobial susceptibility was carried out by disc diffusion method using Muller Hinton agar (MHA) as a medium (Ushimaru et al., 2007).The sensitivity of the isolated bacterial strains was tested against 10 commonly used antibiotics in triplicate.All the media plates were incubated for 24 hours at 37°C.

Antibacterial activity of plant extracts
Agar well diffusion method (Obeidat et al., 2012;Janovska et al., 2003) was applied for evaluation of antimicrobial activity of the leaves extracts of F. adenophylla.Each extract (10 mg) was dissolved in 1 mL of dimethyl sulfoxide to get a concentration of 10 mg/mL.On each sterile Muller Hinton agar plat, 100 µL of standard inoculum (0.5 MacFarland turbidity standards, 10 -6 CFU/mL) of each test bacterial strain was spread with the help of sterilized cotton swabs while sterile borer was used for wells preparation.An amount of 100 µL of each extract i.e. ethanolic extract (EE), methanolic extract (ME), ethylacetate extract (EAE), n-hexane extract (nHE) and water extract (WE) was added through micropipette under aseptic conditions into specifically marked wells and then incubated at 37 ± 2°C for 18 hours.Zone of inhibition (ZI) was then measured to the closest point in millimeters (mm) (Bobbarala et al., 2009).The test was performed in triplicate and results were presented as mean ± standard deviation.

Antibacterial activity profile of the plant extracts
The leaves extracts F. adenophylla were tested against the MDR-UTI pathogens using cefoperazone-sulbactam   ethanol, methanol, ethyl acetate, n-hexane and water, was evaluated and their potency was quantitatively measured by the presence or absence of ZI (Table III).
The leaves extract of F. adenophylla showed significant antibacterial activity.All the test organisms were found sensitive to these extracts except Klebsiella which showed resistance to EE.The methanolic, ethanolic and water extracts have greater antibacterial activity than ethyl acetate and n-hexane extract.
The ethanol extract of F. adenophylla showed significant activity against all organisms except Klebsiella.III).

Discussion
The current results of phytochemical screening of all extracts of F. adenophylla have shown the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, steroids, glycosides, anthracene and reducing sugars which are also reported previously (Kanchanapoom et al., 2001;Muhammad et al., 2012).Results obtained from the FTIR spectra of the extract have showed the presence of many compounds including aldehydes and ketones, amines, amides, imides, alcohols, carboxylic acids, esters and ethers, amino acids, methyl substituted benzenes, alkyl azo compounds, cylclopentadienyls, organosulfonates and many other aliphatic, cyclic and aromatic compounds, which are not reported in the previous studies to the best of our knowledge as no FTIR data has been found in the literature about F. adenophylla.
In the present study eight bacterial strains isolated from urine samples of UTI patients were used to test the antibacterial activities of plant extracts.The bacterial strains were found resistant to most of the antibiotics used in culture sensitivity study.According to standard guidelines (CLSI, 2013;NCCLS, 2012), the organisms were classified as MDR strains.The results showed that E. coli was found to be the most resistant bacteria i.e. to 90% antibiotics, Enterobacter to 70%, Klebsiella and Providencia to 60% and MRSA was found resistant to 50% antibiotics among all the tested bacterial strains.
Citrobacter was resistant to 30% antibiotics, Pseudomonas to 20% and Acinetobacter was found resistant to only 10% antibiotics used in the study.The drug resistance pattern of some of these bacterial strains has also been reported by Ishaq et al. (2014) with little variation, from the same area i.e.Peshawar, Pakistan.In Ishaq et al. (2014) study, E. coli was found 78.6% resistant, Pseudomonas was 50%, Klebsiella and Providencia were 85.7%, S. aureus was 50% and Citrobacter was found 92.8% resistant to all the tested antibiotics.The variation found in the resistance profile of this study as compared to our study may be due to the nature of antibiotics used and sample.
All the leaves extracts of F. adenophylla were found to have good antibacterial activity.All the organisms under test were found sensitive to these extracts except Klebsiella which showed resistance to ethanolic extract.The ethanolic, methanolic and water extracts showed more antibacterial activity than n-hexane and ethyl acetate extracts.The EE showed maximum activity against Enterobacter (27.33 mm ZI) whereas it was found completely ineffective against Klebsiella (no ZI).The ME showed highest activity against Enterobacter (34 mm ZI) and lowest activity against Providencia (14.33 mm ZI).
The EAE showed maximum activity against Enterobacter (26.66 mm ZI) while it was found least effective against Klebsiella (12.66 mm ZI).The nHE also showed optimum activity against Enterobacter (28 mm ZI) and showed minimum activity against Klebsiella (14.66 mm ZI).The WE showed maximum activity against Enterobacter (23.33 mm ZI) and minimum activity against Klebsiella (16 mm ZI).Most of the extracts were very effective against Enterobacter while least effective against Klebsiella.The previous work (Muhammad et al., 2012) on F. adenophylla (leaves and seeds) showed significant antimicrobial activity against different strains of bacteria including B. subtilis, S. aureus, S. epidermidis, P. aeruginosa and E. coli.According to Muhammad et al. (2012) study, extracts of F. adenophylla exhibited optimum activity against S. aureus and E. coli but showed no activity against S. epidermidis.In our study all the extracts showed optimum activity against these microorganisms which is in line with the study of Muhammad et al. (2012).Additionally, our study also covers the MDR bacterial strains in place of normal bacterial strains.There is no previous study on F. adenophylla extracts in which MDR strains have been tested.Furthermore, these MDR strains were isolated from the urine samples of UTI patients which give a new dimension to the present study.
In comparison to the antibiotics used in the culture sensitivity, the plants extracts were very active against all of the tested bacterial strains.In the present study, E. coli was resistant to 90% antibiotics but was found sensitive to EE, ME, EAE, nHE and WE.Enterobacter was found resistant to 70% antibiotics but showed sensitivities towards all extracts.Likewise, Klebsiella was found resistant to 60% antibiotics whereas it was found sensitive to ME, EE, nHE and WE.Similarly, Providencia and MRSA were resistant to 60% and 50% antibiotics respectively but found sensitive to all extracts.Citrobacter, Pseudomonas and Acinetobacter were found resistant to 30, 20 and 10% antibiotics respectively while they were found 100% sensitive to all the extracts.

Conclusion
Extracts of F. adenophylla had valuable phytochemical constituents along with significant antibacterial activity against MDR UTI pathogens.Different extracts have different antibacterial activities against MDR bacterial strains which showed the effectiveness of the plant extracts.

Table II Culture sensitivity of bacterial strains
Similarly Acinetobacter (ZI of 15.66 mm) and Providencia (ZI of 15.33 mm) showed approximately similar sensitivity profile to nHE.The WE was more active against Enterobacter with ZI of 23.33 mm followed by MRSA (20 mm ZI).
Enterobacter was found most sensitive with ZI of 34 mm while Providencia was found least sensitive (14.33 mm ZI).Activity of ME in terms of ZI against other species were, ZI of 17.33 mm for E. coli, 19.33 mm for Pseudomonas, 17.66 mm for Klebsiella, 19 mm for both Acinetobacter and MRSA and 17 mm for Citrobacter.Similarly, EAE also showed variable but significant activities against the tested microorganisms.Enterobacter was the most sensitive among all the strains with ZI of 26.66 mm, whereas Klebsiella was the least sensitive specie with ZI of 12.66 mm.Zone of inhibition for other strains were; 18.33 mm for E. coli, 18 mm for Pseudomonas, 15 mm for both Acinetobacter and Citrobacter, 17 mm for Providencia and 16.33 mm for MRSA.The nHE also showed maximum activity against Enterobacter with ZI of 28 mm while Klebsiella was the least sensitive with ZI of 14.66 mm.E. coli was found the second most sensitive organism with ZI of 18.33 mm while Pseudomonas (ZI of 16.33 mm), MRSA and Citrobacter (ZI of 16 mm each) exhibited almost similar sensitivity to the extract.

Table SII IR spectra of methanol extract for functional groups
-C=C-H, C-H bend Alkynes 594 , C-Br mono subst.alkynes, Alkyl halides

Table SIII IR spectra of ethyl acetate extract for functional groups
Cyclopentadienyls, Alkenes661-C=C-H: C-H bend Alkynes