CPQ Microbiology (2018) 3:1
Research Article

In Vitro Investigation of Antibacterial Activity of Combined Leaves Extracts of Alstonia scholaris and Fernendoa adenophylla against Urinary Tract Infection Pathogens

Muhammad Saqib Ishaq^*, Aqdas Zoreen, Abdul Rehman, Sidra Farooq & Ghadir Ali

Department of Microbiology & Biotechnology, Abasyn University, Peshawar, KP, Pakistan

*Correspondence to: Dr. Muhammad Saqib Ishaq, Department of Microbiology & Biotechnology, Abasyn University, Peshawar, KP, Pakistan.

Copyright © 2018 Dr. Muhammad Saqib Ishaq, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 11 September 2018
Published: 14 December 2018

Keywords: Urinary Tract Infection; Pathogens; Bacterial Strains

Abstract

In the present research study, the antibacterial activity of combined leaves extracts of Alstonia scholaris and Fernandoa adenophylla against urinary tract pathogens was investigated. A total of ten different bacterial strains (E. coli, Klebsiella spp, Proteus spp, Salmonella spp, Morganella morginii, Pseudomonas aeroginosa, Providencia spp, Staphylococcus aureus and Streptococcus pyogene) were isolated from urine samples. Through disc diffusion method, seven different antibiotics were used against the isolated pathogens to determine their culture sensitivity patterns. Among the seven used antibiotics, Meropenem was most effective and produced the largest ZI 44mm, and was 100% active followed by Azithromycin (90%), Pipracillin (90%), Ciprofloxacin (90%), Amikacin (90%) and Nalidixic acid (80%), while Doxycycline showed average activity and was resistant to five tested bacterial strains. Well diffusion method was used to evaluate the antibacterial activity of leaves extracts of both plants. For this purpose, extracts of both plants were mixed together and five combinations were obtained and were tested against the isolated bacterial strains. Among the five combinations, the combination of (n-hexane extract of F. adenophylla + Water extract of A. scholaris) showed broad-spectrum antibacterial activity against all the tested bacterial strains and produced the highest zone of inhibition 18.5mm against Salmonella spp, whereas the combination of Methyl alcohol extract of A. scholars mixed with n-hexane extract of F. adenophylla, showed no activity. The other three combinations showed average activity against the tested bacterial strains. From the present study it can be concluded that the n-hexane extract obtained from the leaf of F. adenophylla mixed with the water extract obtained from the leaf of A. scholaris plant, was found to have antibacterial activity against UTI causing bacterial pathogens. So further study in this regard will be definitely helpful in producing plant-based antimicrobial compounds to replace synthetic antibiotics and suppress the emergence of antibiotic resistance strains.

Introduction
Urinary tract represents a system that collect, store and release urine and include kidneys, ureters, bladder and urethra. Urinary tract infections (UTIs) are infections caused by microorganisms anywhere in the urinary tract [1]. UTI is the second most common type of infection in the body. There is an estimated 150 million urinary tract infections per year worldwide [2].

UTI mostly occurs in women than men, except the extremes of age, after 50 years of age, the incidence of UTI is almost as high in men as in women, because of obstruction from prostatic hypertrophy. The reason that women mostly get UTI infection is that women have shorter urethra than that of men, so the pathogen easily get access to the bladder and cause infection [3].

UTI is mostly caused by gram negative bacteria. Escherichia coli is the most common cause of UTI, accounts for up to 70% of community acquired and 50% of hospital associated UTIs [4]. Other important uropathogens include Proteus mirabilis, Proteus vulgaris, Klebsiella spp, Pseudomonas aeruginosa, Acinetobacter, Serrati spp, Provedincia spp, Morganella morganii [5] Neisseria spp and Salmonella spp [6]. UTI is also caused by some gram positive bacterial species including Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus pyogene, Streptococcus agalactiae, Enterococcus faecalis and Bacillus subtalis [7].

Increasing drug resistance among bacteria that cause UTIs has made therapy of UTI difficult. Bacteria have the genetic ability to transmit and acquire resistance to drugs [8]. The increasing failure of the chemotherapeutic and antibiotic resistance exhibited by pathogenic microbial infectious agents has led to the screening of several medicinal plants for their potential antimicrobial activity [9].

Plants, a key component of traditional medicine, are an important source of valuable medicines. Plants have been used to treat chronic as well as infectious diseases since long time ago [10]. It is estimated that about over 60% of world human population and 80% in the developing countries depends directly on plants for the medicinal purposes [11]. Over 50% of all modern clinical drugs are of natural product origin [12].

The medicinal importance of plants is due to the presence of chemical compounds in them. Some of the important bioactive compounds are Alkaloids, Glycosides, Triterpenoids, Terpenoids, Flavonids, Phenols, Reducing sugars, Saponins and Tannins. Plants containing such type of compounds are used for Ayurvedic, Unani and other treatments in rural areas [13].

Here the plant source is used as Alstonia scholaris and Fernandoa adenophylla. Both are medicinally important plants. In Thai traditional medicine, the leaves of F. adenophylla are used for the treatment of different skin diseases [14]. The fruits are cooked before eating while the flowers are used as such for vegetable and salad purposes [15]. Whereas Alstoniascholaris traditionally being used in debility, arthritis, impotence, wounds and earache, asthma, leucorrhoea, dog bite, fever, cancer, tumor, jaundice, hepatitis, malaria, skin diseases, diarrhea, leprosy, mental disorders, cardiopathy, helminthiasis, pruritus, agalactia, hypertension, dental or gum problem, abdominal pain after delivery and swelling. It is also used as aphrodisiac, antidote to poison, abortifacient, astringent, thermogenic, cardio tonic, stomachic and expectorant. Reports are available on its ethno veterinary use such as fever in cattle. Ayurvedic use is found in phosphaturia and as a blood purifier [16].

Materials and Methods

Plant Collection and Identification
The leaves of Alstonia scholaris and Fernandoa adenophylla were collected from the main campus of University of Peshawar. The leaves were identified in the department of Botany, University of Peshawar while the plants were processed in Microbiology laboratory, Abasyn University Peshawar.

Preparation of Plant Material
The dried leaves of Alstonia scholaris and Fernandoa adenophylla used as plant sample for extract preparation were first thoroughly washed with water and soaked in detergent to remove the microbial load on the surface of leaves sample. These were then powdered in mortar and pestle and were sieved with fine cloth [17].

Extraction of Plant Material
The fine powder of one hundred gram (100g) of both leaves was soaked in separate flasks containing methanol (ME), ethanol (EE), ethyl acetate (EAE), n-hexane (nHE) and distilled water (DWE) and were vigorously shake until powder was mixed well in solvents [18]. After the completion of soaking period, the supernatant solvent was then collected in a conical flask with the help of coarse cloth as a filter medium. The crude extract collected was then shifted to the rotary evaporator and was processed at 40-50°C to afford a blackish green mass. The extracts thus obtained were stored in labelled sterile bottles and preserved at 4°C until further use for the screening of antimicrobial activity. The dried extract was then dissolved (1mg/ml solvent) in Dimethyl Sulfoxide (DMSO) to get semi solid extract [19].

Specimen Collection
Urine samples were collected from patients attending different hospitals and diagnostic centres of Peshawar. Midstream urine samples were collected in sterile containers. The sterile containers containing samples were immediately transported to the Microbiology laboratory, Abasyn University Peshawar for further processing.

Inoculation of Samples
The collected urine samples were inoculated on nutrient agar plates using sterile swabs and were incubated at 37°C for 24 hours.

Isolation and Identification of Bacterial Strains
After 24 hours of incubation, bacterial growth was noticed on the plates. Ten different bacterial strains were isolated and identified i.e E. coli, Klebsiella spp, Proteus spp, Pseudomonas spp, Salmonella spp, Morganella morgini, Providencia spp, Neisseria gonorrhea, Staphylococcus aureus and Streptococcus pyogene. Isolated bacterial strains were sub-cultured on differential and selective media e.g. Cysteine Lactose Electrolyte-Deficient (CLED) agar, MacConkey agar, Eusine methylene blue (EMB) agar, Blood agar and Salmonella Shigella (SS) agar and were identified according to their specific morphological, staining and biochemical reactions up to genus/species levels wherever applicable [20].

Antibiotic Susceptibility Testing
Before the evaluation of antimicrobial activity of plant extracts, all the microorganisms were first tested against commonly used antibiotics. Disc diffusion method was used for measuring the antimicrobial activity and the media used was the Muller Hinton agar. The sensitivity of seven antibiotics was tested against the previously mentioned 10 bacterial strains and the process was repeated thrice. All the plates were incubated at 37°C for 24h [21].

Evaluation of Antimicrobial Activity of Plant Extracts by Mixing the Leaf Extracts of A. scholaris with the Leaf Extracts of F. adenophylla
Leaves extracts from both plants dissolved in different solvents were mixed together and five combinations of extracts were obtained. These five combinations were shortly named as C1, C2, C3, C4 and C5 for our own ease. These five combinations are as follow;

C1) Ethyl alcohol extract of F. adenophylla was mixed with the n-hexane extract of A. scholaris,

C2) Ethyl acetate extract of F. adenophylla was mixed with Ethyl alcohol extract of A. scholaris,

C3) n-hexane extract of F. adenophylla was mixed with the Water extract of A. scholaris,

C4) Methyl alcohol extract of A. scholars was mixed with n-hexane extract of F. adenophylla,

C5) Methyl alcohol extract of F. adenophylla was mixed with Ethyl acetate extract of A. scholaris.

Well diffusion method was followed for the assessment of antimicrobial activities of methanol, ethanol, ethyl acetate, n-hexane and water extracts of A. scholaris and F. adenophylla [22]. Approximately 1mg of the combined plant extracts was dissolved in 1ml of Dimethyl Sulfoxide (DMSO). Pre autoclaved Muller Hinton agar plates were inoculated with a 10-5 dilution of bacterial cultures, using sterile cotton swabs to achieve uniform growth. Sterile cork borer was used to bore wells in the agar, to test the activity of plant extracts. Sixty (60μl) of each extract i.e., C1, C2, C3, C4, and C5 was introduced through micropipette aseptically into specifically marked wells in the agar plates. All the plates were incubated at 37°C for 24h and the process was repeated three times.

Results and Discussion

Antibiotic Susceptibility Testing
The isolated strains were identified as E. coli, Staphylococcus aureus, S. pyogenes, Klebsiella pneumonia, Pseudomonas aeroginosa, Proteus spp, Morganella morginii, Salmonella spp, Providencia spp and Neisseria gonorrhea.

These isolated bacterial strains were tested for antibiotic sensitivity patterns against seven commonly used antibiotics. The used antibiotics showed varying results (Table 2) against the tested bacterial strains. Among the seven antibiotics, Meropenem was the most effective and produce the largest ZI 44mm against Providencia spp and showed 100% broad-spectrum antibacterial activity against all the tested bacterial strains followed by Azithromycin (90%), Pipracillin (90%), Ciprofloxacin (90%), Amikacin (90%) and Nalidixic acid (80%), while Doxycycline showed average activity and was resistant to five tested bacterial strains. Among the ten tested bacterial strains, E. coli was the most resistant specie (71%) followed by Streptococcus pyogenes (28%), Providencia spp (28%), Staphylococcus aureus (15%), Klebsiella spp (15%), Neisseria gonorrhea (15%) likewise Morganella morginii, Pseudomonas aeruginos, Salmonella spp and Proteus spp were 100% sensitive to all the seven antibiotics.

Table 1: Antibiotic sensitivity pattern of tested bacterial strains

Abbreviations: AZM-Azithromycin, MEM-Meropenem, NA-Nalidixic acid, PIP-Pipracillin, CIP-Ciprofloxacin, AK-Amikacin, DO-Doxycycline, mm-millimeter, (-)-No ZI.

Table 2: Standard zone of inhibition of different antibiotics

MDR strains isolated from urine samples were tested against commercially available antibiotics. E.coli and P.auroginosa were 71% resistant to the tested antibiotics, Klebsiella pneumonia, Proteus spp and S.aureus showed 57% resistance, E.fecalis and S.saprophyticus were 42% resistant while S.marcescens was found to be only 14% resistant [23].

Figure 1: Percentage antibiotic resistance and sensitivity of the tested bacterial strains

Evaluation of Antimicrobial Activity of Combined Plant Extracts
Antimicrobial activity of F. adenophylla (leaves & seeds) against normal strains of bacteria including B. subtilis (ATCC 6633), S. aureus (ATCC 25923), S. epidermidis (ATCC 12228), P. aeruginosa (ATCC 27853) and E. coli (ATCC 8739) [24]. Antimicrobial effects of n-hexane fraction of crude methanolic extract of A. scholaris were tested on Shigella dysentery, Enterobacter cloacae, Enterobacteriaceae bacterium and Serratiamarcescens [25]. In the present study, strain specific inhibition zone diameter of leaves extracts of A. scholaris combined with the leave extract of F. adenophylla dissolved in different organic solvents and distilled water tested against the ten tested bacterial strain are shown in table No.3. Among the five combinations obtained from the combination of both plant leaves extracts, C3 (n-hexane extract of F. adenophylla + Water extract of A. scholaris) showed broad-spectrum antibacterial activity against all the tested bacterial strains and produced the highest zone of inhibition 18.5mm against Salmonella spp, 18mm against Pseudomonas areoginosa and Morganella morginii, 17.5mm against Proteus spp, 16.4mm against N. gonorrhea, 16mm against Providencia spp, 15.3mm against S. aureus, 14.3mm against S. pyogene and 13mm against E. coli. The other four combinations were found to be sensitive to most of the tested bacterial strains. C1 produced a 14mm zone against M. morginii and was sensitive to rest of the tested bacterial strains. C2 produced small zones of 10.3mm against E. coli and 11mm against N. gonorrhea while found to be sensitive to rest of the tested pathogens. C4 showed no activity and was completely sensitive to all tested species. Whereas C5 produced small zones of 11.33mm against E. coli and 10mm against M. morginii.

Table 3: Antibacterial activity of combined plant leaves extracts

Abbreviations: mm-millimeter, (-)- No ZI, C1) Ethyl alcohol extract of F.adenophylla + n-hexane extract of A.scholaris, C2)Ethyl acetate extract of F.adenophylla + Ethyl alcohol extract of A.scholaris, C3) n-hexane extract of F.adenophylla + water extract of A.scholaris, C4) Methyl alcohol extract of A.scholaris + n-hexane extract of F.adenophylla, C5) Methyl alcohol extract of F.adenophylla + Ethyl acetate extract of A.scholaris.

Figure 2: Percentage antibacterial activity of combined plant extracts

C1) Ethyl alcohol extract of F.adenophylla + n-hexane extract of A.scholaris.
C2) Ethyl acetate extract of F.adenophylla + Ethyl alcohol extract of A.scholari.,
C3) n-hexane extract of F.adenophylla + Water extract of A.scholaris.
C4) Methyl alcohol extract of A.scholaris + n-hexane extract of F.adenophylla.
C5) Methyl alcohol extracts of F.adenophylla + Ethyl acetate extract of A.scholaris.

Conclusion
It was concluded that Meropenem antibiotic was most effective while Doxycycline showed average activity and was resistant to five tested bacterial strains used against bacterial pathogens isolated from urinary tract infection samples. Among the five combinations, the combination of (n-hexane extract of F. adenophylla + Water extract of A. scholaris) showed broad-spectrum antibacterial activity against all the tested bacterial strains and produced the highest zone of inhibition 18.5mm against Salmonella spp, whereas the combination of Methyl alcohol extract of A. scholars mixed with n-hexane extract of F. adenophylla, showed no activity. From the present study it can be concluded that the n-hexane extract obtained from the leaf of F. adenophylla mixed with the water extract obtained from the leaf of A. scholaris plant, was found to have antibacterial activity against UTI causing bacterial pathogens. So further study in this regard will be definitely helpful in producing plant-based antimicrobial compounds to replace synthetic antibiotics and suppress the emergence of antibiotic resistance strains.

Bibliography