Evaluation of the Antibacterial Activity of Jatropha multifida sap and Artemisia annua Extract on some Clinical Strains Responsible of Urinary Tract Infections

Objectives: The present study aims to evaluate the antibacterial activity of Jatropha multifida sap and aqueous extract of Artemisia annua leaves on some clinical strains responsible of urinary tract infection. Methods: The bacterial strains were isolated after the cytobacteriological examination of the collected urine. The agar and liquid diffusion methods were used for the sensitivity test and the determination of the Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of plant extracts on isolated strains. Findings: Ten bacterial strains isolated from the urine samples were collected. Isolated bacteria exhibited various resistant profiles. As for the antibacterial activity evaluated on the leaves and sap collected, the aqueous extract of the leaves of Artemisia annua had no effect on the strains studied. The MICs and CMBs of Jatropha multifida sap vary according to the bacterial strains studied. Thus, the sap of Jatropha multifida has a bactericidal activity on all strains studied with the exception of the strain of Escherichia coli on which its action is more moderate. At the end of the work, the most isolated bacterial species are respectively Citrobacter freundii (40%), Staphyloccocus aureus (30%) and Klebsiella pneumoniae (20%). All isolated clinical strains are multiresistant. The aqueous extract of Artemisia annua at 600 mg/ml showed no antimicrobial activity on the strains studied. Improvements: The sap of Jatropha multifida shows a bactericidal activity against the various strains tested and could therefore be an alternative to antibiotics following transformations into improved traditional medicines. *Author for correspondence

burning sensation when urinating (the emission of urine), sometimes by abdominal pain and fever 1 . The most commonly involved bacteria are Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa and Staphylococci in descending order 2 . In more than 80% of urinary tract infections, the causative organism is an intestinal Escherichia coli 3 . As for Staphylococcus aureus, it is often diagnosed at an advanced stage of genitourinary infections (43%) 4 .
Antibiotic therapy is the method used to treat this infection. But nowadays, bacterial resistance to antibiotics becomes recurrent and poses a global public health problem. The overall resistance rate of hospital and community enterobacterial strains to amoxicillin associated with the beta-lactamase, quinolone, fluoroquinolone, sulfamethoxazole + trimethoprim and nitrofuran inhibitors is high 5 .
In Africa and Asia, 80% of the population continues to use traditional medicines rather than so-called modern medicines for primary health care. The widespread use of traditional medicine in developing countries is due to the availability of plant resources used and often affordable. Healthcare professionals and the general public worldwide are struggling with the issues of safety, efficacy, quality, availability, preservation and future development of this type of health care 6 . Thus, many medicinal plants are used in traditional medicine among which include Jatropha multifida and Artemisia annua. Jatropha multifida with highly recognized medicinal properties is used to treat various diseases in Africa, Asia or Latin America 7 . The antibacterial effect of leaves, stems and roots has been reported in various studies [7][8][9] . The prescription of its juice in the management of oral candidiasis is a common practice among rural residents of western Nigeria. In Chinese medicine, the bark and leaves are used against itchy skin and eczema 10 . Jatropha multifida Linn. is one of the medicinal plants whose sap is widely used for healing wounds in Benin 11 . As for Artemisia annua L. (Asteraceae), has been used for centuries in traditional Asian medicine for the treatment and prevention of fever and chills. Antioxidant, anti-inflammatory and antimicrobial properties of leaf extracts of Artemisia annua have been reported 12 .
It is in order to find a natural alternative, an affordable solution, accessible to all and effective to fight the resistance of the bacteria responsible for urinary tract infections that the present work is initiated. Thus, the general objective of this study was to evaluate the antibacterial activity of extracts of Jatropha multifida and Artemisia annua on some clinical strains incriminated in urinary tract infections.

Plant Material
The plant material consists of the sap of Jatropha multifida Linn which was collected in the commune of Abomey-Calavi. Sampling was done directly in the sterile tubes after cutting branches of Jatropha multifida Linn and then stored at 4°C in the refrigerator.
With regard to Artemisia annua, the dried leaves and stems of the plant packaged and sold commercially by the house of Artemisia were used.

Biological Material
It consists of 45 urine samples of patients collected for two weeks at the Allada Zone Hospital Laboratory, Menontin Area Hospital and Calavi Area Hospital. All of these patients had come to the laboratory for reasons of infection etiology. Two reference strains (Escherichia coli ATCC 2552 and Staphylococcus aureus ATCC 2552) provided by the laboratory of U.R.M.A.Pha were also exploited.

Other Material and Equipment
The usual culture media in bacteriology have been used for culture and isolation of bacteria. Reagents and dyes such as: Kovacs reagent, gentian violet, lugol, 90° alcohol, fuschine solution diluted 1/10, methylene blue, hydrogen peroxide and fresh plasma rabbit were used to identify bacteria.
Vol 12 (36) | September 2019 | www.indjst.org Several equipment were also used namely: The optical microscope, the refrigerator, the oven, the autoclave, the poupinel, the centrifuge, the balance of precision, the bunsen burner and the rack. Sterile Petri dishes, hemolysis tubes and sterile screw tubes, TPHA plates, swabs, antibiotic discs, anaerobiosis jar and gloves.

Determination of the Resistance Profile of Clinical Strains Isolated from Urinary Tract Infections
The prospective study for analytical purposes was conducted over a period of one month from March 18 to April 18, 2019. It took into account the cytobacteriological examination of 45 urine samples collected from patients received during the period collection. The bacteriological manipulations were carried out in four days. The first day was devoted to the reception of biological samples, the identification of samples, the macroscopic examination, the microscopic examination and the cultivation. The reading of the seeded media, the realization of Gram control and isolation for obtaining pure strain followed on the second day. On the third day, the identification at the classic Leminor gallery and the antibiogram were realized then read on the fourth day.

Antibacterial Activity of Jatropha multifida sap and Artemisia annua Leaf Extract on some Clinical Strains Responsible for Urinary Tract Infection
The sap of Jatropha multifida Linn has been directly tested on isolated strains. As for Artemisia annua, the aqueous extract of the leaves and stems of the plant was prepared and tested on isolated strains. The total aqueous extracts were obtained by an adaptation of the method developed by 13 . Fifty (50) grams of powder was macerated in 500 ml of distilled water. The mixture is stirred continuously (Stuart Bioblock Scientific Fisher shaker) for 72 hours at room temperature. The homogenate obtained was filtered three times on hydrophilic cotton and once on Wattman No. 1 paper. This filtrate was then dried at 45°C. in the oven. The powder thus obtained is the total aqueous extract used.

Preparation of Extracts
The aqueous extract of Artemisia annua was taken up in distilled water at a rate of 100 mg per 1 ml. The stock solutions thus concentrated to 100 mg/ml were then autoclaved at 121°C. for 15 minutes. Sterility of stock extract solutions was verified by inoculating aliquots of each solution onto Mueller Hinton medium and incubated at 37°C for 24 hours. The absence of colonies on the Mueller Hinton medium after 48 hours confirmed the sterility of these mother extracts solutions. Regarding Jatropha multifia, sap was used. The concentration of the whole sap of this plant has been estimated at 320 mg/ml according to the work done by 11

Test of Antimicrobial Activity carried out on Agar by the Diffusion Method
A bacterial preculture (1 colony in 5 ml of sterile distilled water) of 18 h was diluted to obtain a turbidity of 0.5 on the McFarland scale (ie 108 CFU/ml). Each inoculum was seeded by swabbing onto petri dishes containing Mueller Hinton agar (CA-SFM, 2012). Using the sterile pasteur pipette tip 6 mm diameter wells were dug. Then using a sterile cone and a micropipette 50 μl of each extract was deposited in previously dug wells. A well containing sterile distilled water served as a negative control. Standard antibiotic discs have also been used to serve as positive controls. The dishes were left for 15 minutes at room temperature for pre-diffusion before being incubated at 37°C for 24 hours. After the incubation period, the dishes were examined to record any zones of inhibition (diameter measured in mm) ( Table 1). All tests were done in duplicate. (Images 1, 2).

Determination of the Minimal Inhibitory Concentration (MIC) by Micro Dilution and the Minimal Bactericidal Concentration (CMB)
An extract stock solution was prepared at the concentration of 100 mg/ml in distilled water. 100 μl of Mueller-Hinton Broth medium (MHB) were placed in each well of the microplate (wells 1 to 8). 100 μl of the extract stock solution were deposited in the first well.
After homogenization by suction-discharge using a micropipette, 200 μl of a solution of extract at 100 mg/ml are obtained. 100 μl of this new solution were taken and mixed with the MHB medium contained in the 2nd well and this series of dilution of reason 2 from wells to wells is continued until the 6th well, from which the 100 μl are discarded. Finally 100 μl of the bacterial suspension were added to each well. The 7th and 8th wells were respectively the positive control and the negative control and contain 100 μl of MHB + 100 μl of bacterial suspension for the positive control and 100 μl of MHB for the negative control. The microplates were covered for 24 hours in an oven at 37°C.
MICs were estimated visually compared to controls. As for the CMB, it was determined after seeding the contents of each well on the MH agar and incubated at 37°C for 24 hours. CMB was the lowest concentration of extract for which there were no bacterial colonies.

Data Processing and Analysis
The collection of information and the analysis of the tables were carried out using the Excel 2013 software. The graphs were made with the Graph Pad software. Descriptive statistics were performed using SPSS 20.ng.

Bacterial Profile of Strains Isolated from Urine Samples
Of 45 samples collected, 10 were positive (22.22%) and 35 negative (77.78%). The majority of the samples came from women (78%), of which 20% were positive and 58% negative. Only 22% of the samples came from men with 2% positive samples versus 20% negative samples. Citrobacter freundii was the most isolated bacterium (40%) followed by Staphylococcus aureus (30%) ( Figure  1). Table 2 shows the resistance profile of the isolated bacterial strains. It shows that all isolated bacterial strains have resistance to several families of antibiotics, most of which exceed three different families of antibiotics. These strains are therefore all multi-resistant (Table 2).

Extraction Yields
The aqueous extract of the leaves and stems of Artemisia annua has a yield of 23.72%. The sterility tests revealed the absence of contamination in the extract and sap of Jatrophamultifida.

Sensitivity Test
The strains tested, at the concentration of 100 mg/ml showed a variable sensitivity to the extracts tested. The inhibition diameters of the extracts varied between 7 and 21 mm. The sap of Jatropha multifida was active on all bacterial strains tested. In contrast to the aqueous extract of Artemisia annua that showed no antimicrobial activity.
Increasing the concentration of the aqueous extract of Artemisia annua had no effect on the isolated strains including the reference strains. Thus, the aqueous extract tested at various concentrations (100, 200, 400 and 800 mg/ml) on the isolated strains and on the reference strains

Strains
Resistance profile

Minimal Inhibitory Concentration, Minimal Bactericidal Concentration and Antibiotic Potency of Jatropha multifida Sap
The Minimum Inhibitory Concentrations (MIC) obtained is variable depending on the types of strains. The lowest MICs were obtained at a concentration of 40 mg. Minimum Bactericidal Concentrations (MBC) also varied with the type of susceptible strains. Antibiotic potency (a.p) was determined using the CMB/MIC ratio. The CMB/MIC ratio of the Jatrophamultifida sap on Klebsiella pneumoniae, Citrobacter freundii, Staphylococcus aureus and Klebsiella oxytoca is less than 4. The Jatropha multifida sap therefore has a bactericidal effect on these strains (

Discussion
The present study evaluated the antibacterial properties of plant extracts of Artemisia annua (leaf and stem), Jatropha multifida (sap) on bacterial species responsible for urinary tract infections. The study found that the most isolated bacteria are respectively Citrobacter freundii (40%) Staphylococcus aureus (30%), Klebsiella pneumoniae (20%), and Klebsiella oxytoca (10%). The resistance profile of the isolated strains shows that they are all multiresistant. The aqueous extract of leaf and stem of Artemisia annua showed no antibacterial activity on the strains tested regardless of the concentration. On the other hand, mainly with a minimum bactericidal concentration of 40 mg/ml, the sap of Jatropha multifida had a bactericidal activity on the majority of the strains tested despite their multi-resistance to the usual antibiotic disks.
The results obtained from the antibacterial activity showed that the 12 strains studied were sensitive to the sap of J. multifida including the two reference strains but to varying degrees. In fact, the growth of most of the strains studied was totally inhibited by the Jatropha multifida sap with a minimum MIC of 20 mg/ml whereas the Escherichia coli strain was only moderately sensitive. Indeed, the flavonoids, tannins and terpenes identified during phytochemical screening have strong antimicrobial activities 14,15 . These compounds were responsible for the antimicrobial activity of the leaf extract of Melastoma malabathricum because of their antibacterial properties 16 .
Other authors have achieved results similar to ours by studying the antimicrobial activity of different parts of Jatropha multifida. Indeed, Jatropha multifida sap from the University of Ibadan in Nigeria has completely inhibited S. aureus, E. coli and other bacteria 17 . In 18 2007, aqueous and ethanolic extracts of J. multifida leaves were active on E. coli and S. aureus.
Similar results were noted in a study on the leaves, stems, roots of Jatropha multifida 19 . Although these results are similar, MICs differ in terms of effect on strains. In Nigeria, a MIC of 0.78 μg/ml for S. aureus was obtained in 2008 with the leaves and roots of the plant (19) 16 mg/ ml, 66 mg/ml and 263 mg/ml were noted respectively for S. aureus, E. coli and K. pneumoniae with the sap of Jatropha multifida in 2014 20 . The difference in sensitivity obtained between the saps of Nigeria and Benin could be related to environmental factors (pedographic, climatic), the strains tested and the method used.
With regard to the aqueous extract of Artemisia annua, these results are contrary to the results obtained during the work carried out on the antibacterial, antioxidant and anti-inflammatory activity of Artemisinin extracted from Artemisia annualinn 21 . These results are explained by the fact that our study took into account only the aqueous extract of this plant and that the strains tested are different. The quality of the extract varies according to the solvent used during extraction 22 . It will be necessary to make other extracts of the plant with different solvent in addition to distilled water to better evaluate the antibacterial properties of this plant.
Medicinal plants are therefore a good alternative to antibiotics facing the multi-resistance bacterial observed today. It is very important to carry out various investigations on the properties necessary for a better qualification of these plants in order to minimize any risk to health.

Conclusion
At the end of this work, we can remember that the most isolated bacteria are enterobacteria including Citrobacter freundii and Staphylococci. All isolated strains show strong resistance to antibiotics. In the light of the results obtained from the antibacterial activity of the extracts, it should be remembered that the Jatropha multifida sap has good and well antibacterial properties on the multi-resistant strains of K. pneumoniae, K. oxytaoca, C. freundii, S. aureus isolated urine samples which is not the case for the aqueous extract of Artemisia annua (leaves and stems) which had no activity on isolated strains and reference strains.
It is therefore necessary to initiate additional in vitro and in vivo studies necessary to expand the database on the antibacterial properties and toxicity of these plants.
The craze for medicinal plants should shift from a mere fad to a research, investment and sustainable development perspective of aromatic and medicinal plants for health and well-being purposes.