Antimicrobial activity of medicinal plants used for urinary tract infections in pastoralist community in Ethiopia

Background Medicinal plants have wide medicament application used to prevent and management of many ailments. These plants are used for primary health care in pastoralist communities who are deprived of modern medical care. They possess extensive therapeutics bioactive coupled with varied chemical structures. However, scientific validation of efficacy and safety of plants used to treat the urinary tract infections haven’t been fully exploited. The aim of this study was to evaluate antimicrobial activity and screening phytochemicals of medicinal plants used to treat urinary tract infections. Methods In-vitro experimental study was carried out to evaluate the antimicrobial effect and screening phytochemical of Rumex abyssinicus, Cucumis pustulatus, Discopodium penninervium, Lippia adoensis, Euphorbia depauperata, and Cirsium englerianum. Against drug resistance microbes. 80% methanol was used for extraction of the plant parts. The susceptibility tests were investigated using disc diffusion and broth micro-dilution methods. Results The majority of tested extracts showed antimicrobial activity on two or more drug-resistant bacteria with MIC value (1.0–128.0 μg/ml) and 9–27 mm inhibition zone in diameter. Extracts obtained from C.englerianum and E. depauperate showed more potent antibacterial activity on MRSA and Enterococcus faecalis with IZ 25 and 27 mm respectively. E. coli and K. pneumoniae were inhibited by those extracts with IZ ranging 9–25 mm and 11–27 mm respectively. E.faecalis and K. pneumoniae were more susceptible bacteria to the respective extracts. R. abyssinicus showed promising antifungal effect with had 21 mm IZ and MIC range 16-32 μg/ml on C.albicans. Alkaloids, flavonoids, phenolic and terpenoid were common phytochemical characterized in majority of screened plants. Conclusion Tested extracts exhibited significant antibacterial and antifungal activity. Hence, further structural elucidation of bioactive that inhibited the growth of microbes aforementioned plants may be used as precursors for the synthesis of new antibiotics in the future.


Background
Over the past several decades urinary tract infection is one of the most common infections. It is mostly caused by gram-negative bacteria namely E.coli P. aeruginosa and P.neumoniae [1]. There are also urinary tract infection (UTI) caused from S. aureus and E. faecalis and C. albicans in immunocompromised patients and pregnant women [2]. On the top of this, emerging and re-emerging of MRSA, extended-spectrum β-lactamases producing organisms (ESBL), vancomycin-resistant enterococci (VRE), and the carbapenem-resistant organisms (CRE/CRP) is a global public health challenge and imposed socioeconomic crisis worldwide [3,4]. WHO report showed overuse and misuse of available antibiotics and lack of discovery of the new antimicrobials drug by the pharmaceutical industry make the crisis more sever and life threaten condition UTI infected patients [5].
On the other hand, many resistance genes/ gene products are speed in bacteria population through efflux, hyper-mutability and plasmid addiction. Such factors attributed for compromised all or majority of the drugs belonging to a given therapeutic [6]. As a result, urinary tract infections caused with resistant organisms have higher morbidity and mortality, are costlier to treat, result in longer hospital stays and place a greater burden on health systems than infections caused by susceptible organisms. Many studies shown that in United States, at least 2 million people acquire serious infections with bacteria that are resistant to one or more of the antibiotics used for the treatment of infections [7]. The total economic cost of antibiotic resistance was estimated as high as $20 billion in direct healthcare and $35 billion in lost productivity per year. If there were no successful efforts to cure them or no new drugs to combat them, the number of deaths per year would be ten million and the cost would increase up to $100 trillion by 2050 [8]. So, searching for innovative antibiotics from natural products should be ultimately an important segment of modern medicine to overcome the various socioeconomic and health impacts caused by multidrug resistant microbes [9].
Searching of medicinal plants used as alternatives or complementary treatment of emerging and re-emerging of multidrug resistance bacteria is undergoing a revitalization because it is available, accessible, affordable and acceptable to the local population [10]. According to WHO report, medicinal plants can potentially meet communities needs and improve access to safe, quality and culturally sensitive primary health care. Which in turn, they can make a significant contribution to essential health services in the prevention and management of communicable diseases caused by drug resistance bacteria [11]. Many studies have shown that each medicinal plant has many phytochemical compounds namely coumarins, flavonoids, phenolic, alkaloids, terpenoids, tannins, essential oils, lectin, polypeptides, and polyacetylenes. The presence of these bioactive compounds can show bactericidal or bacteriostatic effects on multidrug resistance pathogen bacteria and also as precursor for develop antibiotics for treating infectious agents, mainly from urinary tract infections causing bacteria [12][13][14].
Likewise, antibacterial potent of crude extract obtained from Bidens sulphurea, Bidens pilosa, and Tanacetum vulgare demonstrated antibacterial action on S. aureus, E. faecalis, E.coli, and P. aeruginosa isolated from UTI patients and standard bacterial strains ranged from 7.81-125 mg/l and the MBC values ranged from 7.81-500 mg/l [15]. Calotropis gigantean has shown no antibacterial and antifungal activities for n-hexane fractions against pathogenic microbes; however, its ethyl-acetate fractions how potential inhibitory effects on all tested bacteria and fungi except T.rubrum [16].
Another study conducted on the organic extracts of Phragmanthera capitat had comparative antimicrobial activities against ten pathogen bacteria with a MIC of 1.25-5 mg/ml and MBC of 2.5-10 mg/ml and fungi species with a MIC of less than 0.3125 to 1.25 mg/ml [17]. Likewise, the aqueous extracts of Carum copticum, and Albizia adianthifolia showed significant antibacterial effect against multidrug resistance gram negative human pathogen bacteria such as E.coli, Pseudomonas, K. pneumonia, Salmonella and Proteus species [18,19]. Several aqueous and hydro-alcoholic extracts obtained from different plants were screened for their antibacterial effects against human pathogenic bacteria and demonstrated remarkable antibacterial effects on multidrug resistant bacteria including MRSA and ESBL producing bacteria [20][21][22][23][24]. Moreover, a significant number of plants exhibited a broad antibiotic spectrum against multidrug resistant bacteria, which could help to control the problem of the global threat imposed by infections due to multidrug resistant bacteria [25,26].
It has been pointed out that chemical synthesis and the search for natural products from living organisms (marine and higher plants) are the two major sources of new bioactive compounds. However, less than 1% of plants were isolated and characterized for their secondary metabolites and used for pharmacological utilization [27,28]. Traditional medicinal plants are the most valuable sources of new bioactive chemical entities due to their biodiversity coupled with the chemical diversity found within each species [29][30][31][32][33]. In line with this, C.englerianum, E.depauperata, L adoensis, D.penninervium, C.pustulatus and R.abyssinicus are TMPs used by communities for treatment of different infectious agents. Yet, they haven't been thoroughly examined for their antimicrobial potential and phytochemical entities as therapeutic agents for multidrug resistant bacteria and human pathogen fungi. Gadisa

Study design and period
In vitro, an experimental study was carried out to evaluate the antimicrobial effect and screening phytochemical of C.englerianum, E.depauperata, L adoensis, D.penninervium, C.pustulatus and R.abyssinicus against clinically isolated multidrug-resistant bacteria and their reference strains, and pathogen fungi at KMU, MMHSC, Core Laboratory from January to April, 2020.

Medicinal plants selection criteria
Nowadays, communities are deprived of modern medical care and depend on the traditional medicinal plants to treat many human and animals' ailments such as skin diseases, diabetes, urinary tract infection, hepatitis, sexually transmitted diseases, cancer, hypertension, sexual impotence, and contraceptive. Of which, C.englerianum, E.depauperata, L adoensis, D.penninervium, C.pustulatus and R.abyssinicus are commonly used to treat bacterial infection such as eczema, gonorrhea, syphilis, pneumonia, scabies, skin infection, and superficial mycosis.

Plant collection and extraction
The leaf, bark, or root parts of C.englerianum, E.depauperata, L adoensis, D.penninervium, C.pustulatus and R.abyssinicus was collected and screened for their antibacterial and fungi and phytochemical. Authentication of this plant sample was carried out and deposited at the National Herbarium, Department of Biology, Faculty of Natural Science and computational Science, Addis Ababa University. The fresh parts of the plant were collected and washed thoroughly under running tap water and dried in dark shaded areas. The air-dried plant materials (4 kg each) was powdered and extracted at room temperature with 80% methanol and distilled water. Triplicate of 200 g portions of the dried powdered plant was soaked separately in 1000 ml of distilled water, or methanol for 3 days with frequent agitation and the resulting liquid was filtered using Whatman No.1 filter paper (Whatman Ltd., England). Extraction was repeated seven times and the filtrates of all portions were combined in one vessel. The methanol was removed by evaporation using Rota vapor at 40°C. After the removal of the organic solvent, the aqueous residue was placed in lyophilizer until non-polar solvents removed and dried the extracts. The resulting dried mass packed into a glass vial and stored in a desiccator over silica gel until use [13,32].

Sterility test of extracts
Each extract of methanol and aqueous was tested for the growth of bacteria. This was carried out by inoculating 0.5 ml of each of them on sterile Mueller Hinton Agar and incubated at 37°C for 18-24 h. The plates were observed for growth. No growth in the extracts after incubation indicated that they were sterile and evaluated for antibacterial activity as described by CLSI guideline [32].

Culture media and multidrug bacteria Culture media
Nutrient agar, TSY broth, MacConkey, Muller Hinton agar, Muller Hinton Broth, blood agar, mannitol salt agar, chocolate agar and biochemical reagents for bacteria and sabouraud dextrose agar for fungi were obtained from the Departments of Medical Laboratory Science, Bacteriology Unit.

Screening for multidrug resistant bacteria
Multidrug resistant gram negative and gram-positive bacteria were isolated from different samples (urine, throat swab and blood). All bacterial cultures were first grown on 5% blood agar plates at 37°C for 18-24hrs prior to inoculation onto the MHA. Few colonies (3)(4)(5) of similar morphology of the respective bacteria were transferred with a sterile inoculating loop to a liquid medium until adequate growth of turbidity with McFarland in 0.5. Then the bacterial suspension was streaked on MHA plates using a sterile swab in such a way as to ensure thorough coverage of the plates and a uniform thick lawn of growth following incubation. The susceptibilities of clinical isolates were tested by using the MHA contains a range of antimicrobial agents. Dilutions of overnight broth cultures were inoculated onto antibiotic containing plates to yield final inoculums of approximately 10 6 CFU per spot for Enterobacteriaceae. Selected multidrug resistant K. pneumoniae and E. coli were screened for their resistant for more than two different classes of antibiotics following the disk diffusion method as CLSI guideline [13,31,32].

Determination of MIC and MBC values
After preliminary screening of plants for their antimicrobial activity, those revealed potent antimicrobial effects were further tested to determine MIC and MBC against multidrug resistant gram negative, and gram-positive species. It was determined by MHB broth micro-dilution method. Each 96-well microtiter plate was liquated with 50 μl of MHB; 10th well (sterility control) was added with 100 μl of MHB. And the 9th well (growth control) was added with MHB with 5% DMSO. 50 μl of each extract initially dissolved in 5% DMSO was added into the first well. A serial 2-fold dilution was performed by transferring suspension to the subsequent wells up till the 8th well; this procedure was performed by CLSI guideline and modifying Wiegand protocol [25]. 0.5 McFarland broth inoculum was diluted in the ratio of 1: 100 and added into 1st-8th well in achieving the final inoculum size at 5 x 10 5 CFU/ml [28]. Bacterial cell viability and MIC values were determined by observing the turbidity. The lowest concentration of the extract with clear suspension was considered as the MIC values. The lowest concentration of the extract in the post-incubation suspensions which did not harbor any bacterial growth upon spotting on MHA after overnight incubation at 37°C were considered as the MBC values. The test was performed in triplicates alongside antibiotics ciprofloxacin (5 μg) as a positive control at 0.1 mg/ml [27,28]. MIC values were determined by observing the turbidity. The lowest concentration of the extract with clear suspension was considered as the MIC values. The lowest concentration of the extract in the post-incubation suspensions which did not harbor any bacterial growth upon spotting on MHA after overnight incubation at 37°C were considered as the MBC values. The same method as for bacterial tests was adopted for fungi. However, instead of nutrient agar, sabouraud dextrose agar was used. The inoculated medium was incubated at 37°C for C. albicans. The test was performed in triplicates alongside antibiotics ciprofloxacin (5 μg) as a positive control at a concentration of 0.1 mg/ml for bacteria; whereas, ketoconazole was used as a positive control at a concentration of 0.3 mg/ml for fungi [13,31,32].

Phytochemical screening
The presence of the major phytochemical constituents was identified as alkaloids, flavonoids, saponins, phenolic, tannins, terpenoid, and cardiac glycosides in 80% methanol extracts of each plant using modified standard procedures as mentioned our previous work [13,33].

Statistical analysis
The triplicate data were reading values of inhibition zones in diameter and concentration values (MIC & MBC) analyzed using SPSS, version 21 according to CLSI. Each experiment values were expressed as mean ± SD. Statistical significance was determined by student's T-test. Values with p < 0.05 were considered significance.

Phytochemical screening of medicinal plants
This study revealed that air dried powder form of each medicinal plants has varies yield in 80% methanol extraction. The highest yield was observed for C. englerianum (38%) which used to treat gonorrhea, tonsillitis, syphilis, wound amebiasis, malaria, and diarrhea by pastoralist community. However, the lowest yield was observed for E. depauperata (22%) which was mainly used to treat skin rash, ringworm, bloody diarrhea, gastritis, and constipation (Table 1). On the other hand, the qualitative phytochemical investigation showed that these tested medicinal plants have different phytochemicals namely; saponins, tannins, alkaloids, terpenoids, anthraquinones, phenolics, cardiac glycosides and flavonoids (Table 2). Thus, naturally occurring bioactive attribute significant bactericidal action on (E. coli, K. pneumoniae, S. aureus, and E. faecalis) and fungicidal (C. albicans) properties.

Antibacterial effect of extracts on multidrug resistant bacteria
Almost all extracts showed antibacterial activity against two or more of the multidrug resistant and reference strains of human pathogenic bacteria. The extract of E. depauperata, R. abyssinicus and L. adoensis showed promising antibacterial activity as they inhibited the growth of K. pneumoniae, S. aureus and E. faecalis with zone of inhibition ranging 21-27 mm at their 30 μl/disc. The methanolic extract of C. englerianum, L. adoensis  Table 4). The methanolic extract of E. depauperata had strong antibacterial activity on MSSA and MRSA with inhibition zone of 23 mm (Table 3). This plant extract exhibited remarkable inhibition of the growth of MRSA with MIC and MBC values ranging from 4.0-8.0 μg/ml (Table 4).
This study revealed that E. depauperata, C. englerianum and D. penninervium inhibited the growth of E. coli and K.pneumoniae that attributed for 85% urinary tract infections. In line of this, those medicinal plants inhibited the growth of multidrug resistant K. pneumoniae ranging 11-27 mm in diameter. Of which, L.adoensis and D. penninervium are demonstrated promising treatment for multidrug resistant K.pneumoniae with 27 mm and 26 mm inhibition zone respectively (Table 3). On the other hand, E. depauperata, C. englerianum and D. penninervium demonstrated on the multidrug resistant E. coli with 12 mm, 21 mm and 23 mm inhibition zones in diameter respectively ( Table 3). The second most susceptible bacteria were clinical isolates of multidrug resistant and reference strain of K. pneumoniae with MIC and MBC value ranging from 2.0-16.0 μg/ml and 2.0-32.0 μg/ml respectively (Table 4).
Overall, those medicinal plants have bioactive that able to bactericidal property and used as alternative and/or  complementary treatment for urinary tract infection. This is illustrated by minimum bactericidal concentration as parallel tested with modern antibiotics. As a result, thoroughly exploited traditional medicinal plants is the way to overcome health consequence of drug resistance bacteria caused human and animals' ailments in general and urinary tract infections in particular.

Antifungal activity of medicinal plants
This study revealed that methanolic extracts obtained from C. englerianum, L. adoensis, D. penninervium and R. abyssinicus demonstrating remarkable anti-fungal effect against C. albicans (Table 3). Of which, R. abyssinicus showed promising antifungal effect as compared to others. It had 21 mm inhibition zone in diameter and Mean ± SD, NT Not tested, − = No inhibition zone, C Ciprofloxacin, Ce Cefoxitin, F Ketoconazole, MDR < 16 mm, ATCC > 21 mm *P < 0.05, **P < 0.01 compared to cefoxitin treated MRSA. # P < 0.05 compared to modern drug treated E. faecalis, ¥ P < 0.05 compared to modern drug treated K. pneumoniae  MIC values ranging from 16 to 32 μg/ml. In addition, extracts of D. penninervium and C. englerianum showed inhibitory activity against fungi with MIC/MFC values ranging16-512 μg/ml, whereas those of L. adoensis was found to be less potent for C. albicans with MIC ranging 512 μg/ml.

Discussion
Nowadays, majority of the cases from urinary tract infections caused by multidrug resistance microbes constitutes an important therapeutic challenge. They are widely spread and public health concern at global scale worldwide. Resistance mechanism developed by MDR microbes are transfer from one strain to another bacteria through efflux, hyper-mutability and plasmid addiction. As a result, urinary tract infections caused with MRSA, extended-spectrum β-lactamases producing organisms (ESBL), vancomycin-resistant enterococci (VRE), and the carbapenem-resistant organisms (CRE/CRP) have higher morbidity and mortality. They are costlier to treat, result in longer hospital stays and place a greater burden on health systems than infections caused by susceptible organisms. These multidrug resistant microbes have compromised all or majority of currently accessible and affordable antibiotics widely used in the developing countries. In such case, searching for antibiotic from natural resources in general and medicinal plants in particular is timely concern. Those plants have wide application in the treatment of urinary tract infections caused by multidrug resistant bacteria and fungi. This can be demonstrated in that almost all tested medicinal plants had shown promising antimicrobial activity against the selected human pathogenic microbes. This finding substantiates from previous studies that therapeutic agents derived from plants can be used as an important alternative for the treatment of infectious disease [9][10][11][12].
This study revealed that all species of the plants used here had antibacterial activity and also indicated that E. faecalis was the most susceptible species of all tested human pathogenic bacteria. Moreover, most of the extracts have shown remarkable inhibitory effect on MRSA. Likewise, multidrug resistant and its reference strains of K. pnemoniae were found to be the most sensitive gramnegative bacteria against the tested plant extracts. Implausibly, both multidrug resistant and reference strains of E. coli were the most unresponsive strain of all tested bacteria species. These findings agreed with several reports [13,[19][20][21] which demonstrated that gram positive (S. aureus and E. faecalis) bacteria to be more sensitive than gram negative (E. coli and K. pneumonia) bacteria for the plants' phytochemical components being studied. Moreover, many studies have shown that medicinal plants contain different bioactive ingredients that inhibited the growth of human pathogenic bacteria [22]. On the other hand, researchers demonstrated that E. coli has developed multidrug resistance to many currently available and affordable antibiotics [3,23]. Thus, genetic makeup to E. coli to produce multidrug resistance was favorable to impede the antibiotics through reduced permeability and efflux pump [6,23,26]. As a result, searching for antibacterial activity of the secondary metabolites from medicinal plants is a timely concern [12][13][14]. In this regard, medicinal plants can be used to overcome socioeconomic and health impact caused by multidrug resistant bacteria, including MRSA and multidrug resistant gram-negative bacteria such as E. coli and K. pneumoniae [12][13][14].
Regarding the human pathogen fungi, the presence of bioactive compounds in traditional medicinal plants could inhibit the growth of C. albicans. A study done by Shumaia Parvin shown that C. gigantean had inhibitory potential on all tested human pathogenic bacteria and fungi except T. rubrum [16]. Another study conducted on extracts of Carum copticum and Albizia adianthifolia had exhibited good antimicrobial activities against E. coli, Proteus species, P. aeruginosa, P. neumoniae and Salmonellae species [18,19].
Overall, this study showed that among the tested human pathogenic microbes, fungi were found to be more unaffected by many of the tested extracts than bacteria. These findings demonstrated that C. albicans was less susceptible than those tested pathogen bacteria. The presence of different bioactive constituents can inhibit the growth of not only multidrug resistant bacteria but also inhibit growth of fungi. This difference in effect on the growth of C. albicans indicate the presence of antifungal constituents in the crude extracts of each plant. This agrees with previous findings on the antifungal activity of medicinal plants containing different secondary metabolites that inhibited pathogen fungi [16,17,27].
We can affirm that our preliminary findings suggest most endemic medicinal plants contain flavonoid, anthraquinones, alkaloids, tannin, phenolic and saponin ( Table 2). The presence of these bioactive ingredients can inhibit the growth of not only reference strains but also multidrug resistant microbes. In this regard, many studies demonstrated that medicinal plants contain coumarins, flavonoids, phenolic, alkaloids, terpenoids, tannins and polyacetylenes which can be bactericidal, bacteriostatic or produce fungicidal effect on human pathogenic microbes [11,13,29]. Other researchers proposed that this inhibitory activity of secondary metabolites may be due to sequential inhibition of biochemical pathways, inhibition of protein synthesis and disintegration of the outer membrane of the microbes [20,24]. In line with this, many researchers argued that medicinal plants contain diversified secondary metabolites and this makes them a possible alternative for treating infections caused by microbes including multidrug resistant microbes [13,14,17,28]. Therefore, a lot has to be done to explore the potential of such medicinal plants as a possible source of antimicrobials that can be used against multidrug resistant bacteria and fungi.

Conclusions
Those of tested medicinal plants were found to have more antibacterial effect than antifungal activity. They showed promising antimicrobial effect on multidrug resistant bacteria. Hence, identification and isolation of secondary metabolites from those medicinal plants will help pharmaceutical companies to develop modulator or precursor for the synthesis of new novel antibiotics used to treat diseases caused by pathogens including multidrug resistant bacteria.