In this study, the selected medicinal plants were collected from Berbere district in Bale Zone, being guided by the local traditional healers and resident farmers who utilize these traditional medicines. Berbere district is situated between 06°33ʹ N and 06°75ʹ N and 039°95’ E and 040°29ʹ E. It is located about 526 km away from the Ethiopia capital city (Addis Ababa) in the southeast direction, in Bale Zone of Oromia Regional State. The district has 17 peasant associations, where the northern and southern parts are more of highlands and lowlands respectively. Out of the 17 peasant associations, the medicinal plants were collected from seven peasant associations of Berbere district based on the profundity of traditional knowledge generated during the pilot study interview; namely Burkitu (Lippia adoensis), Chekata (Polysphaeria aethiopica), Darasa (Cucumis pustulatus), Gabe Keka (Discopodium penninervium), Gora Heddo (Rumex abyssinicus), Harawa (Cirsium englerianum), and Sirrima (Euphorbia depauperate).

Berbere district is far away from the Zonal city (Bale-Robe) and gets less coverage of infrastructures (in terms of hospital, transport, electrical power supplies, and other social security rendering institutions). Until the conclusion of these fieldworks (plant collection and interviews), there is only one health center in the district and the residents are imposed to use medicinal plants for the treatment of skin diseases, diabetes, Urinary Tract Infections, hepatitis, Sexually Transmitted Diseases, cancer, hypertension, sexual impotence, and as contraceptives. Of which, P. aethiopica, E. depauperata, C. englerianum, L. adoensis, C. pustulatus, D. penninervium, and R. abyssinicus are used by healers for the treatment of bacterial infection such as eczema, gonorrhea, syphilis, pneumonia, scabies, skin infection, and superficial mycosis.

The leaf, bark, and root parts of seven plants, P. aethiopica, E. depauperata, C. englerianum, L. adoensis, C. pustulatus, D. penninervium, and R. abyssinicus were collected and screened for their phytochemical bioactive ingredients and evaluated for antimicrobial effects on common human pathogens (bacteria and fungi). The collected plant materials were handled with standard storage protocols and transported being wrapped by plastic sheets. Authentication of each plant sample was carried out and deposited at the National Herbarium, Department of Biology, College of Natural and Computational Sciences, Addis Ababa University (AAU), Addis Ababa, Ethiopia.

The collected plant parts were washed thoroughly under running tap water and rinsed in distilled water, air-dried at room temperature under shade (23–27°C), and reduced to appropriate size. The powdered plant materials were packed in a plastic bag and kept until extraction. The powdered plant materials were weighed by sensitive digital weighing balance and a total of 1.2 g of powdered aerial were macerated with 80% methanol (250 g in 1500 mL) in an Erlenmeyer flask and also separately soaked in 500ml of distilled water for three days at room temperature (23–27°C). The extraction process was facilitated by occasional shaking. After three days of frequent agitation, the extract was separated from the marc using gauze and the resulting liquid was filtered using Whatman filter paper No. 3 (Whatman Ltd., England). The residue was re-macerated and repeated three times to exhaustively extract the plant material. The filtrates obtained from the successive maceration were concentrated under reduced pressure using a rotary evaporator (Hamato, Japan) followed by a hot air oven (Medit-Medizin Technik, Germany) set at 40°C. Extraction was repeated seven times and the filtrates of all portions were combined in one vessel. The organic solvent was removed by evaporation using a Rotary evaporator at 40°C. After the removal of the organic solvent, the aqueous residue was placed in a lyophilizer until non-polar solvents were removed and the extracts became dried. The extract was further concentrated to dryness by freeze-drying using a lyophilizer (Labfreez, China). After drying, the amount of dry extract obtained was harvested and the dried extract was transferred into airtight bottles, where the resulting dried mass was packed into a glass vial and stored in a desiccator over silica gel and stored in a refrigerator at −4°C until use. The weight of the dry extract was expressed as a percentage of the total mass of dry plant matter to determine the percentage yield [18, 31].

Each extract of methanol and aqueous was tested for the growth of microbes. This was carried out by inoculating 0.5ml of each of them on sterile Mueller Hinton Agar and incubated at 37°C for 18–24 hrs. The fungal growth was tested on Sabouraud Dextrose Agar for 4 days being incubated at 25°C. The plates were observed for growth. The absence of growth in the extracts after incubation indicates sterility and evaluated for antimicrobial activity as indicated in CLSI guidelines [32].

Nutrient Agar, TSY Broth, MacConkey, Muller Hinton Agar (MHA), Muller Hinton Broth (MHB), Blood Agar (BA), Mannitol Salt Agar (MSA), Chocolate Agar, and biochemical reagents for bacteria and Sabouraud Dextrose Agar (SDA) for fungi were obtained from the Department of Medical Microbiology, Immunology and Parasitology, Addis Ababa University and Tikur Anbessa Specialized Hospital (TASH) Bacteriology and Pharmacology Units.

The reference bacterial species; American type cell culture (ATCC) of Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC700603), Streptococcus pyogenes (ATCC 19615), and Staphylococcus aureus (ATCC 25923) and their respective MDR strains, and Trichophyton mentagrophytes (ATCC 18747) and Candida albicans (ATCC 10535) were collected from Bacteriology Unit of the Microbiology Laboratory of TASH. All laboratory works were performed according to CLSI guidelines [33].

The antimicrobial discs used for this research were the products of Liofilchem® Inc. (Liofilchem, Clinical and Industrial Microbiology Company) produced in 2017. The modern antibiotics discs employed were Ciprofloxacin (5μg/disc), Gentamycin (10μg/disc), Cephalothin (30μg/disc), Cefotaxime (5μg), Ceftazidime (10μg), Cefoxitin (30μg), Ceftriaxone (30μg), Amikacin (30μg), Cefuroxime (5μg), Ceftriaxone (30μg), Cloxacillin (30μg), and Augmentin (30μg) for the bacteria while the modern antifungal drug, Ketoconazole (20μg) was used for C. albicans and T. mentagrophytes as per the CLSI guideline protocols [32, 33].

Multidrug-resistant gram-negative and gram-positive bacteria were isolated from three different samples (urine, throat swab, and blood). All bacterial cultures were first grown on 5% blood agar plates at 37°C for 18–24 hours before inoculation onto the MHA. Few colonies (3 to 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 onto 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 impregnated with a range of antimicrobial agents. Dilutions of overnight broth cultures were inoculated onto antibiotic-containing plates to yield final inoculums of approximately 10⁶ CFU per spot according to CLSI protocol guidelines for Enterobacteriaceae. Selected multidrug-resistant, K. pneumoniae, and E. coli were screened for their resistance for more than two different classes of antibiotics following the disk diffusion method protocol (as in the CLSI guidelines) and WHO recommendations [33].

After preliminary screening of plants for their antimicrobial activity, those which revealed potent antimicrobial effects were further tested to determine MIC and MBC against multidrug-resistant gram-negative, gram-positive, and Candida species. It was determined by the MHB broth micro-dilution method. Each of the 96-well microtiter plates was liquated with 50μl of MHB; while 100μl of MHB was added to the 10th well (sterile control). MHB with 5% DMSO added to the 9th well (growth control). About 50μl of each extract initially dissolved in 5% DMSO, which was dispensed into the first well. A serial two-fold dilution was performed by transferring 50μl of the suspension to the subsequent wells until the 8th well as per the protocol and procedure is given by CLSI guidelines and the modified protocol of Wiegand [32]. About 0.5 McFarland broth inoculum was diluted in the ratio of 1:100 and dispensed to 1 to 8th wells to attain the final inoculum size of 5 x 10⁵ CFU per ml [32–34].

Bacterial cell viability and MIC values were determined by turbidity test. 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 was considered as the MBC values. The method adopted for the fungus was the same as that of the bacteria. Instead of nutrient agar, Sabouraud dextrose agar was used. The inoculated medium was incubated at 25°C for C. albicans and T. mentagrophytes. The test was performed in triplicates alongside antibiotics ciprofloxacin (5μg) as a positive control at a concentration of 0.1mg/ml for bacteria. Ketoconazole was used as a positive control at a concentration of 0.3mg/ml for fungi [33, 35].

Standard preliminary phytochemical qualitative analysis of the extract was carried out for the various plant constituents and screened for the presence or absence of biologically active compounds or secondary metabolites using standard procedures. As a result, the major phytochemical constituents identified were alkaloids, anthraquinones, cardiac glycosides, flavonoids, phenolic compounds, saponins, tannins, and terpenoids in 80% methanol extracts of each plant using modified standard procedures [19, 31, 36], as applied in one of the previous research work on combined antibacterial effect of essential oils [35].

The triplicate data reading values of inhibition zones in diameter and concentration values (MIC and MBC) were analyzed using Statistical Packages for Social Sciences (SPSS) software, Window version 22 according to CLSI. Each experimental value was expressed in terms of mean (SD) (mean and standard deviation). Significance in the difference between the two groups was tested by Student t-test, assessed by comparing the corresponding p-value of the test. The P-values < 0.05 were considered significant for the study.

The Research Ethics Committee under the Office of Vice President for Research of Madda Walabu University, the only University in Bale Zone with its main campus in Bale-Robe and the Berbere district administrative office bilaterally approved the fieldwork of this study. On the other hand, as the research was joint collaborative research between Madda Walabu and Kotebe Metropolitan Universities, the Research Ethics Committee of Menelik Medical and Health Science College, Kotebe Metropolitan University in Addis Ababa, Ethiopia granted an ethical approval letter for the study to make use of the patient samples collected at its disposal and other two hospitals in Addis Ababa under its supervision (Ref. No.: IRB/KMU/8974/12). The ethically cleared study, from which we extracted samples from the human subjects (urine, throat swab, and blood samples) was entitled "Molecular characterization of multidrug-resistant microbes among antenatal care attending pregnant women in Addis Ababa" and principally carried out by one of the co-authors of this study. His research team underwent all the standard procedures of ethical clearance, where informed written consent was obtained from all study participants prior to study participation. For those participants who were below the legal consenting age (15.5 to 18 years), they provided written assent, and additionally, their parents/guardians/spouse provided written informed consent. Furthermore, the current study followed all the standard procedures of random representative sample selection (simple random sampling) for the inclusion of unbiased patient samples collected from Addis Ababa into the study subjects, after we had had full permissions from Menelik Tertiary Level Hospital, Kotebe Metropolitan University.

Almost all extracts showed antibacterial activity against two or more multidrug-resistant and reference strains of human pathogenic bacteria. The extract of E. depauperata, R. abyssinicus, and L. adoensis showed the broadest spectrum of action as they inhibited the growth of E. coli, K. pneumoniae, S. aureus, and S. pyogenes with a zone of inhibition in diameter ranging from 21- 29mm (Table 1). The methanolic extract of C. englerianum, L. adoensis, and E. depauperate showed inhibition zone in diameter values of 28mm, 27mm, and 26mm on the MRSA, respectively. The novel antimicrobial activity was observed from the methanolic extract of C. englerianum against the S. pyogenes. It had an inhibition zone of 29mm against both strains collected from the sample and reference strain. In addition, S. aureus had a remarkable inhibition zone (Fig 1). The methanolic extract obtained from E. depauperata revealed appreciable antibacterial activity against MSSA and MRSA with an inhibition zone of 27mm and 26mm, in that order (Table 1).

On the other hand, MDR gram-negative pathogenic bacteria, K. pneumoniae strains were the second susceptible bacteria. It had an inhibition zone in diameter ranging from 19mm to 29mm. Similarly, another MDR gram-negative pathogenic bacteria, E. coli had shown 15mm, 22mm, and 27mm inhibition zone in diameter when the extract obtained from E. depauperata, C. englerianum, and D. penninervium were applied, respectively (Table 1). However, the extract from P. aethiopica failed to show an observable zone of inhibition against the MDR E. coli bacteria tested (Fig 2).

The MIC value of plant extracts against the tested Gram-positive bacteria ranged from 1.0 to 64.0μg/ml. The lowest MIC and MBC values were observed in the case of S. pyogenes, which were the most inactivated bacteria species by most methanolic extracts. Also, these findings divulge remarkable growth inhibition of both MRSA, with MIC and MBC value ranges of 4.0 to 64.0μg/ml (Table 2). The second most inactivated bacteria were clinical isolate MDR and reference strain of K. pneumoniae, with MIC and MBC value ranges of 2.0 to 64.0 μg/ml and 2.0 to 128.0μg/ml, respectively (Table 2). The minimum bactericidal concentration test was directly proportional to its respective MIC value. The bacteria species inhibited at the lower concentration of the plant extracts also showed no growth in subculture media with relatively lower concentration.

This study revealed that the methanolic extracts obtained from C. englerianum, L. adoensis, D. penninervium, and R. abyssinicus demonstrated remarkable antifungal effects against C. albicans and T. mentagrophytes (Table 1). The methanolic extract of R. abyssinicus showed a promising antifungal effect over others. It had a 26mm inhibition zone in diameter and MIC values ranging from 32 to 64μg/ml. Likewise, extracts of D. penninervium and C. englerianum showed inhibitory activity against fungi at MIC/MFC value range of 16 to 512μg/ml, while L. adoensis was found less effective against human pathogenic fungi in a MIC range of 384 to 512μg/ml. No antifungal activity was observed with extracts of P. aethiopica against C. albicans and T. mentagrophytes (Table 2).

The tested medicinal plants showed significant variation in the percentage yield with 80% methanol extraction by maceration. The highest yield was observed in C. englerianum (38%), while the lowest yield was detected in E. depauperata (22%) (Table 3). In terms of the qualitative phytochemical investigation of the medicinal plants, the medicinal plants extract had different phytochemicals constituents such as saponins, tannins, alkaloids, terpenoids, anthraquinones, phenolic compounds, cardiac glycosides, and flavonoids (Table 4). These bioactive compounds are naturally occurring in most extracts and identified to possess bactericidal or fungicidal properties on the tested human pathogens.