Antimicrobial Properties of Catha edulis (Miraa) against Select Bacterial and Fungal Pathogens, an in-vitro Experimental Study

Background: The increasing risk of emergence of antimicrobial resistance can be addressed by discovering alternatives to antibiotics such as plant-based botanicals. In the present study, the antimicrobial properties of aqueous and methanolic extracts of Catha edulis (Miraa) were tested on select pathogenic bacteria and fungi. Methods: Antimicrobial susceptibility tests were conducted in-vitro using the agar well diffusion method. The aqueous and methanolic extracts were dissolved in water to form 1000 mg/ml, 100 mg/ml, and 10 mg/ml doses. The antimicrobial susceptibility testing was done in appropriate culture media and conditions. Diameters of zones of inhibition were obtained, their means calculated, and t-tests applied to test signicance of differences between means. Results: The aqueous Miraa extracts at all three concentrations signicantly inhibited the growth of all bacterial pathogens except E. coli but did not have an effect on C. albicans. The largest zones of inhibition for the aqueous extracts were observed at 1000 mg/ml against S. pneumoniae (28.41 mm), S. pyogenes ATCC 19615 (24.27 mm), MRSA (21.86 mm), and S. aureus clinical isolate (20.38 mm). Similarly, the largest zones of inhibition for the methanolic extracts were at 1000 mg/ml against S. pneumoniae clinical isolate (26.75 mm), S. pyogenes ATCC 19615 (25.38 mm), S. aureus clinical isolate (19.71 mm), and MRSA ATCC 43300 (16.38 mm). Conclusions: Crude Miraa extracts have signicant antimicrobial effects in vitro against the tested microorganisms. Further studies on Miraa extracts to identify the active phytochemicals and investigate their therapeutic effects in-vivo in animal models are indicated.


Background
The burden of infectious diseases remains a signi cant threat to health, especially in developing countries. Infections affect a large proportion of the world population. For example, about 55 million people have a urinary tract infection at any given time [1]. Approximately 357 million people get sexually transmitted infections annually [2]. Respiratory, gastrointestinal, and wound infections, as well as bacteremia and sepsis, are also a signi cant cause of morbidity disability, and mortality.
Infectious diseases are still a substantial problem despite the rapidly increasing use of antibiotics.
Between 2000 and 2020, the de ned daily doses (DDD) of antibiotics consumed globally increased from 21.1 billion to 34.8 billion, which translates to a 65% increase [3]. The high rates of use of antibiotics are causing the emergence of antibiotic-resistant bacteria. Besides, synthetic antibiotics are associated with several negative features such as short half-life in vivo, toxicity, and high cost of synthesis [4].
There is a need to reduce the use of antibiotics in response to the problem of antimicrobial resistance (AMR) so that the currently available antibiotics stock may not get depleted [5]. Plant-based botanicals are a viable alternative to antibiotics in efforts to address AMR [6]. Their antimicrobial and chemosensitizer effects can be leveraged to optimize the use of the available antibiotics while reducing the pressure on them [7]. Investigating plants used in traditional medicine to treat infectious diseases for antimicrobial effects can provide evidence of their value in managing infectious diseases.
The varieties of C. edulis in Yemen, South Africa, Saudi Arabia, and Lebanon have been shown to have antibiotic effects against various bacteria in in-vitro studies [8][9][10][11][12]. Fatima et al. [8] tested methanol, dimethyl sulfoxide (DMSO), and water extracts of Catha edulis Forsk in Saudi Arabia against Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Candida albicans clinical isolates. The methanol and DMSO extracts had signi cant zones of inhibition in all the bacteria tested while the aqueous extract was only active against gram positive organisms, particularly Staphylococcus aureus [8] Siddiqui [11] tested methanolic crude extracts of C. edulis purchased from a shop in London against various laboratory-stocked microbes (Escherichia coli, Bacillus magaterium, Brevundimonas diminuta, and Micrococcus luteus) using the antibiotic disc diffusion assay. The researcher established that the extracts had signi cant (breakpoint of 14 mm) antimicrobial effect against all the bacteria tested (zones of inhibition >16 mm for B. magaterium; >19mm for M. luteus) except E. coli (zone of inhibition <11 mm) [11]. The variation in E. coli results in the studies by Fatima et al. [8] and Siddiqui [11] imply that Catha edulis grown in different geographical areas could be having varying antimicrobial effects.
Al-hebshi, Al-haroni, and Skaug [9] evaluated the antimicrobial effect of aqueous extracts of Yemen's C. edulis against organisms comprising oral microbiota. The extracts showed more effect on the gramnegative bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia), which are mainly pathogenic in the mouth, compared to the gram-positive bacteria (Streptococci and Actinomyces), which are mostly the normal ora of the oral cavity [9]. Therefore, Catha edulis could be selectively active against pathogens by sparing normal ora.
Miraa, the common name for Catha edulis varieties cultivated in the Igembe region of Meru County in Kenya, is one of the plants used to treat infectious diseases in traditional medicine. Miraa plant is a dicotyledonous shrub in the Celastraceae family whose twigs are harvested and commonly chewed for recreational purposes [13]. There are C. edulis varieties in other parts of the world including Yemen, Ethiopia, Saudi Arabia, South Africa, and Lebanon. According to an ethnobiology study by Kiunga et al. [14], herbalists in Meru use decoctions of the leaves and roots of Miraa to treat oral, respiratory, diarrheal, and urogenital diseases. Miraa can be a suitable source of plant-based botanicals if its antimicrobial properties are established. Its availability is assured given that it is a cultivatable plant without scarcity challenges faced when wild plants are used as sources [15].
The illnesses that herbalists treat using Miraa could be caused by myriad pathogens. S. aureus is a common cause of respiratory, urinary tract, and gastrointestinal infections [16], which herbalists apply Miraa decoctions to treat. Methicillin-resistant S. aureus (MRSA) is rapidly spreading globally amidst the reducing antibiotic options to treat its infections [17]. Streptococcus pyogenes is a common cause of sore throat, one of the respiratory infections that herbalists treat using Miraa decoctions. Its infections have increased in the last three decades and it is commonly developing resistance to antibiotics [18]. Streptococcus pneumoniae, which causes respiratory infections, is a pathogen against which antibiotic resistance is rapidly emerging due to antibiotic selection pressure [19]. Escherichia coli is a common cause of urinary tract, gastrointestinal, and respiratory infections; it is common in outbreaks [20]. Hence, E. coli is a suitable representative of gram-negative bacteria in studies to identify plants that can be sources of antimicrobial botanicals. Candida albicans is a fungus that causes oropharyngeal and vulvovaginal candidiasis. Its multi-drug resistant strains are rapidly emerging. Thus, it is an appropriate representative of pathogenic fungi in this study [21].
Our comprehensive review of the literature did not nd any study investigating the antimicrobial properties of Miraa, the C. edulis cultivated in Kenya. Studying Miraa to determine its antimicrobial effects was essential to generate information on whether it has antimicrobial effects like the varieties tested in other countries. This article reports the in-vitro antimicrobial effects of aqueous and methanolic crude Miraa extracts against Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and Candida albicans clinical isolates, and Streptococcus pyogenes ATCC 19615 and methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 standard strains.

Aim, Design and Setting
The aim of this study was to investigate the antimicrobial effects of Miraa in efforts to explore it as a potential source of plant-based antimicrobial botanicals. An in-vitro experimental study to test the antimicrobial effects of 1000 mg/ml, 100 mg/ml, and 10 mg/ml of Miraa's aquous and methanolic crude extracts on S. aureus, S. pneumoniae, E. coli, and C. albicans clinical isolates, and S. pyogenes ATCC 19615 and MRSA ATCC 43300 standard strains was conducted. The experiments were done in the Drug Analysis and Research Unit (DARU) laboratories in the University of Nairobi, Kenya.
They were transported in a Cooler Ice Box at 2-8 0 C to the Drug Analysis and Research Unit on the same day and stored at -20 0 C until the day of the extraction. A specimen of the twigs and leaves was deposited in the University of Nairobi's School of Biological Science herbarium. It was assigned voucher number DK2020/001. The twigs and leaves were chopped into small pieces using a scissor and ground using a blender after removing debris. The weight of the ground material was 892.24 g.
Five-hundred grams of the ground material were put in a 5-liter conical ask and 99.8% AR/ACS methanol (meets the standard Macron Fine Chemicals™ grade of analytical reagents and the requirements of the American Chemical Society Committee on Analytical Reagents) added and stirred at room temperature for 24 hours for methanolic extraction. Whatman lter papers with particle retention of >11μm and Welch 0.9 CFM DryFast 2 Head PTFE diaphragm vacuum pump were used to lter the extract. The ltrate in a 250 mL round-bottomed ask was reduced in vacuum using a rotary evaporator and xed using dry ice to form a uniform coating on the walls of the ask. It was then freeze-dried using a Heto PowerDryR LL1500 Freeze Dryer. For the aqueous extraction, 300 g of the ground material was mixed with 900 mL of distilled water in a conical ask and the mixture heated for 40 minutes. It was left overnight. It was then ltered, reduced, xed, and freeze-dried for 24 hours like in the methanolic extraction process.

Microbial Strains
The S. aureus, S. pneumoniae, E. coli, and C. albicans were obtained from the stock of clinical isolates in the University of Nairobi's microbiology laboratory. S. pyogenes ATCC 19615 and MRSA ATCC 43300 were standard organisms obtained from the same laboratory. S. aureus, MRSA, and E. coli were provided while subcultured in nutrient agar. S. pyogenes and S. pneumoniae were provided while subcultured in blood agar. C. albicans was in Sabouraud's dextrose agar (SDA).

Antimicrobial Susceptibility Testing
Agar well diffusion method was used. Colonies from the subcultures were suspended in normal saline to attain an equivalent of McFarland's standard 0.5. Two milliliters of each of S. aureus, MRSA, and E. coli suspensions were added to their respective 200 mL of sterile trypticase soy agar (TSA) in liquid form at 50 0 C. Suspensions of S. pyogenes and S. pneumoniae were similarly inoculated but in TSA with 5% de brinated sheep blood. C. albicans was also inoculated following the same procedure but in SDA. The mixtures of media and suspension of the organisms were poured in 20 mL culture media plates. Eight plates were prepared for S. aureus, eight for E. coli, and six for each of the other four organisms. They were left to cool to room temperature. The plates were divided into two equal sets, one for testing the aqueous extracts and the other for the methanolic extracts.
An 8-mm sterile cork borer was used to punch ve uniformly-spaced wells in each of the culture plates. The wells labeled A, B, C, D, and E were for the negative control (A), the positive control (B), and 1000 mg/ml (C), 100 mg/ml (D), and 10 mg/ml (E) concentrations of the extracts. A hundred microliters of distilled water for the negative control, 50 µL of 0.3mg/ml gentamicin (anti-bacterial) or 0.3 mg/ml nystatin (anti-fungal) for the positive control, and 100 µL of each of the three doses of the aqueous and methanolic extracts were added in their respective wells. The plates were left at room temperature for one hour for the preparations to diffuse. The culture plates with S. aureus, MRSA E. coli, and C. albicans were incubated at room temperature for 18 hours. S. pyogenes and S. pneumoniae were incubated in 5% CO 2 at 37 0 C for 18 hours. Diameters of zones of inhibition were measured using a vernier caliper calibrated to give the measurements in a precision of two decimal points.
Mean diameter of zones of inhibition (mDZOI) was calculated for each concentration of the extracts by obtaining the average of the three or four measurements taken from the replicates of the experiments. A mDZOI of 9.0 mm was used as the breakpoint, where <9 mm was interpreted as resistance (R) and >9 mm as susceptibility (S). The independent t-test was used to compare the mDZOI of each of the three dosages of Miraa extracts to the respective mDZOI of the respective negative control. One-way ANOVA was applied to determine whether at least one of the mDZOI was signi cantly different from the mean DZOI of other concentrations of Miraa extracts and the negative control. Tukey's HSD test was applied when ANOVA detected a signi cant difference so that it would be possible to know where the difference was lying. Paired t-test was used to compared the mDZOI of the aqueous Miraa extracts (aME) and the methanolic (mME). A 95% con dence level was used in all the stages of data analysis. Level of signi cance of 5% and power of 80% were applied in all the analyses.

Extraction Yields
The methanolic extraction process yielded 28.93 g from the 500 g ground material used, which was obtained from the Miraa leaves and twigs shown in gure 1. The yield's weight was 5.8% of the weight of the material used. On the other hand, the aqueous extraction method produced 6.75 g of extract from 300 g of the ground material. Thus, the extract's weight was 2.3% the weight of the material used.
Antibacterial Effects against S. aureus Both aqueous Miraa extracts (aME) and methanol Miraa extracts (mME) signi cantly inhibited the growth of S. aureus at the three doses tested. The mDZOI for the 1000 mg/ml, 100 mg/ml., and 10 mg/ ml concentrations of the aME and the corresponding positive and negative controls were as shown in gure 2.
Independent t-test revealed the existence of a signi cant difference between the mDZOI of the 1000 mg/ml, 100 mg/ml, and 10 mg/ml, and the negative control (p<0.05 in all three comparisons). One-way ANOVA test showed that at least one of the mDZOIs for the three doses was signi cantly different (F=64.235, p=0.0001). Tukey HSD posthoc test identi ed the mDZOI of the 1000 mg/ml as the only one signi cantly different from the other two (p=0.0001) for both tests.
For the mME, each of the three doses had a signi cant antimicrobial effect against S. aureus. The mDZOIs were as shown in gure 3. They were all signi cantly different from the mDZOI of the negative control (p=0.0001 for each of the concentrations). They were also signi cantly different from each other (F=438.44, p=0.0001; p=0.0001 for all sets of comparisons).
A paired t-test revealed that only the 10 mg/ml of the aME was different from the corresponding concentration of the mME (p=0.001). The 1000 mg/ml and 100 mg/ml of aME had no signi cantly different effect compared to their respective concentrations of the mME.

Antibacterial Effect of Miraa against MRSA ATCC 43300
Both aME and mME had antibacterial effects against MRSA. The three concentrations of aME signi cantly inhibited the growth of MRSA. The mDZOIs are as shown in gure 4. They were all signi cantly different from the mDZOI of the negative control (p<0.005 for all the comparisons).
Additionally, each of the three concentrations had an effect that was substantially different from the others (F=400.387, P=0.0001; p=0.0001 for all the sets of comparisons).
Similarly, the three concentrations of mME exerted signi cant inhibition against MRSA. The mDZOIs are shown in gure 5. Each of them had an mDZOI different from the negative control's (p=0.0001). Their effects were signi cantly different from each other's (F=214.884, p=0.0001; p<0.003 for all the sets of comparisons). The aME exerted signi cantly greater effects compared to the mME at all three concentrations (p<0.003 for all of them).

Antibacterial Effects of Miraa Extracts against S. pneumoniae
Both the aME and mME at 1000 mg/ml, 100 mg/ml, and 10 mg/ml substantially inhibited the growth of the S. pneumoniae. For both the aME and mME, there were signi cant differences between the mDZOIs of each of the concentrations of extracts and the respective negative control as shown in gure 6 and gure 7 (p=0.0001 for all comparisons). There were also signi cant differences between the mDZOI of the three doses of aME (F=1885.88, p=0.0001; p=0.0001 for all sets of comparisons).
Similarly, the mDZOI of the three doses of mME were signi cantly different (F=2714.72, P=0.0001; p=0.0001 for all sets of comparisons). A comparison of the corresponding concentrations of aME and mME showed that only the mDZOIs of the 10 mg/ml doses were signi cantly different (p=0.01).

Antibacterial Effect of Miraa Extracts against S. Pyogenes
Both aME and mME signi cantly inhibited the growth of S. pyogenes. The mDZOI of each of the concentrations of aME and mME were as shown in gure 8 and 9 respectively. They were signi cantly different from the mDZOIs of the respective negative controls for both the aME and mME (p=0.0001).
The three doses of the aME had signi cantly different mDZOI (F=1568.6, P=0.0001; p=0.0001 for all sets of comparisons). Similarly, the mME doses had mMDZOIs signi cantly different from each other (F=926.76, p=0.0001; p=0.0001 for all sets of comparisons. Comparison of the aME and mME revealed signi cant differences between the 100 mg/ml (p=0.009) and 10 mg/ml (p=0.013) doses.
Neither the aME nor mME inhibited the growth of E. coli and C. albicans.

Discussion
Few antimicrobial studies [8][9][10][11][12] have been done to investigate the antimicrobial properties of C. edulis. However, none of them indicated studying Miraa, the C. edulis cultivated in Kenya. The studied were conducted using C. edulis in Saudi Arabia [ 8], Yemen [9], United Kingdom [11], South Africa [12], and Lebanon [22]. The study by Al-hebshi (9), reported that different cultivars of C. edulis can have varying antimicrobial effects. Hence, it was critical to test whether Miraa, the C. edulis cultivars grown in Kenya, have antimicrobial effects like the cultivars tested in other countries.
In the current study, the methanolic extraction process was more e cient than aqueous extraction. The yield from the methanolic extraction was 5.8% w/w, which was more than double the 2.3% w/w yield of the aqueous extraction. None of the previous studies has reported comparisons of extraction yields; hence this nding could be novel.
Our ndings on the antibacterial effects of Miraa against S. aureus are consistent with the ethnobiology ndings by Kiunga et al. [14]. S. aureus is involved in multiple infections including the urogenital and gastrointestinal infections allegedly treated using Miraa in traditional medicine [14,16]. The results also agree with the study by Fatima et al. [8] which found both aqueous and methanolic extracts of Saudi Arabia's C.edulis to inhibit the growth of S. aureus clinical isolate at 0.125-1 mg/ml. Further, McGaw et al. [12] found ethanol crude extracts of C. edulis in South Africa to have antimicrobial effects against S. aureus clinical isolate with a minimum inhibitory concentration of 0.012 mg/ml. On the other hand, our ndings contradict those of Al-hebshi's who found no growth inhibition with 1.25-20 mg/ml aqueous C. edulis in Yemen against S. aureus ATCC 6538 [9].
The difference in the activity of aME and mME at 10 mg/ml, where the aME exhibited a stronger effect, could be due to the difference in composition of the extraction yields. Fatima et al. [8] demonstrated that aqueous and methanolic extracts of C. edulis have different constituent compounds. The antimicrobial effect against MRSA observed in the current study further points to the potential value of C. edulis in the search for alternatives to treat S. aureus infections. Possibly, the antimicrobial effect is due to 22bhydroxytingenone and tingenone among other phytochemicals in the plant. Elhag et al. [10] extracted them from C. edulis callus cultures and found them to have antimicrobial effect against S. aureus at a MIC of 0.6 µg/ml.
The results of the current study showing signi cant antimicrobial effects of Miraa against S. pyogenes are consistent with the ndings by Kiunga et al. [14] that Miraa treats sore throat in traditional medicine. S. pyogenes cause sore throats and related sequelae [18]. The ndings are also in agreement with those of Al-hebshi et al. [9], who found aqueous extracts of Yemen's C. edulis to have antimicrobial effects against S. pyogenes clinical isolate, MIC of 10-20 mg/ml. The absence of effect observed by Fatima et al. [8] could be due to the low concentrations they tested, 0.125-1 mg/ml. Al-hebshi et al. [9] also noted that at 1.25 mg/ml, the aqueous extracts only stopped the hemolytic effect but not the growth of S. pyogenes. Therefore, the MIC of aqueous crude extracts of C. edulis against S. pyogenes could be between 1.25 mg/ml and 10 mg/ml.
In the study by Fatima et al. [8], methanolic extracts of C. edulis inhibited S. pyogenes at concentrations that aqueous extracts could not (0.125-1 mg/ml). The superior strength of the methanolic extracts against S. pyogenes was also observed in our study. The antimicrobial effect of the extracts is consistent despite the variability of the source of C. edulis cultivars (Kenya, Yemen, and Saudi Arabia) tested in the various research. The types of S. pyogenes used in the multiple studies are also diverse: standard organism, clinical isolate, and multi-drug resistant clinical isolate in: the current study, the research by Alhebshi et al. [9], and the study by Fatima et al. [8] respectively.
The nding that Miraa extracts have substantial antimicrobial properties against S. pneumoniae is consistent with the folklore assertion that Miraa treats respiratory diseases [14]. S. pneumonia is a common cause of respiratory diseases [23]. This nding could be novel; we could not identify a publication of a previous study that reported the antimicrobial effects of C. edulis against S. pneumoniae. Given the rapidly-developing antimicrobial resistance against S. pneumoniae [19], our ndings are valuable as they may aid the identi cation of sources of plant-based botanicals to use as alternatives to conventional antibiotics in treating S. pneumoniae infections.
Our ndings of the absence of an antimicrobial effect of C. edulis against E. coli agree with the results of the study by Siddiqui et al. [11], in which methanolic extracts did not show inhibitory effects against the growth of E. coli K1 strain RS218 clinical isolate. Similarly, McGaw et al. [12] found ethanol extracts of C. edulis to have no antimicrobial effects against E. coli. Elhag et al. [10] also found no effect when they tested 22b-hydroxytingenone and tingenone compounds extracted from C. edulis callus culture against E. coli. Perhaps the urinary tract infections and gastrointestinal infections allegedly treated using Miraa in traditional medicine are caused by other pathogens such as S. aureus, but not E. coli [14].
In contrast, Fatima et al. [8] found 1 mg/ml of C. edulis extracts to inhibit growth of E. coli clinical isolate. However, the fact that 2.5% methanolic solution was used used to dilute their extracts instead of water as in the other studies may explain the variance in the results. Methanol is known to be antibacterial, but Fatima et al. [8] justi ed its use by indicating that is is not inhibitory at low concentrations.
Our ndings that Miraa extracts have no antimicrobial effects against C. albicans are consistent with the ndings by Al-hebshi et al. [9] and Elhag et al. [10]. It is unlikely that the oral and urogenital diseases treated by Miraa in traditional medicine are caused by C. albicans. Notably, just as in E. coli's scenario, our ndings on C. albicans differ from those of Fatima et al. [8], who found the extracts to inhibit the growth of C. albicans. This research was derailed by a few limitations. First, scarcity of resources did not allow the purchase of standard organisms or characterization of the clinical isolates used. The use of available standard organisms and laboratory-stocked clinical isolates partly addressed the challenge. The inadequacy of resources also limited the number of pathogens included in the study and the varieties of extraction methods used. It was also not possible to use a quantitative method for the determination of MICs. The comparison of the effects of C. edulis from various countries was also done with caution because there is no study showing the variability of the genetic makeups of the C. edulis cultivars grown in various countries across the world.

Conclusion
In conclusion, the increasing emergence of antimicrobial resistance, the slowed-down discovery of new antibiotics, and the continued indiscriminate use of antibiotics pose a serious global challenge. The challenge can be partly addressed by exploring alternatives to conventional antibiotics for use in treating infectious diseases. Plant-based botanicals with antimicrobial properties are a suitable alternative.
Investigating the antimicrobial effects of cultivated plants such as Miraa as potential plant-based botanicals is necessary because they show promise of consistent supply. This study showed that aqueous and methanolic crude Miraa extracts have antimicrobial effects against S. aureus, MRSA, S. pyogenes, and S. pneumoniae but not against E. coli and C. albicans. The revealed in-vitro antimicrobial effects provide the basis for testing the crude extracts in in vivo animal studies to help predict whether the crude extracts can have the observed antimicrobial effects in humans. Future studies should also identify the speci c compounds in Miraa imparting the antimicrobial effect and assess the resistancemodifying effects of both the crude extracts and the isolated compounds.

Declarations
Ethics approval and consent to participate Permission to study Catha edulis as part of MSc thesis was provided by Kenya Wildlife Service.

Not applicable
Availability of data and materials The dataset used and analyzed during the current study are available from the corresponding author on reasonable request. 21. Dadar M, Tiwari R, Karthik K, Chakraborty S, Shahali Y, Dhama K. Candida albicans -Biology, molecular characterization, pathogenicity, and advances in diagnosis and control -An update. Miraa leaves and twigs before preparation for extraction