IN VITRO ANTIBACTERIAL ACTIVITY OF MEDICINAL PLANTS IN THE CENTRAL NORTH OF MOROCCO: A POSSIBLE SOURCE OF ALTERNATIVE DRUGS AGAINST METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS

Objective: The present study aims the investigation of the antimicrobial potential of medicinal plants selected in the central north of Morocco against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis strain often involved in dermatitis. Methods: Structured interviews were carried out among 91 herbalists and traditional healers through a specific information questionnaire, the in vitro susceptibility of Staphylococcus strains award ethanol extracts was evaluated using the well-diffusion assay, while the agar-microdilution method was used to determinate the minimal inhibitory concentrations (MIC). The total phenolic and flavonoids contents of all tested extracts were also determined. Results: Based on the ethnobotanical survey, a total of 55 plant species belonging to 30 families were mentioned. The Lamiaceae family was the most represented (18.80%) followed by the Apiaceae family (10.90%). Leaves (45.00%) were the favored used part. Decoction method (48.53%) was the most frequently used to prepare remedies that are taken externally (75.00%). Nine of the 17 most selected species have shown an effective antistaphylococcal activity; the most active extracts were Punica granatum and Rhamnus alaternus with MIC values ranging between 0.25 mg/ml and 2.00 mg/ml. Conclusion: The current data confirm the good antistaphylococcal activity of P. granatum and R. alaternus and suggest that these species could constitute a promoter source for antistaphylococcal drugs with deeply studies.


INTRODUCTION
Staphylococcus species are opportunistic bacteria and the most pathogenic species. Strains of Staphylococcus aureus are able to colonize all tissues [1], secrete various enzymes and toxins [2][3][4] and cause various diseases including skin infections [5,6]. Microbial infections are normally treated using antibiotics, but the development of resistant strains award current antibiotics has become increasingly common which constitutes a growing public health problem, especially since these multi-resistant strains are no longer confined to hospital environments but are also found in the community [7,8]. Moreover, the emergence of strains of S. aureus resistant to methicillin (MRSA) makes the bacterium very dangerous and put a therapeutic problem. This resistance has stepped up the use of vancomycin, main antibiotic used to fight the MRSA infections; in 2002 a study showed that some strains of MRSA were also resistant to vancomycin (RVSA) [9]. The multi-resistance is serious; significant efforts must be invested to search new effective anti-MRSA bioactive molecules. Given that 75% of the drugs against infectious diseases are natural products or natural derivatives [10], herbal medications are more used, and a large number of plants possess antimicrobial activity [11][12][13]. In this context, we target the region of Fez-Meknes located in the center of Morocco known by its particular geographical situation very rich in biodiversity to select anti skin infections medicinal plants and to evaluate theirs in vitro actions against Staphylococcus strains and especially MRSA. The primary objective of the current study was to aid the progressive scientific works related to the antimicrobial activity of plants.

Study area
Located in the central North of Morocco ( Fig. 1), partially integrating the plain of Saïss and along siding the mountain ranges of the Rif and the Middle Atlas, Fez-Meknes region covers an area of 40.075 km² corresponding to 5.7% of the national territory [14]. This region regroups the Prefectures of Fez and Meknes and the provinces of Boulemane, El Hajeb, Ifrane, Moulay Yaâcoub, Sefrou, Taounate and Taza [14].
The region of Fez-Meknes has 4.236.892 inhabitants [15], the density is 105.7 inhabitants per km 2 , very high compared to the national average (47.6 hab/km 2 ). Due to its particular strategic position, the region is characterized by three climatic types; (i) a continental climate in the northern part, very hot and very dry in summer and cold and wet in winter. The winds are dry and cold or cold and wet in winter and hot in summer (Chergui), (ii) a cold and humid climate in a mountainous area, very cold and very snowy in winter and temperate in summer, and (iii) a semi-arid climate in the high hills of Boulemane. Winters are very cold and snowy [14]. The current study was undertaken in the prefectures of Fez and Meknes that are most populated (1,150,131 and 835,695 inhabitants, respectively) [15] and the province of Taounate that 90% of its communes are rural [16], the local population of this province resorts commonly to herbal medicine.

Ethnobotanical survey
Skin infections such as Impetigo or Furunculosis (Fig. 2) are specific infections caused by S. aureus, for this reason, they were the basis of a questionnaire among herbalists. The survey was carried out during the year 2017. In addition to personal sociodemographic data of the interviewers, the questionnaire has also included information about the recommended plants, vernacular names, used parts, preparation, and administration mode. In general, the most recommended plants were collected from the field; voucher specimens of each plant were identified by a specialist (from the Scientific Institute in Rabat-Morocco) and then deposited at a herbarium in Laboratory of Microbial Biotechnology in Faculty of Sciences and Techniques in Sidi Mohamed Ben Abdellah University of Fez-Morocco. The frequency index (FI) was calculated for each plant to evaluate the importance of each plant using the following formula: FI=n/N*100 n: Total number of herbalists who listed a particular plant species. N: Total number of interviewed herbalists.

Plants selection and preparation
Certainly, the local population usually uses the decoction mode to prepare remedies. However, our findings in a previous study show that aqueous extracts have not shown any antibacterial activity due to the influence of solvents nature and polarity. For this reason, ethanol extracts of the seventeen most common plants cited by the healers (FI≥8.88%) have been prepared in accordance to the methods described by Elaloui et al. and Yeo et al. with slight modifications [17,18]. The used part of each plant as mentioned by herbalists was grounded to powder, and then 10 g of the powder of each plant was macerated in 100 ml of ethanol under agitation (500 rpm), at room temperature for 6 h. The resulting mixture was filtered through Whatman filter n°1 then evaporated under vacuum. Dried extracts were stored in a refrigerator at 4°C until further use.

Target microorganisms
The prepared ethanolic extracts have been the objective for the antistaphylococcal activity using agar well diffusion method and the minimum inhibitory concentrations (MIC) determination against strains often involved in cutaneous infections including S. aureus ATCC 29213, S. aureus clinical isolate, and Staphylococcus epidermidis ATCC 12228. The antibiogram profile of strains' bacteria was identified at the Laboratory of Bacteriology in Fez-Morocco, and it has shown that both S. aureus strains are methicillin-resistant (Table 1).

Agar well diffusion method
Revivification of bacteria has been performed by subculturing the agar plate surface Luria-Bertani (LB) pre-poured in Petri dishes and incubated at 37°C for 18 to 24 h. The microbial inoculums were obtained from fresh colonies through the direct colony suspension method. Hence, 1-2 colonies were suspended in sterile saline (NaCl 0.9%) and adjusted to 0.5 McFarland scale (10 8 colony-forming unit/ml). Agar well diffusion method as described in Balouiri et al. [19] was used to evaluate the antimicrobial activity. The agar surface was inoculated     spreading bacterial inoculums. After 30 min of the drying process in ambient temperature and inoculums' diffusion, a hole with a diameter of 6 mm was punched aseptically using a tip, then 80 µl of each extract solution (50 mg/ml) were introduced into the wells. Finally, agar plates were incubated for 24 h at 37°C. Distilled water was used as negative control, while ampicillin (100 µg/ml) was used as positive control. After measuring the diameter of inhibition's zones around the well, means were calculated, and then the active extracts were subjected to the determination of the MIC.

Determination of the MIC
The MIC was determined following the agar dilution method described by Balouiri et al. with slight modifications [19]. It involves the incorporation of varying concentrations of extract as an antimicrobial agent into the agar medium before its solidification. Different concentrations of each extract ranging from 50 to 160 mg/ml per factor of (2) were prepared in dimethyl sulfoxide (20%), and 1 ml of each dilution was incorporated in 9 ml of medium culture (sterile and soft LB). The mixture was grounded carefully and distributed into Petri dishes. After medium's solidification, and from a suspension adjusted to 10 5 UFC/ml, volumes of 5 µl were deposited on agar surface as spots. Finally, the dishes were incubated for 24 h at 37°C. A growth control was prepared without plant extracts.

Total phenolic quantification
The quantification of total phenolic has been evaluated using the Folin-Ciocalteu reagent by introducing 1.5 ml of Folin-Ciocalteu reagent (10%) in 200 µl of extract (1 mg/ml Ethanol), the mixture was agitated carefully and allowed to react for 5 min in dark, then followed by adding 1.5 ml of sodium carbonate (5%). After 2 h of incubation in room temperature and in the dark again, values have been read using spectrophotometer visible-ultraviolet (UV) at 750 nm. Under the same conditions, a calibration range was made using gallic acid with different concentrations ranging between 300 µg/ml and 25 µg/ml. The total phenolic content was expressed as µg gallic acid equivalents per mg dry weight of the extract (µg GA eq mg E).

Total flavonoids quantification
The flavonoid content has been determined as described in Bahorun et al. [20]. Technically, 0.5 ml of each extract was mixed with 0.1 ml of aluminum chloride (10%), 0.1 ml of potassium acetate (1 m), and 4.3 ml of distilled water; after a vigorous agitation, the mixture was incubated for 30 min in ambient temperature. DO's values have been read using spectrophotometer visible-UV at 415 nm. Flavonoid content was expressed as µg Quercetin equivalents per mg dry weight of the extract (µg Quer eq mg E) using a calibration range from 25 to 300 µg/ml.

Data analysis
The data collected from the ethnobotanical surveys have been analyzed using Excel software. The other results have been presented as means values±standard deviation, and statistical analyses were performed using analysis of variance by IBM SPSS Statistics 21. Differences at p<0.05 were considered statistically significant.

Ethnobotanical survey
In the present survey, 91 traditional herbalists and healers (n=91) from provinces of Fez (FS), Meknes (MK) and Taounate (TN) were interviewed. 55 plant species distributed in 30 families were mentioned ( Table 2). The most representative family was the Lamiaceae (18.18%) with 10 species, followed by the Apiaceae (10.91%) with 6 species and the Asteraceae (7.27%) with 4 species. Other families have been presented by one or two species.
On the one hand, leaves were the most frequently cited used part to prepare remedies with 45%, followed by roots or whole plant with 12.5% each, then flowers (11.25%). The remedies were prepared  In the other side, I. viscosa leaves extract has exhibited the same effect with with MIC of 4.00 mg/ml against the three strains which were the same for O. majorana expect for S. epidermidis that its MIC was superior to 16.00 mg/ml. O. europea has also inhibited the growth of S. aureus clinical isolate at 4.00 mg/ml, but it was effective against S. aureus reference at MIC of 08.00 mg/ml and more than 16.00 mg/ml for S. epidermidis. In addition, the MIC values of the other active plant extracts were ranging from 08.00 mg/ml to upper than 16.00 mg/ml against the tested strains.

Quantitative phytochemical assays
The total phenolic and total flavonoids contents of the 17 extracts were presented in Table 5. As can be noted from this

DISCUSSION
The current survey realized in three areas in the central north of Morocco aimed the identification of plants used in the treatment of skin infections, the in vitro evaluation of the most recommended plants against Staphylococcus strains often involved in dermatitis and the analysis of phytochemical compounds that could be responsible for skin care.    18%). This family has been demonstrated to have antibacterial and antifungal activities against skin infections [21]. Furthermore, several pharmacological properties have been attributed to this family due to its richness in active bio-molecules. Admittedly there has been a qualitative and quantitative difference in chemical composition of species belonging to the Lamiaceae family or else the same genus [22], but this family stills generally rich in polyphenols, saponins, irroides, alkaloids, anthocyanins, and aldehydes [23]. Those compounds possess broad-spectrum antimicrobials [24].
The obtained results have also revealed that (i) the leaves were the most used part (45%) which accords other studies [24]. This extensive use could be explained by the abundance of phytochemical compounds in leaves which are the synthesis site of vegetal secondary metabolites [25]; (ii) decoction mode was the most recommended (48.53%). Many investigations concerning the plants' uses in traditional medicine have highlighted the preponderance of decoction method to prepare remedies [26,27] which appears to have a number of advantages like the extraction of the maximum of herbal substances that are soluble in boiling water which makes them easily absorbed and perceived by human body [28]; (iii) external administration route was in the first ring (75.00%) including flushing and cataplasm depending on the patient preference. This is may be explained by the fact that both of methods could be fast and efficient. The internal use of medicinal plants consisted, for example, tract digestive disease, stomachache, or rheumatism pain. However, skin infections need the use of external remedies which is in agreement with literature [29,30].
Nine of the most recommended plant species reported in the current survey were found to be efficient against the three studied Staphylococcus strains.  [32] reported that the aqueous extract of C. oxyacantha leaves has revealed antistaphylococcal activity against S. aureus and S. epidermidis.
A number of studies have also been reported to confirm that P. granatum is effective in skin curing and protection. The extract of P. granatum has been mentioned as one of the ingredients in skin care formulations of an interesting invention [33]. This plant species has been also acclaimed to have a protective effect award Ultraviolet-Irradiated Human Skin Fibroblasts that could cause serious skin disorders [34,35]. The antistaphylococcal activity of P. granatum has been also demonstrated; a previous study has shown that the ethanolic extract prepared from P. granatum pericarp was effective against S. aureus and S. epidermidis [36].
The antibacterial activity of R. alaternus has been also evaluated against pathogenic bacteria; the leaves' extract was efficient against S. aureus [37]. Other in vitro investigations have also confirmed the antibacterial activity (especially against staphylococcus genus) of the extract prepared from the leaves of I. viscosa [38], O. europea [39], O. majorana [40], and the roots of R. tinctorium [41]. Among A. herba alba, it has proven its medicinal use for centuries as an antimicrobial agent [42].
However, our findings have noticed that eight of the most recommended plants have not shown any anti-staphylococcal potent, these extracts may have other biological activities against other parasitic or fungal pathogenic microorganisms that cause dermatitis, or else bacterial genus other than Staphylococcus. Another explanation may justify this result, it was reported in a recent study that not all the preparations could be useful to cure dermatitis. The skin defense is based on the efficacy of the chemical deactivation process through the enzymology mechanisms by broking down the inactive xenobiotics into a more polar inactive metabolite. This is may have place by forming functional groups such as -OH, -NH2, and -SH [43]. Moreover, in our study, the phytochemical analysis could also give an idea about the plant species effectiveness. It was reported that plants containing high contents of phytochemical compounds such as polyphenols and flavonoids have been considered as useful ingredients in skin cosmetic preparations [44]. Many research groups have explained the effectiveness of total phenols and flavonoids by direct action against germs or through the suppression of microbial virulence factors. For instance, it was reported that flavonoids can inhibit some of the bacterial virulence factors, including quorum-sensing signal receptors, enzymes, and toxins that are necessary for bacteria growth and metabolism [45]. Furthermore, it has been documented that the antibacterial activity of different groups of flavonoids can be attributable to numerous mechanisms such as the inhibition of energy metabolism of bacteria, the inhibition of nucleic acid synthesis, and the inhibition of cytoplasmic membrane function [46]. In the case of the Staphylococcus genus, it was demonstrated that flavonoids have an aggregatory effect on whole bacterial cells [47]. In our study, the active plants were rich in total phenols and total flavonoids which may explain their effectiveness against the target Staphylococcus strains. However, we have demonstrated the antistaphylococcal effect of B. hispanica despite the small phytochemical amounts that it contains. Based on an investigation conducted by Musumeci et al. [48], the biological effect of this species could be contributed to the presence of 5'-methoxyhydnocarpine-D and pheophorbide (chlorophyll decomposition products) synthesized by berberine containing in this genus, these substances have no antimicrobial activity, but they are responsible for inhibiting the expression of efflux pumps expression in S. aureus through the extruding of antimicrobial agents from bacterial cells [49].

Zeouk et al.
Numerous causes may justify the variations of total phenolic and flavonoids contents reported in this work. Indeed, the variation of the polyphenolic content of a plant could be influenced by many biotic factors (Plant species, used part, and physiological stage) and abiotic factors (Environment, solvent) which can affect the metabolism of the plant [50].
Based on all results in the present work, complementary studies are necessary to improve and strengthen these current preliminary findings. This is concern bioassay-guided isolation, purification of the bioactive components, in vivo and toxicity assays. Advanced succeeding scientific research could lead to discover novel and cost-effective drugs against Staphylococcus genus and especially methicillin-resistant strains.

CONCLUSION
The ethnobotanical study conducted in Fez, Meknes and Taounat cities belonging to Fez-Meknes region in the central North of Morocco documented 55 plant species belonging to 30 families that were traditionally used by the local populations to cure dermatitis.