Phytochemical Screening, Antibacterial and Synergistic activity of Dittrichia Viscosa Extracts against Multi-Resistant Pathogenic Bacteria

Ennacerie Fatima-Zahra, Rhazi Filali Fouzia, Ed-Dra Abdelaziz, Zekkouri Badr, Bentayeb Amar Microbiology and Health team, Laboratory chemistry Biology applied to the environment, Faculty of Science University Moulay Ismail B.P. 11201 Zitoune, Meknes, Morocco Physicochemistry of Condensed Matter, Faculty of Sciences, Moulay Ismail University BP 11201 Zitoune, Meknes, Morocco *Correspondence Author: Rhazi Filali Fouzia, Microbiology and Health team, Laboratory chemistry Biology applied to the environment, Faculty of Sciences, University Moulay Ismail B.P. 11201 Zitoune, Meknes, Morocco. E-mail: fouzia.filali@yahoo.fr


Introduction
Bacterial resistance to antibiotics appeared with the introduction of antibiotics as a therapeutic treatment as early as the 1940s. Starting in the 2000s, several alert plans were launched to manage the use of these drugs. These actions were answered by a decrease in consumption until 2010, but which did not last long [1] . This issue of bacterial resistance is related to the massive or inadequate use of antibiotics that may even increase the risk of toxicity to the patient, reveal adverse effects, cause drug interactions and super infections, even lead to death [2]. The search for therapeutic alternatives will therefore be necessary to minimize the damage and limit the spread of this problem. Indeed, a series of recent studies have focused on the research of plant-based products, with an important antibacterial power, or also to combine them with antibiotics in order to amplify their antibacterial activities. Certainly, this study is a contribution to the fight against the emergence of multi-resistant bacterial strains and the search for neo-biomolecules for their eradication.
Our work consists of a phytochemical characterization of the Dittrichia viscosa plant and the evaluation of its antibacterial and synergistic activity of its aqueous and ethanolic extracts against multidrug-resistant pathogenic bacteria.
The harvest of leafy stems of D. viscosa was done manually, in May of 2016, from the edges of Oued Rdoum to the city of Sidi Kacem (Latitude: 34.221 °, Longitude: -5.707) in Morocco. The species has been identified at the Plant Biotechnology and Molecular Biology Laboratory of the Faculty of Science of Moulay Ismail University, Meknes [4]. This plant material was sorted and dried in the shade at room temperature. After drying, electric grinding is performed to obtain a fine powder which is used for the preparation of the extracts.

Strains bacterial tested
The evaluation of the antibacterial activity of D. viscosa extracts was performed against five pathogenic bacteria. Four were clinically isolated from different pathological products of a Private Medical Analysis Laboratory, and Listeria monocytogenes was provided by CNRST's Laboratory of Microbiology and Molecular Biology (LMBM). Their removal and isolation were carried out in accordance with the hygiene standards in force [5] and using the appropriate selective culture media.
The strains tested are:

Aqueous extract
One hundred grams of vegetable of the plant are boiled for 15 minutes in one liter of distilled water. This decoction of 10% cooled was filtered successively on hydrophilic cotton and on Wattman paper N° 1. The filtrate obtained was then put in the oven at 55 °C until complete desiccation. The dilution of the residue in water is made to obtain defined concentrations expressed in mg / ml.

Ethanolic extract
The ethanolic extract is obtained with magnetic stirring at room temperature by maceration of 100 g of the vegetable powder in a volume of 300 ml of ethanol for 24 hours. Then, the extract is filtered using the hydrophilic cotton and then Wattman paper N° 1. The filtrate collected is dried in an oven at 50 ° C for the removal of ethanol and obtaining a dry residue expressed in mg. From this residue different concentrations are prepared by its dilution in dimethyl sulfoxide (DMSO), and they are expressed in mg / ml.

Phytochemical Screening
It is a technique based on a set of physicochemical reactions, allowing determining the different chemical groups contained in a plant organ. This study is essential to understand the activities and effects of plants.
The realization of this phytochemical study was established by reactions in solution in test tubes according to the protocol described by Diallo [6], whose main phytochemical groups highlighted are: alkaloids, polyphenols (flavonoids, anthocyanins, and tannins), saponosides, steroids, sterols and triterpenes.

Study of antibacterial activity
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Preparation of the inoculum
The inoculum is prepared from a bacterial culture of 18-24 hours, grown on agar medium and incubated at 37 °C, taking several colonies of the same morphology and suspending them in saline with a sterile loop or a cotton swab. To have an inoculum with a density equivalent to Mc Farland Standard 0.5 [5]. 

Determination of the minimum inhibitory concentration (MIC)
MIC is the minimum concentration of the extract that inhibits the growth of 90% of the bacterial population after incubation for 18 to 24 hours at 37 °C. The determination of the MICs of the plant extracts with respect to the bacterial strains is carried out according to the microtitration technique on microplates described by Ennacerie and his collaborators [7,8,9]. After incubation, the bacterial viability indicator, [2, 3, 5] -triphenyl-2H-tetrazolium chloride (TTC), is added to each well. Wells where bacterial growth has occurred show a pink color. The growth controls are prepared in isolated wells containing the culture medium and the bacterial strains tested, without adding the extract. The number of repetitions is three times for each of the tests performed.

Determination of the minimum bactericidal concentration (MBC)
The minimum bactericidal concentration (MBC) is the lowest concentration of substance that leaves at most 0.01% of surviving germs. It is determined by following the protocol cited by Yao and his collaborators [10,11]. Each experiment is performed three times in three successive experiments.
Referring to the CMB / MIC ratio, the antibacterial effect can be judged; if the ratio CMB/MIC<4 the effect is bactericidal, and if CMB / MIC> 4 the effect is bacteriostatic [12,13].

Synergistic antibacterial interactions between extracts and antibiotics: Disk diffusion method
This technique consists in evaluating the effect of the combination of extracts and antibiotics to which the tested strains are resistant. For each strain two antibiotics to which they are resistant are tested. The test is carried out on a solid medium according to the protocol described by Ennacerie and his collaborators [9,14,15]. Regarding the negative test, it is presented by a disk impregnated with distilled water or DMSO according to the type of aqueous or ethanolic extract. The interaction of the antibiotic and the extract can produce four main types of effects: -Indifference: the activity of the extract has no influence on the activity of the antibiotic; -Addition: the effect of the association is equal to the sum of the effects produced by each of the agents taken separately; -Synergy: the effect of the association is greater than the sum of the effects produced by each of the agents taken separately; -Antagonism: the effect of the combination is less than the sum of the effects produced by each of the antibiotics taken separately [15].

Phytochemical Screening
The preliminary evaluation of the phytochemical composition of the leafy stems of the D. viscosa plant made it possible to obtain the results summarized in Table 1.

Terpene compounds
Steroids -  Phytochemical screening allowed us to highlight the presence of secondary metabolites in the plant tissues of the studied plant. The detection of these chemical compounds is based on tests of constituent solubilities, precipitation and turbidity reactions, and a specific color change.

Saponosides (Foam index) 100
In fact, Dittrichia viscosa leafy stems contain gallic tannins, flavonoids, saponosides, sterols and triterpenes, and they lack alkaloids, steroids, anthocyanins and leucoanthocyanins. Referring to previous studies conducted by Boumaza in Algeria, we note a similarity in results concerning the presence of tannins, flavonoids, saponosides, sterols and triterpenes, but a difference for anthocyanins that are absent in our case [16]. Other studies have identified the phytochemistry of the aerial part of the same plant, they revealed that it contains flavonoids, sesquiterpene acids and triterpenes esters [17]. According to Cohen et al. [18] D. viscosa leaves also contain phenolic compounds, terpenoids and sesquiterpene lactones. The difference in chemical composition is directly related to climatic and soil factors, as well as the physiological characteristics of the plant.

Antibacterial activity of D. viscosa extracts
The profile of antibiotic resistance bacteria tested (Table 2), revealed a high resistance level for the majority of antibiotics prescribed currently in antibiotherapy [19].  The antibacterial effect of the two aqueous and ethanolic extracts prepared from the leafy stems of D. viscosa on the five strains tested is reported in Table 3.

Antibiotic disc
The results of the antibacterial activity reveal that the MICs of the two aqueous and ethanolic extracts range from 0.858 ± 0.29 to 66.66 ± 0.00 mg / ml with a strong activity for the alcoholic extract. The latter showed a stronger inhibition of bacterial growth against Gram-negative than Gram-positive strains. While the decoction generally has the same lower activity against both types of Gram is a MIC of 33.33 ± 0.00 mg / ml, except for Pseudomonas aeruginosa where the MIC is 66.66 ± 0.00 mg / ml.
Regarding MBC,  The comparison of these results with those of other studies shows a correlation in results with those found by Rhimi and his collaborators [20] whose effect of the ethanolic extract was stronger against gram-type bacteria negative. The sensitivity of the latter is related to the characteristics of their membranes and their permeability to bioactive molecules.
Regarding the effect of the extracts of this plant, based on the MBC / CMI ratio, we notice a difference in the bactericidal power between D. viscosa of the province of Sidi Kacem and that collected by Laghrifi and his collaborators around the city of Fez to Ain Taoujtat in Morocco [21]. They revealed a bactericidal effect against Klebsiella pneumoniae. This difference between the results that it is between the two distinct extracts of the same plant, or the same extract of the same species of two different regions, is explained firstly, by the difference in the chemical composition of the extracts which depends on the solvent used and the extraction technique. Secondly, by the chemical composition and the concentration of substances with antibacterial activity which depend on the edaphic and climatic factors as well as the drying technique. It is also reported that despite the low content of active molecules or their low levels, their synergy may be responsible for a strong antibacterial activity.

Synergistic antibacterial interactions between extracts and antibiotics
The results of the combination of decoction 10% and ethanolic extract of D. viscosa with nine antibiotics against the five strains tested are summarized in Table 4.   The analysis of these results shows that the two types of extract can improve the antibacterial power of antibiotics during their association. However, the explanation of this amplifying effect requires in-depth studies that aim to elucidate the detailed mode of action of the active ingredients of each extract on pathogenic bacteria. However, this component is still poorly illustrated.
Referring to the results of other research, that of Side Larbi and his collaborators [22] treating extracts of D. viscosa, they found a synergistic effect of 77% in the case of the association of the methanolic extract of this species harvested from Algeria, with Gentamicin against E. coli. This synergism between antibiotics and extracts is generally related to the destabilization of the bacterial wall. Indeed, it can be assumed that the components of the extracts facilitate the penetration of antibiotic molecules and thus their access to their intracellular targets.
More particularly, according to Esimone et al. [23] polyphenols coupled with β-lactams can enhance antibacterial activity by disrupting cell membrane transpeptidation. As for the combination of imipenem with extracts, it promotes membrane permeability and makes it easier for antibiotic molecules to reach their intracellular target while helping stop bacterial growth. For Macrolides, their association with biologic agents can inhibit bacterial overgrowth by reversible binding to the bacterial ribosome 50s subunit, which can prevent transpeptidation and translocation reactions, resulting in synthesis of RNA-dependent proteins [24,25].
Taking into account the results of the antibacterial activity of D. viscosa extracts against P. aeruginosa resistant to imipenem, it was noted that the aqueous extract was not effective for the inhibition of its growth compared to the ethanol extract and whose explanation can be attributed to the texture of the wall of this bacterium which is provided with an outer membrane rich in phospholipid and forming a barrier impermeable to hydrophobic molecules [26]. In addition, its resistance to imipenem is due to a loss of porin D2 causing a decrease in its permeability, and a production of chromosomal céphalosporinase [27]. The combination of the two extracts with imipenem has produced a restoration of the sensitivity of this strain by an improvement of more than 100% of the antibacterial power of this antibiotic. The inhibitory effect of the growth of this bacterium is most likely due to the creation of porosity and an increase in cell permeability in favor of antibiotic entry. As it can be explained by the breaking of other mechanisms of resistance of this germ. In addition, this surprising result in restoring the weakened efficacy of imipenem on P. aeruginosa, an opportunistic and multidrug-resistant pathogen that is constantly evolving, is a promising and promising addition to antibiotic therapy that is currently therapeutic impasse for certain bacterial strains.
L. monocytogenes, also revealed this synergistic effect with the two antibiotics erythromycin and penicillin whose maximum amplification is evaluated at 471%. The wall of this strain is characterized by a low content of phospholipids hence the ease of contact of the phospholipid bilayer of the bacterial membrane with the hydrophobic compounds. This factor may be responsible for enzymatic system failure, or ion leakage and loss of vital intracellular components [28], hence the amplification of the growth inhibitory power of the antibiotic. Evaluating the effect of extract / antibiotic combinations is a method that can provide therapeutic and socioeconomic solutions. It is an opportunity for valorization and recovery of the effectiveness of antibiotics weakened by the acquisition of multiple resistances. It is also a contribution to reducing the cost of infection medication, which is generally expensive, and the fight against incurable infectious diseases. The interest in results is not limited to antibiotic therapy, but it can be of considerable importance in the pharmaceutical, food and cosmetic industries.

Conclusion
It can be concluded that the two extracts of the D. viscosa medicinal plant are rich in active compounds as they have interesting activities on multiresistant pathogenic bacteria. As well as their combinations with antibiotics of the family Macrolides and beta-lactams amplified their antibacterial effect. So these extracts could be a source of bioactive molecules that can be identified and isolated for use as antibiotic adjuvants against these pathogens.