Biological Systems: Open Access

One of the most problematic and economically relevant nosocomial pathogens in the world is Methicillin-resistant Staphylococcus aureus (MRSA) [1]. The problem has been intensified in last two decades as multidrug-resistant MRSA has emerged with resistance to a wide range of antibiotics and antimicrobial agents [2]. Several classes of drug-resistant S. aureus have been identified. Penicillin G-resistant S. aureus, which produces penicillinase, was identified in the mid-1940s, and multidrug-resistant (MDR) S. aureus, which is resistant to penicillin G, chloramphenicol, tetracycline, and erythromycin, was identified in the late 1950s. Since 1961, MRSA continues to be a life-threatening MDR bacterium, changing its resistance patterns by acquiring resistance to each new antibiotic used against it. Generally, MRSA is a causative agent of many infectious diseases of the skin, soft tissue, respiratory tract, bones and joints, surgical wounds, urinary tract, and bloodstream. Such infections are challenging to treat because they are resistant to several commonly used antibiotics [3].

According to the World Health Organization, medicinal herbs could be important resources for obtaining new antimicrobial drugs [4]. Medicinal plants are certainly better equiped at metabolic bioengineering of its specific active pharmaceutical ingredients than traditional laboratory approaches. Several studies have shown that herbal extracts commonly used in Folkloric Medicine have high potential to be developed as bactericidal or bacteriostatic agents. Medicinal plant extracts have been used as natural therapeutic alternatives to antibiotics in order to reduce or overcome the continuous rise of antibiotic resistance.

The genus Rhazya belongs to the Apocynaceae family [5] and comprises two species: R. stricta Decne (Decaisne) and R. orientalis (Harmal in Arabic). It is a medicinal plant commonly used to cure various ailments in humans and animals [6,7] in many Asian countries, including Saudi Arabia. R. stricta and its metabolites are used to treat an array of diseases including cancer, skin diseases, hypertension, rheumatism, sore throat, syphilis, helminthiasis, inflammatory conditions, and fever [8,9]. Several studies have shown that different parts of R. stricta contain plentiful phytochemical constituents like alkaloids, flavonoids, triterpenes, and volatile bases [9], which have curative properties toward a wide range of ailments [10]. Studies from our lab have shown that the aqueous extract of R. stricta has potential antimicrobial activities against Neisseria meningitides [11]. Recently, Baeshen et al., [10] reviewed several studies on therapeutic potential of R. stricta’s compounds including non-alkaloids such as oleanolic acid. Oleanolic acid is a pentacyclic triterpenoid phyto-compound which showed potential antimicrobial activity against several Gram-positive pathogens [12] predominantly, methicillin-resistant Staphylococcus aureus (MRSA) [13]. Oleanolic acids had been shown to affect peptidoglycan metabolism and prevent peptidoglycan cross-linkage and thus altered bacterial cell morphology, which led to increased autolysis of pathogenic cells [12].

In earlier studies from our laboratory, a significant amount of work has been done on examining the alkaloid extracts of R stricta, therefore, the aim of this study was to explore the antibacterial activities of organic, non-alkaloid extract derived from R. stricta leaves against MRSA using a 1% agarose well-diffusion method as well as transmission electron microscopy (TEM).

Chemicals for antimicrobial assays

Chloroform (Sigma-Aldrich,USA), Ethanol(Sigma-Aldrich,USA), Glacial acetic acid(Sigma-Aldrich,USA), Dimethyl sulphoxide (Sigma- Aldrich,USA), Phosphotungstic acid (PTA; Sigma-Aldrich,USA). Bacterial culture media were purchased from Himedia (India).

Bacterial strains

All S. aureus strains used in this study were obtained from the clinical microbiology laboratory of King Abdulaziz University Hospital, Jeddah, KSA. The S. aureus isolates were obtained from 3 different patients and one reference strain (ATCC #43300). Strains 27, 31, and 39 were obtained from superficial wound samples of different patients, and strain 03 was obtained from an ATCC source. Strains 27 and 39 were found to be MRSA while strain 31 belongs to non-MRSA (Table 1).

Table 1: Antibiogram of clinical Staphylococcus aureus.

Plant material

Leaves of R. stricta were collected from an area near Bahara village along the Jeddah-Makkah road, KSA. The species was identified and authenticated to the genus and species level by Professor Nabih A. Baeshen (Department Biology, Faculty of Science, King Abdulaziz University, Jeddah, K.S.A). R. stricta is also grown in the green house of Department of Biological Sciences, University of Texas at Austin, TX, USA in the laboratory of Prof. Robert Jansen, one of our collaborator.

Preparation of organic, non-alkaloid extract from R. stricta leaves

The leaves were washed and dried under shade and ground to a fine powder. Five hundred grams of powder was measured and kept in 800 ml absolute ethanol (95%) for 2 days. The mixture was then filtered through Whatmann filter paper and evaporated by a rotary evaporator. Twenty-five grams of solid extract was then taken and added to 200 ml 1M HCl and an equal volume of chloroform. The chloroform layer containing non-alkaloids were separated and stored at 4°C.

Bacterial culture and Agarose well diffusion method

Two to three isolated colonies of each S. aureus strain were taken from chocolate agar, and culture suspensions were prepared in Mueller Hinton Broth. The 0.5 McFarland inoculum (1.5 × 10⁸ CFU/ml) was adjusted between 0.08 and 0.13 using a spectrophotometer at 625 nm optical density [14]. The inoculum was spread on Mueller Hinton agar plates using sterile cotton swabs. Wells were made on the agar plates using 10 mm diameter sterile tips. The base of each well was sealed with molten agarose to prevent the loss of extract due to leakage. Each well was then filled with 50 μl extract. Extract concentrations of 4.9, 6.1, 7.3, and 8.5 mg/ml were prepared with 0.04% methanol and tested for antibacterial activity against the S. aureus strains with 0.04% methanol used as a control. All plates were kept at room temperature for 2 h after inoculation with the bacterial strains and the extract. After that, the plates were incubated at 37°C overnight. Zones of inhibition were then measured in mm by a ruler.

Transmission electron microscopy

All mentioned Staphylococcus aureus strains were investigated by TEM using a negative-staining procedure with PTA [15]. Briefly, one drop of MRSA were applied to carbon-coated, 300-mesh copper grids (Electron Microscopy Sciences, Hatfield, UK), incubated for 2 min, and fixed with a 2% PTA for 2 min. The specimens were examined by TEM using a JEM-1011 microscope (JEOL, Tokyo, Japan) at 80 KV accelerated voltage. All the data were presented as the mean ± standard deviation (mean ± SD) where appropriate.

Determination of antibacterial activity using Agarose well diffusion method

The antibacterial activity of the extract depended on the concentration of the test material as well as on the quality of the solid base media. In this study, we used agarose instead of agar. As the Rhazya extract concentration increased, there was an increase in bactericidal activity of the extract, resulting in the enlarged diameter of the inhibition zone (Table 2; Figures 1A-D).

Table 2: Antibacterial activity of non-alkaloid extract from Rhazya stricta leaves.

Figure 1: Inhibition of bacterial growth using a non-alkaloid extract derived from R. stricta leaves. The non-alkaloid extract derived from R. stricta leaves was a potent inhibitor of different isolates of MRSA obtained from a wound sample (A, B, C) and a standard ATCC # 43300 strain (D) at concentrations of 4.9, 6.1, 7.3, and 8.5 mg/ml.

Evaluation of bacterial cellular alterations by TEM

The morphological alterations in the bacteria after exposure to the extract were analyzed by TEM. The MRSA cells exhibited changes compared with the untreated cells (Figures 2A-C). The coccoid shape of the cells was not maintained, and deformations and indentations on the cell surface were clearly observed. Cells with a rough surface were clearly visible (Figure 2B). Shrinkage of the cytoplasm was also observed (Figure 2C).

Figure 2: The non-alkaloid extract caused changes in MRSA cells observed using electron microscopy. The morphology of treated MRSA cells was altered (B, Bar = 0.5 μm; C, Bar = 0.2 μm) compared with that of control cells (A, Bar = 0.5 μm). The coccoid shape of the cells was not maintained, and cells with a rough surface (B) and shrunken cytoplasm (C) were clearly visible.

In the past century or so, natural products have proven to be an important source of new chemical entities, which are now driving pharmaceutical inventions. Plant extracts have been the main source of crude therapeutic formulations for pharmaceutical companies. Several studies have established the antimicrobial properties of plant products [2,4,15-18]. Our study revealed the activity of non-alkaloid extract of R. stricta against MRSA using growth-inhibition assays and morphological assessments. Microbiological examinations revealed zones of inhibition with diameters depending on the extract concentration. Electron microscopy revealed morphological as well as intracellular alterations after cells were treated with the extract. Our results corroborate previous work done by us and other laboratories [4,16,19] and are in general agreement with the current knowledge on the molecular basis of antimicrobial activity. It is possible, however, non-alkaloid extract contained different compounds and different proportions of compounds. To exploit the full therapeutic potential of R. stricta non-alkaloid extracts and the purified compounds that could be derived from R. stricta extracts, additional in vitro and in vivo pharmacological and toxicological studies are required.

The mechanism of the antibacterial activity of the non-alkaloid extract from R. stricta remains largely unknown, but it is likely to be related to the various phytocompounds, including flavonoids, terpinoids, and tannins, that were reported previously by several researchers [20]. Additionally it has been reported that non-alkalods from other plants also show antimicrobial activities [13,21]. It is possible that nonalkaloid extracts might contain different chemical compounds which provide stable antimicrobial activities as seen here against MRSA. An earlier study by Horiuchi et al., [13], revealed that the oleanolic acid, a pentacyclic triterpenoid isolated from the leaves of Salvia officinalis showed potential antimicrobial activity against vancomycin-resistant enterococci (VRE) [13]. Oleanolic acids had been shown to affect peptidoglycan metabolism and prevent peptidoglycan cross-linkage and thus altered bacterial cell morphology, which led to increased autolysis of pathogenic cells [12]. Oleanolic acid is also known to be present in the leaves of R. stricta which might be responsible for the antimicrobial activity of non-alkaloid fraction.

Although the crude extract showed dose-dependent antibacterial activity, the use of crude extracts from R. stricta to control MRSA pathogens requires further research for economic and therapeutic development. Our findings support the overall value of potential medicinal extracts from R. stricta leaves. Further pharmacological and toxicological studies should be performed to determine the purified fractions and bioactive compounds responsible for the antibacterial activities, which could serve as a useful resource for new antibiotics.

The non-alkaloid extract from the leaves of the medicinal plant R. stricta proved to have antimicrobial activities against MRSA clinical isolates based on TEM and 1% agarose well-diffusion method. Those findings indicate that R. stricta leaves might be a source of new antibacterial compounds for the treatment of MRSA infections.