Synergistic activities of methanol leave extracts of Acalypha wilkesiana, Senna alata, Psidium guajava against selected resistant bacteria isolates

Resistant strains of bacteria has over the years rendered conventional antibiotics ineffective. Consequently, this has resulted to severe infection, prolonged treatment, high cost of treatment and often times death. This study aimed to identify reliable alternative sources of bioactive agents with activity against resistant Staphylococcus aureus, Escherichia coli and Salmonella typhi. Methanol extracts of Acalypha wilkesiana (MEAW), Senna alata (MESA) and Psidium guajava (MEPG) were tested alone and in combination against three clinical isolates. Ciprofloxacin was used as the positive control drug. A combination of Microscopic, macroscopic and molecular protocols was used to identify the test isolates. The antibiotic profiles of the isolates E. coli (E1), S. aureus (S4) and S. typhi (St2) indicated MultiDrug-Resisitant status (MDR). All the extracts demonstrated antibacterial activity against the resistant isolates with zones of inhibition that ranged between 3.1 – 25 mm and minimum inhibitory concentration of 12.5 – 200 mg/ml. Amongst the extracts tested, MESA was found to be the most active extract while MEPG was the least active extract. The combination of the different methanol extracts demonstrated synergistic effects against the test organisms with a fractional inhibitory concentration that ranged between 0.06 – 0.8 mg/ml. The observed antibacterial activity may be linked to the presence of some bioactive components such as phenolic compounds and flavonoids present in the extracts. The results of this study suggest A. wilkesiana, S. alata and P. guajava may represent reliable sources of important bioactive compounds for new drug development.


Introduction
Infections caused by resistant strains of bacteria represent a major public health burden in terms of diseases, deaths, increased expenditure on patient management and infection control measures [1]. These pathogenic bacteria have evolved to gain resistance against several conventional antibiotics due to several reasons such as indiscriminate use of antibiotics [2] thus, the search for newer agents has become more expedient. Traditional medicine systems employs the use of herbal preparations made from selected plants with claimed ethno-medicinal properties in the treatment of various diseases [3]. The use of herbal preparations is a global practice and has remained relevant in the primary health care of the indigenous populations worldwide due to some factors such as cultural significance, accessibility, affordability and safety [4; 5]. The World Health Organization (WHO) estimates that 4 billion people, which is about 80% of the world's population, particularly the underdeveloped countries use herbal medicines for various aspects of primary Health care [6; 7]. Also, many countries such as Nigeria still rely on the curative values of herbs and use of medicinal plants for their healing practices [8].
The search for new antimicrobial agents from plants has become more intense due to the continuous spread of multi drug resistance and its attendant challenge [9]. However, some plants have been reported to cause toxic effects resulting from frequent use without available data about their toxicity profile [3; 10]. This has stirred up concerns by researchers with a view to develop new medicines needed to combat the menace of multi antibiotic resistance.

Sample collection
Prior to collection of specimens, verbal informed consent was obtained from all patients who presented themselves to the hospital with complaint of Urinary tract (UTI) and Typhoid fever infections (TFI).

Cultivation, isolation, purification and identification of test bacteria
These was done according to established standard protocols [16] with slight modifications. Briefly, specimens previously cultured in sterile nutrient broth for 24 hr were subcultured on selected selective media such as MacConkey agar for (E. coli), Mannitol salt agar (S. aureus), and Salmonella shigella agar (S. typhi) at 37℃ for 24 hr. Followed by macroscopic, microscopic (Gram stain) and biochemical (confirmatory) tests such as catalase, coagulase, indole and sugar fermentation tests.

DNA isolation and Polymerase chain reaction (PCR)
DNA isolation and amplification were carried out at the Molecular Research Foundation for Students and Scientist Laboratory, Nnamdi Azikiwe University, Awka using standard protocols. Genomic DNA was extracted using Quick-DNATM Miniprep Plus Kit (Zymo Research) according to recommended protocol. The volume of 12.5μl of One Taq Quick-Load 2X Master Mix with Standard buffer (New England Biolabs Inc.); 0.5μl each of forward and reverse primers; 8.5μl of Nuclease free water and 3μl of DNA template was used to prepare 25μl reaction volume of the PCR cocktail. The reaction was gently mixed and transferred to a thermalcycler. Amplification conditions for the PCR were first initial denaturation for 30secs at 94℃, followed by 35 cycles of denaturation at 94 o C for 20secs, primer annealing at 54℃for 45secs and strand extension at 72℃ for 1 min. Final extension at 72 o C for 5 min on an Eppendorf nexus gradient Mastercycler (Germany). PCR products were separated on a 2% Agarose gel and DNA bands were visualized with Ethidium bromide to confirm.

Sequencing
All the PCR products that were sequenced were first cleansed using Exo-SAP-IT (USB, Affymetrix, USA), and 1 ul of the purified product was used as a template for direct sequencing using Big Dye terminator v. 2.0 cycle sequencer, according to manufacturer's instruction. The sequences obtained were blasted in NCBI similarity check platform for proper identification.

Susceptibility test
The antibiotic profile of the test bacteria were determined using the Kirby-Bauer disk diffusion test. Each of the test bacteria was standardized to 0.5 MacFarland standard. A sterile swab was dipped into the bacteria suspension and used to inoculate each of the isolates on sterile Mueller-Hinton agar (MHA). Using a flamed forcep, each multidisc containing: Meropenum (10 μg), Ofloxacin (5 μg), Ceftriaxone (30 μg), Gentamicin (10 μg), Erythromycin (15 μg), Ciprofloxacin (5 μg), Ampicillin (10 μg), Nalidixic acid (30 μg), Cefuroxime (30 μg), Co-trimaxazole (25 μg) and Chloramphenicol (30 μg) was applied on the inoculated MHA plates. Slight pressure was applied on each multidisc to ensure complete contact of the disc with the agar. Plates were allowed to stand on the bench for 15 min to allow for pre-diffusion then inverted and incubated at 37℃ for 18 -24 hr. After incubation, inhibition halos indicating susceptibility were measured in millimeters and results were interpreted referencing the standards of Clinical and Laboratory standards Institute [17].

Collection and preparation of plant extracts
Leaves of Acalypha wilkesiana, Senna alata, Psidium guajava were collected from the botanical garden of Faculty of Pharmaceutical sciences, University of Port Harcourt Nigeria, the plants were identified by Dr Afuoeri of the Department of Pharmacognosy and Phytotherapy, University of Port Harcourt Nigeria and voucher specimens were deposited in the herbarium. The crude extracts from the different leaves were prepared by the marceration method using methanol as the solvent as proposed by Alabi et al. [18]; Cimanga et al. [19], with slight modifications.

Phytochemical analysis
Gas chromatography-flame ionization detector (GC-FID) instrument was used for the analysis and quantification of phytochemicals present in the methanol leaf extracts of Acalypha wilkesiana (MEAW), Senna alata (MESA), Psidium guajava (MEPG). A sample of each of the three methanol extracts was placed in 100 μl of Pyridine, after which 20 μl was placed into a vial on the Gas Chromatography machine for phytochemical analysis. The GC-FID phytochemical analysis was performed on a BUCK M910 Gas Chromatograph (GC) (BUCK Scientific, USA), which is furnished with a flame ionization detector (FID). A RESTEK 15 meter MXT-1 column (15m x 250 μm x 0.15μm) was used. The initial injector temperature was 220 ℃with spring injection of 2 μl of sample and a linear velocity of 30/cms, Helium 5.0 Psi was the carrier gas with a flow rate of 40 ml/min. The oven was heated to 280 ℃at a rate of 3 ℃ /min. Phytochemicals were determined by the ratio between the area and mass of internal standard and the area of the identified phytochemicals.

Agar well diffusion assay
The sensitivity of all the clinical isolates of E. coli (E1-E10), S. aureus (S1-S10), S. typhi (St1-St10) to the methanol leaf extracts of MEAW, MESA, MEPG was determined using the agar well diffusion method as described by Okezie et al., [20]. Each of the prepared bacteria suspension equivalent to 0.5 McFarland Standard was spread onto sterile Mueller-Hinton agar plates using a sterile swab stick. A sterile 6 mm diameter cork borer was used to bore 6 wells into the agar medium. The wells were then filled with approximately 0.1 ml of the extract ranging between 12.5 to 200 mg/ml. The plates were allowed to stand on the laboratory bench for 1 hour to allow proper diffusion of the extracts into the medium after which the plates were incubated at 37℃for 24 hours, and then observed for zones of inhibition. A meter rule was used to measure the inhibition zones in millimeters. Ciprofloxacin 5 µg/ml was used as a positive control, while 10% DMSO served as negative control. The experiment was conducted in triplicate and the average value was calculated.

Synergistic activity
The Checkerboard assay as described by Okore, [21] was employed for the evaluation and measurement of interactive inhibition of synergy between the extracts. This was carried out against each of the representative working isolates (E. coli (E1), S. aureus (S4), S. typhi (St2). Here, the individual MIC were used in preparing the stock solution of each of the extract after which, the solutions were combined in different ratios, adopting the continuous variation model and then diluted using two folds serial dilution in sterile Pyrex test tubes. An aliquot of 60 µL corresponding to 0.06 mL of each of the serially diluted dilutions was transferred into a corresponding well in a sterile MHA plate previously inoculated with 0.5 McFarland standard of the test organism. The plates were incubated at 37℃ for 18-24 hours. The effects of combination were evaluated by calculating the fractional inhibitory concentration (FIC) of each combination.
The sum of the FICs of both extracts gave the FIC index. This can be expressed as in equation 1.1 thus: Where, A' and B' represent Minimal inhibitory concentrations of extracts A and B having inhibitory effects when acting together, while A" and B" stands for the respective MICs of the extracts. The FIC Index is interpreted as synergism if its value is less than 1.0, additivity if it is equal to 1.0, indifference if more than 1.0 and less than 2, and antagonism if more than 2.0.

Statistical analysis
The results were expressed as mean ± standard deviation. Statistical analysis was carried out using one way analysis of variance (ANOVA) and SPSS 17.0 version statistical software package. The measures were done in triplicate (n = 3).

Results and discussion
A total of (30) isolates comprised of [E. coli (E1-E10), S. aureus (S1-S10), S. typhi (St1-St10)] were used for this study. Each of the test bacteria was isolated on their respective selective medium. The macroscopic observation revealed dark pink, yellow, and black colonies on Macconkey agar (MCA), Mannitol salt agar (MSA), and Salmonella shigella agar (SSA) respectively. Microscopic features of isolates on MCA, MSA and SSA were observed to be Gram negative (slender rods); Gram positive (cocci) and Gram negative (rods) respectively, while the biochemical tests conducted showed indole (+ve), citrate (-ve) for E. coli; catalase and coagulase (+ve) S. aureus; while colonies isolated on SSA were catalase (+ve) and produced H2S. Morphological and biochemical characteristic of the bacteria isolates were further confirmed by the sequence of 18S rRNA gene of each bacteria that gave a sequence similarity of 100 % for each of the isolate to those accessible in the National Center for Biotechnology Information (NCBI) data base (GenBank). The molecular characterization also provided the accession numbers of CP0486431.3, CP046291.1 and CP026939.1 for Staphylococcus aureus 4, Salmonella typhi 2 and Escherichia coli 1 respectively.

Discussion
In this study, the clinical strains used such as S. aureus, E. coli and S. typhi were identified based on their reactions to selected biochemical tests: catalase and coagulase; indole and citrate; and hydrogen sulphide production respectively. The Blast outcome of each of the sequence gave a 100% sequence similarity score and accession number CP048643.1, CP046291.1 and CP026939.1 for S. aureus (S4), S. typhi (St2) and E. coli (E1) respectively.
The sensitivity of each of the test organisms S. aureus (S4), S. typhi (St2) and E. coli (E1) to conventional antibiotics was observed to be multidrug resistance. This was based on their observed resistance to most of the antibiotics to which they were exposed to. Ciprofloxacin was the only antibiotic that inhibited the three test organisms. The combination of the different methanol extracts at specific ratios worked synergistically against the MDR S. aureus (S4), S. typhi (St2) and E. coli (E1). Combination of MEPG and MESA demonstrated synergistic effects against E1 at all the combined ratios. Whereas, a combination of MEPG with MEAW had no synergistic effect against St2. Furthermore, a significant decrease in MIC values was observed in the combinations that produced synergistic effect. The FICI of the various combined methanol extracts that produced synergistic effects ranged between 0.06 -0.8 mg/mL. The results strongly suggest a possible synergistic potential of the bioactive compounds in the methanol extracts of the plants under study. Thus, may be developed into new medicines for treatment against infectious caused by MDR organisms. However, some combinations were observed to be antagonistic. The phytoconstituents that may be responsible for the recorded activities were detected using GCFID. The GCFID chromatogram revealed the presence of important biologically active compounds such as proanthocyanin, naringin, anthocyanin, naringenin, flavan-3-ol, sapogenin, phenol, flavonones, catechin, epicatechin, kaempferol and resveratrol.
Several other biological activities including antibacterial and radical scavenging have been reported for flavonoids such as naringin and naringenin [26], catechin & epicatechin [27]. Also, phenols with antibacterial activities have been identified. Activity against Salmonella typhimurium and MRSA have been reported [29]. While Flavonoids have multiple cellular targets including DNA synthesis and have been reported to have broad spectrum activity [26].

Conclusion
In conclusion, MEAW, MESA and MEPG demonstrated antibacterial activity against the multidrug resistant E. coli, S. aureus and S. typhi tested in this work. Also, the combined effects produced synergistic activity against the tested organisms at very low concentrations. The recorded activities may be suggestive of the presence of biologically active phytoconstituents in the extracts previously reported to have varying biological activities including antibacterial activities. Thus, the recorded activity in this work provides additional data on the potential use and development of these plants' bioactive compounds into newer medicines and also these plants can be relied on as reservoir for the detected biologically active compounds.