Antimicrobial activity of Moringa oleifera leaf extracts on multiple drug resistant bacterial isolates from urine samples in Benin City

The aim of this study was to examine the antibacterial effect of Moringa oleifera leaf extracts on selected multiple drug resistant (MDR) bacterial isolates. Escherichia coli , Pseudomonas aeruginosa and Staphylococcus aureus were isolated urine samples from patients attending Lahor Research Medical Centre. Multiple drug resistant bacteria were generated from the isolates by carrying out antibiotic susceptibility testing using the Kirby-Bauer disc diffusion technique. Three isolates each from the multidrug resistant bacteria were selected and molecular characterization was performed for confirming microbial identity. The antibacterial activity of methanol, chloroform and aqueous leaf extracts of M. oleifera at different concentrations (6.25, 12.5, 25, 50 and 100 mg/ml) were analyzed on the selected MDR bacteria using agar disc diffusion method. M. oleifera leaf extracts were observed to inhibit the growth of multidrug resistant bacteria. The highest antibacterial activity 9.32±1.45 mm was observed with the chloroform extracts, while the lowest value of 0.27±0.27 mm was obtained for the aqueous leaf extract. The antibacterial activity examined in this study showed that chloroform and methanol M. oleifera leaf extracts are capable of exerting inhibitory effect on multidrug resistant bacteria. The results obtained in this study indicated that M. oleifera can be a potential source for the treatment of different infections caused by multiple drug resistant bacteria.

than one antibiotic or groups of antibiotics to The leaves were identified at the Department of which it was formally susceptible to either by the Plant Biology and Biotechnology, Faculty of Life production of enzymes that alter the structure of Science, University of Benin, Benin City, Nigeria. the antibiotics, by the modification of the antibiotics, by bypassing certain pathways or by Ethical clearance acquisition of resistant plasmids from other Approval was obtained from the Medical bacteria strain. Resistance can either be plasmid, Directors of the hospitals whose patients chromosome mediated or both plasmid and participated in this study and the patients gave chromosome mediated. This resistance could be their consent after being informed of the ascribed to indiscriminate use of commercial objectives of the study. drugs or not taking an antibiotic prescription according to instruction (Aliero and Afolayan, Bacteriological procedures/identification of 2006). In addition, certain antibiotics present isolates undesirable side effects such as nausea, Specimens were aseptically inoculated depression of bone marrow leading to the onto MacConkey, Blood and Nutrient agars and emergence of previously uncommon diseases o incubated aerobically at 37 C for 24 hours and (Poole, 2002). This has given scientists the drive observed for colonial growth. All isolates were to search for newer and alternative antimicrobial identified based on their colonial appearances on compounds from medicinal plants (Aliero and MacConkey agar and nutrient agar plates. Afolayan, 2006). Besides, the high cost of Isolates were identified using the morphological conventional drugs, particularly in resource and biochemical test (Cheesbrough, 2000) and inadequate communities were access to good confirmation was done using polymerase chain health is costly for the common citizens has led reaction technique. to increased use of plants as alternative for treatment of some diseases. In recent years, Antibiotic susceptibility testing interest has grown in the utilization of what has Susceptibility to antibiotics was assessed come to be known as "multipurpose" plants; one using the Kirby-Bauer disc diffusion method, and of such plants is Moringa oleifera Lam, the most clear area around the antibiotic discs referred to widely cultivated species of a monogeneric as zones of inhibition were read using the Clinical family Moringaceae (Shittu et al., 2017). All parts and Laboratory Standards Institute's guidelines of Moringa tree are edible and have been (CLSI, 2010 Amplicons lysis tube was then centrifuged in a molecular weights were calculated using microcentrifuge at 10,000 x g for 1 minute. molecular weight standard maker. Aliquot of 400 µl of the supernatant was pipetted into a Zymo-spin IV spin filter in a collection tube Preparation of Moringa oleifera leaf extracts and centrifuge at 7,000 rpm for 1 minute. Leaves of M. oleifera were washed with Fungal/Bacterial DNA binding buffer (1,200 µl) running tap water and then air dried at room was added to the filtrate in the collection tube.
temperature for 15 days (Shobayo et al., 2014). The mixture was centrifuged at 10,000 x g for 1 Ten gram (10 g) of the powdered leaves was minute. Pre-wash buffer (200 µl) was added and separately extracted in 500ml conical flasks with centrifuge at 10,000 x g for 1 minute. Aliquot of 100ml of deionised distilled water (aqueous 500 µl of the fungal/bacterial DNA wash buffer extraction), 100ml of 99% methanol (methanolic was added to the Zymo-spin IIC column and extraction) and 100ml of 99% chloroform Centrifuged at 10,000 x g for 1 minute. The (chloroform extraction). The conical flasks were Zymo-spin IIC column was transferred to a clean plugged with rubber corks and allowed to stand 1.5 ml microcentrifuge tube and 80 µl of DNA at room temperature for two weeks with elution buffer was added directly to the column intermittent shaking twice a day (Bukar et al., matrix and centrifuge at 10,000 x g for 30 2010). The extracts were separately filtered seconds to elute the DNA using sterile Whatman No. 1 filter paper. The resulting filtrate obtained were then stored in a o

Polymerase Chain Reaction for the detection of refrigerator at 4 C (Akueshi et al., 2002) which Bacterial isolates
were used as experimental drug for the present study. Amplification of bacteria species-specific genes were carried out using Polymerase Chain Reaction (PCR) technique. The details of the Standardization of inoculums primers used in the study are given in Table 1.
The standard was prepared from the Quick load One Taq one step PCR master mix stock cultures, maintained on nutrient agar slant o (2X) was purchased from lnqaba Biotech, at 4 C and subcultured onto a nutrient broth Hartfield, South Africa Incorporated and used using a sterilized wire loop. The density of the according to the manufacturer's instruction. The suspension inoculated onto the media for PCR was performed in a 25 µl reaction mixture susceptibility test was determined by containing 12.5 µl Quick load One Taq one-step comparison with 0.5 McFarland standard of PCR master mix (2x), 1.25 µl of each species-Barium sulphate solution (Cheesbrough, 2002). specific forward primer (20 µM), 1.25 µl of each species-specific reverse primer (20 µM Makanjuola et al. (2013). Then, 25 ml sterile-1min and a final extension at 72 C for 15 minutes o molten Mueller Hinton Agar (Watin-Biolife, KSA) and final holding at 4 C. Ten microliters (10 µl) of was loaded on the Petri-dish and left at room the amplified PCR products of test isolates and temperature to solidify. After that, wells were positive control strain were fractionated on a punched into the agar with a sterile cork borer (6 1.0% agarose gel containing ethidium bromide mm in diameter). Aliquot of 0.1 ml of the in Tris/Borate/EDTA (TBE) Buffer along side aqueous extract with different concentrations of negative control containing nuclear free water Figure 1: Percentage Bacterial isolation from urine samples The use of conventional antibiotic discs indicated high rate of MDR bacterial isolates. Three isolates among those found to be resistant to multiple antibiotics for each organism were selected and designated as EC1, EC5, EC7 for Escherichia coli, PA2, PA3, PA8 for Pseudomonas aeruginosa and SA1, SA4 and SA6 for Staphylococcus aureus (Table 2). 6.25, 12.50, 25, 50, 100 mg/ml was loaded into Statistical Analysis the wells and same procedure was applied for Experiments were performed in triplicate the methanol and chloroform extract. 100 ìl and results were presented as mean ± standard abolute ethanol and chloroform were used as a error. The data obtained were subjected to positive control. All plates were kept in the parametric and descriptive statistics using the incubator for 24 hours at 37°C. Tests were Statistical Package for the Social Sciences (SPSS) repeated three times and the mean zone of version 20 software (Chicago, IL, USA). Means inhibition was calculated.
were separated by the Tukey's multiple range test when Anova was significant (P<0.05).

Results and Discussion
aeruginosa, which were rod shape, motile, In a total of 70 bacterial cells isolated catalase positive, gram negative, produced pale from urine samples obtained from patients colored colonies on MacConkey agar and attending Lahor Research Medical Centre, 13 greenish colonies on nutrient agar and 8 (18.6%) isolates were Escherichia coli (11.4%) were Staphylococcus aureus isolates Escherichia coli which produces pink colonies on that were found to be non-motile, catalase negative and gram positive cocci, produce MacConkey agar and E. coli appeared colorless golden yellow color on mannitol salt agar plate as on nutrient agar; 10 (14.3%) were Pseudomonas shown in Figure 1.  were categorized into antibiotypes 1, 3 and 5. mm was recorded for EC1. All P. aeruginosa It was observed from the result that isolates showed resistant against five antibiotics SA1 were resistant against six antibiotic discs (AUG, CXM, CRX, CAZ, NIT) and sensitive to OFL, (CAZ, CRX, CTR, ERY, CXC, AUG) used and was GEN, CPR. Highest zone of inhibition of 25.10±0.20 mm for ciprofloxacin were observed sensitive to GEN and OFL (Table 3).  The result of the antibacterial activity of inhibitions were observed but at 50 mg/ml, the effect were same as the control followed by 100 M. oleifera leaf extracts against three isolates of mg/ml with 7.09±0.50 mm zone of inhibition. It MDR E. coli using aqueous, methanol and was clear that there were no effect observed at chloroform at different concentrations (Table 4) 6.25, 12.50 and 25, 50 mg/ml for aqueous exhibited different degrees of antibacterial extract meanwhile, mean zone of inhibition of activities when compared with the control. For 1.37±0.61 were recorded at 100 mg/ml as Chloroform extract, there was considerable shown in Plate 4. Akhtar et al. (2012) reported effect at 6.25 and 12.50 mg/ml with 1.80±0.50 that M. oleifera ethanol leaf extract showed and 3.48±0.86 mm; significant effect was seen moderate effect against all of the isolates and the at 25 and 50 mg/ml which were higher when highest inhibition zone (20.07 ± 0.5 mm) was compared with the control. Highest mean zone found against E. coli at 30 mg/ml concentration, of inhibition of 9.32±1.45 mm was recorded at 100 mg/ml. Methanol extract at concentrations while petroleum ether, acetone and chloroform of 6.25, 12.50 and 25 mg/ml, no zones of extracts did not exhibit any inhibitory activity.  Meanwhile, aqueous extract showed lowest The result obtained for Pseudomonas antibacterial effect among the three extracts aeruginosa revealed that chloroform extracts at used as shown in Plate 5. Emad (2016) shown 6.25, 12.50 and 25 mg/ml was observed to have that at a concentration of 200 mg/ml, ethyl same effect as the control followed by 50 mg/ml acetate extract revealed the highest antibacterial with 3.30±0.48 (Table 5). Highest mean zone of activity against Staphylococcus epidermidis inhibition of 7.32±1.17 mm were recorded at 100 (16.0±0.5 mm), Staphylococcus aureus mg/ml. Methanol extract zones of inhibition at 6.25, 12.5 mg/ml were seen to have lesser effect (13.6±0.3 mm), Pseudomonas aeruginosa than the control but there were no significant (13.3±0.3 mm). The results of this investigation difference at 50 mg/ml effect when compared was in harmony with numerous previous studies with the control, methanol extract at 100mg/ml on M. oleifera leaves with different extraction recorded mean zone of inhibition of 7.15±1.08 methods and solvents such as aqueous, ethanol, mm. Aqueous extract at 6.25, 12.5, 25 and 50 methanol, chloroform and many more reported mg/ml were not significantly different from the interesting antibacterial activity against wide control and aqueous extract at 100 mg/ml were spectrum microorganisms, from both gram observed to show considerable antibacterial positive and gram negative bacteria (Abalaka et activity when compared with the control. al, 2012; Arun and Rao, 2011).  reported that crude extract of M. oleifera had exhibited different levels of antibacterial activity when compared with the control as shown in strong activity against Fusarium solani, Bacillus Table 6 and Plate 6. From the three extracts, subtillis and Staphylococcus aureus. The chloroform performs better hen compared to relatively high potency of the ethanol extract methanol and aqueous extract. Chloroform may be attributed to the dissolving power of extract at 6.25 and 12.50 mg/ml recorded same alcohols (Majorie, 1999). Emad (2016) reported effect as the control while 25 and 50 mg/ml were that Butanol extract was active only against observed to be significantly the same; mean Staphylococcus epidermidis (14.0±0.0 mm) and zone of inhibition of 7.08±0.92 mm were Staphylococcus aureus (10.3±0.3 mm). Water recorded at 100 mg/ml. methanol extract at extract was active only against Staphylococcus 6.25, 12.50 and 25 mg/ml has less effect than epidermidis (12.3±0.6 mm). Chloroform extract the control, at 50 mg/ml, the effect (2.64±2.31 showed antibacterial activity against mm) were not significantly different when Staphylococcus aureus (11.0±0.5 mm). This compared with the control and 100 mg/ml were activity against both gram negative and gram seen to have highest zone of inhibition with positive bacteria may be attributed to presence 4.83±0.75 mm. Aqueous extract at 6.25, 12.50 of some broad-spectrum antibacterial and 25 mg/ml, there were no zone of inhibition compounds (Vinoth et al., 2012).