Comparative evaluation of the antimicrobial profile of Moringa leaf and seed oil extracts against resistant strains of wound pathogens in orthopedic hospitals

1 Department of Microbiology, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria. 2 Deparment of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, Nigeria. 3 Department of Applied Biology and Biotechnology, Enugu State University of Science and Technology, Enugu, Enugu State, Nigeria. 4 Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria. 5 Department of Veterinary Obstetrics and Gynecology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, Nigeria.

treatment of chronic wound infections.Most wounds have been reported to be contaminated by pathogens and body commensals ranging from bacteria, fungi to other parasites (Sani et al., 2012).The common gram positive organisms found in most wounds include βhaemolytic Streptococcus-Streptococcus pyogenes and Staphylococcus aureus.Others such as the gram negative aerobic rods, Pseudomonas aeruginosa have also been implicated in wound infections (Enweani, 1991).The facultative anaerobes like the Enterobacteriaceae species, such as Escherichia coli, Klebsiella and Proteus spp.have also been reported to constitute a serious problem in several wound infections (Mulugeta and Bayeh, 2011).The widespread use of antibiotics, together with the length of time over which they have been available, have led to major problems of resistant organisms, contributing to high levels of morbidity and mortality in patients (Akinjogula et al., 2010).The control of wound infections is becoming more challenging due to widespread bacterial resistance to antibiotics and to a greater incidence of infections caused by methicillin resistant S. aureus (MRSA) and polymicrobic flora (Cheesbrough, 2006).Increased antimicrobial resistance among pathogens of wound infections can result to complications and increase in the cost associated with procedures and treatments.Due to this rising resistance among wound pathogens, there is a need to search for alternative sources of treatment of wound infections in natural compounds from medicinal plants such as Moringa oleifera.
M. oleifera is an ancient tree that is historically known to possess numerous medicinal qualities (Posmontier, 2011).Previous studies have reported the antimicrobial properties of the various parts of Moringa roots, flowers, barks and stem including the seeds (Lockett et al., 2000;Walter et al., 2011).The seed kernel of M. oleifera has been employed in the treatment of bronchial asthma and found to show an appreciable decrease in severity of symptoms of asthma and also simultaneous improvement in respiratory functions (Fahey et al., 2001).The family Moringaceae has been found to be rich in compounds containing rhamnose, and it is rich in a fairly unique group of compounds called glucosinolates and isothiocyanates (Bennett et al., 2003).Moringa is also rich in a number of vitamins and minerals as well as other more commonly recognized phytochemicals such as carotenoids (Siddhuraju and Becker, 2003).M. oleifera has been reported to have natural antioxidant properties and thus enhance the shelf-life of fat-containing foods (Gilani et al., 1994).The roots of Moringa plants have been reported to possess antispasmodic activity through calcium channel blockade which forms basis for its traditional use in diarrhoea (Ghebremichael et al., 2005).The flowers show effective hepatoprotective effect due to the presence of quercetin (Costa-Lotufo et al., 2005).The seed of Moringa is also effective against skin papillomas in mice and the seed ointment had similar effect to neomycin against S. aureus pyodermia in mice (Adegoke et al., 2010).Finding an alternative source of treatment for wound infections using M. oleifera plant extracts could provide the needed replacement for conventional antibiotics since most wound pathogens have appeared to have developed resistance to most of the available antibiotics.The aim of the present study was to investigate the antimicrobial activity of M. oleifera seed and leaf oil extracts against broad spectrum antibiotic resistant microorganisms isolated from wound infections.The study also investigate if the resistance to the conventional antibiotics by some wound pathogens is plasmid associated.

Sample collection (M. oleifera), identification and preparation of the plant extracts
The fresh leaves and seeds of M. oleifera plant (Supplementary Figure 1a and b) were collected from Dekina Local Government Area, Kogi State Nigeria.The plant samples were identified at the Department of Botany, University of Nigeria, Nsukka, Nigeria.For the methanol and ethanol extractions, 50 g each of M. oleifera leaves and seeds were crushed and fed to a lab-scale Soxhlet extractor fitted with a 1 L round-bottom flask and a condenser.About 250 mL of both methanol and ethanol were added to the set up and the extraction was done for 6 h.The solvents were then evaporated under reduced pressure using a rotary evaporator at 45°C.

Aqueous extracts of the fresh leaves and seeds for antimicrobial examination
One hundred grams (100 g) of fresh leaves and seeds of M. oleifera were weighed and crushed directly using electric blender and missed in 400 mL distilled water into a conical flask stoppered with rubber corks and shook for 30 min, after which they were left to stand for 72 h and were shaken intermittently.The mixture was filtered off using sterile filter paper into a clean conical flask.The solvents were allowed to evaporate leaving the concentrated filtrates, which were then stored at 4°C for antimicrobial activity test.

Isolation, characterization and identification of orthopaedic wound samples
Wound samples were collected from one hundred (100) patients at National Orthopaedic Hospital Enugu, using a sterile swab stick and sterile distilled water.Out of the 100 samples, 45 were from postoperative wounds while 55 were from other wound sources.The samples were transferred to the Microbiology Laboratory of the University of Nigeria within 24 h for further analysis.The collected samples were streaked on freshly prepared nutrient agar and MacConkey agar and incubated aerobically at 37°C for 24 h.Discrete colonies differing in size, shape and colour were selected from the different plates and were sub cultured into fresh nutrient agar plates.The colonies were characterized using conventional biochemical tests (indole, catalase, urease, coagulase, sulphide indole motility, sugar fermentation and oxidase tests) following established protocols (Doughari et al., 2007).Further characterization was done using colonial and cell morphology.

Antibiotic susceptibility test
Antimicrobial susceptibility of the bacterial isolates was determined using the discs diffusion method, as described by Doughari et al. (2007).Briefly, the isolates were picked using sterile inoculating loop and inoculated into a sterile test tube with 5 mL of phosphatebuffered saline.The optical density of the organisms at 600 nm wasadjusted to 0.5 McFarland (0.08 to 0.1).Sterile cotton swabs were used to spread the growth evenly on the surface of Mueller-Hilton (MH) agar.The antibiotic discs which contained the following antibiotics, streptomycin, erythromycin, cloxacillin, chloramphenicol, amoxycillin, nitrofurantoin, nalidixic acid, ofloxacid, tetracycline, augmentin, gentamycin and cotrimoxazole were placed on the surface of MH agar plates.The MH agar plates were incubated for 18 to 24 h at 37°C.All the antibiotics were tested on both the gram positive and gram negative organisms.The antibiogram was read and recorded as the diameter of zone of the inhibition (ZI).The resistant isolates were separated from the susceptible ones and further analysis was carried out on them.

Antibacterial activity of the leaf and seed oil extracts
Antimicrobial activity of the aqueous, ethanolic and methanolic oil extracts of the leaves and seeds were assayed using the paper discs diffusion method (Oluma et al., 2000).The concentrated leaf extracts were dissolved in 5% dimethyl sulfoxide (DMSO) and sterile discs, 6 mm (Hi-media, India) in size were each impregnated with 30 μL of 50 μg/mL of each extract.The discs were carefully and firmly placed on the Muller Hinton Agar (MHA) plates earlier seeded with standardized bacterial suspensions (approximately 1.5 × 10 6 CFU/mL).Filter paper discs dipped into sterile distilled water and allowed to dry were used as control.The plates were then incubated at 37°C for 24 h.Antibacterial activity was determined by measurement of ZI around each paper disc.

Determination of minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)
Equal volumes of each oil extract (with different concentrations) and nutrient broths were mixed in 8 test tubes and 0.1 mL of the standardized inoculum was added to each of the tubes.The tubes were incubated aerobically at 37°C for 18 to 24 h.Two control tubes were maintained for each test batch.The MIC was taken as the lowest concentration that prevented bacterial growth.The MBC was determined by sampling all the macroscopically clear tubes and the first turbid tube in the series.Before being sampled, the tubes were gently mixed by flushing them with a sterile pipette, and a 100 mL aliquot was removed from each test tube.Each aliquot was placed on a single antibiotic-free nutrient agar plate in a single streak down the centre of the plate in accordance with the method established by Irkin and Korukluoglu (2007).The samples were allowed to stand for 30 min so that they can be absorbed into the agar until the plate surface appeared dry.The aliquots were then spread over the plate by lawn technique.The growth and sterility controls were sampled in the same manner.The MBC lawn plates were incubated for 24 h at 35°C.After the incubation periods, the lowest concentrations of the extract that did not produce any bacterial growth on the solid medium were regarded as MBC values for the extract (Cruishank et al., 1975).

Time of kill assay
An assay on the rate of killing of S. aureus and Klebsiella spp.by the methanol and aqueous seed oil extracts was carried out using a modified plating technique (Zhou et al., 1990).Briefly, the extracts were incorporated into 10 mL Mueller Hinton broth in McCartney bottles at MIC.Two controls, one Mueller Hinton broth without extract inoculated with test organisms and Mueller Hinton broth incorporated with the extract at the test concentrations without the test organisms, were included.Inoculum density, approximately 10 5 CFU/mL further verified by total viable count was used to inoculate 10 mL volumes of both test and control bottles.The bottles were incubated at 37°C on an orbital shaker at 120 rpm.A 100 μL aliquot was removed from the culture medium at 0, 1, 2, 3 and 4 h for the determination of CFU/mL by the plate count technique (Zhou et al., 1990).About 25 μL of each of the dilutions was plated out and incubated at 37°C for 24 h for emergence of bacterial colonies.The cell counts from the experimental cultures were compared with the controls.

Plasmid isolation and profiling
The bacterial isolates that were resistant to the antibiotic susceptibility test were selected for plasmid isolation and curing.Preparations of plasmid DNA of the resistant isolates was done by alkaline lysis method as described by Taylor and Brose (1988).A single bacterial colony was picked from the clones and grown in nutrient broth at 37°C for 24 h.The cell suspension was transferred to Eppendorf tube using a micropipette and centrifuged at 6000 rpm for 5 min at 4°C.Pelleted cells were re-suspended in 300 µL of the lysis solution (25 mM Tris, 10 mM EDTA, 0.1 N NaOH and 0.5% SDS) and incubated at room temperature for 5 min.Sodium acetate solution was added to the mixture and the solution was mixed properly by whirling before incubation at room temperature for 5 to 10 min.Cold absolute ethanol was used to precipitate the PDNA and the mixture was spun for 10 min at 12000 rpm.The pelleted DNA was washed with 1 mL 70% cold ethanol, air dried for at least 10 min and re-suspended in 20 to 40 µL of distilled water for further analysis.Electrophoresis was done with 2 µL of the extracted plasmid in 0.8% agarose gel for 1 h at 90 V.The gel was stained with ethidium bromide (0.5 µg/mL) and visualized with UV light (302 nm UV transilluminator).

Plasmid DNA curing
Plasmid curing was done with acridine orange (AO) and sodium dodecyl sulphate (SDS) as described in previous studies (Bhalakia, 2005;Isibor et al., 2008).Nutrient broth was prepared and supplemented with 1g of SDS in one batch of 99 ml and 10 g of SDS in a second batch of 90 mL to achieve final concentrations of 1 and 10% (w/v) SDS, respectively.Each of the solutions was adjusted to pH 7.6 with 1 M NAOH and autoclaved at 121°C for 15 min.Selected overnight (O/N) cultures of the isolates were standardized to 0.5 McFarland turbidity standard using sterile saline.In each 5 mL SDS supplemented nutrient broth, 0.1 mL of each O/N culture was inoculated and incubated at 37°C for 24 h.Cultures were later subjected to the same antibiotics to which they were resistant to previously.The curing process was repeated with acridine orange following the standard protocols.

Characterization and antibiotic susceptibility test of the bacterial isolates
A total of 100 wound samples were collected from the National Orthopaedic Hospital Enugu and were identified.From the biochemical test, 43% of the bacterial isolates were S. aureus, 16% were Proteus spp., 15% were Klebsiella spp., 11% were Citrobacter spp.while Escherichia coli and P. aeruginosa were 8 and 6% respectively (Table 1).The result from the biochemical test was further confirmed with the analysis of the colonial and cell morphology of the organisms.Out of the 100 wound isolates used in this study, 98 of them were used for antibiotic susceptibility test, 88.7% of them were resistant to at least 4 or more antibiotics (Table 2).All the organisms were resistant to amoxycillin and cloxacillin.

Bacterial resistance and plasmid association
All the gram negative bacterial wound pathogens (Proteus spp., P. aeruginosa, Klebsiella spp.and E. coli) had 3 plasmid bands each.The first set of bands lie in the region of 23 kb while the two smaller sized plasmids lie within the regions of 2 kb and 500 bp (Figure 1a).For the gram positive wound pathogens which were mainly S. aureus, no bands were observed in two strains (lanes 13 and 72), while the three remaining strains (lanes 16, lane 22 and 45) had 3 plasmid bands each.The first set of bands was 23 kb in size while the other bands lie between the regions of 500 bp and 2 kb (Figure 1b).To analyse if the resistance observed in some multidrug resistant isolates was plasmid associated, a subset of the isolates that showed resistance to the tested antibiotics were subjected to plasmid curing.All the gram negative organisms became susceptible to most of the antibiotics they were previously resistant to after the curing procedure.However, S. aureus remained resistant to all the antibiotics even after plasmid curing.

Antimicrobial activity of the M. oleifera leaves and seed oil extracts
All the leaf oil extracts showed inhibitory effects at the tested except on Proteus spp.whose growth was not  (13,16,22,45 and 72 represent different strains of S. aureus).Electrophoresis was done with 2 µl of the plasmid from each sample in 0.8% agarose gel for 1 h at 90 V.The gel was stained with ethidium bromide (0.5 µg/mL) and visualized with UV light (302 nm UV transilluminator), 23 kb marker was used to determine the size of the isolated pDNA.concentration of 50 µg/mL on all the microorganisms inhibited by the aqueous and ethanolic oil extracts at the tested concentration.The aqueous leaf oil extract had ZI ranging from 3.0 ± 0.0 to 20.3 ± 1.5 mm in Proteus spp.and E. coli respectively (Table 3).The methanolic leaf oil extract had the highest ZI, 22.7 ± 2.1 mm in P. aeruginosa.However, it showed a reduced ZI of 11.0 ± 2.0 mm in Klebsiella spp.The ethanolic leaf oil extracts showed the lowest ZI ranging from 3.0 ± 0.0-12.3± 1.5 mm in Proteus spp and E. coli respectively.In all the extracts, all the isolates tested were more susceptible to the methanolic leaf oil extract (Figure 2) while the ethanolic leaf oil extract had the least antimicrobial activity against the tested organisms (Table 3).The antimicrobial activity of the aqueous oil extract, methanolic and ethanolic oil extracts of M. oleifera seeds respectively is presented in Table 4.All the seed oil extracts showed inhibitory effects at the concentration of 50 µg/mL.The aqueous oil extract of the seeds had more inhibitory activity with ZI ranging from 15.3 ± 2.2 to 22.0 ± 1.3 mm in S. aureus and E. coli respectively.This was followed by methanolic seed oil extract with the ZI ranging from 11.0 ± 3.0-18.7 ± 1.5 mm in S. aureus and E. coli respectively.However, the sensitivity of the isolates to ethanolic seed oil extract was relatively low in most of the isolates tested (Table 4).Three isolates, Citrobacter spp., Proteus spp and E. coli were not sensitive to the ethanolic seed oil extract (Table 4).

Minimal inhibitory concentration of M. oleifera leaves and seeds oil extracts
The MIC for the methanolic leaf oil extract was 20 µg/mL, at this concentration, the growth of all the microorganisms were inhibited.The aqueous leaf oil extract had inhibitory effect on Citrobacter spp and E. coli at a concentration of 20 µg/mL.At a concentration of 30 µg/mL the aqueous leaf oil extract also had inhibitory effect on S. aureus and Klebsiella spp, but did not have any inhibitory effect on P. aeruginosa.A slightly different trend was observed for the seed oil extracts.At the concentration of 20 µg/mL, the aqueous seed oil extract inhibited the growth of all the microorganisms tested except P. aeruginosa that was inhibited at 30 µg/mL.At the concentration of 30 µg/mL, the methanolic seed oil extract had inhibitory effect on Proteus spp, Klebsiella spp and E. coli but it showed inhibition on the growth of S. aureus and P. aeruginosa at concentration of 40 µg/mL.At a higher concentration of 50 µg/mL it inhibited the growth of Citrobacter spp.However, for the ethanolic seed oil extract, the MIC was quite high in most of the bacterial species, 60 µg/mL, 40 µg/mL and 50 µg/mL for S. aureus, Klebsiella spp.and P. aeruginosa respectively (Figure 3).

Minimal bactericidal concentration of M. oleifera leaves and seeds oil extracts
The aqueous leaf oil extract had the highest bactericidal effect, with concentration as low as 20 µg/mL, all the organisms except P. aeruginosa were killed (Figure 4).The methanolic leaf oil extract also had effect on all the tested organisms although at variable concentrations.However M. oleifera ethanolic leaf oil extract had no bactericidal activity on half of the tested bacterial species although at a concentration of 40 µg/mL, Klebsiella spp. was observed to be killed by the extract.A similar trend was observed for the seed oil extracts (data not shown).The aqueous seed oil extract showed the highest bactericidal effect at concentration of 20 µg/mL.At this concentration, Citrobacter species, Proteus spp.and E. coli were killed while S. aureus, Klebsiella spp.and P.   aeruginosa were killed at a concentration of 30 µg/mL.The methanol oil extract had bactericidal effect at 30 µg/mL for E. coli and 50 µg/mL for S. aureus, Proteus species and P. aeruginosa.While ethanol seed oil extract had the lowest bactericidal activity with concentration ranging from 50 µg/mL to 70 µg/mL.

Comparison of the MIC and the MBC from different extraction sources
A comparison of the MIC between leaf and seed oil extracts from the three extraction methods showed that the leaf oil extracts from ethanolic, aqueous and methanolic extractions had more inhibitory activity when compared with the seed extracts from the three sources.
The MIC of the leaf oil extracts for the three extraction sources were the same as the control.However a different trend was observed for the MBC of the leaf and seed oil extracts from the three extraction methods.The seed and leaf oil extracts from the three sources showed variable MBC at relatively high concentrations.For the ethanol seed oil extract, there was no MBC for Citrobacter spp., Proteus and E. coli even at concentrations as high as 70 µg/mL.No MBC was recorded for Proteus spp.for the leaf oil extract as well.A similar trend was observed for the methanolic seed oil extracts.In addition, the methanolic leaf oil extract could not show any observable MBC on Proteus spp.For the aqueous seed oil extract, the leaf oil extract had no MBC on Proteus and Pseudomonas spp.

Reduction of viable bacterial counts by the extracts
The time of kill estimates the time it took the extracts to kill the bacterial pathogens, hence showing a progressive reduction in the number of colonies that survive over a given time frame.The time of kill assayed at a maximum time interval of 0 to 5 h showed a reduction of the Klebsiella spp.from 8.2 to 2.3 CFU/mL by the methanolic leaf oil extract and 9.0 to 4.8 CFU/mL by the aqueous leaf oil extract (Figure 5).For S. aureus, the colonies reduced from 9.4 to 3.2 CFU/mL with the methanolic seed oil extract and 9.6 to 6.4 CFU/mL with the aqueous seed oil extracts at 0 to 5 h (Figure 5).

DISCUSSION
Microorganisms such as bacteria, fungi and other parasites are common pathogens associated with wound infections (Sani et al., 2012).However, the resistance of these microbial agents to conventional antibiotics has  become very alarming.In the present study, an evaluation of the antibiotic susceptibility pattern of microorganisms isolated from orthopaedic wounds and also the antimicrobial potential of M. oleifera leaf and seed oil extracts against orthopaedic wound pathogens were tested.The results from our biochemical analysis showed that gram negative organisms such as Citrobacter spp., Proteus spp., Klebsiella spp., E. coli and P. aeruginosa were the predominant organisms present in the infected wounds.However, S. aureus was the only gram positive organism identified in the samples.This observation corroborates with the findings from other work where S. aureus was reported as the predominant gram positive bacterial isolate in wound infections (Samuel et al., 2010;Magiorakos et al., 2012).The antibiogram of the isolates screened in this study showed that over 90% of the bacterial species showed high level of resistance to most of the tested antibiotics (cloxacillin, erythromycin, amoxycilin, augmentin and tetracycline).However, gentamicin and cotrimoxazole having resistance levels of 70.4 and 75.6% respectively were the antibiotics with the least resistance profiles.The high level of resistance observed among the tested bacterial isolates in this study could be as a result of indiscriminate use of the tested antibiotics.It could also be due to the transfer of resistant genes among organisms as has been reported in microbial communities like biofilms (Andhoga et al., 2002).Although the resistance observed in gentamicin and cotrimoxazole are relatively high, the two antibiotics proved to be the candidates of choice for treatment of orthopaedic wound infections in this study.
Other studies have also reported that gentamicin was the most effective antibiotic against most isolates of gram negative organisms isolated from wound infection (Rathi et al., 2006).The antimicrobial activity of the aqueous, methanolic and ethanolic leave oil extracts of M. oleifera against individual isolates of the bacteria revealed that the zones of inhibition (ZI) of the extracts varied from one bacterial species to the other.Methanolic leaf oil extract showed appreciable antibacterial activity on the orthopedic wound organisms, an indication of its high anti-bacterial potential and effectiveness in the treatment of wound infections.Evaluation of the wound healing property of aqueous oil extracts of leaves of M. oleifera on male Swiss albino mice showed a significant increase in wound closure rate, skin breaking strength, granuloma breaking strength, granuloma dry weight and decrease in scar area (Srinivasan et al., 2001).However, in our study, the aqueous and ethanolic leaf oil extracts showed no activity against Proteus spp., with both extracts showing no ZI against the organism.The variations in the activity of the different leaf oil extracts against individual organisms might be as a result of the differences in the concentration and the amount of phytochemical constituents of each extract.These differences in the phytochemical constituents might have been caused by the difference in polarity of the solvents used in the extraction process (Faizi et al., 1995).Several studies have characterised the chemical compounds present in M. oleifera plant leaves (Fuglie, 1999).Two nitrile glycosides from the ethanolic extracts of M. oleifera leaf and three mustard oil glycosides have been identified (Fuglie, 1999).Although the ethanolic and aqueous leaf oil extracts showed a considerable reduction in the growth of the tested organisms, the methanolic leaf oil extract showed a broader ZI on all the organisms, which could suggest that the method of extraction has a huge effect on the efficacy of the extracts.Additionally, it is also possible that due to the different methods of extraction employed in this work, each extract could possibly have a different biochemical pathway during metabolism in the organisms.For the seed oil extracts, the activity of the individual extracts (ethanolic, methanolic and aqueous) varied amongst the tested organisms.A comparison of the leaf and seed oil extracts showed that the methanolic and ethanolic leaf oil extracts showed relatively higher activity than their corresponding counterparts from the seed oil extracts.A possible explanation for this could be due to the presence of more bioactive phytochemicals in the methanolic and ethanolic leaf oil extracts when compared to the seed extracts.It could also be that the phytochemicals in the two extracts were more soluble due to the polarity of the solvents.Furthermore, the methanol and ethanol have probably eluted more bioactive components from the leaf than in the seed.In contrast to this result, a higher activity of ethanolic leaf oil extract of M. oleifera and a relatively lower activity of the aqueous oil extract was observed at a concentration of 100 µg/mL (Shanholtzer et al., 1992).The reason for this discrepancy in results could not be explained.However, the MIC of the extracts showed that ethanolic leaf oil extract had the lowest activity on the organisms but showed the highest MBC values.MBC values have been shown to be consistently higher than MIC values probably because higher concentrations of antibiotics may be required to kill bacterial organisms than that required to inhibit their growth.The MIC values for the seed oil extracts followed a similar trend with the leaf extracts.Plasmid profiling of the isolates showed the presence of plasmids in all the gram negative organisms tested.These plasmids may be responsible for the observed resistance in the tested isolates as a reasonable number of the gram negative organisms lost their resistance after curing.Most bacterial plasmids harbour antibiotic resistant genes some of which are acquired through horizontal gene transfer.Interestingly, 60% of the gram positive organisms tested in this study had plasmids and they were resistant to the tested antibiotics.However, 40% of the gram positive organisms (S. aureus) had no plasmids but they still showed resistance to most of the tested antibiotics.The presence of resistance in the Staphylococcus strains that had no plasmids could suggest that antibiotic resistance in Staphylococcus spp.and perhaps other bacteria could be of both chromosomal and plasmid origin.From this study, it has been demonstrated that the application of extracts from natural plants such as M. oleifera for the treatment of wound infections can be a promising alternative to conventional antibiotics.The methanolic leaf and aqueous seed oil extracts showed the highest antimicrobial activity when compared to the other extracts.The potential of using these extracts for the treatment of wound infection should be fully harnessed.

Figure 2 .
Figure 2. Susceptibility test of the methanolic leaf oil extract on S. aureus.The susceptibility tests were done using paper discs diffusion methods.Discs were placed on Muller Hinton Agar (MHA) plates earlier seeded with standardized bacterial suspensions (approximately 1.5 × 10 6 CFU/mL).C = Paper discs impregnated with 30 μL of a solution of 50 µg/mL of ciprofloxacin and gentamycin were used as control for comparison.MLE= Methanolic leaf extract, C+ = cotrimoxazole, C= gentamycin.

Figure 3 .
Figure 3.The minimal inhibitory concentration of M. oleifera seed oil extracts against orthopedic wound infection pathogens.Black bars indicate the MIC of M. oleifera aqueous seed oil extracts, green bars indicate the MICs of the methanolic seed oil extract, blue bars show ethanolic seed oil extracts and the yellow bars are the antibiotic control.

Figure 4 .
Figure 4.The MBC of M. oleifera leaf oil extract: yellow bars represent the MBC of the aqueous leaf oil extract, blue bars indicate the methanol leaf oil extract, ash bars represent the ethanolic leaf oil extract while the purple bars are the antibiotic controls.

Figure 5 .
Figure 5.Time of kill of the aqueous and methanolic oil extracts on S. aureus and Klebsiella spp.The blue line represents the time of kill of the methanolic leaf oil extracts on S. aureus, the red line represents the time of kill for the Klebsiella spp., the green line represents the time of kill of the methanolic seed oil extract on S. aureus, the purple line represents the time of kill of the aqueous seed oil extracts on Klebsiella spp., while the light blue one represents the antibiotic control (cotrimazole).The time of kill was measured as the reduction in population of viable cells per hour.

Table 1 .
Biochemical characterization and the identification of bacterial pathogens from orthopaedic wound infections.Different biochemical tests were carried out to characterize and identify each orthopaedic wound samples.The biochemical analysis were able to group the othorpedic wound samples into gram positive (S.aureus) and gram negative organisms (Citrobacter spp, Proteus spp, Klebsiella spp, E. coli and P. aeruginosa).NT = Not tested; -= Negative; + = Positive.

Table 2 .
Descriptive statistics of the antibiotic resistance profile of the different bacterial groups identified in this study.
† = Antibiotics used for the susceptibility study; ‡ = Concentrations of the antibiotics; † † = Number of isolates that were resistant to the antibiotics.ND = Not determined.

Table 3 .
Zones of inhibition of M. oleifera leaf oil extracts on different bacterial species.

Table 4 .
Zones of inhibition of M. oliefera seed oil extracts on different bacterial species.Data are presented as mean ±SD as measurement of inhibition zone (mm); Means and standard deviations determined from 3 biological replications.