Microbes and Infectious Diseases

of


Introduction
Bacterial infection of the wound has been known to be among the leading causes of morbidity and mortality throughout the world [1,2].Wound infection poses a global health challenge leading to delay in wound healing and surgical complications like wound breakdown [3].Wound infection has been identified as the most common nosocomial infection, especially in patients undergoing surgery resulting in a prolonged hospital stay, increased trauma care, treatment costs and more demanding general wound management practices [3].Escherichia coli, species of Staphylococcus, Pseudomonas, Klebsiella, Proteus and anaerobes like Clostridium and Bacteroides species are frequently found in wound infections.
The majority of plants that were used in ethnomedicine have been studied because of their promising effects against various pathogenic microorganisms [4].The use of natural products in combination with antibiotics to enhance treatment efficacy is a new strategy developed to overcome the problem of antibiotic resistance [5].Some of the medicinal plants include Vernonia amygdalina [6], Ocimum gratissimum [7], Andrographis paniculata [8], Polyalthia longifolia [9], among others.
The bitter leaf, V. amygdalina (Delile) which was used in this study has been reported by some researchers to have nutritional and medicinal values [10,11].The plant is also known to possess certain phytochemicals such as sesquiterpene lactones, anthraquinones, terpenoids, saponin, tannin, and flavonoids [12,13].Ruslim et al. [14] stated the wound healing potential of V. amygdalina indicating that the extract of the plant may hasten wound healing.Extracts of V. amygdalina possess antioxidant properties that can hasten wound healing and also has anti-inflammatory and antibacterial effects [15,16].Therefore, the current study was designed to evaluate the antibacterial activity of the stem bark extracts of V. amygdalina on multiple antibiotic resistant bacteria (MARB) isolated from wound samples and characterize the bioactive compounds present in the plant extracts.

Ethical consideration
Ethical approval was obtained from the Health Research Committee, Ministry of Health, Akure, Nigeria.

Isolation and identification of bacteria
A total of 93 wound swab samples were obtained from patients attending the University of Medical Sciences Teaching Hospital (UNIMEDTH), Akure, Nigeria after administering questionnaire.Bacteria was isolated and identified using standard microbiological procedures according to Cheesbrough [17] and Ijabani et al. [18].

Antibiotic sensitivity testing
The inoculum was standardized using 0.5 McFarland's standard as described by Isunu et al.
[19].Antibiotic susceptibility test was performed using Kirby-Bauer disc diffusion method described by Cheesbrough [17].The diameter of zones of inhibition was measured and interpreted using standard interpretative charts as recommended by the Clinical and Laboratory Standards Institute [20].

Collection of plant materials
The stem bark of V. amygdalina were collected from a farm in Akure, Nigeria.The plant samples were air-dried before being used for the research.

Preparation of plant extracts
The air-dried stem bark samples of V. amygdalina were powdered using an electric blender.The powdered plant materials were extracted using methanol, ethyl acetate, hot water, and cold water.A 100g portion of the powdered sample was dissolved in 1000ml of each of the extraction solvents in a conical flask.The mixture was kept in a shaker for 72h after which the extract was drained out using muslin cloth and filtered with Whatman No 1 filter paper.The extracts were concentrated to dryness using a rotary evaporator.The extracts were preserved in air-tight containers at 4 o C for further use [21].

Phytochemical analysis
The presence and amounts of phytochemicals in the different solvent extracts of V. amygdalina stem bark were determined using standard methods described by Trease et al. [22], Sofowora [23] and Harborne [24].

Antibacterial activity of V. amygdalina
The assay for the antibacterial activity of V. amygdalina extracts was carried out using method described by Kone et al. [25].The reconstitution of the extracts was done to give various concentration intended for use in this study.Sterile perforated filter papers were then impregnated with the reconstituted extracts and placed accordingly on Mueller-Hinton agar plates that have been streaked with the test organisms.Ciprofloxacin 2mg/ml and DMSO was used as positive and negative control respectively.The plates were then incubated for 18 hours at 37 ℃ after which the diameter of zones of inhibition was measured in mm.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The MIC assay was carried out using broth dilution method with peptone water broth as described by Kone et al. [25].Test tubes containing different concentrations of the extracts of V. amygdalina, ranging from 6.25 mg/ml to 200 mg/ml were inoculated with the standardized bacteria and incubated for 18 -24 hours.The lowest concentration of V. amygdalina extracts that shows no visible turbidity or growth of the bacterial isolates was recorded as the MIC.
The MBC assay of the extracts was determined using the method described by Kone et al. [25].The test tubes from the MIC test that did not show visible growth were aseptically inoculated on different sterile Muller-Hinton agar plates and incubated at 37 °C for 24 hours.The MBC was chosen as the lowest extracts concentration that resulted in no visible growth of the bacterial isolates on the plate.

Characterization of bioactive compounds in V. amygdalina using GC/MS
The method of Gopu et al. [21] was used for characterization of the bioactive compounds and their percentage abundance using a Varian GC -MS equipment (Varian 4000 mass spectrometer, USA) alongside a mass spectrometer (MS) 3000 equipped with Agilent MS capillary column (30 m length × 0.25 mm diameter × 0.25 µm thickness).

Statistical analysis
Data obtained in this study were subjected to One-Way Analysis of Variance (ANOVA), and differences between means were separated using Duncan's New Multiple Range Test at 5% level of significance using Statistical Package for Social Sciences (SPSS) version 26.0.

Identification of bacterial isolates
The distribution of sample in relation to age and sex is represented in figure (1).Table 1 shows the biochemical characteristics of the bacteria isolated from wound swabs in this study.These include Bacillus subtilis, Escherichia coli (E.coli), Klebsiella pneumoniae (K.pneumoniae), Proteus mirabilis (P.mirabilis), Pseudomonas aeruginosa (P.aeruginosa), Staphylococcus aureus (S. aureus), and Staphylococcus epidermidis.

Percentage occurrence of bacteria isolated from wound swab samples
The percentage occurrence of the bacteria isolated from the wound swab samples is presented in figure (2).The most frequently occurring bacterium in this study was Pseudomonas aeruginosa (36%), followed by Staphylococcus aureus (29%) while Proteus mirabilis (4%) was the least frequently occurring bacterium among the isolates obtained in this study.

Antibiotic sensitivity pattern of bacterial isolates
The antibiotic sensitivity pattern for Gram-positive and Gram-negative bacteria are shown in tables (2 and 3) respectively.The isolates were highly resistant to augmentin and amoxicillin.

Phytochemical constituents of the plant extracts
The qualitative phytochemical analysis revealed that both phlobatannins and anthraquinones are absent in all the extracts.It also confirms the presence of saponin, tannin, flavonoid, terpenoids and cardiac glycosides in all the extracts as presented in table (4).Table 5 shows the quantitative phytochemical constituents and it reveals saponin as the most abundant phytochemical in all the extracts.

Antibacterial activity of V. amygdalina
The antibacterial activities of V. amygdalina stem bark various extracts are shown in tables( 6, 7, and 8) at 100mg/ml, 200mg/ml and 300mg/ml respectively, while ciprofloxacin and DMSO was used as the positive and negative control respectively.

Minimum inhibitory and bactericidal concentration
The MIC and MBC values are given in table (9).The MIC values ranged from 12.5 mg/ml to 100mg/ml across all extracts and organisms tested.

Profile of chemical compounds in the ethyl acetate extract of V. amygdalina
The GC-MS analysis of the purified ethyl acetate extract of V. amygdalina revealed the presence of bioactive compounds such as n-hexadecanoic acid, methyl ester, oleic acid, 9,12-octadecadienoic acid, octadecanoic acid, cyclohexaneethanol, 9hexadecenoic acid among others.The compounds with their molecular formula and structures are shown in table (10) while the GC-MS chromatographic spectra is shown in figure (3).

Discussion
Bacterial wound contamination is a serious problem in the hospital and the treatment of wound infections remains a significant concern for surgeons.This study evaluated the distribution and antimicrobial resistance pattern of bacteria isolated from wound samples among patients attending University of Medical Sciences Teaching Hospital, Akure, Nigeria.In addition, the frequency of occurrence of these bacterial pathogens was estimated.
Patients with wound in this study were found to be mainly adults and the infection was more prevalent among females than males.A high level of resistance of the bacterial isolates to commonly used antibiotics was observed in this study.Escherichia coli were highly resistant to all antibiotics tested.Staphylococcus aureus was resistant to gentamycin.Amoxicillin and highly susceptible to streptomycin and septrin.This is in agreement with Muhammad et al. [28] who reported that S. aureus isolated from wounds were resistant to  -lactam antibiotics, macrolides and aminoglycosides).High rate of resistance demonstrated by the isolated bacteria to antibiotics may be due to practicing self-medication or unavailability of guidelines regarding the selection of drugs thereby leading to inappropriate use of antibiotics.
Phytochemicals such as saponins, tannins, terpenoids, flavonoids and cardiac glycosides were found present in the various extracts.A study by Onifade et al. [29] revealed the absence of alkaloids in the cold and hot water extracts of V. amygdalina which also conforms with the findings of this study.These phytochemicals have been shown to exhibit various pharmacological and biochemical actions and are responsible for the antimicrobial activity of the plant Muhammed et al. [30].
Stem bark extracts of V. amygdalina had antibacterial effects on the isolates at various concentrations.There was significant difference (at p < 0.05) in the activities of the various extracts against the bacterial isolates when the mean values of the zones of inhibition were compared.
Ethyl acetate and hot water extract of V. amygdalina used in this study possess higher antibacterial properties on S. aureus than cold water and methanol extracts.At 300 mg/ml concentration, the ethyl acetate extract mediated a zone of inhibition of 23.17±0.60mm against S. aureus while E. coli has the lowest zone of 12.17±0.44mm.The control was however more effective against the isolate with a mean zone of inhibition 28.67±0.67mm.Similarly, the control antibiotic mediated a wider zone of inhibition against the bacterial isolates.However, at 300mg/ml, the Hot water extract of V. amygdalina mediated a zone of inhibition of 15.97±0.26against P. aeruginosa which is higher than the 12.00±0.58mmmediated by the control antibiotics.The extracts were least effective against P. mirabilis at the different concentrations.The methanol extract was more effective on K. pneumoniae with a zone of 17.83±1.01at 100mg/ml and 22.17±0.44 at 300mg/ml.Similar findings by Asfere et al. [16] revealed the antibacterial activities of the methanolic extracts of the plant against S. aureus, and P. aeruginosa.Another work by Tula et al. [31] revealed that E. coli was not susceptible to the aqueous extract of V. amygdalina stem bark which is in contrary to the findings of this study.The antibacterial activity of V amygdalina was found to be dependent on the nature of the solvent used for extraction and the concentration of the extract.Adetunji et al. [32] reported that V. amygdalina at high concentrations and observable time limit could have a bactericidal effect of the extract on the organism.
The minimum inhibitory concentration (MIC) assay in this study reveals that ethyl acetate, methanol, hot water, and cold water extracts of V. amygdalina stem bark possess in vitro antibacterial activities at varying concentrations.The MBC values were higher than the MIC values in this work.This suggests that the extracts were bacteriostatic at lower concentrations and bactericidal at higher concentrations.
The GC-MS analysis of the purified ethyl acetate extract revealed the presence of Oleic acid which has been previously reported by Rahdar et al. [33] to have some antimicrobial properties, thus justifying why the plant exhibited antibacterial activities against the tested bacterial isolates.Other compounds detected have also been previously reported to have antibacterial properties, and they include hexadecanoic acid, methyl ester, 9octadecenoic acid, 9,12octadecadienoic acid and palmitoleic acid [34].

Conclusion
This study revealed the presence of multiple antibiotic-resistant bacteria in wound infections.It further revealed that the different extracts of the stem bark of V. amygdalina used in this study possess some phytochemicals and bioactive compounds that make them good antibacterial agents against these multiple antibiotic resistant bacteria.Findings from this study have further established that V. amygdalina is a promising candidate and effective alternative treatment means for multiple antibiotic resistant bacteria (MARB) that are associated with wound infections.

Figure 3 .
Figure 3. GC-MS Chromatographic Spectra of Ethyl Acetate Extract of V. amygdalina

Table 1 .
Cultural, Morphological and Biochemical Characteristics of Bacteria Isolated from Wound Swabs.

Table 6 .
Antibacterial Activity of Various Extract of V. amygdalina at 100 mg/ml.Values are presented as mean ± SE of triplicates, values in the same row carrying the same superscript are not significantly different at p < 0.05 according to Duncan's New Multiple Range Test.

Table 7 .
Antibacterial Activity of Various Extract of V. amygdalina at 200 mg/ml.
Values are presented as mean ± SE of triplicates, values in the same row carrying the same superscript are not significantly different at p < 0.05 according to Duncan's New Multiple Range Test.

Table 8 .
Antibacterial Activity of Various Extract of V. amygdalina at 300 mg/ml.
Values are presented as mean ± SE of triplicates, values in the same row carrying the same superscript are not significantly different at p < 0.05 according to Duncan's New Multiple Range Test.

Table 9 .
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Value for Extracts of V. amygdalina

Table 10 .
Compounds Detected in the Gas Chromatography -Mass Spectrometry of V. amygdalina Purified Ethyl Acetate Extract.Percentage Occurrence of Bacteria Isolated from Wound Swab.
Figure 1.Distribution of Samples in Relation to Age and Sex.
to be associated with wound infections.This is similar with a previous study by Babayemi et al.[26]who reported S. aureus, coagulase negative S. aureus, E. coli, K. pneumoniae, P. mirabilis, and S. pyogenes as the bacteria associated with wound infections.Another study by Ijabani et al. [18] reported P. aeruginosa, S. aureus, S. epidermidis, B. subtilis, E. coli, K. pneumoniae, P. vulgaris and S. pyogenes as the bacteria associated with wound infections in a specialist hospital in Jimeta Yola, Adamawa State, Nigeria.P. aeruginosa was the most frequently occurring bacteria isolated from wound swabs in this study followed by S. aureus.Contrary to this study is the work previously done by Ijabani et al. [18] who reported S. aureus as the bacteria with the highest percentage occurrence in wound swabs of patients in Specialist Hospital, Yola, Adamawa State.A recent report by Rashid et al. [27] revealed that P. aeruginosa is one of the most common bacteria and is the cause of nosocomial infection and acquired drug resistance and it accounts for 11% of Hospital Acquired Infections and diseases, particularly in an immunocompromised individual. known