FUDMA Journal of Microbiology Volume 2 number 2, eS Reh. 1, December 2019 p26 - 34 ISSN 2659-1545 JOURNAL OF MICROBIOLOGY (Georg) ANTIBACTERIAL ACTIVITY OF Entada sudanica LEAF AND STEM BARK EXTRACTS AGAINST CLINICAL MULTI BACTERIAL Yusha’ DRUG RESISTANT GRAM NEGATIVE ISOLATES , M.! and Mansir, 1.? Department of Microbiology, Bayero University, P.M.B, 3011 Kano Nigeria Department of Biological Sciences, Federal University of Kashere, P.M.B 0182 Gombe State ABSTRACT Medicinal plants are used worldwide for the treatment of several human ailments including bacterial infections. This study is aimed to evaluate antibacterial activity of £. sudanica extracts against clinical multi drug resistant Gram negative bacterial isolates. Dried leaves and stem barks of E. sudanica were extracted with n-hexane and Methanol using cold maceration method. The extracts were screened for the presence of secondary metabolites using standard procedures. A total number of 87 isolates of E. coli, P. aeruginosa, and K. pneumoniae were collected from specialist hospital Gombe. Identification of bacterial isolates was done using standard biochemical procedures. Sensitivity profiles of the isolates were determined by Kirby Bauer (Disc Diffusion) method and multi drug resistant isolates were tested for sensitivity to the plants extracts using agar well diffusion method. Thin layer chromatography was carried out on each of the extract fractions to separate compounds. Antibacterial activity of separated compounds were carried out using agar overlay method, Phytochemical screening of the plants extracts demonstrated the presence of _ secondary metabolites including; Tannins, flavonoids, anthraquinones, steroids, alkaloids, reducing ‘sugars, cardiac glycosides, saponins, Out of 87 isolated organisms, 24 were E. coli, 28 were P. aeruginosa and 35 were K. pneumoniae, Amongst the isolated organisms, the highest percentage of occurrence of confirmed MDR was observed in E. coli with 12.5% followed by P. aeruginosa, with 10.7% while K. pneumoniae had the least occurrence of 8.6%. From the results of antibacterial activity, stem bark methanolic extracts of E sudanica showed the highest activity against £ coli at 20mg/ml with 19.741.Smm. Thin layer chromatography separation of stem bark provided a maximum of five compounds best visualized under UV light, Agar overlay bioautography of the most active spot amongst Yusha'u and Mansur (2019) 2% separated spots demonstrated only one spots with Re-value of 0.87, and had inhibitory activity agai E.coli with ‘7mm zone of inhi antibacterial activity tests showed that methanolic stem bark extracts of the plants had higher inhibition against the test organisms. Keywords: £. sudanica, E. coli, P. aerugunosa, K. pneumoniae, Kirby Bauer, Multi drug resistant, Clinical Bacterial isolates, Thin layer chromatography, Bioautography INTRODUCTION Herbal medicine, sometimes referred to as ‘Herbalism’ or ‘Botanical medicine’, is the use of herbs for therapeutic or medicinal value. A herb is a plant or plant part valued for its medicinal, aromatic or savory qualities (Kawo ef al., 2011). Herbaceous plants produce and contain a variety of chemical substances that act upon the body for the cure of infections of microbial origin. Many drugs commonly used today are of herbal origin. Indeed, about 25% of the prescription drugs dispensed in the United States contain at least one active ingredient derived from plant material. Some are made from plant extracts; others are synthesized to mimic a natural plant c eae plant compound (Reagor ef Plant-derived produets have been used for medicinal purposes for centuries. At present, itis estimated that about 80.0% of the world Population relies on botanical preparations 4s medicines to meet their health care needs. Herbs and spices are generally considered sale and proved to be effective against certain ailments. They are also extensively used particularly in many Asian, African and other countries (Langner et al, 1998).FUDMA Journal of Microbiology Volume 2 number 2, December 2019 pp26 - 34 Entada sudanica (Tawatsa) is a perennial plant that grows to heights of 4 — 10 m and 90 cm in girth. It is predominantly a West eae Plant that is quite valuable heaves ate y in the treatment of many Sateigy st etude but not tmited to fever, upset and eee arrow poison, stomach Peer eee larthea (Tibi etal, 2010), The - xtracts serve as a tonic and a stimulant while extracts of the stem bark Possess abortifacient properties. The leaves are used for wound dressing among Nigerians and the Ghanians. Literature studies had shown the existence of several classes of bioactive phytoconstituents such as alkaloids, flavonoids, cardiac glycosides, steroids, saponins, terpenoids, coumarins and polyphenols in the methanol extract of E. africana stem bark, Despite the numerous traditional medicinal uses of E. africana, only few reports are found in literature (Montoro et al., 2005). It is also quite important to carry out the phytochemical investigation of E. sudanica in Nigeria, given geographical, regional and seasonal variations in phytoconstituents and their therapeutic efficacy even for the same plant species (Jiofack ef al., 2010). The evaluation of phytochemical constituents, the antibacterial activity and antioxidant property of E, sudanica is necessary to determine some of its specific medicinal values for possible commercial exploitation and hence the needs for this study. ‘Antibacterial therapeutic failure due to the emergence of resistant bacterial strain is a worldwide phenomenon. So, many efforts have been done to enhance antibacterial effectiveness from sources such as plants that have become a necessity to overcome emergent of bacterial resistance in clinical practice (NIAID, 2006). MATERIALS AND METHODS Collection and Identification of Plant Materials The fresh leaves and stem bark of Entada sudanica were collected from various part of Gombe State. The leaves were identified at the botany herbariums with voucher number Yusha'u and Mansur (2019) ISSN 2659 - 1848 157 at the Department of Biological Science, Gombe State University.A soft brush were used to remove any dirtdebris from the plant materials as described by Sarker ef al. (2006). Extraction of Plant Materials Two different organic solvents with varying polarities (high, and low polarity) were used in order to exploit the varying solubility of the plant constituents Sarker ef al. (2006). The two organic solvents chosen were; 1- hexane (low polarity solvent), and hundred percent Methanol (high polarity solvent). Exhaustive extraction using cold maceration procedure was used so as to extract as many compounds as possible from the plant materials adopting the method of Sarker (e al., 2006) as described below; Fifty gram of the pulverized plant materials were soaked separately in different closed conical flasks containing 250mls each, of the two solvents separately at room temperature. ‘The flasks were then kept for 2 weeks, so as to ensure exhaustive extraction. After the 2 weeks, the residual plant material was separated from the solvent by decanting and filtration, and then centrifugation. The filtrate was evaporated to dryness at 40°C using water bath, and then stored in air-tight containers in a refrigerator at 4°C before use. Phytochemical Materials The phytochemical analyses were carried out using standard screening test described by Aiyelaagbe and Osamudiamen, (2009). Collection and Identification of Test Organisms The isolates of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were collected from Specialist Hospital Gombe. The isolates were then be transported within 24hrs to Microbiology Laboratory at Gombe State University. The isolates were then subculture and confirmatory tests were carried out such as Gram staining and other biochemical such as Indole, Oxidase, Urease, Citrate Methyl red test as described by Cheesbrough (2006). Screening of PlantFUDMA Journal uf Microbiology Volume Determination of resistance among the isolates This were done using the Kirby Bauer Disc diffusion method with reference to the Clinical Laboratory Standard Institute (CLS1 performance guidelines for antimicrobial susceptibility testing (CLSI, 2012) Bacteria grown in nutrient broth at 37°C overnight. The suspension were Visually adjusted with normal saline to match 0.5 Maefarland (1.5 X 10°CFU/mL) turbidity standard, Each inoculum was Separately swabbed across the entire surface of Muller Hinton agar plate (Biotech) using sterile swab and the plate were rotated approximately 60°C between streaking to ensure even distribution, Inoculated plates Were left to stand for at least 3 minutes before commercial antibiotics disks (Abtex Biological Ltd) used: Ceftazidime 30yg), Cefuroxime (30g), Cefotaxime (302), Gentamicin (10ug), Ciprofloxacin (Sug), Ofloxacin (Sug), Augumentin (30g), Nitrofuratoin (300g), Ampicilin (10g) and Imipenem (10ug) is placed and incubated within 15 minutes of the application of the disks at 37°C for 24 hours. The inhibition zone diameters around the disks were measured and interpreted according to the CLSI guideline. An isolate considered as MDR if it is resistant to three or more drugs of different classes/group of antibiotics (CLSI 2012). Antibacterial Activity Tests Activity testing of all Crude Extracts This assay was conducted using agar-well diffusion method of Okeke ef al. (2012) as described below. 40 mg/ml concentration of each of the different extracts were constituted by dissolving 0.04g in 2 ml each of 20% viv dimethyl sulfoxide (DMSO) and resultant concentrations were obtain 20, 10, 5, 2.5mg/ml. A portion colony of the test isolate was suspended in 2mls of sterile Muller Hinton Broth, The suspension of the isolate was standardized as stated previously, and used to inoculate the surface of the Muller Hinton agar and the excess fluid was drained into disinfectant jar. The ‘Yusha’u and Mansur (2019) 2 number 2, December 2019 pp26 - 34 | ISSN 2659 - 1545 inoculated agar surfaces Were allowed iy 4 and the plates were appropriately labelleq Using a cork borer, four wells of 6 mm i, diameter were bored in the inocula, Muller Hinton agar. Using a micropiper Sq of each concentration of the 4! extracts were delivered into each well, Th. plates were left on the bench for 30 minyes to allow the extracts to diffuse into the aga, Thereafter, the plates were incubated 9 37°C for 24h. After incubation, the plates were observed for inhibition zones aroung the wells. The diameters of the zones vere measured with meter ruler to the neaes, whole millimeter. Each test were carried oy, thrice and the mean inhibition zone diameter were recorded to the nearest whole millimetre. The test was done separately for all the different plant extracts, Minimum Inhibitory Concentration This test was carried out for extracts that had shown highest activity in the sensitivity tests carried out. The test was carried out using agar dilution method following the procedure outlined by (CLSI, 2012). Foury mg of the extracts (0.04g) was weighed and dissolved in 2mls of 20% viv DMSO to geta stock solution with concentration of 20mg/ml. Sterile test tubes were arranged in a test tube rack and 1 ml of distilled water dispensed into each of them. From the stock solution, Iml was transferred into the first test tube and serial dilution of the extract was carried out until the resultant Concentrations in the test tubes became 20, 10, 5, 2.5, 1.25 and 0.625 mg/ml, Iml each of the extract dilutions was added to separate 19mis of sterile molten Mueller Hinton aga, mixed thoroughly and poured into separate sterile Petri dishes. The plates were allowed Se, and then labelled appropriately. A Fran Colony of the test isolate was picked and ineeateeltred plate with wire loo? to make 4 ne into 2ml sterile normal saline suspension Pension of the test isolate. The Devi Ue Said ass standardized en @ micropipette, 10pl of a Wien Ge ie culture was placed on oe Sof the extract combinatio® " bhFUDMA Journal of Microbiology V1 Plain Mueller Hinton agar (that is, without the extract) was also inoculated and served as negative control. Inoculated plates were incubated at 37°C for 24 h and observed for any visible bacteria growth. MIC was taken as the lowest concentration of extract that resulted in no visibl . le Pre growth on the surface he idal Concentration ee eae Bactericidal Concentration of ditution method cae aes leo (2012), 8 follows; et bY CLSL After completion of the MIC procedure, the agar plates showing no growth in the MIC tests were used for the determination of the MBC. Blocks were cut out from the plates that showed no growth in the MIC test and transferred to corresponding test tubes of fresh nutrient broth that acted as the recovery medium. One test tube containing nutrient broth was left blank to serve as control. Turbidity of the newly inoculated broth media was checked, and then the tubes incubated for 24h at 34°C. At the end of incubation, microbial growth —_was ascertained by checking the turbidity of the media in comparison with the blank nutrient broth test tube. The absence of change in turbidity in the recovery media was used as evidence of total cell death, and vice-versa. Separation of Suspected Most Active Phytochemical Groups Using Thin Layer Chromatography (TLC) Using a method described by Mallikharjuna et al. (2007). Silica gel Precoated TLC ‘Alugram®Sil aluminium plates (20 x 20cm) were used. A line was drawn (3cm from base) at opposite ends of all TLC plates, 125ul of the extract combination (20mg/ml) was then placed on 2 separate positions on 1 line of all TLC plates and allowed to dry with the spots formed not overlapping. 30 mls of each mobile phase was then put in separate 1000ml glass beakers and _ labelled accordingly. One TLC plate was then put into each of the beakers with the mobile Yusha’ and Mansur (2019) ‘olume 2 number 2, December 2019 pp26 - 34 ISSN 2659 - 1545 phase not touching the extract spots and then the beakers were covered with aluminium foil and masking tape to prevent solvent escape. Afier 3h, the TLC plates were removed and allowed to dry for 30 mins. Bands of separation were visible on all TLC plates under normal light. The color and Rr values were recorded for each band. Sensitivity Testing of the Separated Compounds This was done using agar overlay method as described by Runyoro et al. (2006). The TLC plates were used. Molten Mueller- Hinton agar (100 mls) was seeded with 150 ul from a standardized 24 hours broth culture of the test organism and shaken properly. The range of all the separated phytochemical groups were then cut from the TLC plates using scissors and placed inside separate petri dishes and the plates labelled accordingly. Twenty mls of the seeded molten agar was then poured onto each of the plates, allowed to solidify, and then incubated for 24 h at 37°C, The average zones of inhibition around each spot was recorded (Runyoro ef al., 2006). RESULTS In Table 1 the result of phytochemical analysis of methanol extracts showed that E. sudanica leaf extract tested negative for only steroids, reducing sugars, and cardiac glycosides but tannins, flavonoid, anthraquinones, alkaloids, and saponins tested positive. The E. sudanica stem bark extracts tested positive for tanins flavonoids, steroid alkaloids. cardiac glycosides and saponins, but tested negative for anthraquinones, reducing sugars. Hexane extracts shows that E. sudanica leaves extracts tested positive for only tannins flavonoids, steroid and saponins but tested negative for anthraquinones, alkaloids, reducing sugars, cardiac glycosides. The E. ‘sudanica stem bark extracts tested positive for only tannins, flavonoids, and anthraquinones but tested negative for ‘alkaloids, reducing sugars, and cardiac glycosides, steroids, and saponinse -34 ISSN ie FUDMA Journal of Microbiology Volume 2 number 2, December 2019 pp26- 3 ISN 2659 1545 Table 1: phytochemical constituents of the Leaf and Stem Bark extracts of E, sudanicg tochemicals Phytoc! St Al Rs Ce 3 Extract TF __Aa ES ML +t : ES MS + ot + oe ¥ ES HL AG e a - "= ES HS Cee : ae =e Key: ES Mi.=Entada sidanica methanol Teal, ES MS= Entada sudanica methanol stem bay, ES HL= Entada sudanica hexane leaf, ES HS= Entada sudanica hexane stem bar, esent, - =not present T=Tannins, F=flavonoids, Aq=Anthraquinones, S=Steroid. ‘Alkaloids, Rs=Reducing sugars, Cg=Cardiac glycosides, S=saponins, The percentage of occurrence of MDR in test bacterial isolate is presented in a Table 2. Oy, of 87 isolate screened for MDR, 9 were confirmed as MDR giving overall percentage of occurrence 10.3%. The highest percentage of occurrence were found in E. coli with 12.5% followed by P. aeruginosa with 10.7% while K. pneumoniae had 8.6% Table 2: Percentage of occurrence of MDR in test of bacterial isolate Isolates ‘Number of sample Number (%) of MDR sereened isolates E.coli 24 3(12.5) P. aeruginosa 28 3(10.7) K. pneumonia 35 3(8.6) Total 87 931.8) Antibacterial activity methanolic extracts also E, sudanica stem bark extracts had of E. sudanica against MDR isolates The result shows that 20mg/ml, 10mg/ml, Smg/ml and 2.5mg/ml concentrations of methanolic extract of E. sudanica against E. coli, The E. sudanica leaf extracts had produce zone of inhibitions of 18.0+1.0mm, 14.0£1.5mm, 9.7£1.0mm, and 7.0£1.0mm also E, sudanica stem bark extracts had produce zone of inhibitions against the same isolate with 19.741.5mm, 15.3+1.0mm, 8.7£1.2mm, and 7.7+1.0mm, respectively the positive control which is gentamycin used had 27mm zone of inhibition. The same table reveal that 20mg/ml, 10mg/ml, Smg/ml and 2.5mg/ml concentrations of methanilic extract of E, sudanica against P. aeruginosa, The E. sudanica leaf extracts had produce zone of inhibitions of 14.5#1.0mm, 13.041.2mm, 7.7+1.2mm, and 8,0£1.2mm 30 Yusha’u and Mansur (2019) produce zone of inhibitions of 17.71.2mm, 18.3£1.5mm, 8.3£1.0mm, and 70+1.0mm, respectively the positive control which is gentamycin used had 27mm zone of inhibition, The table also shows that 20mg/ml, 10mg/ml, Smg/ml and 2.5mg/ml concentrations of methanolic extract of E sudanica against K. pneumoniae, The E. sudanica leaf extracts had produce zone of inhibitions Of 11.041.2mm, 10.71.2mm, sudanicn ve Mt 7-7#1.2mm also zone of Nt atk extracts had product 14.021, 0mmm i tibitions of 16,02:1.0mm. respectiven” 8.0£1.0mm, and 7.0£1.3mm, gentarn ics the positive control which is cane im used had 27mm zone o is Disa be all the negative control which ee the aot exhibited and activityFUDMA Journal of Table 3: Antibacterial acti Volume 2 number 2, of methanolic extraets of E. sudanica against MDR December 2019 pp26-34 ISSN 2659- 1545 isolates Extracts Concentration (mg/ml) zone of Inhibition (mm) Ctrl gen MDR Isolates 20 10 5 25 40ug ESML E.coli 18.0810 T4321.5 9321.0 7,021.0 26 ESMS E.coli 19.7815 15.0410 8741.2 7.081.027 ES ML P. aeruginosa 14.3415 13.341.2 7541.2 8721.2 26 ES MS P. aeruginosa 17.341.2 18.341.5 8.0210 7.041.026 ESML K. pneumonia 113412 10.741.2 8.01.0 7.741.228 ESMS_K.pnewmonia 16.7815 143415 8.0810 77412 28 Key: ES ML=i Gen= Gentamycin, Antibacterial activity of hexane extracts of E, sudanica against MDR isolates The result shows that 20mg/ml, 10mg/ml, Smg/ml and 2.5mg/ml concentrations of individual hexane extract of E, sudanica against E. coli, The E. sudanica leaf extracts had produce zone of inhibitions of. 12.0£1.0mm, 9.0£1.0mm, 8.7+1.0mm, and 1.0mm also E. sudanica stem bark tracts had produce zone of inhibitions against the same isolate with 13.7+1.2mm, 93£1.0mm, 8.7+1.2mm, and 7.7£1.2mm, respectively the positive control which is gentamycin used had 27mm zone of inhibition. The same table reveal that 20mg/ml, 10mg/ml, Smg/ml and 2.5mg/ml concentrations of individual hexane extract of E. sudanica against P. aeruginosa, The E. sudanica \eaf extracts had produce zone of inhibitions of 12.021.0mm, 9.01.0mm, 8.721.2mm, and 7.0+1.0mm also E. ntada sudanica leaf, ES MS= Entada sudanica stem bark, Ctl= Control, sudanica stem bark extracts had produce zone of inhibitions of 13.7#1.2mm, 9.341.0mm, 8.7£1.2mm, and 7.7+12mm, respectively the positive control which is gentamycin used had 27mm zone of inhibition, The table also shows that 20mg/ml, 10mg/ml, Smg/ml and 2.Smg/ml concentrations of individual hexane extract of E. sudanica against K. pneumoniae, The E, sudanica leaf extracts had produce zone of inhibitions of 11.0#1.0mm, 9.07=1.0mm, 8.0+1.0mm, and 7.7#12mm also E sudanica stem bark extracts had produce zone of inhibitions of 11.051.0mm, 9.041.0mm, 8.0+1.0mm, and 7.0=1.3mm, respectively the positive control which is gentamycin used had 27mm zone of inhibition and all the negative control which is DMSO does not exhibited and activity against all the isolate. Table 4: Antibacterial activity of hexane extracts against of E, sudanica MDR isolates Extracts Concentration (mg/ml) zone of Inhibition Ctrl, gen (mm) 40 ng MDR Isolates 20 10 5 2 ESHL E.coli 12.021.0 9.01.0 8781.2 7.01.0 27 ESHS — E.coli 13.741.2 9,341.0 8.7212 7.081.027 ESHL P. aeruginosa 12.01,0 8,741.2 7,041.0 7.081.027 ESHS —P.aeruginosa —15.741.0 10,7412 8,041.0 8.741.227 ESHL K pneumoniae 11.041.0 9.0410 8,041.0 7,741.2 27 ESHS _K pneumoniae _113+1,0__9.3+1.0___8.7#1.2__ 8.741.227 Key: ES HL= Entada sudanica leaf, ES HS= Entada sudanica stem bark, ct Yusha'u and Mansur (2019)FUDMA Journal of Microbiology Volume 2 Minimum Inhibitory and Bactericidal concentration (MIC/MBC) of E. sudanica methanolic stem bark (ES MS) extracts The results of MIC and MBC test of E. Sudanica stem bark methanol extracts. Table > below reveal that the MIC ES MS against E. coli at 1.25mg/ml while MBC at Smg/ml Table 5: Minimum inhibitory and_bacte Sudanica methanolic stem bark extracts umber 2, December 2019 pp26-34 ISSN 2089-1545 against the same isolate, and the Mic against P. aeriginosa at 1.25mg/ml while MBC at 2.5mg/ml, and it also shows the MIC against K pneumoniae at 2.Smpim) while MBC at Smg/ml. There was also ng growth on the control plate containing gentamycin, ricidal concentration (MIC/MBC) of g Isolates MIC mgiml MBC mg/ml E. coli 1.25 5 K. pneumonia 25 5 P. aeruginosa 1.25 2.5 Thin layer chromatograhy and _— phytochemical groups from the ESMS antibacterial profile of E. sudanica combination shown in Table 6 shows four methanolic stem bark extracts. The results of thin layer chromatography (TLC) separation of suspected most active bands were evident in the TLC plate visualized under UV light Table 6: Thin layer chromatograhy and antibacterial profile of E. sudanica methanolic stem bark extracts, Mean zone of Inhibition (mm) R Values Colour of spot E.coli K. pneumoniae __P. aeruginosa, 0.83 dark green 6 3 | 0.87 Green 5 4 0.70 dark green 4 6 | 0.74 Green 5 4 | DISCUSSION The overall prevalence of MDR in this study was 31.8%, this was however lower than the findings of Ezeonu er al. (2007) who reported overall prevalence of MDR to be 39.2%, the results obtained from this study were higher than those shown by Aibinu er al. (2003) who repotted MDR_ 20.8% in E. coli and Klebsiella spp in Lagos, Nigeria, The highest MDR strain were £. coli (12.5%) followed by P. aeruginosa (10.7%) the least among them is K. pneumoniae which is (8.6%). These variations could be due to difference in sample size, local antibiotics and prescribing habits, antibiotic selection pressure which differ from state to state, institution to institution and from country to country. The antibacterial activity of plants against the tested organisms (MDR E. coli, P. Yusha’u and Mansur (2019) 32 rT aeruginosa and. pneumoniae) respectively. The results from the Tables revealed that all the parts of the plants used showed antibacterial activities against the {ested organisms with the methanol extracts of the plants having the highest activities in both tables, while hexane extracts showed the least antibacterial activities in some parts of the plants used at different concentrations This may be due to its low polarity in nature which make it impossible to extract all the Phytocompounds Present in the parts of the Posen the parts of the plants used, em barks of plants showed the highest vities against the tested (2017) who reported stem bark of antibacterial that the methanolic Sudanica showed strong activities against — isolatesFUDMA Journal of Microbiology Volume 2 number 2, Decemb The broad spectrum activity of the methanolic bark barks extracts of £ fudanica reported in this study are similar to feports that_showed similar antibacterial hetivities of Entada tree plant against pathogenic bacteria The results obtained for the extracts showed that the growth of both MDR E. coli and Klebsiella pneumoniae were inhibited at concentrations ranging from 2.5mg/ml to Smg/ml respectively and were killed at concentrations ranging from 2.Smg/ml to Smg/ml respectively. The MIC test for the extract combination showed that the ES MS. had an MIC values of 2.Smg/ml for both MDR E. coli and Klebsiella pneumoniae respectively, which is lower than the MIC reported by Aliyu et al. (2008). In the agar-overlay bioautography assay, the antibacterial activity of the compounds separated on TLC were determined. Significant antibacterial activity against MDR isolates was demonstrated by compounds with Ry value of 0.87 on agar overlay bioautography, as evident by the significant clear zone of inhibition on the spot. REFERENCES Abbiw, D. K. (1990). Useful Plants of Ghana: West African Uses of Wild and Cultivated Plants, Intermediate Technology Publication and the Royal Botanic Gardens, England 343-432. Aiyelaagbe, O. ., and Osamudiamen, P. M., (2009), Phytochemical Screening for Active Compounds in Mangifera indica Leaves from Ibadan, Oyo State, Plant Sciences Research, 2(1):11-13. Aibinu I. E, Ohaegbulam V. C, Adenipekun E, O, Ogunsola F. T, Odugbemi T. O, Mee B. (2003). Extended- Spectrum Beta-Lactamase Enzymes and MDR in clinical isolates of Enterobacter species from Lagos, Nigeria Journal of Clin. Microbiol; 41: 2197-200. Aliyu, A. B, Musa, A. M, Abdullahi, M. S., Oyewale, A. O, and Gwarzo, U.S Yusha’ and Mansur (2019) ISSN 2689 - 1545 2019 p26 = 34 CONCLUSION The phytochemical screening of Entada sudanica extracts revealed that the plants are rich in secondary metabolites including:Tannins, flavonoids, anthraquinones, steroids, alkaloids, reducing sugars, cardiac glycosides, saponins, and also methanolic extracts of both plants have highest percentage yield. Out of 87 isolated organisms, 24 were E. coli, 28 were P. aeruginosa and 35 were K. pneumoniae. ‘Amongst the isolated organisms, the highest percentage of occurrence confirmed MDR was observed in E. cali with 12.5% followed by P. aeruginosa, with 10.7% while K. ‘pneumoniae had the least occurrence of 8.6%. 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