seahi publications, 2017...
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In vitro Antibacterial Efficacies of Single and Combined
Aqueous Extracts of Ficus exasperata Vahl (Moraceae)
and Tetrapleura tetraptera Taub (Fabaceae) on
Multi-drug Resistant Bacterial Isolates
Akinjogunla, O. J. & Fatunla, O. K.
Department of Microbiology, Faculty of Science,
University of Uyo, P.M.B. 1017, Uyo, Akwa Ibom State, Nigeria
E-mail Address: [email protected]
ABSTRACT
The antibacterial efficacies of single and combined aqueous extracts of Ficus exasperata
(ALEFE) and Tetrapleura tetraptera (AETTP) on multi-drug resistant (MDR) bacterial isolates
were evaluated using disc diffusion and macro broth dilution techniques. Eleven bacterial species
belonging to 9 genera (Escherichia, Staphylococcus, Streptococcus, Serratia, Enterobacter,
Pseudomonas, Enterococcus, Klebsiella and Proteus) were obtained from the clinical samples.
The bio-active constituents detected in the ALEFE and AETTP were alkaloids, flavonoids,
tannins, cardiac glycosides, steroids, saponins, reducing sugar and anthraquinones. The discs
containing 40 mgml-1
ALEFE showed strong inhibitory effects on 58.8 % MDR bacterial isolates
and moderate inhibitory effects on 41.2 % MDR bacterial isolates. The discs containing 40 mgml-
1 AETTP exhibited strong inhibitory effects on 35.3 % MDR bacterial isolates and moderate
inhibitory effects on 64.7 % MDR bacterial isolates with Activity Indices (A.I) ranging from 0.60
to 1.02. MDR bacterial isolates tested were sensitive to growth inhibition of combined ALEFE
and AETTP at concentrations of 20 mgml-1
and 40 mgml-1
with inhibitory zone diameters and
A.I. ranging from 9.5 ± 0.1 mm to 18.3 ± 1.0 mm and 0.59 to 1.14, respectively. The combined
ALEFE and AETTP had synergistic effect against 12 (70.6 %) MDR bacterial isolates with
Growth Inhibitory Indices (GIIs) ranging from ≥ 0.53 to ≥ 0.63. The combined ALEFE and
AETTP had addictive effect against 4 (23.5 %) MDR bacterial isolates with GII of 0.50, while
antagonistic effect of combined ALEFE and AETTP was observed in 1(5.9 %) MDR bacterial
isolate with GII of ≤ 0.49. The MICs (mgml-1
) of ALEFE and AETTP singly ranged from 5 to 20
and 5 to 40, respectively, while the MIC values of combined ALEFE and AETTP were between
2.5 mgml-1
and 10 mgml-1
. The significant antibacterial efficacies of single and combined ALEFE
and AETTP in this study validated their use as antibacterial agents in Nigerian ethno-medicine,
and also suggest their consideration and utilization for production of more potent antibiotics.
Keywords: Ficus exasperata, Tetrapleura tetraptera, Synergistic, Addictive, Resistant,
Antagonistic, Isolates
INTRODUCTION Ficus exasperata Vahl, commonly called forest sand paper tree / plant, is one of the 800 species
of terrestrial plants in the family Moraceae (Odunbaku et al., 2008). F. exasperata inhabits the
secondary rainforest of West Africa and extensively spread in all eco-regions of Nigeria. This
plant is locally called Ewe Ipin (Yoruba), Opoto (Calabar), Anwulinwa (Igbo) and Ijikpi (Igala)
(Adebayo et al., 2009). F. exasperata has been ethnobotanically reported to have varied
therapeutic uses such as treatment / management of hypertension, epilepsy, arthritis (Buniyamin
et al., 2007; Lawal et al., 2009); haemostative, haemorrhoids, cough, intestinal pain and ulcer
(Odunbaku et al., 2008; Sonibare et al., 2008; Adebayo et al., 2009). The leaf is used to scratch
International Journal of Innovative Biosciences Research 5(1):31-47, Jan.-Mar., 2017
© SEAHI PUBLICATIONS, 2017 www.seahipaj.org ISSN:2354-2934
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skin parts affected by ringworm, while the grounded leaves applied topically are used to treat
boils. The viscid non-milky sap of this plant is used to stop bleeding, treat sores eye trouble and
stomach pains (Abbiw, 1990).
Tetrapleura tetraptera Taub is flowering, deciduous forest plant, belonging to the family
Fabaceae (Akin-Idowu et al., 2011; Akinjogunla and Oluyege, 2016). T. tetraptera is locally
called Aridan (Yoruba); Uyayak (Ibibio); Edeminang (Efik); Oshosho (Igbo) and Dawo (Hausa)
(Ojewole and Adewunmi, 2004). The pods of T. tetraptera comprise woody shell, a freshy pulp
and small brownish-black seeds with a pleasant and fragrant odour (Aladesanmi, 2007; Akin-
Idowu et al., 2011). In tropical Africa, the pods (fruits) of T. tetraptera are frequently and
therapeutically employed in the management and / or control of convulsions, asthma, epilepsy,
rheumatoid pains, hypertension, fevers (Ojewole and Adewunmi, 2004) and treatment of skin
infections, leprosy and gastrointestinal related clinical problems (Noamesi et al., 1994;
Gbadamosi and Obogo, 2013).
The resistances of microorganisms to antibiotics have been frequently reported in recent years
from all over the world, predominantly in developing countries, due to unselective use of
antibioitcs in the treatment of infectious diseases (Akinjogunla et al., 2011). Although, the
development of resistance by micro-organisms to antibiotics cannot be stopped, appropriate
action such as the use antibiotic resistant inhibitors of plant origin will significantly decrease the
mortality and health care costs (Ahmad and Beg, 2001). Scientists and pharmaceutical companies
are increasingly rummaging around for novel and efficient antimicrobial substances; turning their
attention to medicinal plants (Akinjogunla and Oluyege, 2016). Plants produce and contain a
diverse range of bioactive components such as alkaloids, tannins, flavonoids, cardiac glycoside
and phenolics (Newman et al., 2000; Akinjogunla et al., 2012).
Exploitation of plants, owing to its availability and affordability, as traditional remedies occupy a
fundamental place in developing countries particularly among a large proportion of low income
rural populace and many plants have shown to be highly helpful for treating sundry ailments
(Planta and Gundersen, 2000). The phenomenon of additive or synergistic effects is often vital to
bioactivity in plant extracts and in some cases; the activity is lost in purified fractions (Aqil et al.,
2006). Development of bacterial resistance to synergistic drug combinations, such as those found
in plants, may be slower than for single drug therapies (Cos et al., 2006). The study aimed at
investigating the antibacterial efficacies of single and combined aqueous extracts of F. exasperata
and T. tetraptera on multidrug resistant (MDR) bacterial isolates.
MATERIAL AND METHODS
Collection of Samples
Thirty (30) clinical samples comprising mid-stream urine (n = 10), stool (n = 10) and wound
swab (n = 10) samples were aseptically collected using sterile, wide-necked, leak-proof universal
bottles and commercially available swab sticks from patients attending a tertiary institution health
centre in Akwa Ibom State. The samples were properly labelled, kept on ice immediately after
collection and transported to the microbiology laboratory for bacteriological analyses.
Bacteriology of the Clinical Samples
One microlitre (μl) of each uncentrifuged, uniformly mixed, mid-stream urine (MSU) sample was
aseptically inoculated onto plates of Cysteine Lactose Electrolyte Deficient (Oxoid, UK). The
stool samples were serially diluted and 0.1 ml of the aliquot was inoculated onto plates of
MacConkey agar, eosin methylene blue agar and cetrimide agar (Oxoid, UK). Each of the wound
swab samples obtained was dropped into each test tube containing 9 ml of sterile distilled water
and was shaken vigorously. Zero point one (0.1) ml was pipetted, inoculated onto plates of
MacConkey agar, eosin methylene blue agar, nutrient agar, blood agar and mannitol salt agar
(Oxoid, UK). All the plates were aerobically incubated overnight at 37ºC. After incubation, the
colonies obtained were subcultured onto nutrient agar plates, incubated overnight at 37ºC, and
streaked onto nutrient agar slant. The isolates were characterized and identified using standard
conventional microbiological techniques.
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Antibiotic Susceptibility Testing
In vitro antibiotic susceptibility of bacterial isolates was determined using Kirby-Bauer disk
diffusion technique. Zero point one (0.1) millilitre of each bacterial isolate prepared directly from
an overnight agar plate adjusted to be equivalent to 0.5 McFarland Standard was inoculated onto
each of the Petri dishes containing Mueller-Hinton Agar (Oxoid, UK). The antibiotics tested
were: Penicillin (PEN, 10µg), Streptomycin (STP, 10µg), Ofloxacin (OFL, 5µg), Ceftriaxone
(CEP, 30µg), Nalidixic Acid (NA, 30µg), Gentamycin (GEN, 10µg), Pefloxacin (PEF, 5µg),
Augmentin (AUG, 30µg), Ciprofloxacin (CIP, 5µg) and Cotrimoxazole (COT, 25µg) (Oxoid,
UK) and were aseptically placed onto the surfaces of the culture plates with a sterile forceps,
gently pressed to ensure even contact and incubated aerobically at 37 °C for 18 hr. After
incubation, the diameters of inhibitory zones around the antibiotic discs were observed and
measured in millimeters using a ruler. The interpretation of the measurement as sensitive and
resistant was made according to the Clinical and Laboratory Standards Institute’s Guidelines.
Determination of Multiple Antibiotic Resistance Index
Multiple antibiotic resistance (MAR) index was determined using the formula MAR=x/y, where
‘x’ was the number of antibiotics to which test isolate displayed resistance and ‘y’ was the
total number of antibiotics to which the test isolates has been evaluated for sensitivity
(Krumpermann, 1983; Akinjogunla and Enabulele. 2010). Isolates that were resistant to three or
more antibiotics were taken to be multiple antibiotic resistant (Jan et al., 2002).
Sources of Medicinal Plants The leaves of F. exasperata and pods of T. tetraptera were obtained in Uyo, Akwa Ibom State.
The F. exasperata (leaves) and T. tetraptera (pods) were authenticated by a taxonomist in
Department of Botany and Ecological Studies and were later transferred to Pharmacognosy and
Natural Medicine Laboratory, Faculty of Pharmacy, University of Uyo for processing. The F.
exasperata (leaves) and T. tetraptera (pods) were separately washed with distilled water so as to
remove extraneous matters, air-dried at room temperature for one month, and pulverized using
mortar and pestle into fine powder. The aqueous extract of F. exasperata (leaves) was prepared
by soaking 2 kg of the powdered leaves into 1 litre of distilled water for 72 hrs with constant
shaking at room temperature. It was then filtered using Whatman No 1 filter paper, the extracted
liquid (filtrate) was evaporated to dryness with steam on water bath (45 °C), and the dried extract
was weighed and stored in a refrigerator at 5oC in screw capped bottle until required for use. The
same procedure was also repeated for powdered pods of T. tetraptera. The graded concentrations
(20 mgml-1
and 40 mgml-1
) of the extracts were aseptically prepared using 100 ml of Dimethyl
sulphoxide (DMSO), Aldrich, Milwaukee, WI, USA) and shaken vigorously to obtain a
homogenous mixture.
Antibacterial Efficacies of Single and Combined ALEFE and AETTP on MDR Bacterial
Isolates
The antibacterial efficacies of aqueous leaf extracts of Ficus exasperata (ALEFE) and aqueous
extracts of T. tetraptera pods (AETTP) on MDR bacterial isolates were determined by disc
diffusion method (Somchit et al., 2004; Sule et al., 2010). Mueller – Hinton Agar (MHA, Difco,
France) was sterilized, cooled to 45 – 50ºC and then poured into sterilized Petri dishes. Sterile
filter paper discs of 6 mm diameter were impregnated with single ALEFE and AETTP solution of
graded concentrations (20 mgml-1
and 40 mgml-1
) and carefully placed onto MHA agar plates
which had previously been inoculated with 0.1 ml of standardized inoculum suspension (106
CFU/ml of MDR-bacteria) using a sterilized forceps and the plates were aerobically incubated
overnight at 37ºC. Also sterile filter paper discs of 6 mm diameter were impregnated with the
combined ALEFE and AETTP of graded concentrations (20 mgml-1
and 40 mgml-1
) prepared in
1:1 by volume and carefully placed onto agar plates which had previously been inoculated with
0.1 ml of standardized inoculum suspension (106 CFU/ml of MDR-bacteria) and the plates were
then aerobically incubated overnight at 37ºC. Control experiments comprising Ciprofloxacin (5
μg / disc) and DMSO were also set up. Assays were performed in triplicate; the diameters of the
inhibitory zones were measured in millimeters and reported as the mean ± standard deviation
(mm ± SD). The ALEFE and AETTP were tested in vitro for purity by plating out on Petri dishes
containing nutrient agar and incubated over night at 37 oC. The scale of measurement used for the
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extracts were as follows: Strong Inhibitory Effect (≥15 mm); Moderate Inhibitory Effect (10-
14 mm) and Mild Inhibitory Effect (˂ 10 mm).
Determination of Growth Inhibitory Index
The growth inhibitory index (GII) was calculated as the mean inhibitory zone diameter (IZD) of
the combined extracts divided by the total mean inhibitory zone diameters (IZD) of the extracts in
single action (Mandal et al., 2010).
GII = IZD of the Combined Extracts
Total IZDs of the Extracts in Single Action
GII ˃ 0.5: Synergistic Activity; GII = 0.5: Additive Activity
GII ˂0.5: Antagonistic Activity.
Determination of Activity Index
Activity Index (A.I) was calculated as the mean inhibitory zone of extract divided by the mean
inhibitory zone of the standard drug used (Ciprofloxacin).
A. I. = Mean inhibitory zone of extract
Mean inhibitory zone of antibiotic (Ciprofloxacin)
A. I ˂ 1: The antibiotic has higher activity than the extract.
A. I = 1: The antibiotic and extract are equally effective.
A. I ˃ 1: The extract has higher activity than the antibiotic.
Determination of Minimum Inhibitory Concentration
The Minimum Inhibitory Concentration (MIC) of the ALEFE and AETTP singly and in
combination was determined for each of the test bacteria in test tubes using macro broth dilution
techniques (Akinyemi et al., 2005; Okwori et al., 2008). Four (4) grams of ALEFE and AETTP
were separately weighed and dissolved into 100 ml of the DMSO to give a concentration of 40
mgml-1
. The 40 mgml-1
was serially diluted by two-fold dilution with pipette to concentrations of
20, 10, 5 and 2.5 mgml-1
. To 0.1 ml of varying concentrations of the combined extracts (2.5, 5,
10, 20 and 40 mgml-1
) in test tubes, nutrient broth (9 ml) was added and then a loopful of the test
organism. A tube containing nutrient broth was only inoculated with the test organism to serve as
control. The same procedure was also repeated for combined ALEFE and AETTP. The culture
tubes were then incubated at 37 oC for overnight. After incubation the tubes were then examined
for microbial growth by observing for turbidity. The MIC was read as the least concentration
that inhibited the growth of the test organism. The antibacterial effect of the combined extracts
was considered synergistic if the MIC value of the combined extracts was lower than the MIC
value of any of the single extract. If the MIC value of the combined extracts was equal to any of
the single extract, the antibacterial effect was considered as additive and if the MIC value of the
combined extracts was higher than the value of any of the single extract, the antibacterial effect
was considered antagonistic.
Phytochemical Screening The phytochemical constituents of the ALEFE and AETTP were analyzed using the methods
described by Sofowora (1993); Trease and Evans (1996).
Test for Saponins: Half a gram (0.5 g) of the filtered ALEFE and AETTP was separately shaken
vigorously with 2ml distilled water in a test tube. Formation of frothing which persisted on
warning was taken as indication for the presence of saponins.
Test for Tannins Half a gram (0.5 g) of the ALEFE and AETTP was separately dissolved in 5 ml
of distilled water and then filtered. One millilitre (1 ml) of the filtrate was treated with a few
drops of % ferric chloride (FeCl3) reagent. Formation of a blue-
green precipitate indicated the presence of tannins.
Test for Cardiac Glycoside: Half a gram (0.5 g) of the filtered ALEFE and AETTP was
separately dissolved in 2 ml of Chloroform. Concentrated sulphuric acid (H2SO4) was carefully
added by running it down the side of the test tube. A reddish-brown colour at the interface
indicated a positive test.
Test for Phlobatanins: Half a gram (0.5g) of the ALEFE and AETTP was separately dissolved in
5ml of distilled water and then filtered. Two millilitres (2ml) of the filtrate was added to 2ml of
diluted HCl and thereafter boiled. Formation of a red precipitate indicated the presence of
phlobatanins.
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Test for Anthraquinones: Half a gram (0.5 g) of the ALEFE and AETTP was separately boiled
with 10 ml of 10 % sulphuric acid (H2SO4) and filtered. The filtrate was shaken with 5 ml
benzene and 10% ammonia (NH3) solution was added to the separate benzene layer. A pink red
or violet colouration in the ammonia (lower) layer indicated the presence of anthraquinones.
Test for Flavonoids: A few fragments of magnesium metal were added to 5 ml of ALEFE and
AETTP filtrate separately, followed by drop-wise addition of concentrated hydrochloric acid
(5 ml) to dissolve the extract. The formation of orange, red crimson or magenta colouration after
few minutes indicated the presence of flavonoids.
Test for Terpenes: Half a gram (0.5 g) of the ALEFE and AETTP was separately dissolved in
3 ml of chloroform and filtered. Ten (10) drops of acetic anhydride and 2 drops of concentrated
sulphuric acid (H2SO4) were added to the filtrate. Pink colour at the interphase was taken as
the positive test.
Test for Deoxy-Sugar: Half a gram (0.5 g) of the ALEFE and AETTP was separately dissolved in
5 ml of distilled water and then filtered. One millilitre (1 ml) of the filtrate was treated with a few
drops of Benedict’s reagent and heated on a water bath. The formation of an orange red
precipitate indicated the presence of reducing sugars.
Test for Alkaloids: Half a gram (0.5 g) of the ALEFE and AETTP was separately dissolved in
5ml of 5% Hydrochloric acid (HCl) and then filtered. One millilitre (1 ml) of the filtrate was
treated with a few drops of saturated picric acid solution. Formation of a yellow coloured
precipitate indicated the presence of alkaloids.
Test for Phenolics: One (1) ml of ferric chloride solution was added to 2 ml of the ALEFE and
AETTP separately. The presence of blue or green colour indicated a positive test.
Fig I: T. tetraptera (Taub) Pods Fig II: Ficus exasperata (Vahl) Leaves
RESULTS
The most frequent bacterial isolates from the MSU samples were Escherichia coli 7 (28.0%) and
Staphylococcus aureus 5 (20.0%). Proteus mirabilis 3 (12.0%), coagulase negative
Staphyloccocus spp. 2 (8.0%), Streptococcus pyogenes 3 (12.0%), Klebsiella pneumoniae 2
(8.0%) and Pseudomonas aeruginosa 3 (12.0%) were also isolated (Table 1). Seven bacterial
species belonging to 6 genera (Escherichia, Serratia, Enterobacter, Pseudomonas, Klebsiella and
Proteus) were obtained from the stool samples. The commonest bacterial isolates were E. coli 9
(31.0%) and P. aeruginosa 6 (20.7%). Other bacterial species from the stool samples with their
respective number and percentage of occurrence were: S. marcescens 2 (6.9%), Enterobacter
spp 2 (6.9%), K. pneumoniae 4 (13.8%), P. mirabilis 3 (10.3%) and P. substilis 3 (10.3%). Gram
negative bacteria were encountered more than Gram positive bacteria in the wound samples. The
occurrence and distribution of the bacterial isolates in the wound samples in increasing order was
as follows: E. faecalis 2 (8.7%), P. mirabilis 2 (8.7%), S. pyogenes 3 (13.0%), E. coli 4
(17.4%), S. aureus 5 (21.7%) and P. aeruginosa 7 (30.4%) (Table 1).
The multidrug resistance patterns of the bacterial isolates from the MSU, stool and wound
samples are presented in Table 2. The multidrug resistance patterns of P. mirabilis PMU5 and
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CON-Staphylococcus spp was PEN-STP-NA-PEF-AUG-COT and PEN-STP-CEP-NA-GEN-
PEF-AUG, respectively. E. coli ECS5 and E. coli ECS9 isolated from stool samples had
multidrug resistance patterns of OFL-CEP-NA-GEN-PEF-AUG-CIP-COT and CEP-NA-PEF-
COT, respectively. The two P. aeruginosa from wound samples were P. aeruginosa PAW3 with
multidrug resistance pattern of PEN-STP-OFL-CEP-AUG-GEN and P. aeruginosa PAW4 with
multidrug resistance patterns of OFL-CEP-NA-GEN-PEF-AUG-CIP-COT (Table 2).
The bio-active constituents detected in a very high concentration in ALEFE were: alkaloids (+++)
and flavonoids (+++). Tannins, cardiac glycosides and steroids were present in a moderately
high concentration (++), while the concentration of saponins and reducing sugar was low (Table
3). Flavonoid was the only bio-active constituent detected in a very high concentration in the
AETTP. Saponins and anthraquinones were present in moderately high concentration (++), while
the concentration of tannins, reducing sugar and cardiac glycosides was low. Phlobatanins was
not detected in both AETTP and ALEFE (Table 3).
The results of the susceptibility of the MDR bacterial isolates to ALEFE are presented in Table 4.
The widest inhibitory zone diameter (IZD) obtained was 17.1 ± 1.5 mm, while the narrowest IZD
was 9.5±0.1mm with activity indices (A.I) ranging from 0.59 to 1.07. The discs containing
40 mgml-1
ALEFE showed strong inhibitory effects on 58.8 % MDR bacterial isolates (IZD: ≥15
mm) and moderate inhibitory effects on 41.2 % MDR bacterial isolates (IZD: 10 to14 mm). The
discs containing 20 mgml-1
ALEFE showed moderate inhibitory effects on 88.2 % MDR bacterial
isolates (IZD: 10 to14 mm) and mild inhibitory effects on 11.8 % MDR bacterial isolates (IZD: ˂
10 mm) (Table 4).
Table 5 shows of the susceptibility of the MDR bacterial isolates to AETTP. The results showed
that 15/17 (88.2%) MDR bacterial isolates tested were sensitive to growth inhibition of 20 mgml-1
AETTP with IZD and A.I. ranging from 9.4 ± 0.2 mm to 13.6 ± 0.5 mm and 0.51 to 0.80,
respectively. The discs containing 40 mgml-1
AETTP exhibited strong inhibitory effects on
35.3 % MDR bacterial isolates (IZD: ≥15 mm) and moderate inhibitory effects on 64.7 % MDR
bacterial isolates (IZD: 10 to14 mm) with A.I ranging from 0.60 to 1.02, while the discs
containing 20 mgml-1
AETTP exhibited moderate inhibitory effects on 76.5 % MDR bacterial
isolates with IZD ranging from 10 to14 mm and mild inhibitory effects on 23.5 % MDR bacterial
isolates (ZI: ˂ 10 mm).
The antibacterial activities of combined ALEFE and AETTP on the 17 MDR bacterial isolates
using disc method are presented in Table 6. The results showed that 17/17(100%) MDR bacterial
isolates tested were sensitive to growth inhibition of combined
ALEFE and AETTP at
concentrations of 20mgml-1
and 40 mgml-1
with IZD and A.I. ranging from 9.5 ± 0.1 mm to 18.3
± 1.0 mm and 0.59 to 1.14, respectively (Table 6). The combined ALEFE and AETTP had mixed
interactions (synergistic, addictive and antagonism) on the MDR bacterial isolates. The combined
ALEFE and AETTP had synergistic effect against 12 (70.6 %) MDR bacterial isolates with GIIs
ranging from ≥ 0.53 to ≥ 0.63. The combination of ALEFE and AETTP had addictive effect
against 4 (23.5 %) MDR bacterial isolates with GII of 0.50, while antagonistic effect of combined
ALEFE and AETTP was observed in 1(5.9 %) MDR bacterial isolate with GII of ≤ 0.49 (Table
7).
The MIC values of the ALEFE and AETTP singly and in combination showed their antibacterial
activities on the bacterial isolates (Table 8). The MIC values of ALEFE ranged from 5 mg/ml (S.
marcescens SMS7, P. aeruginosa PAS2, P. substilis PSS3, E. coli ECS9 and P. aeruginosa
PAW3) to 20 mg/ml (S. aureus SAU10, CON-Staphylococcus spp CNU6 and S. pyogenes
SPW7). The MIC values of AETTP ranged from 5 mg/ml to 40 mg/ml, while the MIC values of
combined ALEFE and AETTP ranged from 2.5 mg/ml to 10 mg/ml. The results of the lower MIC
values obtained for the combination of ALEFE and AETTP showed their synergistic effects
against 12 (70.6%) MDR bacterial isolates, while combination of ALEFE and AETTP produced
additive and antagonistic effects on 4 (23.5%) and 1 (5.9%) MDR bacterial isolates, respectively
(Table 8).
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Table 1: Occurrence of Bacterial Isolates from Clinical Samples
Samples Bacterial Isolates No. of Occurence % of Occurence
Urine
(n=10)
Proteus mirabilis 3 12.0
Staphylococcus aureus 5 20.0
CON-Staphylococcus spp 2 8.0
Streptococcus pyogenes 3 12.0
Klebsiella pneumoniae 2 8.0
Pseudomonas aeruginosa 3 12.0
Escherichia coli 7 28.0
Total 25 100
Stool
(n=10)
Escherichia coli 9 31.0
Serratia marcescens 2 6.9
Enterobacter spp 2 6.9
Pseudomonas aeruginosa 6 20.7
Klebsiella pneumoniae 4 13.8
Proteus mirabilis 3 10.3
Proteus substilis 3 10.3
Total 29 100
Wound
(n=10)
Enterococcus faecalis 2 8.7
Proteus mirabilis 2 8.7
Pseudomonas aeruginosa 7 30.4
Escherichia coli 4 17.4
Staphylococcus aureus 5 21.7
Streptococcus pyogenes 3 13.0
Total 23 100
Key: CON: Coagulase negative
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Table 2: Multidrug Resistance Patterns of the Bacterial Isolates
Keys: PEN: Penicillin; STP: Streptomycin; CEP: Ceftriaxone; OFL: Ofloxacin; NA: Nalidixic Acid
GEN: Gentamycin; PEF: Pefloxacin; AUG: Augmentin; CIP: Ciprofloxacin; COT: Cotrimoxazole.
Bacterial Isolates Source Code Antibiotic Resistant Patterns MAR Index
P. mirabilis Urine PMU5 PEN-STP-NA-PEF-AUG-COT 0.6
S. aureus Urine SAU10 STP-OFL-CEP-NA-PEF-COT 0.6
CON-Staphylococcus spp Urine CNU6 PEN-STP-CEP-NA-GEN-PEF-AUG 0.7
E. coli Urine ECU5 CEP-NA-PEF-AUG-CIP 0.5
E. coli Stool ECS8 NA-PEF-AUG-COT 0.4
Enterobacter spp Stool ESS4 STP-CEP-NA-PEF-CIP 0.5
S. marcescens Stool SMS7 PEN-STP-OFL-CEP-NA-GEN-PEF 0.7
P. aeruginosa Stool PAS2 CEP-NA-PEF-AUG-CIP-COT 0.6
P. mirabilis Stool PMS8 STP-OFL-CEP-AUG-CIP-COT 0.6
P. substilis Stool PSS3 PEN-STP-OFL-NA-GEN-PEF-CIP 0.7
E. coli Stool ECS5 OFL-CEP-NA-GEN-PEF-AUG-CIP-COT 0.8
E. coli Stool ECS9 CEP-NA-PEF-COT 0.4
P. aeruginosa Wound PAW3 PEN-STP-OFL-CEP-AUG-GEN 0.6
P. aeruginosa Wound PAW4 OFL-CEP-NA-GEN-PEF-AUG-CIP-COT 0.8
S. aureus Wound SAW6 PEN-STP-NA-PEF-AUG 0.5
S. pyogenes Wound SPW7 NA-PEF-COT 0.3
E. faecalis Wound EFW5 CEP-NA-GEN-AUG 0.4
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Table 3: Phytochemical Constituents of Aqueous Extracts of F. exasperata Leaf and T. tetraptera
Pods
Plant Extracts Bio-Active Constituents Occurence
F. exasperata
Alkaloids +++
Flavonoids +++
Saponins +
Tannins ++
Cardiac Glycosides ++
Anthraquinones ND
Reducing Sugar +
Phlobatanins ND
Steroids ++
T. tetraptera
Alkaloids +
Flavonoids +++
Saponins ++
Tannins +
Cardiac Glycosides +
Anthraquinones ++
Reducing Sugar +
Phlobatanins ND
Steroids ND
Key: +++: Present in very high concentration; ++: Present in moderately high
concentration; +: Present in low concentration; ND: Not Detected.
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Table 4: Antibacterial Activities of ALEFE on Multidrug Resistant Bacterial Isolates
Bacterial Isolates
Source
Code
Inhibitory Zone
mm ±S.D
(20mg/ml)
A.I
Inhibitory Zone
mm ±S.D
(40mg/ml)
A.I
Ciprofloxacin
mm ±S.D
DMSO
mm ±S.D
P. mirabilis Urine PMU5 11.0±0.5 0.64 15.5±1.5 0.90 17.2±1.0 NZ
S. aureus Urine SAU10 10.2±0.7 0.62 13.1±0.8 0.79 16.5±0.5 NZ
CON-Staphylococcus spp Urine CNU6 9.9±0.5 0.70 14.2±0.5 1.00 14.2±0.3 NZ
E. coli Urine ECU5 12.4±1.0 0.87 14.9±1.0 1.05 14.2±1.0 NZ
E. coli Stool ECS8 13.4±1.0 0.73 16.2±1.2 0.89 18.3±1.5 NZ
Enterobacter spp Stool ESS4 11.8±0.5 0.77 15.4±0.5 1.01 15.2±0.5 NZ
S. marcescens Stool SMS7 12.1±1.1 0.59 16.0±1.1 0.78 20.6±1.0 NZ
P. aeruginosa Stool PAS2 12.0±0.4 0.62 15.6±0.5 0.80 19.5±1.0 NZ
P. mirabilis Stool PMS8 13.1±1.0 0.63 15.2±0.8 0.73 20.9±1.2 NZ
P. substilis Stool PSS3 12.0±0.5 0.62 16.4±1.0 0.84 19.5±0.5 NZ
E. coli Stool ECS5 10.3±0.5 0.74 14.9±0.5 1.07 13.9±0.2 NZ
E. coli Stool ECS9 13.3±0.8 0.72 16.0±0.5 0.87 18.4±0.5 NZ
P. aeruginosa Wound PAW3 12.9±1.0 0.70 15.2±1.0 0.83 18.3±1.2 NZ
P. aeruginosa Wound PAW4 14.5±1.2 0.66 17.1±1.5 0.78 21.9±1.5 NZ
S. aureus Wound SAW6 10.6±0.5 0.61 13.5±0.5 0.77 17.4±1.0 NZ
S. pyogenes Wound SPW7 10.3±0.2 0.67 13.2±0.5 0.86 15.4±0.5 NZ
E. faecalis Wound EFW5 9.5±0.1 0.59 12.9±0.2 0.81 16.0±0.5 NZ
Keys: A.I: Activity Index; NZ: No Zone of Inhibition; mm: mean; S.D: Standard Deviation; DMSO: Dimethyl Sulphoxide; Each inhibitory zone
included 6 mm diameter of the disc. Each value represents the mean of three replicates and standard deviation.
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Table 5: Antibacterial Activities of AETTP on Multidrug Resistant Bacterial Isolates
Bacterial Isolates
Source
Code
Inhibitory Zone
mm ±S.D
(20mg/ml)
A.I
Inhibitory Zone
mm ±S.D
(40mg/ml)
A.I
Ciprofloxacin
mm ±S.D
DMSO
mm ±S.D
P. mirabilis Urine PMU5 10.6±0.6 0.62 13.0±1.0 0.76 17.2±1.0 NZ
S. aureus Urine SAU10 9.6±0.2 0.58 12.9±0.5 0.78 16.5±0.5 NZ
CON-Staphylococcus spp Urine CNU6 NZ - 12.2±0.3 0.86 14.2±0.3 NZ
E. coli Urine ECU5 11.4±0.5 0.80 14.5±1.0 1.02 14.2±1.0 NZ
E. coli Stool ECS8 13.4±1.0 0.73 15.9±1.0 0.87 18.3±1.5 NZ
Enterobacter spp Stool ESS4 11.9±0.4 0.78 15.0±0.7 0.99 15.2±0.5 NZ
S. marcescens Stool SMS7 10.5±0.1 0.51 14.3±0.4 0.69 20.6±1.0 NZ
P. aeruginosa Stool PAS2 12.2±0.2 0.63 15.6±0.2 0.80 19.5±1.0 NZ
P. mirabilis Stool PMS8 12.7±0.5 0.61 15.4±0.5 0.74 20.9±1.2 NZ
P. substilis Stool PSS3 10.5±0.1 0.54 14.0±1.0 0.72 19.5±0.5 NZ
E. coli Stool ECS5 10.8±0.3 0.78 13.6±0.5 0.98 13.9±0.2 NZ
E. coli Stool ECS9 12.5±0.5 0.68 15.7±1.1 0.85 18.4±0.5 NZ
P. aeruginosa Wound PAW3 12.5±1.0 0.68 14.8±1.0 0.81 18.3±1.2 NZ
P. aeruginosa Wound PAW4 13.6±0.5 0.62 15.7±0.5 0.72 21.9±1.5 NZ
S. aureus Wound SAW6 NZ - 10.5±0.1 0.60 17.4±1.0 NZ
S. pyogenes Wound SPW7 11.5±0.2 0.75 12.2±0.2 0.79 15.4±0.5 NZ
E. faecalis Wound EFW5 9.4±0.2 0.59 12.3±0.1 0.77 16.0±0.5 NZ
Keys: A.I: Activity Index; NZ: No Zone of Inhibition; mm: mean; S.D: Standard Deviation; DMSO: Dimethyl Sulphoxide; Each inhibitory zone included 6
mm diameter of the disc. Each value represents the mean of three replicates and standard deviation.
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Table 6: Antibacterial Activities of Combined ALEFE and AETTP on Multidrug Resistant Bacterial Isolates
Bacterial Isolates
Source
Code
Inhibitory Zone
mm ±S.D
(20mg/ml)
A.I
Inhibitory Zone
mm ±S.D
(40mg/ml)
A.I
Ciprofloxacin
mm ±S.D
DMSO
mm ±S.D
P. mirabilis Urine PMU5 12.5±0.5 0.72 16.0±1.0 0.93 17.2±1.0 NZ
S. aureus Urine SAU10 11.5±0.2 0.70 14.5±0.8 0.88 16.5±0.5 NZ
CON-Staphylococcus spp Urine CNU6 11.2±0.5 0.79 15.2±0.5 1.07 14.2±0.3 NZ
E. coli Urine ECU5 14.7±1.0 1.04 15.7±0.3 1.10 14.2±1.0 NZ
E. coli Stool ECS8 13.4±1.0 0.73 16.0±1.0 0.87 18.3±1.5 NZ
Enterobacter spp Stool ESS4 14.9±0.7 0.98 16.9±0.5 1.11 15.2±0.5 NZ
S. marcescens Stool SMS7 15.0±1.1 0.59 16.5±1.0 0.81 20.6±1.0 NZ
P. aeruginosa Stool PAS2 12.2±0.4 0.63 15.7±0.2 0.81 19.5±1.0 NZ
P. mirabilis Stool PMS8 12.9±1.0 0.62 15.4±0.8 0.74 20.9±1.2 NZ
P. substilis Stool PSS3 13.0±0.5 0.67 17.1±1.0 0.88 19.5±0.5 NZ
E. coli Stool ECS5 12.9±0.4 0.93 15.8±0.5 1.14 13.9±0.2 NZ
E. coli Stool ECS9 12.5±0.8 0.68 15.6±1.2 0.85 18.4±0.5 NZ
P. aeruginosa Wound PAW3 15.1±1.0 0.83 18.3±1.0 1.00 18.3±1.1 NZ
P. aeruginosa Wound PAW4 14.9±1.0 0.68 17.8±1.0 0.81 21.9±1.5 NZ
S. aureus Wound SAW6 11.0±0.2 0.63 15.0±0.5 0.86 17.4±1.0 NZ
S. pyogenes Wound SPW7 12.4±0.2 0.81 14.6±0.4 0.95 15.4±0.5 NZ
E. faecalis Wound EFW5 9.5±0.1 0.59 12.7±0.2 0.79 16.0±0.5 NZ
Keys: A.I: Activity Index; NZ: No Zone of Inhibition; mm: mean; S.D: Standard Deviation; DMSO: Dimethyl Sulphoxide; Each
Inhibitory zone included 6 mm diameter of the disc. Each value represents the mean of three replicates and standard deviation.
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Table 7: Growth Inhibitory Indices (GIIs) from the Combined Activities of ALEFE and AETTP
against MDR Bacterial Isolates
Bacterial Isolates Codes GIIs Inference
P. mirabilis PMU5 ≥ 0.56 Synergy
S. aureus SAU10 ≥ 0.56 Synergy
CON-Staphylococcus spp CNU6 ≥ 0.57 Synergy
E. coli ECU5 ≥ 0.53 Synergy
E. coli ECS8 0.50 Addictive
Enterobacter spp ESS4 ≥ 0.56 Synergy
S. marcescens SMS7 ≥ 0.54 Synergy
P. aeruginosa PAS2 0.50 Addictive
P. mirabilis PMS8 0.50 Addictive
P. substilis PSS3 ≥ 0.56 Synergy
E. coli ECS5 ≥ 0.55 Synergy
E. coli ECS9 ≤ 0.49 Antagonism
P. aeruginosa PAW3 ≥ 0.61 Synergy
P. aeruginosa PAW4 ≥ 0.53 Synergy
S. aureus SAW6 ≥ 0.63 Synergy
S. pyogenes SPW7 ≥ 0.57 Synergy
E. faecalis EFW5 0.50 Addictive
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Table 8: Minimum Inhibitory Concentration of Single and Combined ALEFE and AETTP on Multi Drug Resistant Bacterial Isolates
Bacterial Isolates
Code
Minimum Inhibitory Concentration (MIC)
Inference F. exasperata (FE)
(mgml-1
)
T. tetraptera (TT)
(mgml-1
)
FE+TT
(mgml-1
)
P. mirabilis PMU5 10 20 5 Synergy
S. aureus SAU10 20 20 10 Synergy
CON-Staphylococcus spp CNU6 20 40 10 Synergy
E. coli ECU5 10 10 5 Synergy
E. coli ECS8 10 5 5 Addictive
Enterobacter spp ESS4 10 10 5 Synergy
S. marcescens SMS7 5 10 2.5 Synergy
P. aeruginosa PAS2 5 5 5 Addictive
P. mirabilis PMS8 10 10 10 Addictive
P. substilis PSS3 5 10 2.5 Synergy
E. coli ECS5 10 10 5 Synergy
E. coli ECS9 5 5 10 Antagonism
P. aeruginosa PAW3 5 5 2.5 Synergy
P. aeruginosa PAW4 10 10 5 Synergy
S. aureus SAW6 10 40 5 Synergy
S. pyogenes SPW7 20 10 5 Synergy
E. faecalis EFW5 10 10 10 Addictive
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DISCUSSION
The need to develop new antimicrobial agents and antibiotics arises from the fact that
microorganisms are developing resistance to many drugs and the death rates from infectious
diseases have increased tremendously (Lawal et al., 2012). The ALEFE and AETTP exhibited
antibacterial activities against both MDR Gram positive bacteria (S. aureus, S. pyogenes, CON-
Staphylococcus spp and E. faecalis) and MDR Gram negative bacteria (E. coli, P. aeruginosa,
Enterobacter spp., P. mirabilis, P. substilis). The results of the antibacterial activity of ALEFE on
bacterial isolates in this study is similar to the results of Takon et al. (2013). The widest
inhibitory zone was observed when the extracts were tested against multidrug resistant P.
aeruginosa. This widest inhibitory zone exhibited by extracts on MDR P. aeruginosa in this
study suggests that the extracts contained substances which when obtained and purified could
serve as a source of treatment of infections commonly associated with the microorganism. Strong
to moderate inhibitory effects of ALEFE and AETTP at different concentrations against the MDR
bacterial isolates were observed and this corroborated the previous reports of Joe et al. (2009) and
Iram et al, (2012) who reported excellent to moderate inhibitory effects of plant extract at
different concentrations against bacterial strains of E. coli and S. aureus. The MICs of the
combined ALEFE and AETTP on some of the MDR bacterial isolates were almost half of the
MICs of single extracts in this study and this agrees with Baljeet et al. (2015). Synergism
between plant extracts is a novel conception and could be advantageous (synergistic or additive)
or disadvantageous (antagonistic). In this study combined extracts was synergistic, additive and
antagonistic in their effect depending on the MDR bacterial isolates. The synergistic effects of
some plants such as C. sinensis and J. regia against MDR bacteria have been reported by
Farooqui et al. (2015).
The phytochemical screening of ALEFE and AETTP revealed the presence of alkaloids,
anthraquinones, tannins, flavonoids, steroids, reducing sugar, saponins and cardiac glycoside in
varied concentrations. The detection of tannin, cardiac glycoside and in AETTP is in conformity
with results of Achi, (2006). The presence of secondary metabolites in AETTP could provide a
synergistic effect which modifies the bioavailability, effectiveness of the active components and
might be responsible for its antibacterial activities. Flavonoids are hydroxylated phenolic
substances that are synthesized by plants in response to microbial infection. The activity of
flavonoids is possibly due to their ability to complex with bacterial cell walls and disruption of
the microbial membrane (Akinjogunla et al., 2009; Rattnachaikunsopon and Phumkhachorn,
2010). Tannins inactivate microbial adhesion; enzyme and cells envelop transport proteins
(Cowan, 1999). It has also been reported that tannins can precipitate the proteins covering the
surface of the cell or tissue, which acts as a barrier between tissue and irritants, and the
underlying tissue is therefore soothed and protected from further damage, so that healing of
wound can take place (Shivananda et al., 2007). The presence of saponins and cardiac glycosides
is an indication of medicinal significance of extracts. Saponins are glycosides of both triterpenes
and steroids having hypotensive and cardiac depressant properties, and have been detected in over
seventy plant families. Conclusively, this study has provided additional information and
validation on the use of plant extracts, especially ALEFE and AETTP, singly or in combination,
in the treatment of infectious diseases caused by MDR bacterial isolates.
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