seahi publications, 2017...

31

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

http://www.seahipaj.org/

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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.

Akinjogunla & Fatunla .….. Int. J. Innovative Biosciences Res. 5 (1):31-47, 2017

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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)


Serratia marcescens 2 6.9

Enterobacter spp 2 6.9


Klebsiella pneumoniae 4 13.8


Proteus substilis 3 10.3

Total 29 100

Wound

(n=10)

Enterococcus faecalis 2 8.7




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



P. aeruginosa Wound PAW3 12.9±1.0 0.70 15.2±1.0 0.83 18.3±1.2 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



CON-Staphylococcus spp Urine CNU6 NZ - 12.2±0.3 0.86 14.2±0.3 NZ












S. aureus Wound SAW6 NZ - 10.5±0.1 0.60 17.4±1.0 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



CON-Staphylococcus spp Urine CNU6 11.2±0.5 0.79 15.2±0.5 1.07 14.2±0.3 NZ












S. aureus Wound SAW6 11.0±0.2 0.63 15.0±0.5 0.86 17.4±1.0 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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45

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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