The antimicrobial activity synergism

between eucalyptus honey, pomegranate,

date and antibiotics on Escherichia coli
causing diarrhea in children
Cite as: AIP Conference Proceedings 2144, 040003 (2019); https://doi.org/10.1063/1.5123104
Published Online: 23 August 2019

Ali A. Al-Hisnawi, Jassim M. Mustafa, and Yass K. Yasser

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© 2019 Author(s).
 The antimicrobial activity synergism between eucalyptus
honey, pomegranate, date and antibiotics on Escherichia coli
causing diarrhea in children.
Ali A. Al-Hisnawi1a), Jassim M. Mustafa 1), Yass K. Yasser 1)
1
Biology Department, College of Sciences, Kerbala University, Iraq
a)
Corresponding author: A. Al-Hisnawi, E-mail: ali.alhisnawi@yahoo.com

Abstract. The antibiotic resistance of bacteria has been on a steady rise and there is a desperate need to search for
substances with antimicrobial activity as an alternative treatment agents. In spite of the fact that the antimicrobial activity
of syrups has been extensively investigated against a wide range of microbes, it varies according to the type of these syrups.
The objective of this study was to verify the synergistic activity between the eucalyptus honey, pomegranate and date
syrups with antibiotics ampicillin and cefotaxime against Escherichia coli. The antibacterial activity testing of these syrups
and antibiotics was conducted by well diffusion technique using paper disks containing the antibiotic concentrations. In
addition, disk diffusion technique was used to test the activity of antibiotics alone against E. coli. Different concentrations
of syrups were studied against the bacterial strain tested. In both methods Nutrient agar and Brain heart infusion agar were
used. The tested isolate E. coli was supplied by Kerbala Public Health Laboratory, Iraq. The procedures of the current
study were conducted at postgraduate lab, Biology Department, College of Sciences, Kerbala University between
September and December 2017. E. coli was resistant against antibiotics or syrups alone. The synergistic action between
syrups and antibiotics showed antibacterial activity against tested bacteria. In conclusion, honey, pomegranate and date
syrups improved the activity of antibiotics used, and thus, confer assistance for the use of these antibiotics.

INTRODUCTION
Diarrhea is defined as liquid stools which are passed daily more frequently than is normal for the person who
infected by one or more diarrhea 's causative agents. Factors including insufficient drinking water, sanitation and
hygiene are considered to be important risk factors in particular in developed countries. The study of Hay, (2017)
stated that the number of diarrhea deaths in the eastern Mediterranean region in 2015 was 103,692 and the majority
of these deaths were in younger children totaling 65,670 deaths. Clean water, sugar, salt, zinc tablets beside anti-
infective drugs are important choices for treatment of diarrhea infections (Mulu et al., 2004).
However, due to the development and spread of resistant microorganisms, the activity of the antibiotics is reduced
(WHO, 1999). In addition, new antimicrobial agents were failed for developing by the pharmaceutical industry facing
new threats due to the rising costs of drug development and industry. There is desperate need to search for substances
with antimicrobial activity as alternative treatment agents.
Among natural compounds which are used with antimicrobial activity against a wide range of microbes are honey,
pomegranate and date. Compared to other alternative antimicrobial agents, honey has many special characteristics
including lack of any poisonous quality or side effects, low expense of maintenance, and local availability which
provide precious benefits to using honey as alternative antimicrobial therapy (Zainol et al., 2013). The antimicrobial
activity of honey is attributed to different agents including hydrogen peroxide, organic acid (citric and gluconic acids)
and flavonoids and phenolic acids (Weston, 2000, Viuda-Martos et al., 2008).
Water, carbohydrates, proteins, vitamins, amino acids, energy and minerals are considered to be the main
constituents of honey (Gweirif et al., 2015).
The fruit of pomegranate is a rich source of bioactive compounds, minerals, vitamins, polysaccharides and
polyphenols (Bagci, 2014), while pomegranate pericarp (peel, rind) and seeds hold numerous high-value compounds,

The 7th International Conference on Applied Science and Technology (ICAST 2019)
AIP Conf. Proc. 2144, 040003-1–040003-9; https://doi.org/10.1063/1.5123104
Published by AIP Publishing. 978-0-7354-1889-9/$30.00

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especially punicic acid, punicalgins, flavones, flavonones, and flavanols (Dhaouadi et al., 2016, Miklavčič Višnjevec
et al., 2017). Therefore, a wide range of health problems particularly inflammation, diabetes, diarrhea, bacterial
infections, cardiovascular disease, viral and immunosuppressive activities has been prevented and treated by
consuming pomegranate (Aqil et al., 2012, Lin et al., 2013, Orgil et al., 2016, Lytou et al., 2016, John et al., 2017,
Wang et al., 2010).
Date syrup, a food commodity locally known as “Debs” Molasses, is produced in Iraq, Libya and Tunisia from
some local date types. In addition, date syrup has been used for treatment of intestinal disorder (Taleb et al., 2016a),
skin medication (Bauza et al., 2002) also as an antimicrobial agent for wound healing (Tahraoui et al., 2007).
Aboubacar et al., (2010) reported that fruit of date has been used in the industrial production for food as a sweetening
alternative and in the production process of alcohol and beverages. Date syrup is rich in antioxidants (flavonoids,
phenolic acid, ascorbic acid and carotenoids) besides its nutritional compounds including carbohydrate, a good source
of minerals, amino acids, organic acids, polyphenols and carotenoids (Abbès et al., 2013).
The purpose of the current study therefore was to test the antibacterial activity of honey, date syrups and
pomegranate syrup alone or in combination with antibiotics (ampicillin or cefotaxime) in vitro against Escherichia
coli which was isolated from children under 5 years suffering from diarrhea.

MATERIALS AND METHODS

Natural Compound Syrups

Fresh date and pomegranate syrups and honey were purchased from local marked.

Tested Microorganisms
The tested isolate (Escherichia coli) was supplied by Kerbala Public Health Laboratory, Iraq.

Antibiotics
The following antibiotics were used in this study: Ampicillin (Am) 30mcg and Cefotaxime (CTM) 10mcg
(Bioanalyse®).

Preparation Of Syrup Solution

Working solutions of eucalyptus honey were prepared in a suitable volume of distilled water (D.W.) and the
concentrations required 5, 10, and 20 % for the experiments were prepared from this stock solution. The same steps
were followed with date and pomegranate syrups for preparing other concentrations 20, 40, and 80 % (Gweirif et al.,
2015).

Inoculum Preparation
Bacteria supplied were maintained on nutrient agar slant at 4 °C and they were subcultured onto nutrient broth and
incubated at 37°C for 24 hours. Serial tenfold dilution was prepared with phosphate buffered saline (PBS; pH 7.3;
Oxoid, UK) and the turbidity was adjusted by comparison of 0.5 McFarland standards (108 Colony Forming Unit
(CFU)/ml).

Antibacterial Activity
Two types of media Nutrient agar (N.A) and Brain heart infusion agar (B.H.A) were used to evaluate the activity
of the syrups against tested bacteria; this employed the agar-well diffusion technique. Bacterial isolates were prepared
to match 0.5 McFarland standards. Briefly, 100 μl of standardized suspension of examined bacteria 15×108 CFU ml-
1
spread on N.A and B.H.A plates. Plates were left at a room temperature for 15 minutes for adsorption. A 50 μL

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amount of the each diluted working syrup was transferred into each corresponding well in triplicate (four wells were
punched in each of the plate). At the same time 50 μl D.W was transferred to a negative control well. After 24 hours
incubation at 37 °C, the antibacterial activity of honey, date and pomegranate syrups were assessed by determining
the diameter of inhibition zone in centimeter (cm) against the examined bacteria (Taleb et al., 2016b).

Antibiotic Sensitivity Testing

The sensitivity of examined bacteria to antimicrobial agents were tested by employing two types of media (N.A)
and (B.H.A); this was carried out by employing the disk diffusion method using paper disks containing the following
antibiotic concentrations: Ampicillin 30mcg and Cefotaxime 10mcg. Before that, 100 μl of standardized suspension
of examined bacteria (108 CFU ml-1) were spread on N.A and B.H.A plates. Plates were left at a room temperature for
15 minutes for adsorption. Inhibition zones as representing antimicrobial activities were measured from edge to the
edge of inhibition of growth, the result was compared with the standard diameter of inhibition zones for each antibiotic
utilizing the method of Baccer et al., (1966).

Combination Between Syrups And Antibiotics

The antimicrobial combinations between each type of antibiotic and different concentrations of syrups were
examined by the agar diffusion method using paper disks containing the following antibiotic concentrations. Briefly,
100 μl of standardized suspension of examined bacteria (1.5×108 CFU ml-1) was spread on (N.A and B.H.A) plates.
Plates were left at a room temperature for 15 minutes for absorption. A 50 μl amount of a given dilution of the each
syrup was added to each antibiotic test paper disk and combination disks were added to the culture plates. The plates
were incubated aerobically at 37 °C for 24 h. Inhibition zones as representing antimicrobial activities were measured.

STATISTICAL ANALYSIS

Data were presented as means ± standard deviations (SD) of triplicates. Two-way analysis of variance with
Tukey’s test was performed using MiniTab statistical software version 16 (IBM, Pennsylvania, USA), to test for
significant differences between different concentrations in each type of syrup and types of media. Difference of P<0.05
were considered significant.

RESULTS

Combination Between Syrups And Antibiotics

Influence Of Antibiotics And Honey On Bacterial Growth

The synergistic effect of honey in different concentrations with ampicillin and cefotaxime to growth of Escherichia
coli is presented in Figures 1 and 2, respectively. The results of the in vitro susceptibility of the tested microorganisms
to honey samples with the antibiotics were similar. Bacterial strain was sensitive to honey at all concentrations in all
types of media but the concentration of 20% inhibited the growth of bacteria significantly (P =0.000). On the other
hand, the diameter of inhibition zone on nutrient agar at the concentration of 20% of honey was significantly higher
(P =0.000). Furthermore, results showed that the combination of honey with ampicillin displayed better inhibition
zones at all concentrations compared with different concentrations combination of honey with cefotaxime to the
bacterial strain tested.

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 Figure 1: The synergistic effect of eucalyptus honey at different concentrations with ampicillin against growth of Escherichia
coli.
Results are displayed as mean ± SD in each group of concentration (n = 3). Means with different letters indicate
significant differences between concentrations of syrups (P < 0.05). Sample codes, N.A- Nutrient agar, B.H.A- brain
heart infusion, A+ H- Ampicillin+ honey.

Figure 2: The synergistic effect of eucalyptus honey at different concentrations with Cefotaxime against growth of Escherichia
coli.
Results are displayed as mean ± SD in each group of concentration (n = 3). Means with different letters indicate
significant differences between concentrations of syrups (P < 0.05). Sample codes, N.A- Nutrient agar, B.H.A- brain
heart infusion, C+ H- Cefotaxime+ honey.

Influence Of Antibiotics And Date On Bacterial Growth

Figures 3 and 4 show that the antimicrobial combinations assayed included date plus ampicillin or cefotaxime
against bacterial strain tested, respectively. The synergistic effect of the concentration of 80% of date plus ampicillin
on nutrient agar revealed higher significant inhibition zone in comparison with other concentration and media (P

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=0.000) Figure 3. Figure 4 show that the combination of the concentration of 80% of date plus cefotaxime on two
types of media displayed the same diameter of inhibition zone. The same tendency was found in both the combination
of the concentration of 20% of date plus ampicillin or cefotaxime on both media and 40% on brain heart infusion agar
where the bacterial strain was very resistant. On the other hands, Escherichia coli was significantly sensitive on
nutrient agar at concentration of 40%.

Figure 3: The synergistic effect of date at different concentrations with ampicillin against growth of Escherichia coli.
Results are displayed as mean ± SD in each group of concentration (n = 3). Means with different letters indicate
significant differences between concentrations of syrups (P < 0.05). Sample codes, N.A- Nutrient agar, B.H.A- brain
heart infusion, A+ D- Ampicillin+ Date.

Figure 4: The synergistic effect of date at different concentrations with Cefotaxime against growth of Escherichia coli.
Results are displayed as mean ± SD in each group of concentration (n = 3). Means with different letters indicate
significant differences between concentrations of syrups (P < 0.05). Sample codes, N.A- Nutrient agar, B.H.A- brain
heart infusion, C+ D- Cefotaxime + Date.

Influence Of Antibiotics And Pomegranate On Bacterial Growth

The synergistic effect of pomegranate in different concentrations with ampicillin and cefotaxime to growth of
Escherichia coli is presented in Figures 5 and 6, respectively. Results of pomegranate plus ampicillin showed that

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bacterial strain was resistant on nutrient agar at 20% concentration and no inhibition zone was recorded of the
synergistic effect of pomegranate with Cefotaxime on both media at 20% concentration. On both media at
concentration of 80%, the combination of pomegranate plus ampicillin showed a significant decrease in inhibition
zone compared to other concentration and media (P =0.000) Figure 5. Sensitivity of bacterial strain towards the
combination of pomegranate and Cefotaxime on nutrient agar was significantly higher at concentrations of 40% and
80% compared to those on brain heart infusion agar (P =0.000) Figure 6, while the examined synergistic action
showing better efficacy in suppressing tested bacteria was recorded on the nutrient agar.

Figure 5: The synergistic effect of pomegranate at different concentrations with ampicillin against growth of Escherichia coli.
Results are displayed as mean ± SD in each group of concentration (n = 3). Means with different letters indicate
significant differences between concentrations of syrups (P < 0.05). Sample codes, N.A- Nutrient agar, B.H.A- brain
heart infusion, A+ P- Ampicillin + pomegranate.

Figure 6: The synergistic effect of pomegranate at different concentrations with Cefotaxime against growth of Escherichia coli.
Results are displayed as mean ± SD in each group of concentration (n = 3). Means with different letters indicate
significant differences between concentrations of syrups (P < 0.05). Sample codes, N.A- Nutrient agar, B.H.A- brain
heart infusion, C+ P- Cefotaxime + pomegranate.

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 DISCUSSION
In the present study honey, date and pomegranate syrups were tested for their antimicrobial activity on growth of
E.coli alone or in combination with Ampicillin or Cefotaxime. Honey syrup used in this study showed no inhibitory
activity for the tested bacteria. In contrast to the current report, (Nzeako and Hamdi, 2000), tested the antimicrobial
activities of six honey samples against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa and
results showed that honey samples exerted different levels of antibacterial activities. In study of Mulu et al., (2004)
levels of antibacterial activities of honey were observed against a range of standard tested bacteria. The reason for
the results in the present study is not clear. The variation in sensitivity could be attributed to different species of bees
which produce different types of honey and different techniques used (Mulu et al., 2004), it could also be attributed
to different rate of growth of tested bacteria, temperature and nutritional requirements of tested bacteria. Indeed, Gram-
positive bacteria had shown no honey-resistance, but Gram-negative bacteria have found with an ability to be
phenotypical resistant to honey (Henriques, 2006). The same tendency occurred, date and pomegranate syrups were
not found with antibacterial activities against E. coli. Supporting the present findings, Dhaouadi et al., (2010) tested
the antibacterial activity of date syrup against a range of bacteria including Gram- positive Staphylococcus aureus,
Staphylococcus epidermidis and Bacillus cereus, and Gram- negative Pseudomonas aeruginosa, Escherichia coli and
Salmonella spp. Results showed that S. epidermidis was strongly sensitive to date syrup, and the lowest inhibitory
effect was noticed against Salmonella sp. while no antibacterial activity was observed against E. coli. Moreover,
Türkyılmaz et al., (2013), found that among a range of both Gram- negative and Gram- positive bacteria which were
studied for their sensitivity against pomegranate syrup, E. coli was the only bacteria not affected by pomegranate
syrup. In contrast to the current study, previous studies showed that the date and pomegranate syrups exhibited
antimicrobial activities on both Gram- negative and Gram- positive bacteria (Daniyan and Muhammad, 2008,
Višnjevec et al., 2017, Taleb et al., 2016a, Abbès et al., 2013, Al-Hisnawi, 2017). The contradiction in above studies
could be largely influenced by various factors including the differences in solvent type, sterilization method, fruit
varieties, microbe strains and the constituents in the date and pomegranate syrups extracts which also affect their
revealed antimicrobial potentials (Dhaouadi et al., 2010). Cefotaxime and Ampicillin did not show any activity against
bacterial examined strain. These observations clearly indicate that these bacteria revealed antibiotics resistance toward
the two used antibiotics. As far as is known the overuse and misuse of antibiotics can promote the development of
antibiotic-resistant bacteria which nowadays has become a common problem due to transfer of selected resistance
genes among microorganisms to amplify and extend this problem to other microbes (Read and Woods, 2014).
Resistance can also emerge spontaneously through mutation (Read and Woods, 2014). Using different types of
antibiotics including ampicillin plus phenolic-rich maple syrup extract against a wide range of microorganisms
including E.coli, Tufenkji and Maisuria, (2016) showed that this combination led to decrease in biofilm formation of
a bacterial strain and their antibiotics resistance. Literature regarding the effect of combination between tested syrups
and antibiotics against growth of tested bacterial strains is scarce. However, the available literature investigated a
number of combinations between other natural products and antibiotics against different bacterial strain. For instance,
synergistic effect between methanolic extractions of pomegranate and chloramphenicol, gentamicin, ampicillin,
tetracycline, and oxacillin antibiotics against strains of Staphylococcus aureus was evaluated (Braga et al., 2005). A
gnotobiotic mouse model was used to test the synergistic interaction of green tea extract and levofloxacin against
enterohemorrhagic E. coli infection (Isogai et al., 2001). Results showed that mice fed with green tea extract and
levofloxacin exhibited a wide range of protection against an oral challenge with Escherichia coli. Tufenkji and
Maisuria, (2016), stated that the increasing appearance of antibiotic-resistant bacteria and the production of biofilms
are major two factors which implicate in complicating the effectiveness of antibiotic treatments. Numerous strategies
have been conducted to fight antibiotic resistant bacteria including modifying the chemical structure of know
antibiotics and development of new generation of antibiotics (Felder et al., 2017). Indeed, the effective technique is
synergistic combination between antibiotics and natural compounds which prevent the breakdown of the antibiotics
by the resistant microbes. The possible explanation for synergistic action of used natural syrups and cefotaxime or
ampicillin remains unclear, but it is thought to be attributed to the potential to either prevent the efflux pump of
antibiotic or to enhance the influx of the antibiotics (Braga et al., 2005). Further work needs to be conducted to
investigate the synergistic effects of these applied syrups and antibiotics and to clarify the exact reasons behind this
action. Furthermore, constituents of culture media should be taken into consideration for the antimicrobial activity.

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 CONCLUSIONS
The results of the synergistic effects of these syrups and antibiotics when applied at different concentrations against
E. coli showed better efficacy in suppressing these bacteria and could be used as an alternative treatment agent in the
face of antibiotic resistant bacteria. Therefore, the synergistic action of these syrups with antibiotics used in the current
study should be taken into consideration for their antimicrobial activity.

ACKNOWLEDGMENTS
The work was supported by authors themselves.

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