2020, Scienceline Publication  
JWPR  
Poultry Research  
J. World Poult. Res. 10(2S): 278-284, June 14, 2020  
Journal of World’s  
Research Paper, PII: S2322455X2000033-10  
License: CC BY 4.0  
DOI: https://dx.doi.org/10.36380/jwpr.2020.33  
In Vitro Evaluation of Antibacterial Properties of Zinc Oxide  
Nanoparticles alone and in Combination with Antibiotics against  
Avian Pathogenic E. coli  
Mohamed Shakal 1*, Emad Salah2, Maha, A. Saudi1, Eman, A. Morsy3, Shaza Ahmed2 and Ayman Amin4  
1Endemic and Emerging Poultry Diseases Research Center, Cairo University, Egypt  
2Faculty of Biotechnology, October University for Modern Sciences and Arts (MSA), Giza, Egypt  
3Department of poultry diseases, Faculty of Veterinary Medicine, Cairo University, Egypt  
4Department of Plant Physiology, Faculty of Agriculture, Cairo University Giza, Egypt,  
*Corresponding author’s Email: shakal2000@gmail.com; ORCID: 0000-0002-1625-7324  
Received: 11 Feb. 2020  
Accepted: 22 Mar. 2020  
ABSTRACT  
Antibiotic-resistant bacteria have become one of the major issues and concerns worldwide. For the past years,  
scientists have investigated the use of treatments in the nano-scale. Nanomaterials, such as metal oxide nanoparticles,  
have shown promising results due to their antibacterial properties. The aim of this study was to investigate the  
efficiency of in vitro antibacterial activity of zinc oxide nanoparticles (ZnO NPs) alone and in combination with  
different antibiotics against avian pathogenic Escherichia coli. In this study, ZnO NPs were synthesized using direct  
precipitation method. Physical characteristics of ZnO NPs were confirmed using X-ray diffraction and transmission  
electron microscopy. Antibacterial resistance pattern of 10 antibiotics including amoxicillin, ciprofloxacin,  
enrofloxacin, gentamicin, doxycycline, levofloxacin, trimethoprim/sulfamethoxazole, tetracycline, spiramycin, and  
streptomycin, in addition to different concentrations of ZnO NPs, was determined by disc diffusion method on 10  
avian pathogenic E. coli (APEC). The results showed that 50% of the strains were resistant to all antibiotics, while  
the rest were found to be sensitive to one or two antibiotics. The best concentration of ZnO NPs was 50 mg/disk,  
which showed greater zones than that of other used concentrations (25, 12.5, 6.25, 3.125, and 1.56 mg/disk). The  
combination of spiramycin and gentamycin with ZnO NPs showed a synergistic effect while the combination of ZnO  
NPs with ciprofloxacin, enrofloxacin, and streptomycin showed an antagonistic effect. No antibacterial effect was  
observed in combination of ZnO NPs with other used antibiotics. This study recommends in vivo evaluations to  
confirm the results.  
Keywords: Antibiotic, Escherichia coli, Nanoparticle, Zinc Oxide  
INTRODUCTION  
the use of antimicrobial therapy is a key tool in decreasing  
the incidence and mortality of avian colibacillosis (Dheilly  
et al., 2012). Unfortunately, there are increasing numbers  
of E. coli strains that have become resistant to antibiotics.  
Escherichia coli is a bacterial species identified as a  
normal inhabitant of the gastrointestinal tract of humans  
and animals. It is also a part of normal intestinal  
microflora in avian species (De Carli et al., 2015). Certain  
pathogenic strains of E. coli invade several organs of birds  
and causes localized or systemic infections, collectively  
named as colibacillosis (Matter et al., 2011 and De Carli et  
al., 2015) which, caused by Avian Pathogenic E. coli  
(APEC) (Matin et al., 2017). Colibacillosis is responsible  
for significant economic losses in the poultry industry  
globally (Ewers et al., 2003 and Raji et al., 2007). The  
escalation of colibacillosis in both the incidence and  
severity indicates that it is likely to continue and become  
an even larger problem in the poultry industry. However,  
Antibiotic-resistant bacteria have become  
a global  
problem, which has been threatening public health in the  
last few years. To overcome this problem, nanomaterials,  
such as metal oxide nanoparticles, have appeared as  
promising candidates. Nanoparticles are a distinctive  
group of materials with unique structures and a wide range  
of applications in various disciplines (Matei et al., 2008).  
Furthermore, nanotechnology has considerably progressed  
due to its vast applications and uses (Suresh et al., 2016).  
Zinc oxide nanoparticles (ZnO NPs) signify a  
significant class of commercially sustainable products.  
ZnO NPs have several characteristics that allow it to have  
To cite this paper: Shakal M, Salah E, Saudi MA, Morsy EA, Ahmed Sh, and Amin A (2020). In Vitro Evaluation of Antibacterial Properties of Zinc Oxide Nanoparticles alone and  
in Combination with Antibiotics against Avian Pathogenic E. coli. J. World Poult. Res., 10 (2S): 278-284. DOI: https://dx.doi.org/10.36380/jwpr.2020.33  
278  
J. World Poult. Res., 10(2S): 278-284, 2020  
numerous advantages in their use. One of their major uses  
1. X-Ray Diffraction analysis  
is being an antimicrobial agent due to their high efficiency  
on resistant strains of microbial pathogens, reduced  
toxicity and heat resistant properties (Jin et al., 2009;  
Rizwan et al., 2010). Additionally, ZnO NPs have other  
significant features such as physical and chemical  
stability, high catalysis activity as well as intensive  
ultraviolet and infrared adsorption with a wide range of  
applications as semiconductors, sensors, transparent  
electrodes (Matei et al., 2008; Kalyani et al., 2006).  
Moreover, ZnO has received substantial attention in recent  
years because of its distinctive magnetic, optical, and  
piezoelectric properties (Marcus and Paul, 2007). For  
these reasons, the present study aimed to examine the  
antibacterial effect of different ZnO NPs concentrations on  
E. coli different serotypes as well as examine the effect of  
the combination between antibiotic and ZnO NPs on E.  
coli antibacterial sensitivity.  
X-ray diffraction was carried using Rigaku X-ray  
diffractometer system over 20 < 2θ < 80 using Cu-Kα  
radiation of wavelength λ = 0.154 nm.  
2. Transmission Electron Microscope  
Transmission electron microscope was used for  
further structural characterization. A small amount of ZnO  
NPs was dispersed in alcohol by ultra-sonication. A drop  
of the previous solution was taken on a carbon-coated grid  
for TEM imaging.  
Bacterial Strain  
Ten E. coli isolates (previously isolated from broiler  
chicks suffered from high mortalities). (Khelfa and Morsy  
2015). These isolates are E. coli O6, O26, O27, O78, O114  
,
O119, O142, O158, and O159. All serotypes were grown  
aerobically in nutrient broth at 37°C for 24 h before using  
as target organisms. The density of bacterial isolates was  
adjusted to an optical density of 0.5 McFarland standards.  
MATERIALS AND METHODS  
Antimicrobial sensitivity test  
The study was carried out in Endemic and Emerging  
Poultry Diseases Research Center, Cairo University,  
Sheikh Zayed, 6th of October, Giza, Egypt throughout the  
last half of 2019.  
Antibiotic sensitivity test of the E. coli isolates was  
tested against 10 antibiotic disks from (Oxoid, Hampshire,  
UK): amoxicillin (AMC, 10 µg), ciprofloxacin (CIP, 5  
µg), enrofloxacin (EX, 5 µg), gentamicin (CN, 10 µg),  
doxycycline (Do, 30 µg), levofloxacin (LEV, 5 µg),  
trimethoprim/sulfamethoxazole (SXT, 1.25/23.75μg),  
tetracycline (TE, 30 µg), spiramycin (SR, 100 µg) and  
streptomycin (S, 10 µg). These antibiotics were selected  
due to their extensive consumption in the poultry feed for  
treatment of colibacillosis and other avian diseases. The  
test performed following a modified Kirby-bauer disk  
diffusion method as recommended by the Clinical and  
Laboratory Standards Institute (CLSI, 2014).  
Preparation of zinc oxide nanoparticles  
Zinc Oxide Nanoparticles (ZnO NPs) were  
synthesized by direct precipitation, using zinc nitrate and  
KOH as precursors. In this work, the aqueous solution  
(0.2M) of zinc nitrate (Zn (NO3)2.6H2O) and the solution  
(0.4 M) of KOH were prepared with deionized water,  
respectively. The KOH solution was slowly added into  
zinc nitrate solution at room temperature under vigorous  
stirring, which resulted in the formation of a white  
suspension. The white product was centrifuged at 5000  
rpm for 20 min and washed 3 times with distilled water  
and washed with absolute alcohol at last. The obtained  
product was calcined at 500 °C in air atmosphere for 3 hr.  
To examine the antibacterial activity of the ZnO  
nanoparticles on the E. coli microorganisms, ZnO  
nanoparticles were suspended in sterile normal saline and  
constantly stirring until a uniform colloidal suspension  
was formed to yield a powder concentration of 1000  
mg/ml. Two-fold serial dilution was made and the first 5  
concentrations were tested. 0.05 ml of various  
concentrations of ZnO nanoparticle was added in discs.  
After the inoculation and cultivation of E. coli on top of  
nutrient agar, discs were placed in selected areas on  
different plates. The zone of inhibition (ZOI) was  
measured after 24 h incubation. The antibacterial activity  
of ZnO NPs alone and with antibiotics was compared.  
Characterization of zinc oxide nanoparticles  
To confirm the physical characteristics of ZnO NPs,  
the following techniques were performed: X-Ray  
Diffraction (XRD) and Transmission Electron Microscopy  
(TEM) XRD was performed at Central laboratory, Tanta  
University. TEM was performed in the National Research  
Center, Giza, Egypt.  
279  
Shakal et al., 2020  
RESULTS  
O119 were sensitive to gentamycin, E. coli O27 and O 159  
were sensitive to tetracycline, while O26 and O44 were  
sensitive to ciprofloxacin and levofloxacin, respectively as  
represented in table 1. The antibacterial effect of ZnO NPs  
showed that the concentration contributed to greater ZOI  
was 50 mg/disk, the zone is decreased in size as the  
concentration of ZnO NPs decreased until no ZOI at 1.56  
mg/disk was detected as represented in table 2. The  
combination of ZnO NPs concentration that contributed to  
a broader zone (50 mg) and antibiotics showed different  
results between the different antibiotics; that is, there was  
a synergistic effect between ZnO NPs and gentamycin  
which gave ZOI of 22.1, while ZnO NPs alone gave ZOI  
of 19.8. Similarly, spiramycin gave ZOI of 21.5 in  
combination with ZnO NPs. In contrast, the combination  
with ciprofloxacin, streptomycin, and enrofloxacin gave  
ZOI of 15.6, 15.2 and 13.4, respectively which were less  
than that of ZnO NPs alone and that may indicate the  
antagonistic effect between those antibiotic types and ZnO  
NPs as represented in table 3.  
Different techniques were used to characterize the  
synthesized ZnO NPs. Crystal structure and primary  
crystal size were characterized using XRD. Other than  
that, the morphological features especially the size and the  
shape of ZnO NPs were determined using Transmission  
Electron Microscope (TEM).  
The XRD represented in Figure 1 showed broad  
diffraction peaks at 2θ values 31.70, 34.37, 36.19, 56.51  
and 62.78 which are typical for the ZnO structure. Notable  
line broadening of diffraction peaks is an indication that  
the synthesized materials are in the nanometer range  
(Reference code. 96-900-4180). Furthermore, TEM  
images of ZnO confirmed that the particles are almost  
hexagonal. The average particle size was found to be 25-  
32 nm (Figure 2) reveals that most of the ZnO NPs are  
hexagonal in shape with average particles of the size 28.  
Antibiotic sensitivity test showed E. coli resistance  
against all tested antibiotics except E. coli O6, O44 and  
Figure 1. X-ray diffraction pattern of synthesized zinc oxide nanoparticles  
280  
J. World Poult. Res., 10(2S): 278-284, 2020  
Figure 2. Transmission Electron Microscopy image of synthesized zinc oxide nanoparticles  
Table 1. Results of antibiotic sensitivity test for Escherichia coli serotypes  
Antibacterial agent  
Sensitivity  
percentage  
AML  
CIP  
Ex  
Cn  
Do  
LEV  
SXT  
SR  
TE  
S
Strain  
E. coli O158  
E. coli O78  
E. coli O114  
E. coli O44  
E. coli O26  
E. coli O119  
E. coli O55  
E. coli O159  
E. coli O27  
E. coli O6  
r
r
r
r
r
r
r
r
r
r
r
r
i
r
r
i
r
r
r
s
i
r
r
r
r
r
r
r
r
r
r
r
r
r
s
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
i
r
r
r
r
r
r
r
s
s
r
r
r
r
r
r
r
r
r
r
r
0%  
0%  
0%  
r
s
i
r
i
20%  
10%  
10%  
0%  
r
r
r
r
r
s
r
r
i
r
r
r
r
10%  
10%  
10%  
s
i= intermediate; r= resistance; s= sensitive AML: amoxicillin, CIP: ciprofloxacin, Ex: enrofloxacin, Cn: gentamicin, Do: doxycycline, LEV: levofloxacin,  
SXT: trimethoprim/sulfamethoxazole, SR: spiramycin, TE: tetracycline, S: streptomycin  
Table 2. Zone of inhibition produced by different concentrations of zinc oxide nanoparticles for Escherichia coli serotypes  
ZnO concentration/disk  
50 mg  
25 mg  
12.5 mg  
6.25 mg  
3.125 mg  
1.56 mg  
E.coli strain  
E. coli O158  
E. coli O78  
E. coli O114  
E. coli O44  
E. coli O26  
E. coli O119  
E. coli O55  
E. coli O159  
E. coli O27  
E. coli O6  
20 mm  
19 mm  
17 mm  
22 mm  
16 mm  
20 mm  
23 mm  
24 mm  
19 mm  
18 mm  
0
14 mm  
15 mm  
14 mm  
16 mm  
10 mm  
15 mm  
20 mm  
20 mm  
15 mm  
15 mm  
0
11 mm  
10 mm  
10 mm  
12 mm  
0
8mm  
7 mm  
0
8 mm  
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
11 mm  
14 mm  
15 mm  
9 mm  
12 mm  
0
8 mm  
10 mm  
10 mm  
0
10 mm  
0
0
7 mm  
8 mm  
0
0
0
Control  
ZnO: zinc oxide  
281  
 
Shakal et al., 2020  
Table 3. Zone of inhibition (mm) of combination of zinc oxide nanoparticles (50 mg/disk) with different antibiotics for  
Escherichia coli serotypes  
Antibacterial agents  
AMC+  
ZnO  
NPs  
CIP+  
ZnO  
NPs  
EX+  
ZnO  
NPs  
CN+  
ZnO  
NPs  
DO+  
ZnO  
NPs  
LEV+  
ZnO  
NPs  
SXT+  
ZnO  
NPs  
SR+  
ZnO  
NPs  
TE+  
ZnO  
NPs  
S+  
ZnO  
NPs  
ZnO  
NPs  
Strain  
E. coli O158  
E. coli O78  
E. coli O114  
E. coli O44  
E. coli O26  
E. coli O119  
E. coli O55  
E. coli O159  
E. coli O27  
E. coli O6  
Control  
20  
19  
17  
22  
16  
20  
23  
24  
19  
18  
0
18  
20  
17  
21  
16  
21  
22  
23  
19  
18  
0
16  
13  
15  
17  
13  
16  
20  
19  
14  
13  
0
13  
10  
14  
16  
10  
13  
17  
18  
12  
11  
0
24  
22  
19  
23  
20  
22  
24  
22  
23  
22  
0
19  
18  
16  
22  
17  
19  
21  
23  
20  
17  
0
19  
19  
15  
21  
18  
20  
20  
24  
20  
19  
0
18  
15  
19  
20  
18  
21  
22  
23  
19  
16  
0
23  
24  
20  
20  
19  
21  
24  
23  
22  
19  
0
19  
20  
16  
20  
18  
22  
21  
23  
17  
17  
0
15  
16  
12  
18  
10  
19  
18  
16  
15  
13  
0
Mean  
19.8  
19.5  
15.6  
13.4  
22.1  
19.2  
19.5  
19.1  
21.5  
19.3  
15.2  
ZnO NPs: zinc oxide nanoparticles, amoxicillin (AMC), ciprofloxacin (CIP), enrofloxacin (EX,), gentamicin (CN), doxycycline (Do), levofloxacin (LEV),  
trimethoprim/sulfamethoxazole (SXT), tetracycline (TE), spiramycin (SR) and streptomycin (S)  
NPs. The results showed a prominent increase in the  
inhibition zone, starting with the concentration of 50  
mg/disk, the inhibition zone was observed in all the  
bacterial strains at this concentration but in different sizes.  
The concentration of 25 mg/disk showed an inhibition  
zone in all the E. coli strains. At the concentration of 12.5  
mg/disk, the inhibition zone was observed in all types of  
bacterial strains except O26. The results indicated the  
ability of ZnO NPs to inhibit E.coli through its  
antibacterial property.  
In the concentration of 6.25 mg/disk, the number of  
resistant strains was three. No inhibition zone was detected  
in concentrations of 3.125mg/disk and 1.56mg/disk. The  
results showed that ZnO NPs (50 mg/disk, 25 mg/disk, and  
12 mg/disk) enhanced antibacterial effects while lower  
concentrations had low or no effect.  
The obtained results in the present study are  
supported by another research conducted by Rauf et al.  
(2017) who stated that ZnO NPs possess effective  
antibacterial activity against E. coli. The ZnO NPs  
antibacterial effect has been associated with bacterial  
exterior membranes decomposition by reactive oxygen  
species (ROS), mainly by the hydroxyl radicals (OH),  
which lead to phospholipid peroxidation and eventually  
kill bacteria. Rauf et al. (2017) stated that nanoparticles  
have a physical property that allows them to adhere to a  
cell and kill the bacteria if they come in contact with it.  
Another similar research conducted by Rizwan et al.  
(2010) stated that increasing the concentration of ZnO NPs  
increases the antibacterial activity. The inhibition zone  
DISCUSSION  
The present study demonstrated the potential of using ZnO  
NPs as an antibacterial agent as well as their efficiency to  
develop  
a synergistic effect with antibiotics. The  
employed methodology of the ZnO NP production was  
based on structural and compositional characterization; the  
in situ produced ZnO NPs were interpreted using electron  
microscopy and XRD analysis.  
The selected 10 types of antibiotics were used in  
combination with ZnO NPs against 10 strains of avian E.  
coli in order to inhibit its growth. The sensitivity test was  
performed primarily as control by using the antibiotics  
alone without the nanoparticles. The use of gentamicin  
showed that all the bacterial strains have resistance against  
this type of antibiotic except O6, O44, and O119; these  
three strains of E. coli were sensitive to gentamicin. All  
bacterial strains were resistant against tetracycline except  
O27 and O159. While O26 and O44 were sensitive to  
ciprofloxacin and levofloxacin. The results are in  
agreement with findings of a study conducted by Kibret  
and Abera (2011) who explained the antimicrobial  
sensitivity patterns of E. coli from human samples against  
the selected antibiotics used in the present study and  
reported high resistance rates to amoxicillin and  
tetracycline. In addition, gentamicin and ciprofloxacin  
produced high ZOI.  
The experiment was followed by using the ZnO NPs  
with different concentrations as an antibacterial agent.  
Resistant strains were used to evaluate the effect of ZnO  
282  
J. World Poult. Res., 10(2S): 278-284, 2020  
size was divergent according to bacterial strain, size, and  
antibacterial on Gram-negative bacteria. The same results  
were confirmed in the study of Zhongbing et al. (2008) in  
which Gram-negative membrane and Gram-positive  
membrane disorganization was approved by transmission  
electron microscopy of bacteria ultrathin sections.  
In addition, Nazoori and Kariminik (2018) stated that  
antibacterial activity of ZnO NPs showed notable  
decreasing activity. The inhibition of growth was observed  
in a concentration-dependent manner for all bacteria which  
were statistically significant inhibitory effects compared  
with the control (general antibiotics) in this condition.  
Further studies should be performed investigating the toxic  
effect of ZnO NPs on bacteria.  
ZnO NPs concentration. Colonies’ number forming unit  
(cfu) of E. coli and S. aureus were incubated overnight  
with different concentrations of ZnO NPs. The least  
concentration of ZnO NPs that inhibited the growth of  
bacteria was 3.1 mg/ml for E. coli and 1.5 mg/ml for  
S.aureus. The current study validates earlier researches  
and proposes that ZnO NPs in high concentrations have an  
antibacterial effect against resistant strains of E. coli.  
The experiment followed was the evaluation of the  
effect of ZnO NPs with the antibiotics. The results  
indicated the ability of ZnO NPs’ effect with 10 studied  
antibiotics as presented in table 3. The results showed that  
the average size of inhibition zone caused by ZnO NPs  
was 19.8mm. When combining different antibiotics with  
ZnO NPs, gentamicin and ZnO NPs in concentration of  
50mg resulted in the size of inhibition zone to be increased  
to 22.1 mm; while using a combination of Spiramycin and  
ZnO NPs led to an inhibition zone of 21.5 mm, which  
indicates a synergistic effect between ZnO NPs and  
(gentamicin and spiramycin). These two antibiotics with  
ZnO NPs have a prominent effect in inhibiting avian E.  
coli.  
The other types of antibiotic such as ciprofloxacin,  
streptomycin, and enrofloxacin resulted in 15.6 mm, 15.2  
mm, and 13.4 mm of the inhibition zone, respectively. The  
previous inhibition zones were smaller than the size of  
inhibition zone of ZnO NPs alone, so the combinations  
were not effective compared to the effect of ZnO NPs with  
gentamicin and spiramycin. This result could be explained  
by Rauf et al. (2017) who proposed combination of ZnO-  
NPs with different antibiotics could using the disc  
diffusion method results could differ in efficiency due to  
the variances in fold increase among these antibiotics as  
well as their variance in their mechanism of action.  
CONCLUSION  
The present study demonstrated the antibacterial activity  
of the addition of ZnO NPs to some antibiotics. The result  
showed a synergistic and antagonistic effect between ZnO  
NPs and some antibiotics on different avian E.coli strains.  
In conclusion, the study showed promising results to  
eradicate the issue of antibiotic resistance. This study  
recommends in vivo studies to confirm the obtained  
results.  
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