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African Journal of Pig Farming ISSN 2375-0731 Vol. 8 (2), pp. 001-007, February, 2020. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 

 

Antibacterial effects of zinc oxide nanoparticles 
on Escherichia coli K88 

 
Chao Wang, Lian-Long Liu, Ai-Ting Zhang, Peng Xie, Jian-Jun Lu* and Xiao-Ting Zou* 

 
Institute of Feed Science, College of Animal Science, Zhejiang University, Key Laboratory of Molecular Animal 

Nutrition, Ministry of Education, Hangzhou Zhejiang Province, People’s Republic of China 310058. 
 

Accepted 14 October, 2019 
 
This study was conducted to evaluate the antibacterial effects of zinc oxide nanoparticles in vitro. Escherichia coli 

K88 was chosen as an indicator of pathogenic bacteria, because it could cause diarrhea in both children and in 

early-weaned piglets. In this study, the characterization of the nanoparticles was examined. Antibacterial activities 

against E. coli K88 were evaluated by determining the minimum inhibitory concentration (MIC) and minimum 

bactericidal concentration (MBC) and observing the effects on the values of the optical density (OD) at 620 nm and 

the populations. Results indicate that zinc oxide nanoparticles had strong antibacterial activity against E. coli K88. 

The activity increased as the concentration of the nanoparticles increased. The MIC and MBC were 0.1 and 0.8 

µg/ml, respectively. To study the antibacterial mechanisms, atomic force microscopy (AFM) and scanning electron 

microscopy (SEM) were used to observe morphological changes of E. coli K88 treated with 0.8 µg/ml zinc oxide 

nanoparticles. The results reveal that zinc oxide nanoparticles could damage cell membranes, lead to leakage of 

cytoplasm and kill the bacterial cells. Our study indicates that zinc oxide nanoparticles could potentially be an 

antibacterial reagent to treat diseases caused by bacteria. 

 
Key words: Zinc oxide, nanoparticle, Escherichia coli K88, antibacterial activity, atomic force microscopy (AFM). 

 
INTRODUCTION 

 
Escherichia coli K88 is an important pathogen, which could 

cause diarrhea in both children and early-weaned piglets 

(Guo et al., 2005; Yu et al., 2011). Antibiotics are the most 

common drugs to inhibit the growth and propagation of E. 

coli K88. However, the use of antibiotics has various side 

effects, such as the increase in bacterial resistance 

(Baquero et al., 2011). The exploitations of novel substitutes 

to antibiotics, especially on inorganic nanoparticles, have 

recently attracted more attention.  
Zinc oxide is an important inorganic material, which has 

multiple properties, such as semiconducting properties, 

antibacterial activity and growth promoter. It is widely applied 

in the field of optoelectronics (Yu et al., 2003; Gao 
 
 

 
*Corresponding author. E-mail: jjlu@zju.edu.cn, xtzou  
@zju.edu.cn. Tel: 86-571-88982729. Fax: 86-571-88982650. 

 
Abbreviations: MIC, Minimum inhibitory concentration; MBC, 
Minimum bactericidal; OD, optical density; AFM, atomic force 
microscopy; SEM, scanning electron microscopy. 

 
 
 
et al., 2005), pharmaceutics (Baldwin et al., 2001), 
cosmetics (Sheldon et al., 2000; Mitchnick et al., 1999), 
food science (Daniel et al., 2003) and agriculture (Smith 
et al., 1997; Carlson et al., 1999). The antibacterial 
activity of zinc oxide has been widely explored. It has 
been documented that concentration, size and healing 
temperature can affect the antibacterial activity. Zinc 
oxide as an inorganic antibacterial reagent is more stable 
than the organic reagents (Yamamoto, 2001; Sawai, 
2003; Sawai et al., 1996a). Several antimicrobial 
mechanisms of zinc oxide were supposed; i) hydrogen 
peroxide, which is generated from the surface of zinc 
oxide, can penetrate through the cell membrane, produce 
some type of injury, and inhibit the growth of the cells 
(Yamamoto, 2001; Sawai, 2003; Sawai et al., 1996b, 
1998); ii) the affinity between zinc oxide and bacterial 
cells is an important factor for antibacterial activity 
(Stoimenov et al., 2002).  

Combined with nanotechnology, zinc oxide 
nanoparticles can be prepared, which possess some 
unique characters, such as small particle size and large 
area surface. Zinc oxide nanoparticles may exhibit 



 
 
 

 

stronger antibacterial activity than zinc oxide itself 
(Yamamoto, 2001). Therefore, the interactions of 
nanoparticles with microorganisms have recently 
attracted more attention and a wide range of antibacterial 
effects of zinc oxide nanoparticles have been reported 
(Jones et al., 2008; Reddy et al., 2007). Moreover, zinc 
oxide nanoparticles have selective toxicity and are 
generally regarded as a safe reagent to humans and 
animals (Reddy et al., 2007; Liu et al., 2009; Fu et al., 
2005; Berube, 2008), which could be an ideal potential 
antibacterial reagent to replace some antibiotics. 
However, few studies have been conducted to evaluate 
the antibacterial effects of zinc oxide nanoparticles on E. 
coli K88. Moreover, the antibacterial mechanism of zinc 
oxide nanoparticles is still unclear. Therefore, our study 
was undertaken to investigate the antibacterial activity 
against E. coli K88 in vitro. The atomic force microscope 
(AFM) and scanning electron microscopy (SEM) were 
used to study the mechanism. 

 

MATERIALS AND METHODS 
 
Characterization of zinc oxide nanoparticles 
 
Zinc oxide nanoparticles were provided by Institute of Feed 
Science, Zhejiang University, Zhejiang, China. The particle sizes of 
zinc oxide nanoparticles were determined by AFM (AFM, SPM-
9500J3, Shimadzu CO., Japan) and Zetasizer Nano-ZS90 (Malvern 
Instruments) by the method of Du et al. (2008). For AFM, the zinc 
oxide nanoparticles suspended into water were placed onto cleaved 
mica and observed in contact mode with Si3N4 probes. For 
Zetasizer Nano-ZS90, the analysis was performed at a scanning 
angle of 90° at 25°C using samples diluted with water. The water 
used throughout this study was the reagent-grade water produced 
by Milli-Q SP Ultra-Pure-Water Purification System of Nihon 
Millipore Ltd. (Tokyo, Japan). 

 
Determination of minimum inhibitory concentration (MIC) and 
minimum bactericidal concentration (MBC) 
 
The minimum inhibitory concentration (MIC) and minimum 
bactericidal concentration (MBC) were determined by a method 
recommended in NCCLS (2000) with some modifications. Briefly, 
the sterile tubes were incubated aerobically at 37°C for 24 h, which 
contained 5 ml Muller- Hinton (MH) broth (Difco, USA) with 

approximate 5×10
9
 CFU bacterial cells and 0 (the control group), 

0.025, 0.05, 0.1, 0.2, 0.4, and 0.8 µg/ml zinc oxide nanoparticles. 
The concentration of tube without visible growth of the bacterial 
cells was the MIC. To evaluate the MBC, 100 µl of sample from 
each tube without visible growth was transferred into MH agar plate 
(Difco, USA), and then incubated aerobically for another 24 h. The 
concentration of the tube without growth was the MBC (in this test, 
the population in agar plate less than 10 was regarded no growth). 
All the measures were triplicate. 

 
Antibacterial effects on values of optical density (OD) at 620 
nm and the populations 
 
The sterile tubes contained 5 ml Luria–Bertani (LB) culture fluids, 

approximate 5×10
9
 CFU bacterial cells and 0 (the control group), 

0.025, 0.05, 0.1, 0.2, 0.4, 0.8 µg/ml zinc oxide nanoparticles, and 
were incubated aerobically at 37°C for 24 h. Then, samples were 

  
  

 
 

 
collected to measure the values of OD at 620 nm and bacterial 
populations. Values of OD at 620 nm were determined using an 
ultra violet visible (UV-Vis) spectrophotometer (Hitachi Ltd, Tokyo, 
Japan). A sample of 1 ml from each tube was serially diluted using 
phosphate buffer solution (PBS) for enumeration of bacterial 
populations. Bacterial populations were enumerated on LB agar 
plate. All the measures were in triplicates. 

 

Morphological changes of E. coli K88 
 
AFM (SPM-9500J3, Shimadzu CO., Japan) and SEM (XL30-ESEM 
Philips company, Japan) were used to examine morphological 
changes of E. coli K88 treated with 0.8 µg/ml zinc oxide 
nanoparticles. For AFM, the morphological changes were 
determined as described by Du et al. (2008). Briefly, the sterile 
tubes, which contained 5 ml, 0.8 µg/ml zinc oxide nanoparticles LB 

culture fluid and approximate 5×10
9
 CFU bacterial cells, were 

cultured aerobically in a shaken thermostat (200 rmp/min) at 37°C. 
Samples were collected from the tubes at 0 (the control), 0.5, 1.0, 
2.0, 3.0, 4.0 h, dried on slices, and observed by AFM in contact 
mode with Si3N4 probes.  

For SEM, samples were collected from the tubes at 0 (the 
control) and 0.5 h. The samples were immersed in the solution of 
2.5% glutaraldehyde (Merck EM grade, Auer Bittman Soulie AG, 
Basel, Switzerland) for 24 h and were washed by PBS. Then, they 
were fixed by 1% OsO4 solution for 2 h and dehydrated in a grade 
series of ethanol (50, 70, 80, 90, 95, 100, and 100%) for 15 min at 
each step. Subsequently, the samples were treated with solution of 
ethanol and iso-amyl acetate(1:1) for 30 min, then treated with pure 
iso-amyl acetate for 2 h and dried by Hitachi HCP-2 critical point 
dryer (Hitachi Ltd, Tokyo, Japan). The samples were then coated 
with gold using Eiko IB-5 ion coater (Eiko Engineering Co. Ltd., 
Tokyo, Japan), after which, they were observed with SEM. 

 

Statistical analysis 
 
Data were analyzed statistically by one-way analyses of variance 
(ANOVA), using the SPSS statistical software package for 
Windows (version 16.0, SPSS, Chicago, USA). Probability values 
below 0.05 were considered to be statistically significant. 
 

 

RESULTS AND DISCUSSION 

 

Particle size 

 

The primary sizes of zinc oxide nanoparticles are one of 
the important factors for the antibacterial activity (Zhang 
et al., 2007a). In our study, zinc oxide nanoparticles were 
characterized by Zetasizer Nano-ZS90 (Malvern 
Instruments) and AFM. The sizes of zinc oxide 
nanoparticles were 75±20 nm as determined by Zetasizer 
Nano-ZS90, which indicated that the zinc oxide 
nanoparticles in this study were small sized and could 
exhibit antibacterial activity of nanoparticles. Images 
observed by AFM are shown in Figure 1. The images 
show that the zinc oxide nanoparticles were small sized, 
which were in line with the results of Zetasizer Nano-
ZS90. Moreover, Figure 1b reveals that the morphologies 
of the zinc oxide nanoparticles were not quasi-spherical 
morphology, as reported by Wu et al. (2010). The 
nonspherical morphology coming from the 



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 1. AFM images of mica surface (a) and zinc oxide nanopartcles (b). 

 
 

 

preparation method may influence the antibacterial 
activity, which could be studied in the future. 
 

 

Antibacterial activity 

 

In this study, results of MIC and MBC show that the MIC 
and MBC were 0.1 and 0.8 µg/ml, respectively. The ratio 
of MBC/MIC was 8. These results indicate that zinc oxide 
nanopaticles had antibacterial effects on E. coli K88, 
which is partly in accordance with the reports by 
Soderberg et al. (1990), Sawai (2003), Hernandez-Sierra 
et al. (2008), and Liu et al. (2009). They reported that zinc 
oxide or its nanoparticles had antibacterial activity against 
Staphylococcus aureus, Streptococcus, E. coli 745 and 
E. coli O157:H7. However, the antibacterial 
concentrations were inconsistent. Soderberg et al. (1990) 
revealed that 179 and 1790 µg/ml zinc oxide could exhibit 
a clear antibacterial effect on S. aureus. Results of Liu et 
al. (2009) indicated that 3 mmol/l zinc oxide nanoparticles 
could inhibit the growth of E. coli O157:H7 and 12 mmol/l 
or higher concentrations completely inhibited the growth. 
The main factors for the variable antibacterial 
concentrations may be that the different microbes and 
zinc oxide or its nanoparticles were used. Interestingly, 
Roselli et al. (2003) reported that 1 mmol/l zinc oxide 
(greatly exceeding the level of 0.8 µg/ml zinc oxide 
nanoparticles) did not affect the growth of E. coli K88, 
which partly explained the reports that zinc oxide 
nanoparticles exhibited stronger antibacterial activity than 
zinc oxide (Yamamoto, 2001). It has been reported that 
the values of OD at 620 nm can be an important indicator 

 
 
 

 

for bacterial growth (Missotten et al., 2009; Zhang et al., 
2007b). Therefore, effects of zinc oxide nanoparticles on 
values of OD at 620 nm were measured and are shown 
in Figure 2a, which indicated that 0.025 and 0.05 µg/ml 
zinc oxide nanoparticles did not affect the bacterial 
growth. Compared with the control group, 0.1, 0.2, 0.4, 
and 0.8 µg/ml zinc oxide nanoparticles could significantly 
decrease the values of OD at 620 nm, especially the 0.8 
µg/ml. These results are consistent with the results of 
MIC and MBC. Figure 2a also reveals that the 
antibacterial activities increased as the concentration of 
zinc oxide nanoparticles increased, which are in line with 
results of Brayner et al. (2006) and Liu et al. (2009).  

Effects of zinc oxide nanoparticles on the populations of 

E. coli K88 are shown in Figure 2b, which indicates that 

compared with the control group, 0.05, 0.1, 0.2, 0.4 and 0.8 

µg/ml zinc oxide nanoparticles significantly decreased the 

population of E. coli K88. These results are partly 

inconsistent with the results of the MIC and effects on 

values of OD at 620 nm, in which 0.05 µg/ml zinc oxide 

nanoparticles did not affect the bacterial growth. This 

difference may indicate that concentrations of zinc oxide 

nanoparticles in MH broth or LB media could affect the 

results of the MIC and values of OD at 620 nm. 
 

 

Morphological changes of E. coli K88 

 
The mechanisms of antibacterial activities of zinc oxide or its 

nanoparticles against some microbes have been explored. 

However, no study has been reported to investigate the 

mechanisms of antibacterial effects of zinc 



   
 
 
 
 
 
 

 

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Concentration of zinc oxide nanoparticles (μg/ml) 

 
Figure 2. Effects of zinc oxide nanoparticles on values of OD at 620 nm (a) and populations (b) 
of E. coli K88. Data given represents means ± standard error of mean (n=3). 

 
 

 

oxide nanoparticles on E. coli K88. Therefore, 
morphological changes were observed by AFM and SEM 
to investigate the preliminary mechanisms.  

The AFM is a scanning probe technique. The probe 
allows imaging in any environment instead of only 
vacuum for conventional probes (Binning et al., 1986). 

 
 
 

 

AFM has been widely used in researches for yeast cells 
(Touhami et al., 2003), biopolymers (Morris et al., 2001), 
proteins (Yan et al., 2003) and bacteria (Kasas et al., 
1994; Doctycz et al., 2003; Du et al., 2008; Kailas et al., 
2009). It is an ideal tool to determine the morphological 
changes of cells (Camesano et al., 2000). In our present 



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. AFM images of E. coli K88 treated by zinc oxide nanoparticles for 0.0 (a), 0.5 (b), 1.0 (c), 2.0 (d), 3.0 (e), and 4.0 h (f). 

 
 

 

study, AFM in contact mode was used to examine 
the morphological changes of E. coli K88. The 
images are shown in Figure 3. Figure 3a shows 
that E. coli K88 was a rod shape of about 1 µm 
wide and 2 µm long, and the membrane was 

 
 
 

 

intact. This result is similar to previous report (Du et 

al., 2008). After 0.5 h of treatment with 0.8 µg/ml 

zinc oxide nanoparticles, the membrane of E. coli 

K88 was affected, and the boundary became blurry 

as shown in Figure 3b. When treated for 

 
 
 

 

1.0 h, the membrane of E. coli K88 was further 
affected, some cell contents leaked out of the cell 
and a lot of debris was found around the cell 
(Figure 3c). Results of the cells treated for 2.0 h 
are similar to the results for 1.0 h, as shown in 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 4. SEM images of E. coli K88 without (a) and with (b) treatment of zinc oxide nanoparticles. 

 
 

 

Figure 3d. After 3.0 h of treatment, the boundary of the 
cell was further damaged and almost disappeared, and 
only high concentrations of debris were left (Figure 3e). 
When treated for 4.0 h, debris filled in the vision field 
instead of bacterial cells (Figure 3f).  

In agreement with the AFM image (Figure 3a), SEM 
image in Figure 4a shows that most of the E. coli K88 in 
our present study were 1.0×2.0 µm rod shape and had no 
flagellum. However, after 0.5 h of treatment with 0.8 
µg/ml zinc oxide nanoparticles, no intact cell was found 
as shown in Figure 4b, which was different from the AFM 
image (Figure 3b). One of the reasons for no intact cell 
may be that the complicated treatments for SEM images 
further damaged the structures of bacterial cells, and led 
them to debris.  

Results of morphological changes show that zinc oxide 

nanoparticles could damage the membrane of E. coli K88, 

lead to the leakage of cytosolic components and kill the 

bacterial cells, which is partly consistent with the previous 

reports of Sawai et al. (1996b, 1998), Yamamoto (2001), 

Stoimenov et al. (2002) and Sawai (2003). They reported 

that zinc oxide could damage the membrane of bacterial cell 

by hydrogen peroxide or the affinity between zinc oxide and 

bacteria surface. From these studies, we preliminarily 

supposed that the mechanisms of antibacterial activities of 

zinc oxide nanoparticles against E. coli K88 were closely in 

line with those against other microbes. The unique 

characters of nanoparticles largely increased the surface of 

zinc oxide, or enhanced the affinity, so, zinc oxide 

nanoparticles could exhibit stronger antibacterial activity 

than zinc oxide (Yamamoto et al., 1998; Yamamoto, 2001). 

However, the concentration of nanoparticles should also 

exceed the MIC to produce enough hydrogen peroxide or 

the affinity to damage the bacterial membrane. Interestingly, 

results of SEM images observed by Liu et al. (2009) 

indicated that 12 mmol/l zinc oxide nanoparticles (the 

concentration which completely 

 
 
 

 

inhibited the bacterial growth) did not result in the 
morphological changes, which was inconsistent with our 
present results. Reasons for the difference of the images 
are still unknown, which require further research. In 
addition, the mechanisms underlying the damage of 
bacterial membrane are yet to be studied. 
 

 

Conclusion 

 
Results in our present study indicate that zinc oxide 

nanoparticles had strong antibacterial activity against E. coli 

K88 and the activity increased as the concentration of zinc 

oxide nanopartices increased. The mechanisms may be that 

zinc oxide nanoparticles could damage the membrane, lead 

to the leakage of cytosolic components and kill the bacterial 

cells. In summary, our study reveals that zinc oxide 

nanoparticles could potentially be an antibacterial reagent to 

treat diseases caused by bacteria. In future, these 

nanoparticles might replace conventional antibiotics in 

humans and animals. However, antibacterial effects, safety, 

and detailed mechanisms of zinc oxide nanoparticles should 

be further studied in vitro and in vivo. 
 

 

ACKNOWLEDGEMENT 

 

The authors gratefully acknowledge the financially 
support of Hangzhou King Techina Technology CO., Ltd. 
 

 
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