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Research Paper
Bacteriostatic Effect of Quercetin as an Antibiotic Alternative In
Vivo and Its Antibacterial Mechanism In Vitro
SHENGNAN WANG,
1
JIAYING YAO,
1
BO ZHOU,
1
JIAXIN YANG,
1
MARIA T. CHAUDRY,
1
MI WANG,
1
FENGLIN XIAO,
1
YAO LI,
1
*AND WENZHE YIN
2
1
Institute of Animal Nutrition, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, People’s Republic of China; and
2
Second Affiliated
Hospital, Harbin Medical University, Harbin, Heilongjiang Province 150086, People’s Republic of China
MS 17-214: Received 1 June 2017/Accepted 18 August 2017/Published Online 22 December 2017
ABSTRACT
Quercetin, a ubiquitous flavonoid, is known to have antibacterial effects. The purpose of this study was to investigate the
effect of quercetin on cecal microbiota of Arbor Acre (AA) broiler chickens in vivo and the bacteriostatic effect and antibacterial
mechanism of quercetin in vitro. In vivo, 480 AA broilers (1 day old) were randomly allotted to four treatments (negative control
and 0.2, 0.4, or 0.6 g of quercetin per kg of diet) for 42 days. Cecal microbial population and distribution were measured at the
end of the experiment. The cecal microflora in these broilers included Proteobacteria, Fimicutes, Bacteroidetes, and
Deferribacteres. Compared with the negative control, quercetin significantly decreased the copies of Pseudomonas aeruginosa (P
,0.05), Salmonella enterica serotype Typhimurium (P,0.01), Staphylococcus aureus (P,0.01), and Escherichia coli (P,
0.01) but significantly increased the copies of Lactobacillus (P,0.01), Bifidobacterium (P,0.01), and total bacteria (P,
0.01). In vitro, we investigated the bacteriostatic effect of quercetin on four kinds of bacteria (E. coli, P. aeruginosa, S. enterica
Typhimurium, and S. aureus) and the antibacterial mechanism of quercetin in E. coli and S. aureus. The bacteriostatic effect of
quercetin was stronger on gram-positive bacteria than on gram-negative bacteria. Quercetin damaged the cell walls and
membranes of E. coli (at 50 3MIC) and S. aureus (at 10 3MIC). Compared with the control, the activity of the extracellular
alkaline phosphatase and b-galactosidase and concentrations of soluble protein in E. coli and S. aureus were significantly
increased (all P,0.01), and the activity of ATP in S. aureus was significantly increased (P,0.01); however, no significant
change in ATP activity in E. coli was observed (P.0.05). These results suggest that quercetin has potential as an alternative
antibiotic feed additive in animal production.
Key words: Antibacterial mechanism; Bacteriostasis; Cecal microbiota; Food safety; Quercetin
The main purpose of livestock production is to provide
safe and healthy food for human consumers while taking
into account animal welfare, public health, environmental
issues, etc. Since antibiotic growth promoters were devel-
oped in 1940s, these products have been widely used as feed
additives (5). However, long-term use of antibiotics can
induce mutations in antibiotic resistance genes in the
intestines of livestock (17), consequently producing antibi-
otic-resistant strains (18). Antibiotic resistance can spread to
other animals and humans by direct contact and indirectly
via the food chain, water, air, and soils (25). Antibiotics are
poorly absorbed in the intestines of animals and humans and
can lead to environmental pollution after excretion (32). The
inclusion of antibiotics in animal feeds is considered a public
health issue by the World Health Organization (38, 39), and
the use of antibiotics as growth promoters in the production
of food animals was banned by the European Union in 2006
(5). Therefore, demand is high for the development of
antibiotic alternatives.
The intestinal microbiome is the largest microecosystem
in the body, and the distribution and quantity of microor-
ganisms directly influence host health. The normal micro-
biota play an important part in intestine, involving energy
transfer, metabolism, growth, and reproduction. When the
intestinal environment is suitable for growth of beneficial
bacteria, general health, absorption of nutrients, and
performance are improved. In contrast, high levels of
harmful bacteria impair animal health and cause disease.
The main way to improve intestinal microbiota is to use feed
additives. Flavonoids can be a safe feed additive for
improving intestinal microbiota in animal production. The
antibacterial activity of the diprenylated flavone kuwanon C
has been widely investigated using broth microdilution
methods. This flavone had strong activity against both gram-
negative bacteria (Escherichia coli and Salmonella enterica
serotype Typhimurium) and gram-positive bacteria (Staph-
ylococcus epidermis and Staphylococcus aureus)(36).
Quercetin, a flavonoid found in fruits and vegetables,
contains the basic flavonoid structure of 15 carbon atoms
arranged in three rings (C6-C3-C6) and has unique
biological properties that may improve physical perfor-
* Author for correspondence. Tel: þ86 147 4515 6908; Fax: þ86 451
8725 3030; E-mail: liyaolzw@163.com.
68
Journal of Food Protection, Vol. 81, No. 1, 2018, Pages 68–78
doi:10.4315/0362-028X.JFP-17-214
Copyright Ó, International Association for Food Protection
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mance (anticarcinogenic, anti-inflammatory, antiviral, anti-
oxidant, and psychostimulant activity) and inhibit lipid
peroxidation (22). A quercetin dosage of approximately
0.367 to 0.369 g/kg improved performance by modulating
the intestinal environment in laying hens (23). However,
little research has been done on the potential of quercetin to
improve the microbiota in intestines of Arbor Acre (AA)
broiler chickens and on the antimicrobial mechanism.
Therefore, well-designed clinical trials are needed to further
investigate the bacteriostatic effects of quercetin. The
objective of this study was to investigate the effect of
dietary quercetin on the cecal microbiota of AA broilers and
the antibacterial mechanism in vitro.
MATERIALS AND METHODS
All procedures were performed in accordance with the
guidelines set forth by the Animal Welfare Committee of Northeast
Agricultural University (Harbin, People’s Republic of China).
Birds, diets, and experimental treatment. Four hundred
eighty AA broilers (1 day old) were obtained from a commercial
facility (Yinong Poultry, Harbin, People’s Republic of China).
Birds were randomly allotted to four experimental treatments
comprising six replicates of 20 birds in each treatment. All birds
were raised in stainless steel cages (316 by 400 by 400 mm) under
continuous light in a controlled room for 42 days. The room
temperature was maintained at 338C for the first 3 days, and then
the temperature was reduced to 248C until the end of the
experiment. Water and experimental diets were provided ad
libitum.
The experimental diets were based on corn and soybean meal,
and quercetin was added at four concentrations: 0, 0.2, 0.4, and 0.6
g/kg of diet. Feeding was divided into two phases: the starter phase
from 1 to 21 days and the grower phase from 21 to 42 days. The
basal diet was formulated to meet the nutritional requirements
suggested by the National Research Council (27) (Table 1). Diets
containing quercetin were mixed in basal diet and quercetin
dihydrate powder with 97% purity (Sigma-Aldrich, St. Louis,
MO).
Denaturing gradient gel electrophoresis (DGGE). On day
42, six birds from each group were randomly chosen, and cecal
contents were aseptically harvested for DNA extraction. About 20
g of cecal contents was stored at 808C in a sterile frozen tube for
further analyses. Total DNA was extracted from 200 mg of cecal
contents using a TIANamp Stool DNA Kit (Tiangen, Beijing,
People’s Republic of China). Concentration and purity of DNA
were measured with a NanoPhotometer P-Class (Implen GmbH,
Munich, Germany). The 50-lL PCR volume contained 45 ng of
template DNA and 5 lmol/L concentrations of each of the primers
(F: CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG
GCA CGG GGG GCC TAC GGG AGG CAG CAG; R: ATT
ACC GCG GCT GCT GG) (26), which amplify the 233-bp V3
region of the 16S rRNA gene. The reaction mixture consisted of 2
lL of template DNA, 5 lLof103Taq buffer with (NH
4
)
2
SO
4
,1
lL of deoxynucleoside triphosphates, 0.5 lLofTaq DNA
polymerase, 4 lL of MgCl
2
, and 2 lL of each primer, for a final
volume of 50 lL with the addition of sterilized double-distilled
water (Sangon Biotech, Shanghai, People’s Republic of China).
The PCR assay was denatured at 958C for 5 min and kept at 958C
for 1 min. The temperature was subsequently dropped to 548C for 1
min followed by an elongation step of 728C for 1 min. After the
first products were generated, 35 cycles of 958C for 1 min, 548C for
1 min, and 728C for 1 min were completed followed by a final
elongation step was at 728C for 30 min and holding at 48C for 5
min. Aliquots of 3 lL for each amplification product was separated
in 13Tris-acetate-EDTA (TAE) buffer by 1.5% agarose gel
electrophoresis.
DGGE was performed using a DCode Universal Mutation
Detection System. The PCR products were applied on 8%
polyacrylamide gels in 13TAE with gradients formed with 8%
acrylamide stock solutions and contained 35 to 60% denaturant.
Preelectrophoresis was performed at 200 V for 10 min at 608C.
Electrophoresis was then performed at 90 V for 12 h at the same
temperature. After electrophoresis, the gels were stained with
AgNO
3
.
The representative bacterial groups were determined from the
main DNA bands in DGGE gels. These bands were excised with a
razor blade, and the DNA was eluted overnight at 48C in a 1.5-mL
tube containing 100 lL of sterilized double-distilled water. The
DNA was amplified by PCR with primers without a GC clamp for
DGGE as described above. The DNA products were purified with a
SanPrep Column PCR Product Purification Kit (Sangon Biotech).
PMD18-T (TaKaRa Biotechnology, Dalian, People’s Republic of
China) was used to ligate the purified PCR products, which were
then transformed into E. coli DH5acells (TaKaRa Biotechnology).
Positive clones were chosen using ampicillin and blue-white
screening, and three positive clones of each band were selected
randomly for sequence analysis at Sangon Biotech. The DNA-
MAN tool (Lynnon Biosoft, San Ramon, CA) was used to check
TABLE 1. Calculated composition of basal diets and nutrient
content
Composition
% (air-dry basis)
1–21 days 21–42 days
Ingredient
Corn 57.50 62.30
Soybean meal 34.50 30.00
Fish meal 1.00 1.00
Soybean oil 3.00 3.00
Sodium chloride 0.30 0.30
Dicalcium phosphate 1.65 1.70
Limestone 1.52 1.17
Methionine 0.20 0.20
Choline 0.10 0.10
Multivitamin premix
a
0.03 0.03
Mineral premix
b
0.20 0.20
Nutrient
Metabolizable energy (MJ/kg) 12.33 12.50
Crude protein 21.75 19.72
Lysine 1.18 1.04
Methionine þcysteine 0.91 0.86
Ca 1.07 0.96
Total P 0.70 0.68
Available P 0.46 0.45
a
Content per kilogram of diet: 1,500 IU of vitamin A, 3,200 IU of
vitamin D
3
, 10 IU of vitamin E, 0.5 mg of vitamin K, 1.8 mg of
vitamin B
1
, 3.6 mg of vitamin B
2
, 3.5 mg of vitamin B
6
, 0.01 mg
of vitamin B
12
, 0.15 mg of biotin, 0.55 mg of folic acid, 30 mg of
niacin, and 10 mg of pantothenic acid.
b
Content: 8 mg of Cu (CuSO
4
5H
2
O), 0.35 mg of I (KI), 80 mg of
Fe (FeSO
4
7H
2
O), 60 mg of Mn (MnSO
4
H
2
O), 0.15 mg of Se
(NaSeO
3
), and 40 mg of Zn (ZnO).
J. Food Prot., Vol. 81, No. 1 BACTERIOSTASIS AND ANTIBACTERIAL MECHANISM OF QUERCETIN 69
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the chimeric contructs of sequences deposited in the National
Center for Biotechnology Information database (NCBI; Bethesda,
MD).
RT-qPCR. According to these sequence results of the DGGE,
seven kinds of main bacteria in cecal microbiota were determined
using real-time quantitative PCR (RT-qPCR) (Table 2). The
quantification of DNA by RT-qPCR was performed using the ABI
Prism 7500 sequence detection system (Roche, Mannheim,
Germany) with an initial denaturation at 958C for 30 s followed
by 40 cycles of 958C for 5 s and 608C for 34 s. The 10-lL reaction
mixture contained 5 lL of SYBR Premix Ex Taq, 0.2 lL of ROX
Reference Dye II, 0.2 lL of each primer (10 lmol/L), 3.4 lLof
sterilized double-distilled water, and 1 lL of template DNA. Each
sample was subjected to RT-qPCR in duplicate, and the mean
cycle threshold value of duplicates was used for subsequent
calculations.
Agarose gel electrophoresis was performed to further confirm
the specific PCR products. The PCR products in solution were
combined and electrophoresed on an agarose gel, the bands were
quickly cut out under UV light, and the PCR products were
extracted with a SanPrep Column DNA Gel Extraction Kit
(Sangon Biotech). The concentration of the extracted products
was determined by spectrophotometer, and the copy number was
calculated in terms of the product size. The extracted products were
serially diluted to 10
7
,10
5
,10
3
, 10, 10
1
,10
3
,10
5
,10
7
, and a
standard curve was established.
Antibacterial activity. To further verify the bacteriostatic
effect of quercetin on bacteria in poultry, four common bacteria
were chosen from the above in vivo results: E. coli (ATCC 25922),
S. enterica Typhimurium (ATCC 14028), and S. aureus (ATCC
29213) (Prof. Xu, Northeast Agricultural University) and Pseudo-
monas aeruginosa (ATCC 27853) (Prof. Duo, Harbin Medical
University, Harbin, People’s Republic of China). The antibacterial
activity of these four bacteria was determined using Oxford cup
assays. A quercetin antibacterial solution was prepared at 25 lmol/
mL with DMSO and then diluted to 0.006 lmol/mL using a double
dilution method. One hundred microliters of the bacterial
suspension was added on specific culture media. One hundred
microliters of the antibacterial solutions was added to Oxford cups,
which were then placed at equal distances on the agar surface. The
diameter of the inhibition zone for each cup was measured after 24
h of incubation at 378C. The same procedure was repeated in
triplicate. Chloramphenicol, gentamicin, and penicillin were used
as positive controls. Distilled water was used as the negative
control. After 24 h of incubation, the diameter of each inhibition
zone was measured. All tests were performed in triplicate.
MIC. The MICs of quercetin for E. coli, S. enterica
Typhimurium, S. aureus, and P. aeruginosa were also determined
using Oxford cups. One hundred microliters of suspension
containing bacteria at 1 310
4
CFU/mL was added to specific
culture medium. Various solutions of quercetin were prepared by
serial dilution (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 lmol/mL).
Oxford cups were placed on the inoculated agar, and 100 lLof
quercetin solution was added to each cup and incubated at 378C.
The diameter of the inhibition zone was determined initially and
after 24 h. Distilled water was used as the negative control. The
regression curve was established according to the diameter of the
24-h zone and the quercetin concentration, and the MICs were
calculated.
TEM. One gram-negative bacterium (E. coli) and one gram-
positive bacterium (S. aureus) were selected as model bacteria to
evaluate the antibacterial mechanism of quercetin. Quercetin
solutions of 1.0 3the MIC, 103MIC, 503MIC, 1003MIC,
and 5003MIC were prepared. Bacterial suspensions containing 1
310
7
CFU/mL were then added to the quercetin solutions, and the
cultures were kept in a constant temperature shaking incubator at
378C with shaking at 220 rpm. After 4.5 h, the suspensions were
centrifuged at 10,000 3gfor 10 min. The precipitate was washed
with phosphate buffer three times and fixed in buffer containing
1% glutaraldehyde for 2 h. Each sample was observed by
transmission electron microscopy (TEM; HITACHIH-7650, To-
kyo, Japan). The bacterial suspension containing 1 310
7
CFU/mL
in DMSO without quercetin was used as a negative control. The
bacterial suspension containing 1 310
7
CFU/mL in antibiotic
solution was used as a positive control.
Permeability of bacterial cell wall. E. coli and S. aureus
were inoculated into liquid medium, and the culture was incubated
at 378C with shaking at 220 rpm for 24 h. Two milliliters of
bacterial suspension was added to eight centrifuge tubes, and
distilled water was added to the first tube as a blank control,
DMSO was added to the second tube as a negative control, and
antibiotic solution was added to the third tube as a positive control.
One milliliter of quercetin solutions of 1.03MIC, 103MIC, 503
MIC, 1003MIC, and 5003MIC were added to the other tubes. All
TABLE 2. PCR primers
Target bacteria Primer sets Product size (bp)
Total bacteria F: 50-CCTACGGGAGGCAGCAG-30205
R: 50-ATTACCGCGGCTGCTGG-30
P. aeruginosa F: 50-AGACACCGTCCAGACTCCTAC-3 0277
R: 50-CCAACTTGCTGAACCACCTAC-30
S. enterica serotype Typhimurium F: 50-GTGGCGGACGGGTGAGTAA-3 0231
R: 50-CCGTGCTTCAGTTCCAGTGTG-30
E. coli F: 50-CATTGACGTTACCCGCAGAAGAAGC-3 0195
R: 50-CTCTACGAGACTCAAGCTTGC-30
S. aureus F: 50-TGGAGAGTTTGACCTGGCTCAG-30513
R: 50-TACCGCGGCTGCTGGCAC-30
Lactobacillus F: 50-AGCAGTAGGGAATCTTCCA-30341
R: 50-CACCGCTACACATGGAG-30
Bifidobacterium F: 50-TCGCGTCCGGTGTGAAAG-3 0243
R: 50-CCACATCCAGCATCCAC-30
70 WANG ET AL. J. Food Prot., Vol. 81, No. 1
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tubes were incubated at 378C. After 24 h, these suspensions were
centrifuged at 3,500 rpm for 10 min. The supernatant was
collected, and the activity of alkaline phosphatase (ALP) was
determined with an ALP assay kit (Nanjing Jiancheng Bioengi-
neering Institute, Nanjing, People’s Republic of China). All tests
were performed in triplicate.
Permeability of bacterial cell membrane. The pretreatment
for this test was the same as that for testing the permeability of the
cell wall. After culture 24 h, these bacterial suspensions in tubes
were centrifuged at 3,500 rpm for 10 min. The supernatant was
collected, and the activity of b-galactosidase was determined by
UV-visible spectrophotometry. The precipitate was washed with
stroke-physiological saline solution (SPSS) three times and then
resuspended in SPSS. The activity of bacterial ATP was
determined with an ATP assay kit (Nanjing Jiancheng Bioengi-
neering). All tests were performed in triplicate.
For determination of the soluble protein concentration,
Coomassie brilliant blue G-250 dye was prepared with 95%
ethanol and 85% phosphoric acid, and the 1.0 mg/mL standard
protein solution was prepared with bovine serum albumin (BSA).
The absorbance of the blank control, the negative control, and the
experimental groups was determined at 595 nm. The protein
concentration of each experimental group was calculated according
to the standard curve established with BSA.
Statistical analysis. The data were subjected to a one-way
analysis of variance as a completely randomized design with four
treatments and six replicates for each treatment using SPSS 20.0
(SPSS, IBM, Armonk, NY). Treatment means were tested using
orthogonal polynomial contrasts for evaluation of the linear and
quadratic effects of the dietary supplement. Statistical significance
was established at P,0.05.
RESULTS
Analysis of cecal microbiota using a PCR-based
DGGE. The 233-bp product was amplified via PCR from all
cecal samples prior to DGGE. Bands from sample treated
with four concentrations of quercetin were separated by
DGGE (Fig. 1). Some bands were found randomly among
these four treatments. The DGGE image reveals little
difference among the four treatments, suggesting that cecal
microbial species in these four treatment groups were not
significantly different. Thus, quercetin appeared to have no
significant effect on cecal microbial species. Based on an
NCBI BLAST analysis of the microbial sequences, the main
screened cecal microbiota in AA broilers in this experiment
were Proteobacteria (Gammaproteobacterales, Helicobac-
ter, and Campylobacter jejuni), Firmicutes (Clostridium),
Bacteroidetes (Bacteroides), and Deferribacteres (Deferri-
bacterales) (Table 3).
Quantitative analysis of dominant bacteria in the
cecal community. To determine the quantitative change of
the bacterial community in the cecum of AA broilers treated
with quercetin, total bacterial populations and populations of
some major cecal bacterial groups were determined using
RT-qPCR. Compared with supplementation with no quer-
cetin, supplementation with 0.2 g/kg quercetin significantly
decreased the copies of P. aeruginosa (P,0.05), S.
enterica Typhimurium (P,0.01), S. aureus (P,0.01),
and E. coli (P,0.01) but significantly increased the copies
of Lactobacillus (P,0.01). Supplementation with 0.4 g/kg
quercetin significantly decreased the copies of P. aeruginosa
(P,0.05), S. enterica Typhimurium (P,0.05), and S.
aureus (P,0.01) but significantly increased the copies of
Bifidobacterium (P,0.01). Supplementation with 0.6 g/kg
quercetin significantly decreased the copies of S. aureus (P
,0.05) but significantly increased the copies of total
bacteria (P,0.01) and Bifidobacterium (P,0.05) (Table
4).
Bacteriostatic effect of quercetin on four kinds of
bacteria in vitro. To further verify the bacteriostatic effect
of quercetin in vitro we tested four bacteria: E. coli, S.
enterica Typhimurium, S. aureus, and P. aeruginosa at
FIGURE 1. DGGE gel of DNA from cecal
contents treated with quercetin at (A) 0 g/
kg, (B) 0.2 g/kg, (C) 0.4 g/kg, and (D) 0.6
g/kg. Numbers 1 through 29 indicate mean
bands (Table 3).
J. Food Prot., Vol. 81, No. 1 BACTERIOSTASIS AND ANTIBACTERIAL MECHANISM OF QUERCETIN 71
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initial levels of 2.2 310
9
, 1.4 310
9
, 2.3 310
9
, and 2.1 3
10
9
CFU/mL, respectively. Antibacterial solutions with
quercetin were mixed with the four bacterial suspensions
to determine their antibacterial activity. Results revealed that
the initial level of bacteria has no significant effect on the
bacteriostatic effect of quercetin (P.0.05).
The diameters of the inhibition zones for the four tested
bacterial strains significantly increased (P,0.01) with
increasing quercetin concentrations (Table 5 and Fig. 2). The
MICs of quercetin for E. coli, S. enterica Typhimurium, S.
aureus, and P. aeruginosa were 0.0082, 0.0072, 0.0068, and
0.0085 lmol/mL, respectively. These results indicate that
the bacteriostatic effect of quercetin on gram-positive
bacteria was stronger than that on gram-negative bacteria
and that gram-positive bacteria are highly sensitive to
quercetin.
Comparison of bacteriostatic effect between quer-
cetin and antibiotics. Specific antibiotics were used as
positive controls, and regression curves were drawn for
different concentrations of quercetin and the diameters of the
inhibition zones. The MICs of antibiotics were higher than
those of quercetin for four of the tested strains (Table 6).
Effects of quercetin on cell wall ultrastructure. E.
coli and S. aureus were treated with quercetin at 503MIC
and 103MIC, respectively, for 4.5 h. TEM images revealed
that untreated S. aureus and E. coli had normal morphology
(Fig. 3A and 3C). However, in E. coli treated with quercetin
at 503MIC, the cell wall was damaged and exhibited
abnormalities including separation of the cytoplasmic
membrane from the cell wall, cell wall lysis, leakage and
polarization of cytoplasmic contents, and cell distortion (Fig.
3B). The cell walls of S. aureus were damaged by 103MIC
quercetin; the cytoplasmic membranes were thin and
difficult to distinguish from the cell wall, and the
endochylema density was uneven (Fig. 3D).
Effects of quercetin on cell membrane ultrastruc-
ture. The TEM images revealed that untreated S. aureus and
E. coli cell remained intact and had clearly discernible cell
membranes with uniformly distributed cytochylema (Fig.
4A and 4D). However, in E. coli treated with quercetin at
503MIC, the structural integrity and cell membrane were
damaged (Fig. 4B and 4C), the endochylema density was
uneven, cytoplasmic contents leaked (Fig. 4B and 4C), and
cell cavitation was evident (Fig. 4C) compared with the
control. In S. aureus treated with quercetin at 103MIC, the
extracellular pili of were shed, the cell membrane was
damaged, and the endochylema density was uneven (Fig.
4E) compared with the control, and endochylema contents,
chromatin lysis (Fig. 4F), and nuclear region cavitation (Fig.
4F) were visible.
TABLE 3. Sequencing results for DGGE
Band
no.
a
Closest sequence relative
Identity
(%) Accession no.
1Bacteroides 95 KP944149.1
2 Uncultured Bacteroides 86 EF706917.1
3 Uncultured Bacteroides 88 GU957712.1
4 Uncultured Firmicutes 92 KF506901.1
5Bacteroides 94 LT615364.1
6 Uncultured Bacteroides 98 KT963780.1
7Bacteroides 90 LT615364.1
8 Uncultured bacterium 81 JQ155669.1
9 Uncultured Bacteroides 97 GU222204.1
10 Bacteroides 100 AB910339.1
11 Uncultured bacterium 100 KF945015.1
12 Bacteroides 93 LN998056.1
13 Helicobacter 93 LC028024.1
14 Uncultured bacterium 87 HE602143.1
15 Uncultured Clostridium 88 AB234488.1
16 Uncultured bacterium 82 JQ135413.1
17 Uncultured Deferribacterales 90 KF150651.1
18 Uncultured c-proteobacterium 88 HQ845612.1
19 Bacteroides 98 AB910339.1
20 Uncultured Bacteroides 94 GU939593.1
21 Bacteroides 98 AB910339.1
22 Bacteroides 87 AB910339.1
23 Uncultured bacterium 90 KC662086.1
24 Campylobacter jejuni 98 CP017859.1
25 Uncultured Firmicutes 98 EU281978.1
26 Bacteroides 86 LT631521.1
27 Uncultured bacterium 81 KU764676.1
28 Uncultured bacterium 95 EF400759.1
29 Uncultured Clostridium 82 KP107264.1
a
Band number as shown in Figure 1.
TABLE 4. Effects of quercetin on DNA copies of cecal microbiota
Item
Mean 6SD no. of copies (log copies/g) at quercetin concn of
a
:
0 g/kg 0.2 g/kg 0.4 g/kg 0.6 g/kg
Total bacteria 27.65 61.97 A27.73 61.79 A28.22 61.62 A31.24 60.72 B
P. aeruginosa 15.51 60.22 a AB 15.06 60.15 b B15.11 60.42 b B15.63 60.34 a A
S. enterica serotype Typhimurium 16.31 60.42 a A15.30 60.41 b B15.50 60.81 bc AB 15.99 60.67 ac AB
E. coli 16.78 60.51 a A15.20 60.94 b B16.29 61.13 a AB 16.39 60.74 a AB
S. aureus 16.29 60.61 a A15.10 60.28 b B15.27 60.66 b B15.62 60.57 b AB
Lactobacillus 13.93 60.55 A14.76 60.29 B14.22 60.43 AB 13.92 60.54 A
Bifidobacterium 14.07 60.21 a A14.71 60.63 ac AB 15.11 60.77 c B14.99 60.49 c AB
a
Within a row, means with different lowercase letters are significantly different at P,0.05, means with different uppercase letters are
significantly different at P,0.01, and means with the same lowercase letter are not significantly different at P.0.05.
72 WANG ET AL. J. Food Prot., Vol. 81, No. 1
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Effects of quercetin on the permeability of bacterial
cell walls. Compared with the control, the extracellular ALP
activity of E. coli and S. aureus significantly increased with
increasing quercetin concentrations (P,0.01). The
permeability of the cell wall was higher in treatments with
503MIC quercetin than in treatments with antibiotics (Table
7).
Effects of quercetin on the permeability of bacterial
cell membranes. Compared with the control, the b-
galactosidase activity and concentrations of soluble protein
in E. coli and S. aureus significantly increased (P,0.01)
with increasing quercetin concentrations (Tables 8 and 9).
Quercetin had no significant effect on ATP activity of E.
coli; however, the ATP activity of S. aureus significantly
increased (P,0.01) with increasing quercetin concentra-
tions (Table 10).
The changes in b-galactosidase activity, soluble protein
concentrations, and ATP activity of both tested bacteria
revealed that the permeability of the cell membrane was
affected by quercetin. The effect of 1003MIC quercetin on
the activity of b-galactosidase was higher than that of
antibiotics. For soluble protein concentrations, the effects of
503MIC quercetin were greater than that of antibiotics for
both bacteria, and for ATP activity, the effects of quercetin
at 103MIC (E. coli) and 1003MIC (S. aureus) were greater
than that of antibiotics.
DISCUSSION
Effects of quercetin on the main cecal microflora in
AA broilers. The composition of the intestinal microflora is
closely correlated with nutrition in animals and humans. The
amount and distribution of beneficial bacteria and harmful
bacteria in the digestive tract directly affects the absorption
of nutrients and feed conversion rates. The beneficial
intestinal microorganisms promote the development of
animal health when they represent a large proportion of
the gut microflora (14, 28); however, animal growth will be
inhibited when harmful bacteria are dominant in the gut (20,
35). In poultry, the main beneficial microorganisms in the
intestine are Bifidobacterium and Lactobacillus (3), and the
main harmful bacteria are E. coli. Quercetin is a typical
representative of flavonols, which have strong antibacterial
activity. The broad-spectrum antibacterial effect of quercetin
can be used to prevent and treat various infectious bacterial
diseases. In the present study, copies of E. coli, S. enterica
Typhimurium, P. aeruginosa, and S. aureus in the three
groups treated with quercetin were significantly lower than
those in the group not treated with quercetin, and the number
of copies of Lactobacillus and Bifidobacterium in the three
treated groups was significantly higher. These results are
consistent with those of previous studies in which quercetin
and other flavonoids had an inhibitory effect on E. coli (23),
P. aeruginosa (9), S. aureus (13), and S. enterica
Typhimurium (10); however, quercetin and other flavonoids
play an important role in promoting growth of Bifidobacte-
rium (23) and Lactobacillus (16). Quercetin may act as a
metabolic prebiotic and thus significantly improve the
intestinal environment by promoting the growth of beneficial
bacteria and inhibiting the growth of harmful bacteria. With
ingestion of quercetin, a microecological protection barrier
TABLE 5. MIC of quercetin for four bacterial strains
Strain MIC (lmol/mL)
E. coli 0.0082
S. enterica serotype Typhimurium 0.0072
S. aureus 0.0068
P. aeruginosa 0.0085
FIGURE 2. Bacteriostatic effect of quer-
cetin on E. coli, S. enterica Typhimurium,
S. aureus, and P. aeruginosa in vitro.
TABLE 6. Comparison of bacteriostatic effects of quercetin and
antibiotics
a
Strain
MIC (lmol/mL)
Antibiotic Quercetin
E. coli 0.8345 0.0082
S. enterica serotype Typhimurium 3.1060 0.0072
S. aureus 8.7040 0.0068
P. aeruginosa 0.2618 0.0085
a
Chloromycetin was the positive control for E. coli and S. enterica
Typhimurium; gentamicin was the positive control for S. aureus
and P. aeruginosa.
J. Food Prot., Vol. 81, No. 1 BACTERIOSTASIS AND ANTIBACTERIAL MECHANISM OF QUERCETIN 73
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FIGURE 3. Effects of quercetin on cell
wall ultrastructure of E. coli and S. aureus.
(A) E. coli without quercetin; (B) E. coli
with quercetin; (C) S. aureus without
quercetin; (D) S. aureus with quercetin.
FIGURE 4. Effects of quercetin on cell membrane ultrastructure of E. coli and S. aureus. (A) E. coli without quercetin; (B, C) E. coli with
quercetin; (D) S. aureus without quercetin; (E, F) S. aureus with quercetin.
74 WANG ET AL. J. Food Prot., Vol. 81, No. 1
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was formed, the distribution of intestinal flora imbalance
was improved, gastrointestinal disease was prevented, and
absorption of nutrients was increased.
Bacteriostatic effect of quercetin on four kinds of
bacteria in vitro. E. coli, S. enterica Typhimurium, S.
aureus, and P. aeruginosa infections are primary causes of
chicken mortality (2), which may result in huge economic
losses to the breeding industry. E. coli can cause
inflammation of the fallopian tubes and abdominal mucosa
(21) and air sacs (24) in poultry. Salmonella Gallinarum
biovar Pullorum is the causative agent of pullorum disease
in poultry, an acute systemic disease that results in high
mortality in young chickens (11) and decreases the laying
rate and egg quality of laying hens (8). S. aureus mainly
damages skin (37) and the abdominal cavity of diseased
chickens, with resulting caused traumatic infection and death
of sick chickens. Cyanomycosis, caused by P. aeruginosa,
can be a local or systemic infection. Because of intensive
feeding practices, the incidence of cyanomycosis is
gradually increasing. The devastating effects of common
infectious bacterial diseases in poultry should not be
ignored. Therefore, based on the results of in vivo
experiments, we tested the effects of quercetin on four
common bacteria in vitro: E. coli, S. enterica Typhimurium,
S. aureus, and P. aeruginosa.
Qin et al. (30) reported that quercetin significantly
inhibited S. aureus, E. coli, and P. aeruginosa with MICs of
0.0061, 0.0242, and 0.0121 lmol/mL, respectively. Sugar-
cane bagasse extract had bacteriostatic activity against the
growth of S. aureus, E. coli, and S. enterica Typhimurium
with MICs of 0.625 to 2.5 mg/mL (44). Hossion et al. (15)
found that the novel artificially designed and synthesized
quercetin acyl glucosides effectively inhibited the growth of
E. coli, S. aureus, and P. aeruginosa. Bayberry fruit extract
had significant antibacterial activity against Salmonella,
Listeria, and Shigella with MICs of 2.07 to 8.28 mg/mL
(43). These results indicate that flavonoids can have a
significant inhibitory effect on the four tested strains.
In our study, quercetin significantly inhibited the four
tested strains, and the bacteriostatic effect was stronger on
gram-positive bacteria than on gram-negative bacteria,
possibly because of the differences in the structure and
composition of the gram-positive and gram-negative cell
walls and membranes, as indicated previously (1, 19, 34).
TABLE 7. Effects of quercetin on extracellular ALP activity in E.
coli and S. aureus
Group
Mean 6SD ALP activity (King unit/mL)
E. coli S. aureus
Antibiotic 8.68 60.46 a A1.54 60.06 a A
Quercetin
1.03MIC 7.42 60.30 b AB 0.90 60.10 b B
103MIC 8.15 60.41 ab A0.99 60.12 b B
503MIC 10.54 60.80 c C1.06 60.09 b B
1003MIC 14.31 60.87 d D1.41 60.15 a A
5003MIC 16.57 60.50 e E2.48 60.18 c C
a
Within a row, means with different lowercase letters are
significantly different at P,0.05, means with different
uppercase letters are significantly different at P,0.01, and
means with the same lowercase letter are not significantly
different at P.0.05.
TABLE 8. Effects of quercetin on b-galactosidase activity in E.
coli and S. aureus
Group
Mean 6SD b-galactosidase activity (U)
E. coli S. aureus
Antibiotic 0.16 60.04 a A0.44 61.71 A
Quercetin
1.03MIC 0.14 60.12 b B0.33 60.30 B
103MIC 0.14 60.04 bc BC 0.55 60.46 C
503MIC 0.15 60.08 c C0.74 60.92 D
1003MIC 0.23 60.63 d D0.85 62.26 E
5003MIC 1.66 60.10 e E0.95 61.90 F
a
Within a row, means with different lowercase letters are
significantly different at P,0.05, means with different
uppercase letters are significantly different at P,0.01, and
means with the same lowercase letter are not significantly
different at P.0.05.
TABLE 9. Effects of quercetin on concentration of soluble protein
of E. coli and S. aureus
Group
Mean 6SD soluble protein concn (mg/mL)
E. coli S. aureus
Antibiotic 0.35 60.89 a AB 0.32 60.49 A
Quercetin
1.03MIC 0.33 60.22 b A0.31 60.98 A
103MIC 0.35 60.59 ac B0.32 60.22 A
503MIC 0.37 60.22 c B0.37 60.30 B
1003MIC 0.42 60.34 d C0.53 60.45 C
5003MIC 0.53 60.87 e D0.72 60.56 D
a
Within a row, means with different lowercase letters are
significantly different at P,0.05, means with different
uppercase letters are significantly different at P,0.01, and
means with the same lowercase letter are not significantly
different at P.0.05.
TABLE 10. Effects of quercetin on ATP activity of E. coli and S.
aureus
Group
Mean 6SD ATP activity (lmol Pi/mg protein/h)
E. coli S. aureus
Antibiotic 12.88 60.0043 14.87 60.0005 A
Quercetin
1.03MIC 12.97 60.0011 11.20 60.0041 B
103MIC 12.88 60.0010 11.85 60.0016 C
503MIC 13.01 60.0004 14.21 60.0004 D
1003MIC 12.97 60.0017 17.14 60.0023 E
5003MIC 12.99 60.0004 21.30 60.0006 F
a
Means with different uppercase letters are significantly different
at P,0.01.
J. Food Prot., Vol. 81, No. 1 BACTERIOSTASIS AND ANTIBACTERIAL MECHANISM OF QUERCETIN 75
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Quercetin may result in bacteriostasis by damaging cell
walls and cell membranes. To clarify the inhibitory
mechanism of quercetin, the effects of quercetin on the cell
walls and cell membranes of gram-positive and gram-
negative bacteria were further investigated.
Antibacterial mechanism of quercetin. To investi-
gate the antibacterial mechanism of quercetin, we used both
gram-negative (E. coli) and gram-positive (S. aureus)
bacteria. Bacteria are mainly composed of three parts: cell
wall, cell membrane, and cytoplasm. Some bacteria have
flagella, capsules, pili, and other special structures. The
gram-positive bacterial cell wall is thick with abundant
peptidoglycan. The gram-negative bacterial cell wall is
thinner, with low peptidoglycan and high lipid concentra-
tions.
The effects of drugs on the morphology and ultrastruc-
ture of bacteria has been observed with TEM (7). In both E.
coli and S. aureus treated with the sugarcane bagasse
extract, TEM revealed cell wall degradation, envelope
disruption, and leakage of cytoplasmic content (44). E. coli
and S. aureus treated with cinnamaldehyde exhibited
numerous abnormalities, including cytoplasmic membrane
separation from the cell wall, cell wall and cell membrane
lysis, cytoplasmic content leakage, cytoplasmic content
polarization, cell distortion, and cytoplasmic content con-
densation (33).
An understanding of the changes in microbial cell walls
and cell membranes at the cellular level is the basis for
exploring the antibacterial mechanism of any drug. The
TEM images revealed that the cell wall and membrane of S.
aureus cells were damaged by 103MIC quercetin, and
treatment of E. coli cells with 503MIC quercetin eventually
resulted in cavitation and death. These results suggest
quercetin at specific doses may damage the cell wall
ultrastructure and cell membrane integrity of these two
bacterial strains. This antibacterial mechanism is similar to
that of other flavonoids (33, 44).
Leakage of cytoplasmic contents is a classic indication
of damage to the bacterial cytoplasmic membrane. The cell
membrane is a structural component that may be compro-
mised during biocidal challenges, such as exposure to an
antibacterial agent. Therefore, release of intracellular
components is a good indicator of lack of membrane
integrity. ALP is found between the cell wall and the cell
membrane of bacteria, and ALP activity does not occur
extracellularly. However, when the cell wall is damaged and
the permeability of cell wall is increased, the ALP leaks out
of the cell. Therefore, the changes of cell wall permeability
may be indicated by the changes in extracellular ALP
activity (12). The activity of extracellular ALP in E. coli
increased after treatment with propolis extract, indicating
that propolis extract significantly affected the permeability
of the cell wall (45). Phytic acid damaged the cell wall of
Shewanella putrefaciens, resulting in significantly increased
activity of extracellular ALP (40). The extracellular ALP
activity of S. aureus was increased by the addition of
cryptotanshinone (6). In the present study, quercetin
significantly increased the extracellular ALP activity of E.
coli and S. aureus, which suggests that quercetin signifi-
cantly influenced cell wall permeability. These results are
consistent with those reported for other flavonoids (6, 40,
45).
b-Galactosidase is an intracellular enzyme, which
hydrolyses lactose into glucose and galactose. Normally it
does not leak out of the cell. However, an increase in cell
membrane permeability will allow b-galactosidase to be
released into the surrounding medium (31). In the present
study, the b-galactosidase activity in the E. coli and S.
aureus culture medium increased significantly with increas-
ing concentrations of quercetin, indicating that cell mem-
brane permeability of significantly increased. We also found
that the effect of quercetin on the cell membrane
permeability was stronger on gram-positive bacteria than
on gram-negative bacteria. This effect may result from the
difference in cell wall composition of the two types of
bacteria. The peptidoglycan concentration in the bacterial
cell wall is the main factor affected by quercetin. The
peptidoglycan concentration in the cell wall of gram-positive
bacteria is higher than that in the cell wall of gram-negative
bacteria; therefore, gram-positive bacteria were more
sensitive to quercetin. The permeability of the cell
membrane was increased, thus b-galactosidase activity in
the culture medium also increased. Therefore, quercetin
inhibited bacterial growth and reproduction by damaging the
cell structure.
The bacterial cell membrane is a functional unit and an
important part of the cell. It regulates the penetration of
foreign matter into the cell and participates in important
processes such as energy and material transfer and
information exchange. The permeability of the bacterial cell
membrane was damaged by cryptotanshinone, resulting in
leakage of cell contents, destruction of the normal
metabolism of the cell, decreased growth and reproduction,
and even cell death (6).
In the present study, the soluble protein concentration
was determined using Coomassie brilliant blue G-250 stain.
The soluble protein in E. coli and S. aureus cells increased
significantly with increasing quercetin concentration, which
suggests that the permeability of the cell membrane
increased. These results indicate that quercetin affected the
permeability of the cell membrane of these two bacterial
strains, leading to leakage of intracellular protein into the
culture medium and increasing the soluble protein concen-
tration in the medium. These results are consistent with the
antibacterial properties described for other flavonoids (6,
41).
The antibacterial mechanism of flavonoids may be
associated with the inhibition of nuclein synthesis, and the
flavonoid B-ring plays an important role in this inhibition
(42). As one of the major flavonoids, quercetin may inhibit
the biosynthesis of nucleotides and the activity of ATP (4).
ATP is presented on the membrane of tissue cells and
organelles and are important for transporting materials,
exchanging information, and transferring energy. Because
ATP activity varies with the environment of the bacteria, the
bacterial response to external environmental disturbances
may be determined by detecting changes in ATP activity.
76 WANG ET AL. J. Food Prot., Vol. 81, No. 1
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Plaper et al. (29) found that quercetin altered the activity of
ATP, thereby affecting the growth of E. coli.
In the present study, the ATP activity in S. aureus
increased significantly with increasing quercetin concentra-
tions; however, the effect of quercetin on the ATP activity of
E. coli did not change significantly. Thus, quercetin may
change the permeability of the S. aureus cell membrane and
affect the ATP activity. The differences in the results for E.
coli may be related to its structure and metabolic pathways.
Further research on the cytoplasm of microorganisms could
provide additional information about the extent of damage
and the mode of action of quercetin.
In the present study, the MIC of quercetin was lower for
gram-positive bacteria than for gram-negative bacteria, and
the damaging effect on the ultrastructure and permeability of
the cell wall and membrane was greater for gram-positive
bacteria than for gram-negative bacteria. These data confirm
that the bacteriostatic effect of quercetin is stronger on gram-
positive bacteria than on gram-negative bacteria.
In summary, quercetin inhibited growth of E. coli and S.
aureus in vitro. Quercetin damaged the structure of the
bacterial cell wall and cell membrane, leading to increased
permeability of these structures. The endochylema contents
of the cell were released and the activity of ATP was
affected. We concluded that quercetin decreased the
synthesis of bacterial proteins, affected the expression of
proteins in the cell, and finally resulted in cell lysis and
death.
ACKNOWLEDGMENTS
This study was supported by the Heilongjiang Provincial Government
of China (C2016017) and the Harbin Science and Technology Bureau of
China (2015RQXXJ014).
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