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Bacteriostatic Effect of Quercetin as an Antibiotic Alternative In Vivo and Its Antibacterial Mechanism In Vitro

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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 × MIC) and S. aureus (at 10 × MIC). Compared with the control, the activity of the extracellular alkaline phosphatase and β-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.
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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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... ONPG can only reach cytoplasm evading membrane barrier and be hydrolyzed by cytoplasmic β-galactosidase when the inner membrane permeability is compromised (Wang et al., 2018;Yun et al., 2018). However, the cytoplasmic βgalactosidase may also be leaked out and hydrolyze ONPG in the surrounding medium when the inner membrane permeabilization significantly increases, or otherwise, the membrane integrity is completely disrupted (Wang et al., 2018;Zheng et al., 2019). ...
... ONPG can only reach cytoplasm evading membrane barrier and be hydrolyzed by cytoplasmic β-galactosidase when the inner membrane permeability is compromised (Wang et al., 2018;Yun et al., 2018). However, the cytoplasmic βgalactosidase may also be leaked out and hydrolyze ONPG in the surrounding medium when the inner membrane permeabilization significantly increases, or otherwise, the membrane integrity is completely disrupted (Wang et al., 2018;Zheng et al., 2019). Likewise, the present study observed that extracellular hydrolysis of ONPG was luteolin concentrations-dependent in the non-lytic investigation, which is found to be similar to a previous study on the membrane-damaging effect of luteolin (Guo et al., 2020). ...
Thesis
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Over the last few years, interest in antibacterial and antibiofilm phytochemicals has been trending in next-generation antimicrobial development research and food safety management. However, the mechanistic actions of phytochemicals remain mostly unknown, which is considered a critical obstacle to their selective applications against particular pathogenic bacterial biofilms. Likewise, the biomechanics of biofilm inhibitory actions, along with the antibacterial activities of a potential flavone, luteolin, remain mostly elusive. While several recent studies reported the antibiofilm and antibacterial potential of luteolin against several pathogens, there is a notable lack of comprehensive knowledge regarding two detrimental foodborne pathogens, Salmonella Typhimurium and Escherichia coli, and their biofilms. Remarkably, no study has yet reported the antibiofilm and antibacterial actions of luteolin against S. Typhimurium. Henceforth, the study attempted to figure out the biofilm inhibitory biomechanics of luteolin by evaluating its antibiofilm efficacy against these foodborne pathogens using biotic (eggshell) and abiotic (stainless steel and silicon rubber) surfaces and further investigating its antibacterial actions on adhesion ability (surface hydrophobicity), membrane properties (permeability and integrity), and energy metabolism (metabolic activity, cellular respiration, and ATP biosynthesis) of planktonic cells. The study findings showed that luteolin has strong antibiofilm efficacy against S. Typhimurium and E. coli, evidenced by complete inhibition of biofilm formation by both pathogens on food-contact surfaces after 24h incubation with different MICs (ranging from 1/8 to 2 MIC) of luteolin. Luteolin could also significantly (P < 0.05) eliminate young (24-h) biofilms of both pathogens from the contact surfaces while treated with high concentrations (above MIC) of luteolin for 4h. Further investigation revealed that luteolin exerted its antibiofilm efficacy by significantly (P < 0.05) disrupting bacterial adhesion ability to contact surfaces via altering cell surface hydrophobicity and interfering with bacterial cell-contact surface interactions. The comprehensive investigations on luteolin and its antibacterial actions on planktonic cells further revealed that luteolin mainly exhibited its bactericidal activities by posing enormous ROS-mediated oxidative stress to cells in a dose-dependent manner that ultimately severely damaged the cellular structure and energy metabolic systems. The investigation of membrane-targeting actions of luteolin figured out that higher MICs of luteolin (1/2 and 1MIC) could significantly (P < 0.05) alter membrane permeability and drastically damage membrane integrity, evidenced by the leakage of intracellular components (e.g., cytoplasmic proteins and β-galactosidase). Further analysis of energy metabolism in cells demonstrated that luteolin-mediated oxidative stress significantly (P < 0.05) inhibits metabolic activity by dysregulating the cellular respiratory electron transport chain and suppressing cell bioenergy (ATP) production. Overall, the finding portrays the potency of luteolin as a potent antibacterial and antibiofilm agent, which would be efficiently applicable in designing next-generation antimicrobials and food safety management.
... Quercetin's antioxidative activities have been extensively studied and demonstrated through various pathways, both in vivo and in vitro (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Firstly, it exerts antioxidative effects by directly neutralizing free radicals, thereby reducing potential damage to important biological components such as proteins, DNA, and lipids (22)(23)(24). ...
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Purpose The objective of this study was to investigate the impact of quercetin, a potent antioxidant, on tendon healing utilizing a rat Achilles tendon injury model. Materials and methods The study involved 32 male Wistar-Albino rats, randomly split into experimental (quercetin) and control groups, each with 16 rats. A bilateral Achilles tenotomy model was applied, with the experimental group receiving quercetin and the control group receiving corn oil via oral gavage from surgery until sacrifice. One Achilles tendon per rat underwent histopathological and immunohistochemical evaluations, while the other underwent biomechanical analysis. Results Tendons were evaluated histopathologically in terms of tenocyte, ground substance, collagen, and vascularity, and quercetin was observed to significantly increase tendon healing in the experimental group ( p -values = 0.0232, 0.0128, 0.0272, 0.0307, respectively). In the immunohistochemical analysis, type I collagen, type III collagen, alpha smooth muscle actin (SMA), and Galectin-3 were evaluated, and it was observed that quercetin increased tendon healing ( p -values = 0.0166, 0.0036, 0.0323, 0.0295, respectively). In the biomechanical analysis, the rupture strength was evaluated with six parameters (failure load, maximum energy, displacement, stiffness, ultimate stress, and strain), and it was observed that quercetin significantly increased the rupture strength ( p -values = 0.032, 0.014, 0.026, 0.025, 0.045, 0.012, respectively). Conclusion Quercetin significantly enhanced tendon healing both biomechanically and immunohistochemically. However, further clinical studies are needed to understand its effects on human tendon healing, as this is the first study of its kind.
... Nguyen and Bhattacharya (2022) also reported that quercetin possesses the property to significantly disrupt the bacterial cell membrane integrity, thereby inhibiting bacterial growth, including E. coli. In another study Wang et al. (2018) also claimed that the inhibition mechanism of quercetin and their conjugates damage the structure of bacterial cell walls and cell membranes of E. coli, leading to an increase in the permeability of their structure. This condition also leads to the endochylema content releasing and affects ATP activity, thus protein synthesis of E. coli bacteria decreases, thereby affecting protein expression in the cell, and eventually, the cell lysed and experiences death. ...
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This study identifies the phytochemical compounds in ethanolic extracts of Syzygium myrtifolium leaves, applies it to developing composite bioplastics as a natural antibacterial agent, and compares it with composite bioplastics prepared with sodium benzoate, particularly regarding inhibition zone diameter and microstructure. The results showed that the phytochemicals were identified in the ethanolic extract of Syzygium myrtifolium leaves, like flavonoids, alkaloids, tannins, phenolics, terpenoids, and saponins. LC-MS confirming bioactive in it as auraptenol, calopiptin, quercetin-3-O-β-D-glucuronide, and quercetin-3-O-L-arabinopyranoside. Moreover, in vitro tests showed that composite bioplastics with the ethanolic extracts of Syzygium myrtifolium had inhibition zone diameter against E. coli, similar to those with sodium benzoate added. Additionally, the microstructure of the composite bioplastics with the ethanolic extracts of Syzygium myrtifolium was rougher, irregular, and more porous than those of another. It indicated that the ethanolic extract of Syzygium myrtifolium leaf could be used as a natural antibacterial agent to replace the chemical agent.
... Therefore, quercetin/colistin combination can be considered as an effective and less toxic solution for MDR treatment. Apart from anti-quorum sensing effect, quercetin can alter the bacterial cell wall ultrastructure and cell membrane integrity of the bacteria [23]. The increase in cell membrane permeability makes the combination treatment more effective. ...
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Quercetin is one of the flavonoids of a large group of phenolic compounds, which are secondary metabolites of plants and have a wide range of biological activity. A plant pigment, which is a powerful antioxidant flavonoid, found mainly in onions, grapes, berries, cherries, broccoli and citrus fruits. The flavonoid quercetin, due to its antioxidant, anti-inflammatory, antitumor, antimicrobial and other beneficial properties, is a promising biologically active substance and the subject of attention of many scientists around the world. In connection with the global problem of antimicrobial resistance, the scientific community is actively investigating the possibilities of using quercetin to create new antimicrobial drugs, especially against resistant strains of bacteria. Its combination with antibiotics can increase the effectiveness of standard antibacterial therapy. The use of quercetin in the fight against biofilms of microorganisms is also a promising area of research. The history of the study of quercetin began in 1814, and in 1854 the glycosidic nature of this substance was established. However, the world's interest in flavonoids, including quercetin, appeared only in 1936 after it was found that the sum of flavonoids of lemon peel has P-vitamin activity and strengthens the walls of blood vessels. Quercetin is synthesized in more than 400 plants and this is a large field of research for the scientific community, overcoming the low bioavailability of quercetin when administered orally and studying the passage of various forms of quercetin through the digestive tract, bioavailability – a task to be solved. Thus, quercetin is a promising bioactive compound with a wide range of medicinal properties. Its use in medicine can be the largest direction for combating statistical infectious, inflammatory and cancer diseases, as well as for improving general health and supporting the immune system. Research and development of quercetin-based drugs is a relevant area of modern science and human and veterinary medicine.
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Introduction: Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are manufactured fluorinated chemicals, including Perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA), they cause dormant environmental toxicity. Quercetin (QE), one of the major flavonoids, present in numerous food, has anti-oxidants and anti-inflammatory properties. Aim of the Study: The study aimed to clarify the potential hepatoprotective role of QE against PFOS-induced liver histological and immunohistochemical changes in adult male albino rats. Materials and Methods: Thirty-six adult male albino rats were randomly and equally distributed into three groups: Control group, PFOS-treated group: were received PFOS (20 mg/kg/day) by oral gavage for 28 days and PFOS+QE group: were received PFOS (20 mg/kg/day) and QE (75 mg/kg/day) by oral gavage for 28 days. At the end of the experiment, the rats in all groups were anesthetized, sacrificed, and the livers were processed for biochemical, histological, and immunohistochemical study. Results: In the PFOS-treated group, body weight, superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalyze (CAT) levels significantly decreased while liver weight, liver function tests (LFTs), malondialdehyde (MDA), and C-reactive protein (CRP) levels were significantly increased. Microscopically, liver sections of the PFOS-treated group exhibited inflammatory cellular infiltration, hepatocytes with vacuolation, abnormally shaped nuclei and swollen mitochondria. Also, strong positive reactions for Caspase 3 and tumor necrosis factor-alpha (TNF-α) were detected. The PFOS+QE group displayed a significant resetting of the biochemical parameters and a partial extenuation of the light, electron, and immunohistochemical changes. Conclusion: This work could highlight the possibility of using QE as a preventative strategy for potential PFOS-induced hepatic toxicity.
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Quercetin, a flavonoid found in vegetables and fruits, has been extensively studied for its health benefits and disease management. Its role in the prevention of various pathogenesis has been well-documented, primarily through its ability to inhibit oxidative stress, inflammation, and enhance the endogenous antioxidant defense mechanisms. Electronic databases such as Google Scholar, Scopus, PubMed, Medline, and Web of Science were searched for information regarding quercetin and its role in various pathogeneses. The included literature comprised experimental studies, randomized controlled trials, and epidemiological studies related to quercetin, while editorials, case analyses, theses, and letters were excluded. It has been reported to have a wide range of health benefits including hepatoprotective, antidiabetic, anti-obesity, neuroprotective, cardioprotective, wound healing, antimicrobial, and immunomodulatory effects, achieved through the modulation of various biological activities. Additionally, numerous in vitro and in vivo studies have shown that quercetin’s efficacies in cancer management involve inhibiting cell signaling pathways, such as inflammation, cell cycle, and angiogenesis, activating cell signaling pathways including tumor suppressor genes, and inducing apoptosis. This review aims to provide a comprehensive understanding of the health benefits of quercetin in various pathogeneses. Additionally, this review outlines the sources of quercetin, nanoformulations, and its applications in health management, along with key findings from important clinical trial studies. Limited clinical data regarding quercetin’s safety and mechanism of action are available. It is important to conduct more clinical trials to gain a deeper understanding of the disease-preventive potential, mechanisms of action, safety, and optimal therapeutic dosages. Furthermore, more research based on nanoformulations should be performed to minimize/overcome the hindrance associated with bioavailability, rapid degradation, and toxicity.
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In vitro and some animal models have shown that quercetin, a polyphenol derived from plants, has a wide range of biological actions including anti-carcinogenic, anti-inflammatory and antiviral activities; as well as attenuating lipid peroxidation, platelet aggregation and capillary permeability. This review focuses on the physicochemical properties, dietary sources, absorption, bioavailability and metabolism of quercetin, especially main effects of quercetin on inflammation and immune function. According to the results obtained both in vitro and in vivo, good perspectives have been opened for quercetin. Nevertheless, further studies are needed to better characterize the mechanisms of action underlying the beneficial effects of quercetin on inflammation and immunity.
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SUMMARY Salmonella contamination of laying hen flocks and shell eggs is associated with various management and environmental factors. Foodborne outbreaks of human salmonellosis have been traced back to consumption of Salmonella-contaminated shell eggs. In the present study, a systematic literature review was conducted to identify and provide an evidence-based overview of potential risk factors of Salmonella contamination of laying hens, layer premises, and shell eggs. This systematic literature search was conducted using AGRICOLA, CAB Abstracts, and PubMed databases. Observational studies that identified risk factors for Salmonella contamination of layer flocks and shell eggs were selected, and best evidence was synthesized to summarize the results. Altogether, 13 cross-sectional studies and four longitudinal studies published in English were included in the review. Evidence scores were assigned based on the study design and quality of the study to grade the evidence level. The strength of association of a risk factor was determined according to the odds ratios. In this systematic review, the presence of previous Salmonella infection, absence of cleaning and disinfection, presence of rodents, induced molting, larger flock size (>30,000 hens), multiage management, cage housing systems, in-line egg processing, rearing pullets on the floor, pests with access to feed prior to movement to the feed trough, visitors allowed in the layer houses, and trucks near farms and air inlets were identified as the risk factors associated with Salmonella contamination of laying hen premises, whereas high level of manure contamination, middle and late phase of production, high degree of egg-handling equipment contamination, flock size of >30,000, and egg production rate of >96% were identified as the risk factors associated with Salmonella contamination of shell eggs. These risk factors demonstrated strong to moderate evidence of association with Salmonella contamination of laying hens and shell eggs. Eggshells testing positive for Salmonella were 59 times higher when fecal samples were positive and nine times higher when floor dust samples were positive. Risk factors associated with Salmonella Enteritidis infection in laying hens were flock size, housing system, and farms with hens of different ages. As a summary, this systematic review demonstrated that Salmonella contamination of laying hen flocks and shell eggs in layer production systems is multifactorial. This study provides a knowledge base for the implementation of targeted intervention strategies to control Salmonella contamination of laying hen flocks and shell eggs.
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Gunnison sage-grouse (Centrocercus minimus) are distributed across southwestern Colorado and southeastern Utah, United States. Their distribution has decreased over the past century and the species has been listed as threatened by the U.S. Fish and Wildlife Service. Reduced genetic diversity, small population size, and isolation may affect Gunnison sage-grouse population persistence. Population augmentation can be used to counteract or mitigate these issues, but traditional translocation efforts have yielded mixed, and mostly unsuccessful, results. Captive-rearing is a viable, although much debated, conservation approach to bolster wild conservation-reliant species. Although there have been captive-rearing efforts with greater sage-grouse (C. urophasianus), to date, no information exists about captive-rearing methods for Gunnison sage-grouse. Therefore, we investigated techniques for egg collection, artificial incubation, hatch, and captive-rearing of chicks, juveniles, subadults, and adults for Gunnison sage-grouse. In 2009 we established a captive flock that produced viable eggs. From 2009-2011, we collected and artificially incubated 206 Gunnison sage-grouse eggs from 23 wild and 14 captive females. Our hatchability was 90%. Wild-produced eggs were heavier than captive-produced eggs and lost mass similarly during incubation. We produced 148 chicks in captivity and fed them a variety of food sources (e.g. invertebrates to commercial chow). Bacterial infections were the primary cause of chick mortality, but we successfully reduced the overall mortality rate during the course of our study. Conservationists and managers should consider the utility in developing a captive-rearing program or creating a captive population as part of a proactive conservation effort for the conservation-reliant Gunnison sage-grouse. Zoo Biol. XX:XXX-XXX, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
Article
Correct spelling of co-author's Kalab first name is Milos or Miloslav. The Chinese green tea extract was found to strongly inhibit the growth of major food-borne pathogens, Escherichia coli O157:H7, Salmonella Typhimurium DT104, Listeria monocytogenes, Staphylococcus aureus, and a diarrhoea food-poisoning pathogen Bacillus cereus, by 44–100% with the highest activity found against S. aureus and lowest against E. coli O157:H7. A bioassay-guided fractionation technique was used for identifying the principal active component. A simple and efficient reversed-phase high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of four bioactive polyphenol compounds, epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epicatechin (EC), and caffeine (CN). The structures of these polyphenols were confirmed with mass spectrometry. Among the four compounds, ECG and EGCG were the most active, particularly EGCG against S. aureus. EGCG had the lowest MIC90 values against S. aureus (MSSA) (58 mg/L) and its methicilin-resistant S. aureus (MRSA) (37 mg/L). Scanning electron microscopy (SEM) studies showed that these two compounds altered bacterial cell morphology, which might have resulted from disturbed cell division. This study demonstrated a direct link between the antimicrobial activity of tea and its specific polyphenolic compositions. The activity of tea polyphenols, particularly EGCG on antibiotics-resistant strains of S. aureus, suggests that these compounds are potential natural alternatives for the control of bovine mastitis and food poisoning caused by S. aureus.