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.
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 developed 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 antibiotic-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 microorganisms directly influence host health. The normal microbiota 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 (Staphylococcus 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 performance (anticarcinogenic, anti-inflammatory, antiviral, antioxidant, 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 33°C for the first 3 days, and then the temperature was reduced to 24°C 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 −80°C 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-μL PCR volume contained 45 ng of template DNA and 5 μmol/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 μL of template DNA, 5 μL of 10× Taq buffer with (NH4)2SO4, 1 μL of deoxynucleoside triphosphates, 0.5 μL of Taq DNA polymerase, 4 μL of MgCl2, and 2 μL of each primer, for a final volume of 50 μL with the addition of sterilized double-distilled water (Sangon Biotech, Shanghai, People's Republic of China). The PCR assay was denatured at 95°C for 5 min and kept at 95°C for 1 min. The temperature was subsequently dropped to 54°C for 1 min followed by an elongation step of 72°C for 1 min. After the first products were generated, 35 cycles of 95°C for 1 min, 54°C for 1 min, and 72°C for 1 min were completed followed by a final elongation step was at 72°C for 30 min and holding at 4°C for 5 min. Aliquots of 3 μL for each amplification product was separated in 1× Tris-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 1× TAE with gradients formed with 8% acrylamide stock solutions and contained 35 to 60% denaturant. Preelectrophoresis was performed at 200 V for 10 min at 60°C. Electrophoresis was then performed at 90 V for 12 h at the same temperature. After electrophoresis, the gels were stained with AgNO3.
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 4°C in a 1.5-mL tube containing 100 μL 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 DH5α cells (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 DNAMAN tool (Lynnon Biosoft, San Ramon, CA) was used to check the chimeric contructs of sequences deposited in the National Center for Biotechnology Information database (NCBI; Bethesda, MD).
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 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. The 10-μL reaction mixture contained 5 μL of SYBR Premix Ex Taq, 0.2 μL of ROX Reference Dye II, 0.2 μL of each primer (10 μmol/L), 3.4 μL of sterilized double-distilled water, and 1 μL 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 107, 105, 103, 10, 10−1, 10−3, 10−5, 10−7, and a standard curve was established.
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 Pseudomonas 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 μmol/mL with DMSO and then diluted to 0.006 μmol/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 37°C. 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.
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 × 104 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 μmol/mL). Oxford cups were placed on the inoculated agar, and 100 μL of quercetin solution was added to each cup and incubated at 37°C. 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.
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 × the MIC, 10× MIC, 50× MIC, 100× MIC, and 500× MIC were prepared. Bacterial suspensions containing 1 × 107 CFU/mL were then added to the quercetin solutions, and the cultures were kept in a constant temperature shaking incubator at 37°C with shaking at 220 rpm. After 4.5 h, the suspensions were centrifuged at 10,000 × g for 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, Tokyo, Japan). The bacterial suspension containing 1 × 107 CFU/mL in DMSO without quercetin was used as a negative control. The bacterial suspension containing 1 × 107 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 37°C 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 1.0× MIC, 10× MIC, 50× MIC, 100× MIC, and 500× MIC were added to the other tubes. All tubes were incubated at 37°C. 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 Bioengineering 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 β-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 Bioengineering). 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.
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.
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, Helicobacter, and Campylobacter jejuni), Firmicutes (Clostridium), Bacteroidetes (Bacteroides), and Deferribacteres (Deferribacterales) (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 quercetin, 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 initial levels of 2.2 × 109, 1.4 × 109, 2.3 × 109, and 2.1 × 109 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 μmol/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 quercetin 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 50× MIC and 10× MIC, 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 50× MIC, 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 10× MIC 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 ultrastructure
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 50× MIC, 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 10× MIC, 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.
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 50× MIC quercetin than in treatments with antibiotics (Table 7).
Effects of quercetin on the permeability of bacterial cell membranes
Compared with the control, the β-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 concentrations (Table 10).
The changes in β-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 100× MIC quercetin on the activity of β-galactosidase was higher than that of antibiotics. For soluble protein concentrations, the effects of 50× MIC quercetin were greater than that of antibiotics for both bacteria, and for ATP activity, the effects of quercetin at 10× MIC (E. coli) and 100× MIC (S. aureus) were greater than that of antibiotics.
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 Bifidobacterium (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 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 μmol/mL, respectively. Sugarcane 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). 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 investigate 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 concentrations.
The effects of drugs on the morphology and ultrastructure 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 condensation (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 10× MIC quercetin, and treatment of E. coli cells with 50× MIC 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 compromised 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 significantly influenced cell wall permeability. These results are consistent with those reported for other flavonoids (6, 40, 45).
β-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 β-galactosidase to be released into the surrounding medium (31). In the present study, the β-galactosidase activity in the E. coli and S. aureus culture medium increased significantly with increasing concentrations of quercetin, indicating that cell membrane 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 β-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 concentration 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. 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 concentrations; 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.
This study was supported by the Heilongjiang Provincial Government of China (C2016017) and the Harbin Science and Technology Bureau of China (2015RQXXJ014).