CN115998807A - Application of tea polyphenol in preparation of medicines for inhibiting bacteria from generating antibiotic resistance - Google Patents

Abstract

The invention provides application of tea polyphenol in preparing a medicament for inhibiting bacteria from generating antibiotic resistance, and belongs to the technical field of medicaments. The invention proves that the tea polyphenol can reduce the mutation frequency of bacterial drug resistance induced by antibiotics and non-antibiotics and prevent the generation of multiple antibiotic drug resistant bacteria. The invention discloses the effect and application of tea polyphenol in preventing the generation of multiple antibiotic drug-resistant bacteria for the first time, and provides a new way for the anti-infection treatment of the drug-resistant bacteria.

Description

Application of tea polyphenol in preparation of medicines for inhibiting bacteria from generating antibiotic resistance

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of tea polyphenol in preparation of medicines for inhibiting bacteria from generating antibiotic resistance.

Background

Bacterial antibiotic resistance is a global public health problem. In a clinical setting, antibiotic-resistant bacterial infections reduce alternative effective treatment regimens, and thereby increase morbidity and mortality, as compared to bacterial infections caused by susceptible bacteria. The lancet reported in 2022 that antibiotic-resistant bacteria infection directly caused about 127 tens of thousands of deaths worldwide in 2019, and 1000 tens of thousands of deaths were expected every year by 2050.

Antibiotic abuse and long-term administration are the main reasons for the massive emergence of antibiotic-resistant bacteria. Common cold, enteritis, tonsillitis, and infections are all listed as antibiotic indications. In addition, more and more studies report that, in addition to antibiotics inducing bacterial resistance, non-antibiotic drugs (e.g., metformin, fluoxetine, duloxetine, etc.) can also induce intestinal bacteria to develop antibiotic resistance; in 2018, nature reported that 270 or more non-antibiotic drugs can inhibit the growth of at least one microorganism in the gut, and it was thought that these non-antibiotic drugs could have the potential to promote antibiotic resistance by bacteria, and even cause global public health problems.

Bacterial resistance is a serious problem, and presents a great challenge to antibiotic therapy, which poses a serious threat to human health. In order to effectively control the generation and transmission of clinical antibiotic resistant bacteria, prevention and control drugs of the antibiotic resistant bacteria must be developed. At present, aiming at antibiotic resistant bacteria clinically, treatment means including antibiotic combination therapy, new drug research and development and the like are often adopted, and as the development speed of bacterial drug resistance is far faster than that of new drugs, the control effect of the antibiotic combination therapy on super resistant bacteria is limited, so that searching for effective means for preventing or inhibiting bacteria from generating antibiotic resistance is an important way for coping with multi-drug resistant bacterial infection, however, the related technology has not been reported yet.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of tea polyphenol in preparing a medicament for inhibiting bacteria from generating antibiotic resistance.

The invention provides application of tea polyphenol in preparing a medicament for inhibiting bacteria from generating antibiotic resistance.

Preferably, the tea polyphenols include flavanols, anthocyanins, flavonoids, flavonols and phenolic acids.

Preferably, the concentration of the tea polyphenol in the medicine is 200-800 mg/g.

Preferably, the bacteria include pathogenic bacteria that develop antibiotic resistance caused by clinical drugs;

the clinical drugs include antibiotic and non-antibiotic drugs.

Preferably, the antibiotics include three or more of the following antibiotics: iminothiolane, tetracycline, gentamicin, chloramphenicol, azithromycin, cefotaxime, ceftazidime, cefazolin, cefoxitin, ciprofloxacin, doripenem, ertapenem, tigecycline, polymyxin B, cefepime, tobramycin, levofloxacin, doxycycline, aztreonam, minocycline, meropenem, and amikacin.

Preferably, the non-antibiotic drug comprises any one or more of the following: metformin, fluoxetine and duloxetine.

Preferably, the pathogenic bacteria include the genera Escherichia (Escherichia Castellani and Chalmers), klebsiella (Klebsiella), and Pseudomonas (Pseudomonas).

Preferably, the genus Escherichia comprises Escherichia coli;

the genus Ehrlichia includes Klebsiella pneumoniae (Klebsiella pneumoniae);

the genus pseudomonas includes pseudomonas aeruginosa (Pseudomonas aeruginosa);

the invention provides a medicine for preventing antibiotic drug-resistant bacteria, which comprises tea polyphenol and components for causing the bacteria to generate antibiotic drug resistance;

the mass ratio of the tea polyphenol to the components which cause the bacteria to generate antibiotic resistance is 0.8-1.2:0.8-1.2.

Preferably, the components that cause the bacteria to develop antibiotic resistance include antibiotics and non-antibiotic drugs;

the antibiotics preferably include three or more of the following antibiotics: iminothiolane, tetracycline, gentamicin, chloramphenicol, azithromycin, cefotaxime, ceftazidime, cefazolin, cefoxitin, ciprofloxacin, doripenem, ertapenem, tigecycline, polymyxin B, cefepime, tobramycin, levofloxacin, doxycycline, aztreonam, minocycline, meropenem, and amikacin;

the non-antibiotic drug preferably comprises any one or two or more of the following: metformin, fluoxetine and duloxetine.

The invention provides application of tea polyphenol in preparing a medicament for inhibiting bacteria from generating antibiotic resistance. Experiments prove that the metformin, the chloramphenicol, the ceftazidime and the cefazolin can induce bacteria to generate multiple antibiotic resistance, and the tea polyphenol can effectively inhibit bacteria from generating antibiotic resistance in vivo and in vitro. The results of the embodiment of the invention show that compared with the mutation frequency of bacteria in a control group (a sterile water treatment group), the metformin, chloramphenicol, ceftazidime and cefazolin can obviously raise the mutation frequency of the bacteria to generate multiple antibiotic resistant bacteria; the mutation frequencies of the groups after the addition of the tea polyphenol are not significantly different from those of the control group, so that the tea polyphenol can inhibit the generation of bacterial antibiotic resistance induced by non-antibiotics represented by metformin and antibiotics represented by chloramphenicol, ceftazidime and cefazolin. The invention discloses the effect and application of tea polyphenol in inhibiting bacterial antibiotic resistance for the first time, and provides a new way for anti-infection treatment of drug-resistant bacteria.

Drawings

FIG. 1 shows the results of the frequency of chloramphenicol-resistant mutations of enteric bacteria under metformin and chloramphenicol exposure; wherein p represents a value < 0.05;

FIG. 2 shows the results of antibiotic susceptibility of an enteric bacterial chloramphenicol resistant isolate;

FIG. 3 shows the results of chloramphenicol resistance mutation frequency of enterobacteria under tea polyphenol exposure, which shows p value < 0.05; # represents p value > 0.05;

FIG. 4 shows the frequency of chloramphenicol-resistant mutations in E.coli under individual and combined exposure of each drug; * Represents a p value < 0.05; # represents p value > 0.05;

FIG. 5 shows the antibiotic susceptibility results of E.coli chloramphenicol resistant mutants;

FIG. 6 shows the frequency of chloramphenicol resistance mutations in Pseudomonas aeruginosa with individual and combined exposures, with p values < 0.05; # represents p value > 0.05;

FIG. 7 shows the results of antibiotic susceptibility of Pseudomonas aeruginosa chloramphenicol resistant mutants;

FIG. 8 shows the results of chloramphenicol resistance mutation frequencies of Klebsiella pneumoniae with individual and combined exposures, with p values < 0.05; # represents p value > 0.05;

FIG. 9 shows the antibiotic susceptibility results of Klebsiella pneumoniae chloramphenicol resistant mutants.

Detailed Description

The invention provides application of tea polyphenol in preparing a medicament for inhibiting bacteria from generating antibiotic resistance.

In the present invention, the tea polyphenols preferably include flavanols, anthocyanins, flavonoids, flavonols, and phenolic acids. The effective concentration of the tea polyphenol is preferably 5-15 mg/L in vitro, more preferably 10mg/L; the body weight is preferably 100 to 300mg/kg, more preferably 200 mg/kg. The animal is preferably a rat. In the present invention, the tea polyphenol is purchased from Shanghai source leaf biotechnology Co.

In the present invention, clinical drugs that cause bacteria to develop antibiotic resistance preferably include bacteria that cause antibiotic resistance by antibiotic and/or non-antibiotic drugs. The antibiotics preferably include three or more of the following antibiotics: iminothiolane, tetracycline, gentamicin, chloramphenicol, azithromycin, cefotaxime, ceftazidime, cefazolin, cefoxitin, ciprofloxacin, doripenem, ertapenem, tigecycline, polymyxin B, cefepime, tobramycin, levofloxacin, doxycycline, aztreonam, minocycline, meropenem, and amikacin; more preferably, chloramphenicol, ceftazidime, and cefazolin are included. The effective concentrations of chloramphenicol, ceftazidime and cefazolin are preferably 2-8 mg/L, more preferably 4mg/L, 2mg/L and 4mg/L, respectively. The non-antibiotic drugs preferably include metformin, fluoxetine, and duloxetine, more preferably include metformin. The effective concentration of the metformin in vitro is preferably 1-100 mg/L, more preferably 10mg/L; the effective in vivo concentration of metformin is preferably 150 to 250mg/kg, more preferably 200mg/kg body weight. The bacteria include enteric bacteria such as Escherichia, eggeratia and Pseudomonas. The genus escherichia preferably includes escherichia coli; the genus Ehrlichia preferably comprises Klebsiella pneumoniae; the genus pseudomonas preferably comprises pseudomonas aeruginosa.

In the embodiment of the invention, in vivo experiments, the metformin and chloramphenicol gastric lavage rats are separated to obtain chloramphenicol resistant isolates, and drug sensitivity experiments show that the metformin and chloramphenicol can cause intestinal bacteria to generate multiple antibiotic resistance, wherein the MIC of the chloramphenicol and the macrolide azithromycin in tetracyclines, doxycycline and minocycline in chloramphenicol exceeds the drug resistance limit. In vitro experiments, escherichia coli, pseudomonas aeruginosa and klebsiella pneumoniae are respectively exposed in LB culture media with certain concentrations of metformin and chloramphenicol (ceftazidime and cefazolin), and compared with unexposed bacteria, the exposure experiment results show that the mutation frequency of the bacteria and the MIC of various antibiotics are obviously improved. In the embodiment of the invention, the mutation frequency and the antibiotic sensitivity are used as indexes to indicate the antibiotic resistance condition of bacteria, and in-vivo and in-vitro exposure experimental results show that the tea polyphenol can obviously inhibit the generation of the antibiotic resistance of bacteria after being added, thus indicating that the tea polyphenol can prevent the bacteria from generating the antibiotic resistance.

In the invention, the tea polyphenol can effectively inhibit bacteria from generating antibiotic resistance under both in-vivo and in-vitro conditions.

The invention provides a medicine for preventing antibiotic drug-resistant bacteria, which comprises tea polyphenol and components for causing the bacteria to generate antibiotic drug resistance; the mass ratio of the tea polyphenol to the components which cause the bacteria to generate antibiotic resistance is 0.8-1.2:0.8-1.2.

In the present invention, the components that cause the bacteria to develop antibiotic resistance preferably include antibiotics and non-antibiotic drugs. The antibiotics preferably include three or more of the following antibiotics: iminothiolane, tetracycline, gentamicin, chloramphenicol, azithromycin, cefotaxime, ceftazidime, cefazolin, cefoxitin, ciprofloxacin, doripenem, ertapenem, tigecycline, polymyxin B, cefepime, tobramycin, levofloxacin, doxycycline, aztreonam, minocycline, meropenem, and amikacin, more preferably including chloramphenicol, ceftazidime, and cefazolin. The non-antibiotic drug preferably comprises any one or two or more of the following: metformin, fluoxetine and duloxetine, more preferably comprises metformin. The mass ratio of tea polyphenols to the components responsible for the development of antibiotic resistance of the bacteria is preferably 1:1. The preparation method of the medicine is not particularly limited, and conventional preparation methods of clinical medicines known in the art can be adopted.

The use of tea polyphenols according to the present invention for the manufacture of a medicament for inhibiting the development of antibiotic resistance in bacteria is described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Metformin and chloramphenicol induce intestinal bacteria to develop multiple antibiotic resistance

The model mouse of the ZDF male diabetes of 13-14 weeks old (ZDF) is selected ^fa/fa ) 15 rats purchased from Beijing Veitz laboratory animal technologies Inc. and having been assayed for fasting blood glucose levels prior to delivery to ensure successful molding, rats with no related infectious disease and no limb insufficiency deformity. The experimental animals are kept in SPF-class animal houses, two animals are kept in one cage, free drinking and eating are ensured in the experimental process, the circadian rhythm is 12h, the ambient temperature is (25.0+/-0.5) DEG C, and the relative humidity is 40% -60%. ZDF rats were grouped and numbered according to the random number method, 5 per group, divided into 3 groups: control group (sterile water treatment), metformin group (200 mg/kg) and chloramphenicol group (160 mg/kg). The stomach was irrigated once daily for 8 weeks continuously during the experiment. Feces were picked once a week during the experiment and chloramphenicol resistant mutants were isolated and mutation frequencies were calculated according to formula I.

Mutation frequency = N ₀ N formula I

Wherein N is ₀ The colony number of the chloramphenicol-resistant isolated strain obtained by screening the chloramphenicol-free far rattan culture medium in 100 mu L of the bacterial liquid is multiplied by the dilution factor, and N is the total colony number in the far rattan culture medium without chloramphenicol at each concentration in 100 mu L of the bacterial liquid is multiplied by the dilution factor. When N is ₀ When=1, the spontaneous mutation frequency was noted.

The mutation frequencies of intestinal coliform are shown in FIG. 1, and the results show that the mutation frequencies of the metformin group and the chloramphenicol group reach the highest after 8 weeks of continuous gastric lavage of diabetic rats, namely 5.89×10 respectively ^-5 And 2.45X10 ^-4 Mutation frequency (< 2.30X10) compared with control group ^-7 ) In comparison, the resistance mutation frequency of chloramphenicol increased 255.97-fold and 1065.22-fold (< 0.05). The results show that the metformin and the chloramphenicol can obviously improve the mutation frequency of the intestinal coliform of the ZDF rat on the resistance to the chloramphenicol and promote the intestinal bacteria to generate antibiotic resistance.

For the above isolated metformin group (MET-CHL ^r ) And chloramphenicol group (CHL-CHL) ^r ) Is subjected to antibiotic susceptibility testing. The antibiotic susceptibility test is specifically as follows:

antibiotic susceptibility experiments were performed with a turbidimeter, full automatic sampler AIM, microbiological identification and drug susceptibility analyzer manufactured by Thermo Fisher Scientific company, with reference to Mueller-Hinton (MH) microbulk dilution recommended by the american clinical laboratory standardization institute (Clinical and Laboratory Standards Institute, CLSI). The plates (CHNM 4F and GNX2F, respectively) can detect 22 antibiotics, namely, iminothiolane (IPM), tetracycline (tetracycloine, TET), gentamicin (Gentamicin, GEN), chloramphenicol (Chloramphhexiol, CHL), azithromycin (Azithromycin, AZI), cefotaxime (Cefotazime, FOT), ceftazidine (Cefttazidime, TAZ), cefazolin (Cefazoline, FAZ), cefoxitin (CefoX), ciprofloxacin (CIP), doripenem (Dotapenem, DOR), ertapenem (ETR), tigecycline (Tigecyclomycin, C), polymyxin B (PolyxinB, AZI), cefpiramide (Cefotame), toxel (FEP), levalacycline (Levalnem), and doxorubicin (Levalnem, paxin, levalnem).

The method comprises the following specific steps: (1) Selecting 3 strains of the strain to be detected, taking wild escherichia coli K12 as a control (3 strains), and simultaneously adding the strains into a sterile LB liquid culture medium without antibiotics, and culturing at 37 ℃ for 150r/min overnight;

(2) Streaking the overnight cultured mutant strain into LB agar medium without antibiotics, and culturing for 24 hours at 37 ℃ in an inverted way;

(3) Preparing a bacterial suspension: picking the mutant strain cultured overnight on a flat plate by using an inoculating loop into 5mL distilled water, and shaking and mixing uniformly;

(4) The turbidimeter power supply is inserted, the standard tube is gently and reversely mixed for 3 to 5 times, and the standard tube is inserted into a detection hole for calibration, and the standard tube is 0.5 McFarland (MCF) standard turbidity;

(5) Placing the prepared bacterial suspension test tube into a detection hole for calibration;

(6) mu.L of the calibrated bacterial suspension (0.5 MCF) was pipetted into a tube containing 11mL of MH broth, at which point an inoculation density of 5X 10 was obtained ⁵ CFU/mL of bacterial suspension;

(7)Sensititre

the sample adding head replaces the test tube cover and is transferred to the AIM of the full-automatic sample adding instrument;

(8) Loading the drug sensitive plate onto an AIM loading frame, wherein the bar code of the drug sensitive plate faces to the drug sensitive plate;

(9) Selecting a 50 mu L loading volume according to the instrument instruction;

(10) After sample addition is completed, a sealing film is stuck in time, and the sample is cultured for 24 hours at 37 ℃ and then is detected by a machine;

(11) After the cultivation is completed, the drug sensitive plate is placed in a to-be-detected area of the drug sensitive detector, and antibiotic sensitivity detection is carried out according to SWIN software operation instructions.

As shown in FIG. 2, the minimum inhibitory concentrations (Minimal Inhibitory Concentration, MIC) of the chloramphenicol-resistant isolates of the metformin group and the chloramphenicol group against iminothiolane, tetracycline, chloramphenicol, azithromycin, cefotaxime, ceftazidime, cefazolin, ciprofloxacin, tigecycline, cefepime, doxycycline and minocycline were all increased to a different extent (2-32 times) compared to the MIC of the wild strain of E.coli and were resistant to tetracyclines, doxycycline and minocycline in tetracyclines, chloramphenicol in chloramphenicol, and the azithromycin of macrolides were all over the resistance limit, with the MIC of doxycycline being increased up to 32 times, indicating that the metformin and chloramphenicol-induced intestinal chloramphenicol-resistant isolates were resistant to tetracyclines, chloramphenicol and macrolide antibiotics, which were multiple antibiotics resistant.

Example 2

Tea polyphenols can inhibit intestinal bacteria from generating multiple antibiotic resistance

The model mouse of the ZDF male diabetes of 13-14 weeks old (ZDF) is selected ^fa/fa ) 45 rats purchased from Beijing Veitz laboratory animal technologies Inc. and having measured fasting blood glucose levels prior to delivery ensured successful molding, and rats had no related infectious disease and no limb insufficiency deformity. The experimental animals are raised in SPF-class animal houses, two animals are housed in one cage, and in the experimental processEnsuring free drinking and eating, circadian rhythm for 12h, environment temperature of (25.0+/-0.5) DEG C and relative humidity of 40-60 percent. ZDF rats were grouped and numbered according to the random number method, 5 per group, divided into 6 groups altogether: control (sterile water treatment), metformin (200 mg/kg), chloramphenicol (160 mg/kg), tea polyphenols (200 mg/kg) +metformin (200 mg/kg), tea polyphenols (200 mg/kg) +chloramphenicol (160 mg/kg). The stomach was irrigated once daily for 8 weeks continuously during the experiment. Feces were collected once a week during the experiment and chloramphenicol resistant mutants were isolated and mutation frequencies were calculated as in example 1.

The mutation frequencies of intestinal coliform are shown in FIG. 3, and the results show that the mutation frequencies of the intestinal coliform are (< 2.35×10) compared with those of a control group ^-7 ) In comparison, the continuous administration of metformin and chloramphenicol for 8 weeks can significantly increase the mutation frequency of intestinal coliform (5.43X10) ^-5 And 2.25X10 ^-4 ) The method comprises the steps of carrying out a first treatment on the surface of the In contrast, the mutation frequencies of the tea polyphenol+chloramphenicol group and the tea polyphenol+metformin group were not significantly different from those of the control group (#p > 0.05), which suggests that tea polyphenol can prevent the occurrence of intestinal bacterial multi-antibiotic resistance.

Example 3

Tea polyphenols can inhibit antibiotic resistance of Escherichia coli

E.coli K12MG1665 (strain No. ATCC 700926) was inoculated into LB medium and incubated on a shaker at 150rpm and 37℃for 12 hours to give a bacterial solution having a concentration of 10 ⁸ ～10 ⁹ CFU/mL. Then, 30. Mu.L of fresh bacterial culture was transferred to 2970. Mu.L of LB liquid medium containing different drugs, the drug classes include metformin (purchased from beijing solebao technologies, inc.), chloramphenicol (purchased from the biotechnology (Shanghai) stock, tea polyphenols (purchased from Shanghai source leaf biotechnology, inc.), blueberry extract (purchased from Shanghai source leaf biotechnology, inc.), grape seed extract (purchased from Shanghai source leaf biotechnology, inc.), anthocyanins (purchased from Shanghai source leaf biotechnology, inc.) and astaxanthin (purchased from Shanghai source leaf biotechnology, inc.).

The experiments were divided into 18 groups, namely a control group (sterile water treatment), a metformin group (10 mg/L), a chloramphenicol group (4 mg/L), a tea polyphenol group (10 mg/L), a blueberry extract group (10 mg/L), a grape seed extract group (10 mg/L), a anthocyanin group (10 mg/L), an astaxanthin group (10 mg/L), a metformin+tea polyphenol (10 mg/L), a metformin+blueberry extract (10 mg/L), a metformin (10 mg/L) +grape seed extract (10 mg/L), a metformin (10 mg/L) +anthocyanin (10 mg/L), a metformin (10 mg/L) +astaxanthin (10 mg/L), a chloramphenicol (4 mg/L) +tea polyphenol (10 mg/L), a chloramphenicol (4 mg/L) +blueberry extract (10 mg/L), a chloramphenicol (4 mg/L) +grape seed extract (10 mg/L), a chloramphenicol (4 mg/L) +astaxanthin (10 mg/L) and a chloramphenicol (10 mg/L). After incubation on a shaker at 150rpm for 24h at 37℃100. Mu.L of drug-exposed E.coli culture was pipetted onto a far rattan agar (BD, USA) plate containing 16mg/L chloramphenicol, the presence or absence of antibiotic-resistant bacteria was observed and antibiotic susceptibility experiments were performed.

The results of mutation frequency and antibiotic sensitivity are shown in fig. 4 and 5, and the results show that metformin and chloramphenicol can induce escherichia coli to generate antibiotic resistance (p < 0.05); the tea polyphenol can inhibit the generation of antibiotic resistance of escherichia coli after being added, which indicates that the tea polyphenol can prevent the generation of antibiotic resistance of escherichia coli.

Example 4

Tea polyphenols can inhibit Pseudomonas aeruginosa from producing antibiotic resistance

Pseudomonas aeruginosa (strain number ATCC 27853) is inoculated into LB culture medium and incubated for 12h on a shaking table at 150rpm and 37 ℃ to reach the bacterial concentration of 10 ⁸ ～10 ⁹ CFU/mL. Then, 30. Mu.L of fresh bacterial culture was transferred to 2970. Mu.L of LB liquid medium containing different drugs, including metformin, ceftazidime (purchased from Biotechnology (Shanghai) Co., ltd.), tea polyphenols, blueberry extract, grape seed extract, anthocyanidin and astaxanthin.

The total composition is 18 groups, namely a control group (sterile water treatment), a metformin group (10 mg/L), a ceftazidime group (2 mg/L), a tea polyphenol group (10 mg/L), a blueberry extract group (10 mg/L), a grape seed extract group (10 mg/L), a anthocyanin group (10 mg/L), an astaxanthin group (10 mg/L), a metformin (10 mg/L) +tea polyphenol (10 mg/L), a metformin (10 mg/L) +blueberry extract (10 mg/L), a metformin (10 mg/L) +grape seed extract (10 mg/L), a metformin (10 mg/L) +anthocyanin (10 mg/L), a metformin (10 mg/L) +astaxanthin (10 mg/L), a ceftazidime (2 mg/L) +tea polyphenol (10 mg/L), a ceftazidime (2 mg/L) +blueberry extract (10 mg/L), a ceftazidime (10 mg/L) +ceftazidime (10 mg/L), and a ceftazidime (10 mg/L) of the other than are respectively. After 24h incubation on a shaker at 150rpm at 37 ℃, 100 μl of drug-exposed pseudomonas aeruginosa culture was pipetted into a pseudomonas agar basal medium (BD, USA) containing 8mg/L ceftazidime, and antibiotic resistance bacteria were observed for the presence or absence of antibiotic resistance bacteria, and antibiotic susceptibility experiments were performed.

The results of mutation frequency and antibiotic sensitivity are shown in fig. 6 and 7, and the results show that metformin and ceftazidime can induce pseudomonas aeruginosa to generate multiple antibiotic resistance (p < 0.05); the tea polyphenol can inhibit the pseudomonas aeruginosa from generating antibiotic resistance after being added, which indicates that the tea polyphenol can prevent the pseudomonas aeruginosa from generating antibiotic resistance.

Example 5

Tea polyphenols can inhibit antibiotic resistance of Klebsiella pneumoniae

Klebsiella pneumoniae (strain number ATCC 700603) is inoculated into LB liquid medium and incubated for 12h on a shaking table at 150rpm and 37 ℃ to lead the concentration of bacterial liquid to reach 10 ⁸ ～10 ⁹ CFU/mL. Then, 30. Mu.L of fresh bacterial culture was transferred to 2970. Mu.L of LB liquid medium containing different drugs, including metformin, cefazolin (available from Biotechnology (Shanghai) Co., ltd.), tea polyphenols, blueberry extract, grape seed extract, anthocyanidin and astaxanthin.

The total composition is 18 groups, namely a control group, a metformin group (10 mg/L), a cefazolin group (4 mg/L), a tea polyphenol group (10 mg/L), a blueberry extract group (10 mg/L), a grape seed extract group (10 mg/L), a anthocyanin group (10 mg/L), an astaxanthin group (10 mg/L), a metformin (10 mg/L) +tea polyphenol group, a metformin (10 mg/L) +blueberry extract (10 mg/L), a metformin (10 mg/L) +grape seed extract (10 mg/L), a metformin (10 mg/L) +anthocyanin (10 mg/L), a metformin (10 mg/L) +astaxanthin (10 mg/L), a cefazolin (4 mg/L) +tea polyphenol (10 mg/L), a cefazolin (4 mg/L) +blueberry extract (10 mg/L), a cefazolin (4 mg/L) +2 mg/L, a cefazolin (10 mg/L) +4 mg/L), and a cefazolin (10 mg/L). After incubation on a shaker at 150rpm for 24h at 37 ℃, 100 μl of drug-exposed klebsiella pneumoniae cultures were pipetted onto a far rattan agar (BD, USA) plate containing 16mg/L cefazolin, and antibiotic-resistant bacteria were observed for the presence or absence of antibiotic-resistant bacteria, and antibiotic susceptibility experiments were performed.

The results of mutation frequency and antibiotic sensitivity are shown in fig. 8 and 9, and the results show that metformin and cefazolin can induce klebsiella pneumoniae to generate multiple antibiotic resistance (p < 0.05); the tea polyphenol can inhibit the generation of antibiotic resistance of klebsiella pneumoniae after being added, which shows that the tea polyphenol can prevent the generation of antibiotic resistance of klebsiella pneumoniae.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. The application of tea polyphenols in preparing medicine for inhibiting bacteria from generating antibiotic resistance is provided.

2. The use according to claim 1, wherein the tea polyphenols comprise flavanols, anthocyanins, flavonoids, flavonols and phenolics.

3. The use according to claim 1 or 2, wherein the concentration of tea polyphenols in the medicament is 200-800 mg/g.

4. The use according to claim 1, wherein the bacteria comprise pathogenic bacteria responsible for the development of antibiotic resistance caused by clinical drugs;

the clinical drugs include antibiotic and non-antibiotic drugs.

5. The use according to claim 1 or 4, wherein the antibiotics comprise more than three of the following antibiotics: iminothiolane, tetracycline, gentamicin, chloramphenicol, azithromycin, cefotaxime, ceftazidime, cefazolin, cefoxitin, ciprofloxacin, doripenem, ertapenem, tigecycline, polymyxin B, cefepime, tobramycin, levofloxacin, doxycycline, aztreonam, minocycline, meropenem, and amikacin.

6. The use according to claim 4, wherein the non-antibiotic medicament comprises any one or more of the following: metformin, fluoxetine and duloxetine.

7. The use according to claim 4, wherein the pathogenic bacteria comprise the genera escherichia (Escherichia Castellani and Chalmers), klebsiella (Klebsiella) and Pseudomonas (Pseudomonas).

8. The use according to claim 7, wherein the genus Escherichia comprises Escherichia coli (Escherichia coli);

the genus Eklebsiella includes Klebsiella pneumoniae (Klebsiella pneumaroniae);

the genus Pseudomonas includes Pseudomonas aeruginosa (Pseudomonas aeruginosa).

9. A medicament for preventing the development of antibiotic-resistant bacteria, comprising tea polyphenols and a component that causes the bacteria to develop antibiotic resistance;

the mass ratio of the tea polyphenol to the components which cause the bacteria to generate antibiotic resistance is 0.8-1.2:0.8-1.2.

10. The medicament of claim 9, wherein the components that cause the bacteria to develop antibiotic resistance include antibiotic and non-antibiotic drugs;

the antibiotics preferably include three or more of the following antibiotics: iminothiolane, tetracycline, gentamicin, chloramphenicol, azithromycin, cefotaxime, ceftazidime, cefazolin, cefoxitin, ciprofloxacin, doripenem, ertapenem, tigecycline, polymyxin B, cefepime, tobramycin, levofloxacin, doxycycline, aztreonam, minocycline, meropenem, and amikacin;

the non-antibiotic drug preferably comprises any one or two or more of the following: metformin, fluoxetine and duloxetine.

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