WO2016106712A1

WO2016106712A1 - Small molecule metabolite for improving antibiotic clearance of pathogenic bacteria

Info

Publication number: WO2016106712A1
Application number: PCT/CN2014/095975
Authority: WO
Inventors: 彭宣宪; 李惠; 苏玉斌
Original assignee: 中山大学
Priority date: 2014-12-31
Filing date: 2014-12-31
Publication date: 2016-07-07

Abstract

The present invention belongs to the field of biological medicines. Particularly disclosed is a novel use of a small molecule compound alanine. Through study, it is discovered in the present invention that alanine can enhance the effect of antibiotics on killing pathogenic bacteria including drug-resistant bacteria. Therefore the small molecule metabolite alanine provided by the present invention can be used as a medicament for enhancing the bactericidal function of antibiotics.

Description

A small molecule metabolite that enhances the elimination of pathogens by antibiotics

Technical field

The invention belongs to the technical field of medicine, and particularly relates to a small molecule metabolite for improving antibiotics to remove pathogenic bacteria.

Background technique

Pathogens seriously endanger human health and require effective measures for prevention and treatment. Although antibiotics are effective in killing bacteria, bacteria develop resistance due to the abuse of antibiotics. Bacterial bacteria reduce or lose the effectiveness of the originally effective antibiotic treatment, making infection difficult to control. Therefore, it is important to use new methods to control the infection of bacteria, especially resistant bacteria.

Enhancing the sensitivity of bacteria to antibiotics is currently a hot research area for controlling multi-drug resistant bacteria. This antibacterial pathway needs to be based on overcoming resistance mechanisms. For the bete-lactamase produced by bacteria, synthetic enzyme inhibitors can be studied in order to combine the enzyme inhibitor with the antibacterial agent to achieve the purpose of sterilizing the antibacterial drug while overcoming the bacterial resistance. Currently, there are clinically available combinations of bete-lactamase inhibitors and penicillin (or cephalosporin). Further research on drugs that promote the sensitivity of bacteria to antibiotics, together with antibiotics to prepare a compound preparation, is very important for controlling the infection of bacteria, especially resistant bacteria.

Alanine is the basic unit of protein and one of the 20 amino acids that make up human proteins. Its molecular formula is C ₃ H ₇ O ₂ N, and there are two isomers of α-alanine and β-alanine. Alanine has been disclosed in the prior art to prevent kidney stones, assist in the metabolism of glucose, and to help alleviate hypoglycemia and improve body energy. Studies on whether alanine can improve antibiotics and host clearance of pathogens have not been reported.

Summary of the invention

The object of the present invention is to provide alanine (Alaine) as a technical method for controlling the small molecule metabolites of antibiotics to eliminate pathogenic bacteria and to control the infection of bacteria, especially resistant bacteria.

The invention finds that after the addition of alanine, the bactericidal effect of kanamycin on the delayed Edward-resistant bacteria is significantly improved, showing kanamycin concentration effect, alanine concentration dependence and time-dependent correlation. These results indicate that alanine can enhance the sensitivity of delayed Edwardian resistant bacteria to kanamycin. Non-resistant bacteria as a control group also have increased sensitivity to antibiotics.

By adding alanine, the invention improves the sensitivity of the resistant strain of Edwards edulis multidrug resistant bacteria, ampicillin, balofloxacin and tetracycline to kanamycin, indicating that this effect can be applied to different drug resistance Mechanism of resistant bacteria.

The invention increases the retardation of the multi-drug resistant bacteria of Edwards to gentamicin by adding alanine to different degrees. The sensitivity of neomycin, ceftazidime, aztreonam, cefazolin sodium, balofloxacin and guitarmycin suggests that this effect can be applied to different antibiotics.

The invention increases the retardation of Edwards, Vibrio parahaemolyticus, Streptococcus aureus, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae to Qingda by adding alanine to different degrees. The sensitivity of themycin indicates that this effect can be applied to different pathogens.

In the present invention, after adding glucose, the survival rate of the retarded Edwards-resistant bacteria is significantly decreased when treated with kanamycin, indicating that glucose can increase the sensitivity of kanamycin-resistant bacteria to kanamycin. Further, through the addition test of a combination of alanine and glucose, it was found that the two added substances have a remarkable synergistic effect.

Further, a mouse model of chronic urinary tract infection was used, and a drug-resistant biofilm was implanted in the urethra, followed by injection of kanamycin with alanine or/and glucose. The results showed that the treatment group with added substances had obvious bactericidal effect, and the combination of two small molecular substances with antibiotics was the best. At the same time, the bacterial content in the kidney tissue was detected. It was found that the number of bacteria in the kanamycin and alanine or / and glucose treatment groups decreased significantly, and the combination of the two small molecules with the antibiotic was the best. These results indicate that kanamycin combined with alanine or / and glucose can effectively eliminate drug-resistant bacteria in animal bodies.

The present invention finds that alanine promotes the antibiotic resistance of bacteria by increasing the proton dynamic potential (PMF) of bacteria, promoting the entry of antibiotics into the cells, and increasing the content of intracellular antibiotics.

In summary, the addition of alanine to antibiotics can significantly improve the sensitivity of drug-resistant and non-resistant bacteria to antibiotics, providing a new technical method for the treatment of drug-resistant bacteria.

Thus, the invention discloses and protects the use of alanine in increasing the sensitivity of bacteria to antibiotics. It can be used to prepare antibacterial or bactericidal drugs to further enhance the sensitivity of bacteria or resistant bacteria to antibiotics.

At the same time, the invention discloses and protects a method for increasing the sensitivity of bacteria to antibiotics, characterized in that alanine is used in combination with an antibiotic.

The bacteria include, but are not limited to, Edwards edulis, Vibrio parahaemolyticus, Streptococcus mutans, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae. Because these bacteria are common human and farm animal pathogens, among which Staphylococcus aureus and beta-hemolytic streptococcus are Gram-positive bacteria, slow-edged Edwardia, Vibrio parahaemolyticus and Klebsiella pneumoniae are Gram Negative bacteria. These bacteria can be either resistant or non-resistant. These bacteria are common pathogens, and their resistant strains are common, so these bacteria are better representative bacteria of resistant and non-resistant bacteria.

The antibiotic is selected from the group consisting of, but not limited to, kanamycin, gentamicin, neomycin, ceftazidime, aztreonam, cefazolin sodium, balofloxacin, and guitarmycin. Because aztreonam, ceftazidime and cefazolin sodium are β-lactam antibiotics; balofloxacin is a quinolone antibiotic; gentamicin, kanamycin and neomycin are aminoglycoside antibiotics; Otamycin is a chloramphenicol antibiotic. These include the major types of antibiotics currently in clinical use.

The dose ratio of alanine to antibiotic is 1:0.0015-300 by weight.

When the above method is used to increase the sensitivity of bacteria to antibiotics, alanine is used at a concentration of 3 mg to 30 g per administration.

Through the disclosure of the present invention, a new antibacterial or bactericidal agent can be prepared, which comprises an antibiotic and an alanine; or a preparation for improving antibacterial or bactericidal action of an antibiotic against a resistant bacterium, mainly The ingredients are alanine and antibiotics.

Although in the examples of the present invention, the listed bacteria include E. edulis, Vibrio parahaemolyticus, Streptococcus mutans, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae. In particular, most of the verification tests of the present invention are based on the delayed Edwardian bacteria. However, these bacteria are not intended to limit the scope of the present invention. This is because 1) Staphylococcus aureus is a model strain for studying resistance mechanisms. 2) The above bacteria belong to Gram-negative and positive bacteria, respectively, wherein Staphylococcus aureus and Streptococcus hemolyticus are Gram-positive bacteria, slow-edged Edwardia, Vibrio parahaemolyticus and Klebsiella pneumoniae are Gram Negative bacteria. All human and farm animal pathogens can be classified according to the staining, so the above bacteria are well represented. 3) Bacteria can have resistant and non-resistant states, ie resistant and non-resistant strains of the same bacteria, while the control strains of the present invention are relatively non-resistant, and the antibiotics are also increased after the addition of alanine. Sensitivity. Therefore, it is inferred from these strains based on the above principle that more strains are also suitable for the concept of the present invention.

The antibiotics listed in the examples of the present invention are kanamycin, gentamicin, neomycin, ceftazidime, aztreonam, cefazolin sodium, balofloxacin and guitarmycin. However, these antibiotics are not intended to limit the scope of the present invention. This is because although there are hundreds of antibiotics, they can be classified according to their chemical structure and antibacterial mechanism. Similar chemical structures have the same antibacterial mechanism, so there is no need to verify them one by one. At present, the main commonly used antibiotics are: penicillin antibiotics, cephalosporin antibiotics, quinolone antibiotics, aminoglycoside antibiotics, etc. Aztreonam, ceftazidime and cefazolin sodium are β-lactam antibiotics; balofloxacin is a quinolone antibiotic; gentamicin, kanamycin and neomycin are aminoglycoside antibiotics; Chloramphenicol antibiotics. These include the major types of antibiotics currently in clinical use. Therefore, it has a good representation of antibiotics. Those skilled in the art, based on the teachings of the present invention, can readily infer that the remaining various antibiotics in the clinic are equally applicable to the methods described herein.

The invention also found that the combination of alanine and glucose has a significant synergistic effect in increasing the sensitivity of bacteria to antibiotics.

Preferably, the weight ratio of alanine to glucose is from 1:0.0001 to 10,000.

Preferably, the antibiotic is selected from the group consisting of kanamycin, gentamicin, neomycin, ceftazidime, aztreonam, and cephalosporin Zolinium sodium, balofloxacin and guitarmycin.

DRAWINGS

Figure 1 shows the effect of alanine on the sensitivity of delayed Edwards-resistant bacteria to kanamycin. The A, B and C plots show that this effect has alanine concentration-dependent, kanamycin concentration effects and The action time is related.

Figure 2 shows the results of MIC determination of ampicillin-resistant bacteria, balofloxacin-resistant bacteria and tetracycline-resistant bacteria of Edwards.

Figure 3 shows the results of MIC determination of various antibiotics by Edwards.

Figure 4 shows the results of alanine increasing the sensitivity of delayed Edwards and its resistant bacteria to kanamycin (A) and increasing the sensitivity of various pathogens to gentamicin (B).

Figure 5 shows the results of alanine increasing the sensitivity of delayed Edwards to various antibiotics.

Figure 6 shows the results of alanine assay to improve the sensitivity of bacteria to antibiotics by increasing bacterial PMF (A) to increase intracellular antibiotic levels (B).

Figure 7 shows the results of a synergistic effect of alanine and glucose on the sensitivity of resistant bacteria to kanamycin. The A, B, and C plots show that glucose increases sensitivity results, alanine synergizes with glucose (B), and glucose synergizes with alanine (C) to increase sensitivity results.

Figure 8 shows that alanine and glucose increase the clearance of E. faecalis in mice. A is the scavenging effect on the biofilm, and B is the mouse kidney scavenging effect.

detailed description

The invention is further described in detail below with reference to the drawings and specific embodiments. The experimental methods used below, unless otherwise specified, are conventional methods in the art, and the ingredients or materials used are commercially available ingredients or materials unless otherwise specified. The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements without departing from the principles of the present invention. The scope of protection.

Example 1 Obtaining the resistant strain of Edwardian artificial passage and confirming the wild resistant bacteria

1. Obtaining the LTB4 resistant strain of Edwardella sinensis by artificial passage: The minimum inhibitory concentration of the delayed strain of E. eutropha LTB4 (designated LTB4-S) against Kanamycin was tested by two-fold dilution method. Then, LTB4-S was continuously cultured for 10 passages in a medium containing 1/2 of the minimum inhibitory concentration kanamycin in a number of 10 ⁵ colony forming units/ml, and the minimum inhibitory concentration was measured. If the minimum inhibitory concentration/initial minimum inhibitory concentration is >4, the strain exhibits resistance to the antibiotic, which is a resistant strain. The results showed that the selected strain had a minimum inhibitory concentration of 200 μg/mL, which was 64 times that of the original strain LTB4-S (3.125 μg/mL), indicating that a kanamycin-resistant strain of the delayed E. eutropha LTB4 was obtained ( Named LTB4-R).

2. Confirmation of resistant strains of wild-type Edwards kanamycin: Strains of E. sinensis EIB202 stored in the laboratory at 80 ° C were streaked on LB plates and placed in a 30 ° C incubator for 24 hours; Single colonies were taken, inoculated in 5 mL of LB medium, and cultured to saturation at 30 ° C, 200 rpm; inoculated in 5 mL of LB liquid medium at a ratio of 1:100 (v/v), and cultured at 30 ° C until the OD ₆₀₀ value was 0.5. , the minimum inhibitory concentration was determined. The minimum inhibitory concentration of EIB202 was 12.5 μg/mL, which was 4 times that of LTB4-S (3.125 μg/mL), indicating that EIB202 is a wild-resistant strain resistant to kanamycin.

The following in vitro tests were performed using drug-resistant artificial passage resistant bacteria (LTB4-R) and wild-resistant bacteria (EIB202).

3. Culture of the test bacteria and preparation of the sample: Pick the Latvia E. sinensis LTB4-R and EIB202 plate monoclonals in 100 mL LB liquid medium, and incubate at 30 ° C, 200 rpm until saturated; collect the appropriate amount of saturated cells, 8000 rpm Centrifuge for 2 min. Then, the cells were washed 3 times with sterile physiological saline; the washed cells were adjusted to an OD value of 0.2 with 1×M9 medium, and then 5 mL of the bacterial solution was dispensed into a test tube for use.

Example 2 Alanine Improves Sensitivity of Delayed Edwardian Resistance to Kanamycin

1. The sensitivity of drug-resistant bacteria to kanamycin increases with the increase of alanine concentration: the final concentration of 40μg/mL (EIB202) or 800μg/mL (LTB4-R) card is added to the prepared 5mL bacterial solution. At the same time, do not add or add different concentrations of alanine, the final concentration of alanine is 0, 5, 10, 20, 40 and 80 mM; culture at 30 ° C, 200 rpm for 6 hours, use a flat plate to detect The number of viable cells was calculated to calculate the survival rate at different metabolite concentrations. The calculation formula is: survival rate (%) = (number of viable cells after adding alanine / number of viable bacteria without alanine) × 100%, and the results are shown in Fig. 1A. As can be seen from Fig. 1A, even if the lowest concentration of alanine was added, the number of viable bacteria in the resistant bacteria was significantly reduced compared with the control alone, and the number of viable cells was decreased as the concentration of the added substance was increased. These results indicate that different concentrations of alanine can increase the sensitivity of drug-resistant bacteria to kanamycin. At 40 mM alanine, the sensitivity of EIB202 to kanamycin was increased by about 120-fold, and the sensitivity of LTB4-R to kanamycin was increased about 60-fold.

2. The sensitivity of the resistant bacteria to kanamycin was concentration-dependent: 5 mL of the prepared bacterial samples were divided into two groups with or without a final concentration of 40 mM alanine. The two groups were added with different concentrations of kanamycin, wherein the final concentrations of EIB202 were 0, 10, 20, 30, 40 and 50 μg/mL, respectively; the final concentrations of LTB4-R were 0, 200, 400, 600, respectively. 800 and 1000 μg/mL. Then, it was cultured at 30 ° C, 200 rpm for 6 hours, and the viable cell count was measured with a plate, and then the survival rate under different antibiotic concentrations was calculated. The calculation formula is: survival rate (%) = (number of viable bacteria after adding antibiotics / number of antibiotics without antibiotics) × 100%, the results are shown in Figure 1B. As can be seen from Fig. 1B, even without the use of alanine, only a small amount of resistant bacteria can be killed even if the highest concentration of kanamycin is used. After the addition of alanine, the lowest concentration of kanamycin can significantly kill these resistant bacteria, and with the increase in the amount of antibiotics added, the fewer viable bacteria, the higher the sensitivity to antibiotics. This result indicates that alanine can enhance resistant bacteria Sensitivity to kanamycin; and sensitivity is related to antibiotic concentration: the higher the antibiotic concentration, the higher the sensitivity of the resistant bacteria. For EIB202, it can be increased by about 120 times at 40 μg/mL kanamycin and about 150 times at 50 μg/mL kanamycin; for LTB4-R, it can be increased at 800 μg/mL kanamycin. 60 times, about 1000 times higher at 1000 μg/mL kanamycin.

3. Alanine has a time-dependent correlation between the sensitivity of drug-resistant bacteria to kanamycin: the final concentration of the prepared 5 mL bacterial solution is 40 μg/mL (EIB202) or 800 μg/mL (LTB4, respectively). -R) kanamycin and 40 mM alanine. The cells were cultured at 30 ° C and 200 rpm for 8 hours, and the number of viable cells was measured with a plate every 1 hour to calculate the survival rate at different times. The calculation formula is: survival rate (%) = (number of viable cells at a certain point after adding alanine / number of viable cells at a certain time point without alanine) × 100%, and the results are shown in Fig. 1C. As can be seen from Figure 1C, the number of bacterial viability begins to decrease after 1 hour of alanine addition compared to the control alone, and the lower the number of viable cells over time. These results indicate that alanine can increase the sensitivity of drug-resistant bacteria to kanamycin, and the longer the time of adding substances, the more obvious the effect.

Example 3 Alanine enhances the sensitivity of drug-resistant bacteria to kanamycin

1. Screening and identification of other antibiotic-resistant strains of Edwards: use the two-fold dilution method to detect the minimum of ampicillin, balofloxacin and tetracycline in the delayed strain of E. faecalis LTB-4 (designated LTB-S) Inhibitory concentration. LTB-S was continuously cultured for 10 passages in a medium containing 1/2 minimum inhibitory concentration of ampicillin or balofloxacin or tetracycline in a volume of 10 ⁵ colony forming units/ml, and the minimum inhibitory concentration was re-measured. If the re-measured minimum inhibitory concentration/initial minimum inhibitory concentration is >4, the strain is considered to exhibit resistance to the antibiotic. From the measurement results of Fig. 2, it was found that resistant strains of ampicillin, balofloxacin and tetracycline were screened.

2. Determination of the resistance of Edwards to various antibiotics: the six strains of L. edulis strains (LTB4-S, LTB4-R, EIB202, WY37, WY28, ATCC15947) kept in the laboratory were as in Example 1. In the first step, the minimum inhibitory concentrations against kanamycin, ampicillin, tetracycline, streptomycin, erythromycin, chloramphenicol and rifampicin were determined, and the results are shown in Fig. 3. As can be seen from Figure 3, these delayed Edwards are multi-drug resistant bacteria.

3. Alanine increases the sensitivity of wild resistance to Edwards and its resistant bacteria to kanamycin: other strains of Edwards will be delayed (LTB4-S, LTB4-R, EIB202, WY37, WY28, ATCC15947, ampicillin) Test samples for penicillin, balofloxacin and tetracycline were prepared according to the procedure of the third step in Example 1. Then 40 mM alanine was added or not added to each sample with the addition of 40 μg/mL kanamycin (except LTB4-R was 800 μg/mL). After incubation at 30 ° C and 200 rpm for 6 hours, the viable cell count was measured with a plate, and the survival rate after the addition of the substance was calculated. The calculation formula is: survival rate (%) = (number of viable cells after adding metabolites / number of viable cells without metabolites) × 100%. The results are shown in Fig. 4A. It can be seen from the figure that the number of viable cells is significantly reduced when the substance is added, and the number of viable cells is reduced by 5 to 123 times for alanine. This result indicates that alanine can not only improve the sensitivity of delayed Edward's multi-drug resistant bacteria to kanamycin, but also improve the retardation of love. The sensitivity of other antibiotic-resistant bacteria to Chinese kanamycin indicates that alanine enhances the sensitivity of bacteria to kanamycin.

4, alanine can improve the sensitivity of a variety of pathogenic bacteria to gentamicin: a variety of other pathogens (Vibrio parahaemolyticus, Streptococcus mutans, Staphylococcus aureus, MASA and Klebsiella pneumoniae) according to the implementation The test sample was prepared separately in the third step of the test procedure in Example 1. Then, after adding appropriate amount of gentamicin (Vibrio parahaemolyticus 25 μg/mL, Streptococcus mutans 25 μg/mL, Staphylococcus aureus 8 μg/mL, MASA 6.4 μg/mL, and Klebsiella pneumoniae 1 μg/mL) 40 mM alanine was added or not added to each sample. After incubation at 30 ° C and 200 rpm for 6 hours, the viable cell count was measured with a plate, and the survival rate after the addition of the substance was calculated. The calculation formula is: survival rate (%) = (number of viable cells after adding alanine / number of viable cells without alanine) × 100%. The result is shown in Figure 4B. It can be seen from the figure that when alanine is added, the number of viable cells of different pathogenic bacteria is reduced. For Vibrio parahaemolyticus, the number of viable bacteria was reduced by nearly 1000 times; for Streptococcus B, the number of viable bacteria was reduced by nearly 30 times; for Staphylococcus aureus, the number of viable bacteria was reduced by nearly 3 times; for MASA and pneumonia The number of live bacteria was reduced by nearly 20% by R. rapae; this result indicates that alanine can increase the sensitivity of various pathogens to gentamicin.

Example 4 Alanine Improves Sensitivity of Delayed Edwards to Multiple Antibiotics

The prepared EIB202 samples were separately treated with gentamicin (16 μg/mL), ceftazidime (3.2 μg/mL), balofloxacin (1.6 μg/mL), aztreonam (32 μg/mL), and cefazolin sodium. (256 μg/mL), neomycin (6.4 μg/mL) and guitarmycin (1.6 μg/mL) were treated with 7 antibiotics. Each antibiotic treatment component was divided into two groups, a control group supplemented with antibiotics and a test group supplemented with antibiotics and 40 mM alanine. After incubation at 30 ° C and 200 rpm for 6 hours, the viable cell count was measured with a plate, and the survival rate after the addition of the substance was calculated. The calculation formula is: survival rate (%) = (number of viable cells after adding substances / number of viable cells without substances) × 100%. It can be seen from the results in Fig. 5 that the number of viable cells after adding the substance is lower than that of the antibiotic-only control group, but the effects of different antibiotics are different. For gentamicin, the effect is very significant, after adding alanine, it can be increased by more than 300 times; neomycin can be increased by 5 times, and the rest can be increased by nearly 1.5 times.

Example 5 Alanine increases bacterial sensitivity to antibiotics by increasing bacterial PMF to increase intracellular antibiotic content

PMF (proton dynamic potential) determination: The prepared EIB202 bacterial samples were divided into two groups: a saline group and a 40 mM alanine group. After 6 hours of incubation at 30 ° C and 200 rpm, PMF was measured using a BacLight bacterial membrane potential kit (Invitrogen) kit. The process is as follows: 10 ⁶ CFU of each of the above-mentioned treatment liquids, 10 μL of 3 mM DiOC ₂ (3,3'-diethyloxa-carbocyanine iodide), incubation at room temperature for 30 minutes, and flow cytometry FACSCalibur flow cytometer (Becton Dickinson) , San Jose, CA, USA) The fluorescence intensity of red and green light was measured at 488 nm. The ratio of red light intensity to green light intensity indicates the intensity of the film potential. The PMF value is calculated as Log (10 ^3/2 × red fluorescence intensity / green fluorescence intensity). The results of the PMF measurement are shown in Figure 6A. From this result, it was found that the PMF was increased by about 10 times after the addition of the substance.

Kanamycin content determination: The prepared EIB202 bacterial samples were divided into three groups: a saline group, a 40 μg/mL kanamycin group, and a 40 μg/mL kanamycin plus 40 mM alanine group. After incubation at 30 ° C, 200 rpm for 6 hours, the ELISA rapid detection kit (Beijing Zhongwei Weikang Technology Co., Ltd.) was used to detect the kanamycin content in bacteria.

1) Determine the standard curve of the kanamycin standard: Dilute the kanamycin standard into different concentration gradients and add them to different wells of the microplate; add 50 μL of the enzyme antigen and 50 μL of antibody to each well. Incubate, incubate at 37 °C for 30 minutes; pour out the liquid in the well, wash it 4 times with washing solution; add 150 μL of chromogenic substrate to each well, incubate at 37 °C for 10 minutes in the dark; add 50 μL of reaction stop solution per well, mix After averaging, the OD value at 450 nm was measured by a microplate reader, and a standard curve of kanamycin concentration and absorbance was plotted.

2) Sample measurement: The bacteria in various treatment groups were collected by centrifugation, washed with physiological saline for 3 times, resuspended in physiological saline, adjusted to OD ₆₀₀ to 1.0, and 1 ml of the cells were sonicated for 3 minutes, and 50 μL of supernatant was taken after centrifugation. The OD value at 450 nm was measured in the same manner as above. The kanamycin content of each sample was calculated based on the standard curve, and the results are shown in Fig. 6B. From this result, it can be seen that when only kanamycin was added, the antibiotic in the bacteria was only 40 ng/ml, and when alanine was added, the amount of antibiotics entering the bacteria was significantly increased by 4 times.

Example 6 synergistic effect of alanine and glucose can significantly improve the sensitivity of drug-resistant bacteria to kanamycin

1. Glucose can increase the sensitivity of drug-resistant bacteria to kanamycin: in the prepared bacterial sample (5mL bacterial solution), the final concentration is 40μg/mL (EIB202) or 800μg/mL (LTB4-R). At the same time, kanamycin did not add or add different concentrations of glucose to a final concentration of 0, 1.25, 2.5, 5, 10 and 20 mM, respectively. Then, the cells were cultured at 30 ° C and 200 rpm for 6 hours, and the viable cell count was measured with a plate to calculate the survival rate at different glucose concentrations. The calculation formula is: survival rate (%) = (number of viable cells after adding glucose / number of viable cells without glucose) × 100%, and the results are shown in Fig. 7A. As can be seen from Fig. 7A, the number of viable bacteria surviving bacteria was significantly reduced even when the lowest concentration of glucose was added, and the bactericidal ability was increased as the concentration of the added substance was increased. These results indicate that glucose can increase the sensitivity of drug-resistant bacteria to kanamycin; and sensitivity is related to glucose concentration: the higher the glucose concentration, the higher the sensitivity of drug-resistant bacteria. The sensitivity of EIB202 and LTB4-R to kanamycin was increased by nearly 400 and 300 times, respectively, at 10 mM glucose.

2, the synergy of alanine and glucose can significantly improve the sensitivity of drug-resistant bacteria to antibiotics: the prepared bacterial samples are divided into two groups: one group is added to 5mL bacterial solution to a final concentration of 40μg / mL kanamycin ( Under the premise of EIB202) or 800 μg/mL kanamycin (LTB4-R) and 10 mM glucose, alanine was added to give final concentrations of 0, 10, 20, 40 and 80 mM respectively; one group was in 5 mL of bacterial solution. Glucose was added to the final concentration of 0, 2.5, and 5, respectively, at a final concentration of 40 μg/mL kanamycin (EIB202) or 800 μg/mL kanamycin (LTB4-R) and 40 mM alanine. , 10 and 20 mM. After incubation at 30 ° C and 200 rpm for 6 hours, the viable cell count was measured with a plate, and the survival rate after the addition of the substance was calculated. The calculation formula is: survival rate (%) = (number of viable cells after addition of metabolites / number of viable cells without metabolites) × 100%. The results of alanine synergy are shown in Figure 7B, and the results of glucose synergy are shown in Figure 7C. As can be seen from Fig. 7B, after the addition of alanine, the number of viable bacteria in the resistant bacteria was significantly reduced compared with the glucose only in the control, and the effect of 10 mM alanine synergistically with glucose was increased by nearly 10 times; The number of viable bacteria is getting less and less. When the alanine was 40 mM, the effect was increased by 150 times. It can be seen from Fig. 7C that after adding glucose, compared with the control group only adding alanine, the number of viable bacteria in the resistant bacteria was also significantly reduced, and the effect of 2.5 mM alanine synergistically with glucose was increased by 50 times; The number of viable bacteria is also decreasing. When the glucose is 10 mM, the effect is increased by 500 times. These two results fully demonstrate that the combination of alanine and glucose is significantly better than either substance alone in improving the kanamycin sensitivity of drug-resistant bacteria.

Example 7 Alanine Improves Clearance of Delayed Edwards in Mice

Individuals of EIB202 and ATCC15947 were picked and cultured in LB medium for 24 h, then transferred to 2 mL of fresh LB medium at 1:100, and added to a 6 mm PE-50 biological catheter sterilized by UV for 3 hours. They were cultured in an incubator at 30 ° C and 37 ° C for 72 hours, respectively, during which half of the fresh medium was changed every day. The prepared bacterial biofilm catheter was placed in a 1.5 mL EP tube, washed 5 times with physiological saline, and then implanted into the urethra of female BALB/c mice (6 weeks, weighing about 18-20 g). After 48 hours, the mice were randomly divided into 2 large groups (16 each), ie, no kanamycin and kanamycin group. Each large group was divided into four groups: saline, alanine, glucose, alanine + glucose. 8 mice per group. Intraperitoneal injections were given twice daily. The doses were 3 g kg ^-1 alanine and 1.5 g kg ^-1 glucose, respectively, and the antibiotic dose was 3 mg kg ^-1 . Continuous treatment for 3 days. Twenty-four hours after the last treatment, the biotube bacteria were suspended from the catheter tube in physiological saline, diluted in a gradient and plate counted to calculate the bacterial survival rate on the catheter biofilm. The calculation formula is the number of live bacteria in the injection group/the number of live bacteria in the control group × 100%. Through the analysis of the results, it was found that the survival rate of EIB202 after adding kanamycin was 75.82%, 91.05% when only alanine was added, and 3.79% when kanamycin was added; 80.19% when only glucose was added. The kanamycin decreased to 5.43%; the alanine and glucose alone were 78.06%, and the kanamycin decreased by 2.99%; the results are shown in Figure 8A. The survival rate of ATCC15947 after adding kanamycin was 89.04%, 87.38% when only alanine was added, and the decrease of kanamycin was 6.09%. When only glucose was added, it was 91.87%, and kanamycin was added. The decrease was 12.65%; the addition of alanine and glucose was 81.79%, and the kanamycin decreased by 4.11%; the results are shown in Figure 8A. From these results, it can be seen that 1) the test group with the addition of antibiotics and the addition of alanine showed a significant reduction in bacteria on the biofilm, and for EIB202, it was reduced by 24 and 20 times, respectively, compared with the addition of only alanine and antibiotics; For ATCC15947, compared with the addition of only alanine and antibiotics, the reduction was 14 times; 2) when adding antibiotics, the addition of glucose can also significantly improve the ability to remove resistant bacteria, for EIB202, compared with the addition of only glucose Reduced by 14 times; for ATCC15947, compared with adding only glucose, it is reduced by 7 times; 3) When alanine and glucose are used together, the effect of eliminating drug-resistant bacteria is obviously better than using only one substance, and its efficiency is Can be increased by 26 times and 19 times respectively.

At the same time, the kidneys of each mouse were thoroughly ground and homogenized by adding an appropriate amount of physiological saline, and the bacterial content (live bacteria/gram) in the kidney tissue was measured by a plate count. The statistical results are shown in Figure 8B. After analysis and comparison, it was found that 1) the number of viable bacteria in the kidneys of the experimental group supplemented with antibiotics and alanine was significantly reduced, and the number of viable cells was reduced by 2 times compared with the control group supplemented with only alanine and antibiotics. 2) When adding antibiotics, the addition of glucose can also significantly eliminate the drug-resistant bacteria, which is twice as efficient; 3) when alanine and glucose are used in combination, the effect of eliminating drug-resistant bacteria is significantly better than using only one substance. Its efficiency is increased by 5 times.

Based on the above animal test results, alanine can increase the sensitivity of kanamycin-resistant bacteria to kanamycin in the body. Moreover, the effects of alanine and glucose are synergistic and are more effective when used in combination.

Claims

Alanine is used to increase the sensitivity of bacteria to antibiotics.
A bacteriostatic or bactericidal drug comprising an antibiotic and an alanine.
A method for increasing the sensitivity of bacteria to antibiotics to remove pathogenic bacteria, characterized in that alanine is used in combination with an antibiotic.
The method according to claim 3, wherein said bacteria are delayed Edward, Vibrio parahaemolyticus, Streptococcus aureus, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae. bacteria.
The method according to claim 3 or 4, wherein the bacteria are sensitive bacteria and resistant bacteria.
The method according to claim 3, characterized in that the dose ratio of alanine to antibiotic is 1:0.0015 to 300 by weight.
The method according to claim 3 or 4 or 6, wherein the antibiotic is selected from the group consisting of kanamycin, gentamicin, neomycin, ceftazidime, aztreonam, cefazolin sodium, and balo Shaxing and guitarmycin.
The method according to claim 3, wherein said alanine is used in an amount of from 3 mg to 30 g per administration.
The combination of alanine and glucose is used to increase the sensitivity of bacteria to antibiotics to eliminate pathogens.
The use according to claim 9, wherein the weight ratio of alanine to glucose is from 1:0.0001 to 10,000.