CN111956642A - Method for improving sterilization efficiency of aminoglycoside antibiotics by indole and derivatives thereof - Google Patents

Abstract

The invention discloses a method for improving the bactericidal efficiency of aminoglycoside antibiotics by indole and derivatives thereof. The method comprises: adding auxiliary agents and aminoglycoside antibiotics to bacterial liquid containing bacteria to be sterilized to obtain a bacterial liquid treatment liquid, The adjuvant is indole or an indole derivative. Experiments have proved that the method of the present invention can greatly improve the sterilization efficiency of existing aminoglycoside antibiotics to bacteria, effectively reduce the risk of pathogenic bacteria producing drug resistance, and at the same time, under the premise of achieving the same therapeutic effect, the dosage and administration can be reduced. medication time, thereby reducing its side effects.

Description

Method for improving the bactericidal efficiency of aminoglycoside antibiotics by indole and its derivatives

technical field

The invention relates to the field of biotechnology, in particular to a method for improving the bactericidal efficiency of aminoglycoside antibiotics by indole and its derivatives.

Background technique

On a global scale, the trend of bacterial resistance is becoming more and more severe, and it is a major public health problem facing the world in the 21st century. The resistance of bacteria to antibiotics is a common phenomenon that causes nosocomial infections and community-acquired diseases. In addition to serious threats to human life, it also causes serious losses to the economic and property of society, especially in underdeveloped countries. The resulting increase in morbidity and mortality, and high health care costs are one of the reasons for long-term poverty. At the same time, bacterial resistance can also increase people's health care costs, reduce livestock production, increase extreme poverty, and reduce per capita GDP. The reasons for the widespread global spread of bacterial resistance may include: extensive clinical use of broad-spectrum antibiotics; overuse of antibiotics in aquaculture; poor medical systems; Internal transmission; lack of antibacterial vaccines; high densities of drug-resistant bacteria on medical equipment and facilities; increase in high-risk patient groups and patients not fully complying with infection control measures; .

The increase of bacterial resistance reduces the bactericidal efficacy of the original antibiotics, but the development of new antibiotics is difficult, costly and time-consuming. The FDA approval rate for new antibiotics has dropped by 90 percent over the past 30 years. As of December 2018, 45 new antibiotics were in clinical trials in the United States, some of which were discovered in the last century. Therefore, screening other compounds with antibacterial activity and improving the bactericidal efficiency of existing antibiotics to kill pathogenic bacteria quickly and efficiently is an important means to reduce the risk of bacterial resistance.

For a long time, Staphylococcus aureus is an important cause of hospital and community infections, and the plateau Staphylococcus aureus shows obvious resistance to a variety of antibiotics. Staphylococcus aureus can cause chronic and recurrent infections, including osteomyelitis, endocarditis, and recurrent abscesses, and can recur long after infection without symptoms. In recent years, there have been more and more reports on Meticillin-resistant Staphylococcus aureus (MRSA). MRSA has increased virulence, drug resistance and colonization ability, and is prone to be infected in vulnerable or other high-risk groups. erupted.

Aminoglycoside antibiotics are currently important drugs for the treatment of bacterial infections and are bactericidal antibiotics. A class of broad-spectrum antibiotics that are effective against both Gram-negative and Gram-positive bacteria. Named because it is a glycoside formed by amino sugars (monosaccharides or disaccharides) and aminocyclohexane polyols. Antibiotics such as tobramycin and gentamicin contain a 2-deoxystreptamine core, while streptomycin does not have a 2-deoxystreptamine structure. Tobramycin and streptomycin have different effects on different types of bacteria. Aminoglycoside antibiotics mainly act on bacterial ribosomes and affect many aspects of bacterial protein synthesis. Such antibiotics first inhibit the formation of the 70S initiating complex, and then combine with the 30S small subunit of the bacterial ribosome, causing the bacteria to synthesize the wrong protein and make the synthesized peptide chain unable to be released, resulting in the depletion of the bacterial endosome. This inhibits the synthesis of bacterial proteins and ultimately kills the bacteria. However, the clinical application of these antibiotics has been limited by many factors, mainly including the growing phenomenon of bacterial resistance and the nephrotoxicity and ototoxicity of these antibiotics. Aminoglycoside antibiotics currently used in clinical medicine or in aquaculture mainly include: tobramycin, streptomycin, gentamicin, kanamycin, neomycin, amikacin, apramycin, diabemycin Su, netilmicin, sisomicin and so on.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to improve the bactericidal efficiency of the existing antibiotics.

In order to solve the above technical problems, the present invention provides a method for improving the bactericidal efficiency of aminoglycoside antibiotics by using indole and its derivatives.

The method for improving the bactericidal efficiency of aminoglycoside antibiotics: adding auxiliary agents and aminoglycoside antibiotics to the bacterial liquid containing the bacteria to be sterilized to obtain a bacterial liquid treatment liquid, and then subjecting the bacterial liquid treatment liquid to shaking table culture, the auxiliary The agent is indole or indole derivatives, and the indole derivatives are 2-methyl indole and 5-methyl indole.

The shaker incubation temperature is 37°C, and the time is 1-5 hours.

Further, the final concentration of the adjuvant in the bacterial solution treatment solution is 1mM-4mM.

Further, the bacterium is Staphylococcus aureus, and the aminoglycoside antibiotic is tobramycin. The final concentration of the tobramycin in the bacterial liquid treatment solution is 50-500 μg/mL.

Further, the bacteria are Staphylococcus aureus, and the aminoglycoside antibiotics are streptomycin, gentamicin and kanamycin. The final concentration of the streptomycin in the bacterial solution treatment solution is 2000 μg/ml, the final concentration of gentamicin in the bacterial solution treatment solution is 500 μg/ml, and the final concentration of kanastreptomycin in the bacterial solution treatment solution. The concentration was 1000 μg/ml.

Further, the bacteria are methicillin-resistant Staphylococcus aureus, the aminoglycoside antibiotic is streptomycin, and the final concentration of the streptomycin in the bacterial solution treatment solution is 250-1000 μg/mL.

Experiments show that the method of the present invention can improve the killing efficiency of bacteria. For Staphylococcus aureus, the sterilization efficiency of adjuvant combined with 250μg/ml tobramycin can be improved by 6 orders of magnitude; the sterilization efficiency of adjuvant combined with 2000μg/ml streptomycin has been improved to varying degrees; The bactericidal efficiency of adjuvant combined with 500μg/ml gentamicin can be improved by 5 orders of magnitude; the bactericidal efficiency of adjuvant combined with 1000μg/ml kanamycin has been improved to varying degrees; for MRSA, the use of adjuvant The bactericidal efficiency of the agent combined with streptomycin can be improved by 3 orders of magnitude. This shows that the method of the present invention can greatly improve the bactericidal efficiency of aminoglycoside antibiotics, which will effectively reduce the risk of drug resistance of pathogenic bacteria. its side effects.

Description of drawings

Figure 1 is a comparison of the bactericidal efficiency of 4mM adjuvant combined with 250μg/ml tobramycin to kill Staphylococcus aureus after treatment at 37°C for 3h.

Figure 2 is a graph showing the bactericidal effect of 4mM adjuvant combined with 50μg/ml and 100μg/ml tobramycin on Staphylococcus aureus after treatment at 37°C for 3h, respectively.

Figure 3 is a line graph of tobramycin concentration and bactericidal efficiency.

Figure 4 is a graph showing the bactericidal effect of 4mM adjuvant combined with 2000ug/ml streptomycin, 500ug/ml gentamicin and 1000μg/ml kanamycin on Staphylococcus aureus after treatment at 37°C for 3h respectively.

Figure 5 is a comparison of the bactericidal efficiency of 4mM adjuvant combined with 1000μg/ml streptomycin to kill MRSA.

Figure 6 is a graph showing the bactericidal effect of 4mM adjuvant combined with 250μg/ml and 500μg/ml streptomycin to kill MRSA, respectively.

Figure 7 is a line graph of streptomycin concentration and bactericidal efficiency.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

Staphylococcus aureus ATCC 25923 and methicillin-resistant Staphylococcus aureus MRSA ATCC43300 in the following examples, the public can obtain the biological materials from the applicant, and the biological materials are only used for repeating the relevant experiments of the present invention, and cannot be used as other biological materials. Use S. aurus ATCC 25923 is a standard strain of Staphylococcus aureus widely used in biological laboratories. References can be found in Treangen TJ. et al. (2014) Complete genome sequence of the quality control strain Staphylococcus aureus subsp. aureus ATCC 25923. GenomeAnnounc. 2(6):e01110–e01114.doi:10.1128/genomeA.01110-14.MRSA ATCC 43300 is a standard strain of mecillin-resistant Staphylococcus aureus widely used in biological laboratories. References are available from Tan Honglue.etal. (2012 ).The use of quaternised chitosan-loaded PMMA to inhibit biofilmformation and downregulate the virulence-associated gene expression of antibiotic resistant staphylococcus.Biomaterials,33(2),365-77.doi:10.1016/j.biomaterials.2011.09.084).

Embodiment 1 Auxiliary agent cooperates with tobramycin to kill plateau Staphylococcus aureus

1. Activated bacteria: Staphylococcus aureus (S. aureus ATCC 25923), draw 1 μl of the 20% glycerol bacterial solution of Staphylococcus aureus ATCC 25923 stored in a -80°C refrigerator, add it to 1 ml of LB liquid medium, and add it to 1 ml of LB liquid medium. 37°C shaker (250rpm) was cultured to the plateau phase, the obtained bacterial solution was diluted 1000 times and then inoculated into 30ml LB liquid medium, and 37°C shaker (250rpm) was cultured to the plateau phase (24h) to obtain Staphylococcus aureus. culture fluid.

2. Use dimethyl sulfoxide (DMSO) to prepare indole, 2-methyl indole and 5-methyl indole mother liquor with a concentration of 1M, and then dilute the mother liquor with DMSO to prepare a working solution with a concentration of 200mM. The whole preparation process should be avoided. Light.

3. Control group: Take 6 ml of the plateau Staphylococcus aureus culture solution obtained in step 1, and distribute it into 12 sterile glass test tubes, each tube containing 500 μl of bacterial solution; The test tubes were randomly divided into 4 groups, namely no adjuvant group, indole group, 2-methyl indole group, and 5-methyl indole group;

No adjuvant was added to the 3 test tubes in the adjuvant-free group, which were marked as A-1, A-2, and A-3;

Each test tube in the indole group was added with indole, and the concentration of indole in the bacterial solution was set to 4 mM; the three test tubes were marked as I-1, I-2 and I-3 (I represents the addition of indole);

In the 2-methyl indole group, 2-methyl indole was added to each test tube, and the concentration of 2-methyl indole in the bacterial solution was set to 4mM; the three test tubes were marked as 2MI-1 and 2MI-2 respectively. and 2MI-3 (2MI represents the addition of 2-methyl indole);

In the 5-methyl indole group, 5-methyl indole was added to each test tube, and the concentration of 5-methyl indole in the bacterial solution was set to 4mM; the three test tubes were marked as 5MI-1 and 5MI-2 respectively. and 5MI-3 (5MI represents the addition of 5-methyl indole);

4. Place the test tube of the control group in step 3 at 37°C and incubate in a 250rpm shaker for 3h in the dark.

5. Experimental group: Take 7.5 ml of the plateau Staphylococcus aureus culture solution obtained in step 1, and distribute it into 15 sterile glass test tubes, each tube containing 500 μl of bacterial liquid; The glass tubes were randomly divided into 5 groups, and each group had 3 glass test tubes containing bacterial liquid. They were the untreated group, the tobramycin-treated group, the tobramycin combined with indole group, the tobramycin combined with 2-methyl indole group, and the tobramycin combined with 5-methyl indole group.

Untreated group: neither adjuvant nor tobramycin was added to the bacterial solution, 3 test tubes containing bacterial solution (denoted as B1, B2 and B3);

Tobramycin treatment group: Tobramycin was added to the glass tube in the group, and the concentration of tobramycin in the bacterial solution was set to 250ug/ml; the three test tubes were recorded as T-250-1, T- 250-2 and T-250-3 (T means adding tobramycin, 250 means adding tobramycin at a concentration of 250 μg/ml);

Indole combined with tobramycin group: add indole and tobramycin to the glass tube in the group, the concentration of indole in the bacterial solution is set to 4mM; the concentration of tobramycin in the bacterial solution is set to 250ug /ml; a set of 3 glass test tubes, record the 3 test tubes as I+T-250-1, I+T-250-2 and I+T-250-3 (I means adding indole, T means adding Tobramycin, 250 means adding tobramycin at a concentration of 250 μg/ml);

2-methyl indole combined with tobramycin group: 2-methyl indole and tobramycin were added to the glass tube in the group, and the concentration of 2-methyl indole in the bacterial solution was set to 4 mM; The concentration of bruomycin in the bacterial solution was set to 250 μg/ml; a group of 3 glass test tubes were recorded as 2MI+T-250-1, 2MI+T-250-2 and 2MI+T-250 -3 (2MI means adding 2-methyl indole, T means adding tobramycin, 250 means adding tobramycin at a concentration of 250 μg/ml);

5-methyl indole combined with tobramycin group: 5-methyl indole and tobramycin were added to the glass tube in the group, and the concentration of 5-methyl indole in the bacterial solution was set to 4 mM; The concentration of bromycin in the bacterial solution was set to 250 μg/ml; a group of 3 glass test tubes were recorded as 5MI+T-250-1, 5MI+T-250-2 and 5MI+T-250 -3 (5MI means adding 5-methylindole, T means adding tobramycin, 250 means adding tobramycin at a concentration of 250 μg/ml).

6. Incubate the test tube of step 5 in a shaker at 37°C and 250rpm in the dark for 3h.

7. Take out the bacterial liquid incubated in the dark for 3 hours in steps 4 and 6, take 100ul of bacterial liquid and centrifuge (10000g, 2min), remove the supernatant, and then use 100μl of 100mM sterile phosphate buffer (pH is 7.4) The cells were resuspended, washed twice, and then resuspended in 100 μl of 100 mM sterile phosphate buffer (pH 7.4).

8. After step 7 is completed, dilute the obtained bacterial solution with 100 mM sterile phosphate buffer (pH 7.4) according to a 10-fold gradient each time, and the dilution gradient is 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 4 μl of bacterial droplets for each dilution were placed on a six-square plate of LB solid medium, placed in a constant temperature incubator at 37 °C for 12 hours, and the bacterial death was observed, and the colonies were counted to calculate the staphylococcus aureus by The survival rate after treatment, the results are shown in Figure 1.

9. Repeat the above steps 5, 6, 7 and 8 with different concentrations of tobramycin respectively. The concentration of tobramycin is 50 μg/ml and 100 μg/ml; The survival rate of Staphylococcus aureus after treatment, and the tobramycin concentration-surviving colony number curve was drawn. The results are shown in Figures 2 and 3.

Table 1 Comparison of adjuvant to improve the efficiency of tobramycin in killing Staphylococcus aureus

Note: Relative bactericidal efficiency = survival rate with adjuvant without tobramycin × survival rate with tobramycin/survival rate with adjuvant and tobramycin

The results showed that the bactericidal efficiency of tobramycin was one order of magnitude after adding tobramycin to the bacterial solution for 3 hours. The adjuvant (indole and its derivatives) combined with tobramycin was added to the bacterial solution for 3 hours. Compared with the group without adjuvant, 4mM adjuvant could increase the mortality of Staphylococcus aureus by 6 orders of magnitude. Kill the plateau Staphylococcus aureus to the lower limit of detection. There is a concentration-dependent relationship between the bactericidal efficiency of this method and the antibiotic concentration.

Embodiment 2 Auxiliary agent cooperates with streptomycin, gentamicin, kanamycin to kill plateau stage Staphylococcus aureus

1. Activated bacteria: Staphylococcus aureus (S. aureus ATCC 25923), draw 1 μl of the 20% glycerol bacterial solution of Staphylococcus aureus ATCC 25923 stored in a -80°C refrigerator, add it to 1 ml of LB liquid medium, and add it to 1 ml of LB liquid medium. 37°C shaker (250rpm) was cultured to the plateau phase, the obtained bacterial solution was diluted 1000 times and then inoculated into 30ml LB liquid medium, and 37°C shaker (250rpm) was cultured to the plateau phase (24h) to obtain Staphylococcus aureus. culture fluid.

2. Use dimethyl sulfoxide (DMSO) to prepare indole, 2-methyl indole and 5-methyl indole mother liquor with a concentration of 1M, and then dilute the mother liquor with DMSO to prepare a working solution with a concentration of 200mM. The whole preparation process should be avoided. Light.

3. Additives combined with streptomycin to treat plateau Staphylococcus aureus: take 7.5ml of the plateau Staphylococcus aureus culture solution obtained in step 1, and distribute them into 15 sterile glass test tubes, each containing 500μl of bacterial solution; The 15 glass tubes containing bacterial liquid were randomly divided into 5 groups, and each group had three glass test tubes containing bacterial liquid. They were untreated group, streptomycin-treated group, indole-streptomycin group, 2-methylindole-streptomycin group, and 5-methylindole-streptomycin group.

Untreated group: neither adjuvant nor streptomycin was added to the bacterial solution, 3 test tubes containing bacterial solution (denoted as C1, C2 and C3);

Streptomycin-treated group: add streptomycin to the glass tube in the group, and set the concentration of streptomycin in bacterial solution to 2000 μg/ml; mark the three test tubes as S-2000-1 and S-2000-2 respectively And S-2000-3 (S means adding streptomycin, 2000 means adding streptomycin concentration is 2000μg/ml);

Indole combined with streptomycin group: add indole and streptomycin to the glass tube in the group, and set the concentration of indole in the bacterial solution to 4 mM; the concentration of streptomycin in the bacterial solution is set to 2000 μg/ml; A set of 3 glass test tubes, record the 3 test tubes as I+S-2000-1, I+S-2000-2 and I+S-2000-3 (I means adding indole, S means adding streptomycin , 2000 means that the concentration of added streptomycin is 2000 μg/ml);

2-methyl indole combined with streptomycin group: add 2-methyl indole and streptomycin to the glass tube in the group, and set the concentration of 2-methyl indole in the bacterial solution to 4 mM; streptomycin The concentration in the bacterial solution was set to 2000μg/ml; a group of 3 glass test tubes were recorded as 2MI+S-2000-1, 2MI+S-2000-2 and 2MI+S-2000-3 ( 2MI means adding 2-methyl indole, S means adding streptomycin, 2000 means adding streptomycin concentration of 2000 μg/ml);

5-methyl indole combined with streptomycin group: add 5-methyl indole and streptomycin to the glass tube in the group, and set the concentration of 5-methyl indole in the bacterial solution to 4 mM; streptomycin The concentration in the bacterial solution was set to 2000 μg/ml; a group of 3 glass test tubes were recorded as 5MI+S-2000-1, 5MI+S-2000-2 and 5MI+S-2000-3 ( 5MI means adding 5-methyl indole, S means adding streptomycin, 2000 means adding streptomycin concentration of 2000 μg/ml).

4. Additives and gentamicin to treat plateau Staphylococcus aureus: Take 7.5ml of the plateau Staphylococcus aureus culture solution obtained in step 1, and distribute them into 15 sterile glass test tubes, each containing 500μl of bacterial solution ; The 15 glass tubes containing bacterial liquid were randomly divided into 5 groups, each group of 3 glass test tubes containing bacterial liquid. They were untreated group, gentamicin treatment group, indole combined with gentamicin group, 2-methyl indole combined with gentamicin group, and 5-methyl indole combined with gentamicin group.

Untreated group: neither adjuvant nor gentamicin was added to the bacterial solution, 3 test tubes containing bacterial solution (denoted as D1, D2 and D3);

Gentamicin treatment group: add gentamicin to the glass tube in the group, the concentration of gentamicin in the bacterial solution is set to 500 μg/ml; the three test tubes are recorded as G-500-1, G- 500-2 and G-500-3 (G means adding gentamicin, 500 means adding gentamicin at a concentration of 500 μg/ml);

Indole combined with gentamicin group: add indole and gentamicin to the glass tube in the group, the concentration of indole in the bacterial solution is set to 4 mM; /ml; a set of 3 glass test tubes, record the 3 test tubes as I+G-500-1, I+G-500-2 and I+G-500-3 (I means adding indole, G means adding Gentamicin, 500 means that the concentration of added gentamicin is 500 μg/ml);

2-methyl indole combined with gentamicin group: 2-methyl indole and gentamicin were added to the glass tube in the group, and the concentration of 2-methyl indole in the bacterial solution was set to 4 mM; The concentration of damycin in the bacterial solution was set to 500 μg/ml; a group of 3 glass test tubes were recorded as 2MI+G-500-1, 2MI+G-500-2 and 2MI+G-500 -3 (2MI means adding 2-methyl indole, G means adding gentamicin, 500 means adding gentamicin concentration of 500μg/ml);

5-methyl indole combined with gentamicin group: 5-methyl indole and gentamicin were added to the glass tube in the group, and the concentration of 5-methyl indole in the bacterial solution was set to 4 mM; The concentration of damycin in the bacterial solution was set to 500 μg/ml; a group of 3 glass test tubes were recorded as 5MI+G-500-1, 5MI+G-500-2 and 5MI+G-500 -3 (5MI means adding 5-methyl indole, G means adding gentamicin, 500 means adding gentamicin at a concentration of 500 μg/ml).

5. Additives and kanamycin to treat plateau Staphylococcus aureus: Take 7.5ml of the plateau Staphylococcus aureus culture solution obtained in step 1, and distribute them into 15 sterile glass test tubes, each containing 1000μl of bacterial solution ; The 15 glass tubes containing bacterial liquid were randomly divided into 5 groups, each group of 3 glass test tubes containing bacterial liquid. The groups were untreated group, kanamycin-treated group, indole combined with kanamycin group, 2-methyl indole combined with kanamycin group, and 5-methyl indole combined with kanamycin group.

Untreated group: neither adjuvant nor kanamycin was added to the bacterial solution, 3 test tubes containing bacterial solution (denoted as E1, E2 and E3);

Kanamycin-treated group: add kanamycin to the glass tube in the group, and set the concentration of kanamycin in the bacterial solution to 1000 μg/ml; mark the three test tubes as K-1000-1, K- 1000-2 and K-1000-3 (K means adding kanamycin, 1000 means adding kanamycin at a concentration of 1000 μg/ml);

Indole combined with kanamycin group: add indole and kanamycin to the glass tube in the group, the concentration of indole in the bacterial solution is set to 4 mM; /ml; a set of 3 glass test tubes, record the 3 test tubes as I+K-1000-1, I+K-1000-2 and I+K-1000-3 (I means adding indole, K means adding kanamycin, 1000 means adding kanamycin at a concentration of 1000 μg/ml);

2-methyl indole combined with kanamycin group: add 2-methyl indole and kanamycin to the glass tube in the group, and set the concentration of 2-methyl indole in the bacterial solution to 4 mM; The concentration of namycin in the bacterial solution was set to 1000 μg/ml; a group of 3 glass test tubes were recorded as 2MI+K-1000-1, 2MI+K-1000-2 and 2MI+K-1000 -3 (2MI means adding 2-methyl indole, K means adding kanamycin, 1000 means adding kanamycin at a concentration of 1000 μg/ml);

5-methyl indole combined with kanamycin group: add 5-methyl indole and kanamycin to the glass tube in the group, and set the concentration of 5-methyl indole in the bacterial solution to 4 mM; The concentration of namycin in the bacterial solution was set to 1000 μg/ml; a group of 3 glass test tubes were recorded as 5MI+K-1000-1, 5MI+K-1000-2 and 5MI+K-1000 -3 (5MI means adding 5-methylindole, K means adding kanamycin, 1000 means adding kanamycin at a concentration of 1000 μg/ml).

6. Incubate the test tubes of step 3, step 4 and step 5 in a shaker at 37°C and 250rpm in the dark for 3h.

7. Take out the bacterial solution after incubation in the dark for 3h in steps 3, 4 and 5, take 100ul bacterial solution and centrifuge (10000g, 2min) respectively, remove the supernatant, and then use 100μl 100mM sterile phosphate buffer ( pH 7.4) to resuspend the bacteria, and after washing twice, resuspend the bacteria with 100 μl of 100 mM sterile phosphate buffer (pH 7.4).

8. After step 7 is completed, dilute the obtained bacterial solution with 100 mM sterile phosphate buffer (pH 7.4) according to a 10-fold gradient each time, and the dilution gradient is 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , take 4 μl of bacterial droplets for each dilution on the six-square plate of LB solid medium, and place them in a 37°C constant temperature incubator for 12 hours to observe the bacterial death and count the colonies. The results are shown in Figure 4. .

The results showed that streptomycin had an order of magnitude bactericidal efficiency when streptomycin was added to the bacterial solution of Staphylococcus aureus at the plateau stage for 3 hours. The adjuvant (indole and its derivatives) combined with streptomycin was added to the bacterial solution for 3 hours. Compared with the group without adjuvant, 4mM indole could increase the mortality of Staphylococcus aureus by 3 orders of magnitude, 4mM 2-methylindole at 4 mM increased S. aureus mortality by 4 orders of magnitude, and 4 mM 5-methyl indole increased S. aureus mortality by 5 orders of magnitude. Among them, the auxiliary agent 5-methyl indole had better bactericidal effect than 2-methyl indole and indole.

The bacterial solution of Staphylococcus aureus in the plateau stage was treated with gentamicin for 3 hours, and the bactericidal efficiency of gentamicin was two orders of magnitude. The adjuvant (indole and its derivatives) combined with gentamicin was added to the bacterial solution for 3 hours. Compared with the group without adjuvant, 4mM adjuvant could increase the mortality of Staphylococcus aureus by 5 orders of magnitude.

When kanamycin was added to the bacterial solution of Staphylococcus aureus at the plateau stage for 3 hours, the bactericidal efficiency of kanamycin was one order of magnitude. The adjuvant (indole and its derivatives) combined with kanamycin was added to the bacterial solution for 3 hours. Compared with the group without adjuvant, 4mM indole could increase the mortality rate of Staphylococcus aureus by one order of magnitude. 4 mM 2-methyl indole increased S. aureus mortality by 3 orders of magnitude, and 4 mM 5-methyl indole increased S. aureus mortality by 4 orders of magnitude. The bactericidal effect of the adjuvant is: 5-methyl indole>2-methyl indole>indole.

Example 3 Auxiliary agent combined with streptomycin to kill platform MRSA

1. Activated bacteria: Methicillin-resistant Staphylococcus aureus (MRSA ATCC43300), draw 1 μl of MRSA ATCC43300 20% glycerol bacterial solution stored in a -80 °C refrigerator, add it to 1 ml of LB liquid medium, and shake at 37 °C Inoculated into 30 ml of LB liquid medium after being diluted 1000 times in the bed (250 rpm) to the plateau phase, and cultivated at 37°C on a shaker (250 rpm) to the plateau phase (24 h) to obtain MRSA culture medium.

2. Use dimethyl sulfoxide (DMSO) to prepare indole, 2-methyl indole and 5-methyl indole mother liquor with a concentration of 1M, and then dilute the mother liquor with DMSO to prepare a working solution with a concentration of 200mM. The whole preparation process should be avoided. Light.

3. Control group: Take 6ml of the MRSA culture medium in the plateau phase obtained in step 1, and distribute it into 12 sterile glass test tubes, each of which is filled with 500 μl of bacterial liquid; these 12 glass test tubes containing bacterial liquid are randomly divided into There were 4 groups, namely no adjuvant group, indole group, 2-methyl indole group, and 5-methyl indole group.

No adjuvant was added to the 3 test tubes in the no adjuvant group, which were marked as C-1, C-2, and C-3 respectively;

Indole was added to each test tube in the indole group, and the concentration of indole in the bacterial solution was set to 4 mM; the three test tubes were marked as I-1, I-2 and I-3 (I represents the addition of indole);

In the 2-methyl indole group, 2-methyl indole was added to each test tube, and the concentration of 2-methyl indole in the bacterial solution was set to 4mM; the three test tubes were marked as 2MI-1 and 2MI-2 respectively. and 2MI-3 (2MI represents the addition of 2-methyl indole);

In the 5-methyl indole group, 5-methyl indole was added to each test tube, and the concentration of 5-methyl indole in the bacterial solution was set to 4 mM; the three test tubes were marked as 5MI-1 and 5MI-2 respectively. and 5MI-3 (5MI represents the addition of 5-methyl indole);

4. Place the test tube of the control group in step 3 at 37°C and incubate in a 250rpm shaker for 3h in the dark.

5. Experimental group: Take 7.5ml of the platform MRSA culture solution obtained in step 1 and distribute it into 15 sterile glass test tubes, each tube containing 500μl of bacterial liquid; these 15 glass tubes containing bacterial liquid were randomly Divide into 5 groups, each group has 3 glass test tubes containing bacterial liquid. They were untreated group, streptomycin-treated group, indole-streptomycin group, 2-methylindole-streptomycin group, and 5-methylindole-streptomycin group.

Untreated group: neither adjuvant nor streptomycin was added to the bacterial solution, and the three test tubes in this group were marked as F1, F2 and F3;

Streptomycin-treated group: add streptomycin to the glass tube in the group, and set the concentration of streptomycin in the bacterial solution to 1000 μg/ml; mark the three test tubes as S-1000-1 and S-1000-2 respectively and S-1000-3 (S means adding streptomycin, 1000 means adding streptomycin concentration of 1000 μg/ml);

Indole combined with streptomycin group: add indole and streptomycin to the glass tube in the group, the concentration of indole in the bacterial solution is set to 4mM; the concentration of streptomycin in the bacterial solution is set to 1000μg/ml; The three test tubes in this group are marked as I+S-1000-1, I+S-1000-2 and I+S-1000-3 (I means adding indole, T means adding streptomycin, 1000 means adding streptomycin 1000μg/ml);

2-methyl indole combined with streptomycin group: add 2-methyl indole and streptomycin to the glass tube in the group, and set the concentration of 2-methyl indole in the bacterial solution to 4 mM; The concentration in the bacterial solution was set to 1000 μg/ml; the 3 test tubes in this group were recorded as 2MI+S-1000-1, 2MI+S-1000-2 and 2MI+S-1000-3 (2MI means adding 2-formaldehyde Indole, S means adding streptomycin, 1000 means adding streptomycin concentration is 1000μg/ml);

5-methyl indole combined with streptomycin group: 5-methyl indole and streptomycin were added to the glass tube in the group, and the concentration of 5-methyl indole in the bacterial solution was set to 4 mM; The concentration in the bacterial solution was set to 1000 μg/ml; the 3 test tubes in this group were recorded as 5MI+S-1000-1, 5MI+S-1000-2 and 5MI+S-1000-3 (5MI means adding 5-methyl) Indole, S means adding streptomycin, 1000 means adding streptomycin concentration is 1000μg/ml);

6. Incubate the test tube of step 5 in a shaker at 37°C and 250rpm in the dark for 3h.

7. Take out the bacterial liquid incubated in the dark for 3 hours in steps 4 and 6, take 100ul of bacterial liquid and centrifuge (10000g, 2min), remove the supernatant, and then use 100μl of 100mM sterile phosphate buffer (pH is 7.4) ) to resuspend the bacteria, and after washing twice, resuspend the bacteria with 100 μl of 100 mM sterile phosphate buffer (pH 7.4).

8. After step 7 is completed, dilute the obtained bacterial solution with 100 mM sterile phosphate buffer (pH 7.4) according to a 10-fold gradient each time, and the dilution gradient is 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 4 μl of bacterial droplets for each dilution were placed on a six-square plate of LB solid medium, placed in a 37°C constant temperature incubator for 12 hours, and the bacterial death was observed, and colony counts were performed to calculate the survival of MRSA after treatment. rate, and the results are shown in Figure 5.

9. Repeat the above steps 5, 6, 7 and 8 with different concentrations of streptomycin respectively. The streptomycin concentrations are 250 μg/ml and 500 μg/ml; take the average of the number of surviving colonies in the three groups of experiments, and calculate the treated MRSA and plotted the streptomycin concentration-survival colony number curve, the results are shown in Figure 6 and Figure 7.

Table 2 Comparison of adjuvant to improve the efficiency of streptomycin to kill MRSA in plateau stage

Note: Relative bactericidal efficiency = survival rate with adjuvant without streptomycin × survival rate with streptomycin/survival rate with adjuvant and streptomycin

The results showed that streptomycin was added to the bacterial solution, and streptomycin had almost no bactericidal efficiency after 3 hours of treatment. 4 mM adjuvant alone barely kills plateau MRSA. The adjuvant (indole and its derivatives) combined with streptomycin was added to the MRSA bacterial solution for treatment for 3 hours. Compared with the group without adjuvant, adding 4mM adjuvant improved the bactericidal efficiency of streptomycin respectively: indole can The mortality of MRSA in the plateau phase increased by one order of magnitude, 2-methyl indole could increase the mortality of MRSA in the plateau phase by three orders of magnitude, and 5-methyl indole could increase the mortality rate of MRSA in the plateau phase by two orders of magnitude. After treatment with this method, the bactericidal efficiency of the adjuvant combined with streptomycin was concentration dependent on the streptomycin concentration.

Claims (9)

1. improve the method for the bactericidal efficiency of aminoglycoside antibiotics, it is characterized in that: in the bacterial liquid containing bacteria to be sterilized, add auxiliary agent and aminoglycoside antibiotics, obtain bacterial liquid treatment liquid, and described auxiliary agent is indole or indole derivative. 2 . The method for improving the bactericidal efficiency of aminoglycoside antibiotics according to claim 1 , wherein the bacterial liquid treatment solution is subjected to shaking culture. 3 . 3 . The method for improving the bactericidal efficiency of aminoglycoside antibiotics according to claim 2 , wherein the temperature of the shaker incubation is 37° C. and the time is 1-5 hours. 4 . 4. The method for improving the bactericidal efficiency of aminoglycoside antibiotics according to claim 1, wherein the indole derivative is 2-methyl indole or 5-methyl indole. 5 . The method for improving the bactericidal efficiency of aminoglycoside antibiotics according to claim 1 , wherein the final concentration of the adjuvant in the bacterial liquid treatment solution is 1 mM to 4 mM. 6 . 6. The method for improving the bactericidal efficiency of aminoglycoside antibiotics according to claim 1, wherein the bacteria is Staphylococcus aureus, and the aminoglycoside antibiotics are tobramycin. 7 . The method for improving the bactericidal efficiency of aminoglycoside antibiotics according to claim 6 , wherein the final concentration of the tobramycin in the bacterial liquid treatment solution is 50-500 mg/mL. 8 . 8. The method for improving the bactericidal efficiency of aminoglycoside antibiotics according to claim 1, wherein the bacteria are methicillin-resistant Staphylococcus aureus, and the aminoglycoside antibiotics are streptomycin. 9 . The method for improving the bactericidal efficiency of aminoglycoside antibiotics according to claim 8 , wherein the final concentration of the streptomycin in the bacterial solution treatment solution is 250-1000 mg/mL. 10 .

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