CN111956642A - Method for improving sterilization efficiency of aminoglycoside antibiotics by indole and derivatives thereof - Google Patents
Method for improving sterilization efficiency of aminoglycoside antibiotics by indole and derivatives thereof Download PDFInfo
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- CN111956642A CN111956642A CN202010980865.4A CN202010980865A CN111956642A CN 111956642 A CN111956642 A CN 111956642A CN 202010980865 A CN202010980865 A CN 202010980865A CN 111956642 A CN111956642 A CN 111956642A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Abstract
The invention discloses a method for improving the sterilization efficiency of aminoglycoside antibiotics by indole and derivatives thereof, which comprises the following steps: adding an auxiliary agent and aminoglycoside antibiotics into the bacterial liquid containing bacteria to be killed to obtain a bacterial liquid treatment liquid, wherein the auxiliary agent is indole or indole derivatives. Experiments prove that the method can greatly improve the sterilization efficiency of the prior aminoglycoside antibiotics to bacteria, effectively reduce the risk of pathogenic bacteria for generating drug resistance, and reduce the dosage and the administration time on the premise of achieving the same treatment effect, thereby reducing the side effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for improving the sterilization efficiency of aminoglycoside antibiotics by indole and derivatives thereof.
Background
The trend of bacterial resistance is becoming more severe worldwide and is a major public health problem facing the world in the 21 st century. The drug resistance of bacteria to antibiotics is a common phenomenon causing nosocomial infection and community acquired diseases, besides causing serious threats to human life, serious losses are caused to social economic property, especially in underdeveloped countries, the increase of morbidity and mortality caused by the drug resistance of bacteria and high health care cost are one of the reasons causing long-term poverty; meanwhile, the bacterial drug resistance can also increase the medical care cost of people, reduce the livestock yield, increase extreme poverty and reduce the GDP per capita. The reasons for the wide spread of bacterial resistance worldwide may be: the clinical use of broad spectrum antibiotics in large quantities; overuse of antibiotics in the aquaculture industry; poor medical systems; the rise of global travel drives the spread of drug-resistant bacteria worldwide; absence of antibacterial vaccines; the density of the drug-resistant flora on medical equipment facilities is higher; the increase in the high risk patient population and inadequate patient compliance with infection control measures; antibiotics are not used properly in the clinic and lack of rapid diagnostic reagents to guide the correct use of antibiotics.
The increase of the drug resistance of bacteria causes the reduction of the sterilization efficacy of the original antibiotics, but the difficulty of developing new antibiotics is high, the development cost is high, and the time is long. Over the past 30 years, the U.S. FDA's approval rate for new antibiotics has decreased by 90%. By 12 months 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 sterilization efficiency of the existing antibiotics to quickly and efficiently kill pathogenic bacteria are important means for reducing the risk of bacterial drug resistance.
Staphylococcus aureus has long been a significant cause of nosocomial and community infections, and staphylococcus aureus has shown significant resistance to a variety of antibiotics in the plateau phase. Staphylococcus aureus can cause chronic and recurrent infections, including osteomyelitis, endocarditis, recurrent abscesses, and the like, and can recur in the long-term, asymptomatic situation following infection. In recent years, there have been more and more reports on methicillin-resistant Staphylococcus aureus (MRSA), which has increased toxicity, drug resistance and colonization ability and is likely to outbreak in vulnerable people or other high-risk people.
Aminoglycoside antibiotics are important drugs for treating bacterial infection at present and belong to bactericidal antibiotics. Effective against both gram-negative and gram-positive bacteria is a class of broad-spectrum antibiotics. They are named because they are glycosides formed from amino sugars (mono-or disaccharides) and aminocyclohexyl polyols. Antibiotics such as tobramycin and gentamicin contain a 2-desmethoxystreptomycin core, whereas streptomycin does not have a 2-desmethoxystreptomycin structure. Tobramycin and streptomycin act differently on different species of bacteria. Aminoglycoside antibiotics act mainly on bacterial ribosomes and affect multiple links in the bacterial protein synthesis process. The antibiotics firstly inhibit the formation of 70S initiating complex, then are combined with bacterial ribosome 30S small subunit, which causes the bacteria to synthesize wrong protein, and can not release synthesized peptide chain, which causes the exhaustion of lactoprotein body in the thallus, thereby inhibiting the synthesis of bacterial protein, and finally killing the bacteria. However, clinical applications of these antibiotics are limited, mainly due to the increasingly severe phenomenon of bacterial resistance and the nephrotoxicity and ototoxicity of these antibiotics. The aminoglycoside antibiotics currently used in clinical medicine or breeding industry mainly include: tobramycin, streptomycin, gentamicin, kanamycin, neomycin, amikacin, apramycin, dalbenamycin, netilmicin, sisomicin, and the like.
Disclosure of Invention
The invention aims to solve the technical problem of how to improve the sterilization efficiency of the existing antibiotics.
In order to solve the technical problems, the invention provides a method for improving the sterilization efficiency of aminoglycoside antibiotics by adopting indole and derivatives thereof.
The method for improving the sterilization efficiency of the aminoglycoside antibiotics comprises the following steps: adding an auxiliary agent and aminoglycoside antibiotics into a bacterial liquid containing bacteria to be killed to obtain a bacterial liquid treatment liquid, and then carrying out shake culture on the bacterial liquid treatment liquid, wherein the auxiliary agent is indole or indole derivatives, and the indole derivatives are 2-methylindole and 5-methylindole.
The temperature of shaking table culture is 37 ℃, and the time is 1-5 hours.
Further, the final concentration of the auxiliary agent in the bacterial liquid treatment liquid is 1mM-4 mM.
Further, the bacteria is staphylococcus aureus, and the aminoglycoside antibiotic is tobramycin. The final concentration of the tobramycin in the bacterial liquid treatment liquid is 50-500 mug/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 liquid treatment liquid is 2000 mug/ml, the final concentration of the gentamicin in the bacterial liquid treatment liquid is 500 mug/ml, and the final concentration of the kanamycin streptomycin in the bacterial liquid treatment liquid is 1000 mug/ml.
Further, the bacteria is methicillin-resistant staphylococcus aureus, the aminoglycoside antibiotic is streptomycin, and the final concentration of the streptomycin in the bacterial liquid treatment liquid is 250-.
Experiments prove that the method can improve the killing efficiency of bacteria. Aiming at staphylococcus aureus, the sterilization efficiency of the treatment by adopting the adjuvant and matching with 250 mu g/ml tobramycin can be improved by 6 orders of magnitude; the sterilization efficiency of the adjuvant matched with 2000 mug/ml streptomycin is improved to different degrees; the sterilization efficiency of the adjuvant matched with 500 mu g/ml gentamicin for treatment can be improved by 5 orders of magnitude; the sterilization efficiency of the treatment by adopting the adjuvant and 1000 mug/ml kanamycin is improved to different degrees; for MRSA, the sterilization efficiency of the treatment by adopting the adjuvant and the streptomycin can be improved by 3 orders of magnitude. Therefore, the method provided by the invention can be used for greatly improving the sterilization efficiency of the aminoglycoside antibiotic, effectively reducing the risk of drug resistance of pathogenic bacteria, and reducing the dosage and administration time on the premise of achieving the same treatment effect, thereby reducing the side effect.
Drawings
FIG. 1 shows the comparison of the bactericidal efficiency of 4mM adjuvant in combination with 250. mu.g/ml tobramycin against Staphylococcus aureus after 3 hours of treatment at 37 ℃.
FIG. 2 is a graph showing the bactericidal effect of 4mM adjuvant in combination with 50. mu.g/ml tobramycin and 100. mu.g/ml tobramycin on killing Staphylococcus aureus after treatment at 37 ℃ for 3 hours, respectively.
Fig. 3 is a line graph of tobramycin concentration versus bactericidal efficiency.
FIG. 4 is a graph showing the bactericidal effect of 4mM adjuvant in combination with 2000ug/ml streptomycin, 500ug/ml gentamicin and 1000 ug/ml kanamycin on killing Staphylococcus aureus after treatment at 37 deg.C for 3h, respectively.
FIG. 5 is a comparison of the bactericidal efficiency of 4mM adjuvant in combination with 1000. mu.g/ml streptomycin for MRSA.
FIG. 6 is a graph showing the bactericidal effect of 4mM adjuvant in combination with 250. mu.g/ml and 500. mu.g/ml streptomycin on MRSA, respectively.
FIG. 7 is a line graph showing streptomycin concentration versus bactericidal efficiency.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Staphylococcus aureus ATCC 25923 and methicillin-resistant Staphylococcus aureus ATCC43300 in The following examples, which are available to The public from The applicant only for repeating The relevant experiments of The present invention, and S.aureus ATCC 25923, which is a standard strain of Staphylococcus aureus widely used in biological laboratories, are not available for other uses, and Treanggen TJ.et al (2014) Complete Genome sequence of The quality control strain of Staphylococcus aureus, Aurea ATCC 25923, Genome Annouc.2 (6) e 01110-e01114. doi:10.1128/Genome A.01110-14.MRSA ATCC43300, which is a standard strain of Staphylococcus aureus resistant to methicillin, which is widely used in biological laboratories, can be used in Tan Honghuo.et al (use of The same family restriction of microorganisms of The same organism PMMA, 33(2),365-77.doi:10.1016/j. biology materials.2011.09.084).
EXAMPLE 1 adjuvant in combination with tobramycin to kill plateau Staphylococcus aureus
1. Activating bacteria: the method comprises the following steps of (1) sucking 1 mu l of 20% glycerol bacterial liquid of staphylococcus aureus (S.aureus ATCC 25923) stored in a refrigerator at the temperature of-80 ℃, adding the glycerol bacterial liquid into 1ml of LB liquid culture medium, culturing the glycerol bacterial liquid to the stage by a shaking table (250rpm) at the temperature of 37 ℃, diluting the obtained bacterial liquid by 1000 times, inoculating the diluted bacterial liquid into 30ml of LB liquid culture medium, and culturing the glycerol bacterial liquid to the stage by the shaking table (250rpm) at the temperature of 37 ℃ for 24 hours to obtain a staphylococcus aureus culture solution.
2. The mother liquor of indole, 2-methylindole and 5-methylindole is prepared by dimethyl sulfoxide (DMSO) with the concentration of 1M, the mother liquor is diluted by DMSO to prepare the working solution with the concentration of 200mM, and the whole preparation process is protected from light.
3. Control group: taking 6ml of the platform-stage staphylococcus aureus culture solution obtained in the step 1, and subpackaging the 6ml of the platform-stage staphylococcus aureus culture solution into 12 sterile glass test tubes, wherein each tube is subpackaged with 500 mu l of a bacterial solution; randomly dividing the 12 glass test tubes filled with the bacterial liquid into 4 groups, namely an adjuvant-free group, an indole group, a 2-methylindole group and a 5-methylindole group;
no adjuvant is added in 3 test tubes of the adjuvant-free group, and the test tubes are marked as A-1, A-2 and A-3 respectively;
adding indole into each test tube in the indole group, wherein the concentration of the indole in a bacterial liquid is set to be 4 mM; 3 tubes were designated I-1, I-2 and I-3, respectively (I indicates the addition of indole);
2-methylindole is added into each test tube in the 2-methylindole group, and the concentration of the 2-methylindole in a bacterial liquid is set to be 4 mM; 3 tubes were scored as 2MI-1, 2MI-2 and 2MI-3(2MI for 2-methylindole addition);
5-methylindole is added into each test tube in the 5-methylindole group, and the concentration of the 5-methylindole in the bacterial liquid is set to be 4 mM; 3 tubes were scored as 5MI-1, 5MI-2 and 5MI-3(5MI for 5-methylindole addition);
4. the control test tubes of step 3 were incubated at 37 ℃ in a shaker at 250rpm for 3h in the dark.
5. Experimental groups: taking 7.5ml of the platform-stage staphylococcus aureus culture solution obtained in the step 1, and subpackaging the culture solution into 15 sterile glass test tubes, wherein each tube is subpackaged with 500 mu l of bacterial solution; the 15 glass tubes containing the bacterial liquid are randomly divided into 5 groups, and each group comprises 3 glass test tubes containing the bacterial liquid. The treatment groups were an untreated group, a tobramycin-treated group, a tobramycin-indole group, a tobramycin-2-methylindole group, and a tobramycin-5-methylindole group.
Untreated group: adding neither adjuvant nor tobramycin to the bacterial liquid, and 3 test tubes (marked as B1, B2 and B3) containing the bacterial liquid;
tobramycin treatment group: adding tobramycin into the glass tube in the group, wherein the concentration of the tobramycin in the bacterial liquid is set to be 250 ug/ml; marking 3 test tubes as T-250-1, T-250-2 and T-250-3(T represents adding tobramycin, 250 represents adding tobramycin concentration of 250 mug/ml);
indole-tobramycin group: adding indole and tobramycin into the glass tube in the group, wherein the concentration of the indole in the bacterial liquid is set to be 4 mM; the concentration of the tobramycin in the bacterial liquid is set to be 250 ug/ml; a group of 3 glass test tubes, wherein 3 test tubes are respectively marked as I + T-250-1, I + T-250-2 and I + T-250-3(I represents adding indole, T represents adding tobramycin, and 250 represents adding tobramycin with the concentration of 250 mu g/ml);
2-methylindole with tobramycin group: adding 2-methylindole and tobramycin into the glass tube in the group, wherein the concentration of the 2-methylindole in the bacterial liquid is set to be 4 mM; the concentration of tobramycin in the bacterial liquid is set to be 250 mug/ml; a set of 3 glass tubes, 3 tubes were designated as 2MI + T-250-1, 2MI + T-250-2 and 2MI + T-250-3(2MI for 2-methylindole addition, T for tobramycin addition, 250 for tobramycin addition at a concentration of 250. mu.g/ml);
group of 5-methylindole with tobramycin: adding 5-methylindole and tobramycin into the glass tube in the group, wherein the concentration of the 5-methylindole in the bacterial liquid is set to be 4 mM; the concentration of tobramycin in the bacterial liquid is set to be 250 mug/ml; a set of 3 glass tubes was identified as 5MI + T-250-1, 5MI + T-250-2 and 5MI + T-250-3(5MI for 5-methylindole addition, T for tobramycin addition and 250 for tobramycin addition at a concentration of 250. mu.g/ml).
6. The test tubes of the experimental group of step 5 were incubated at 37 ℃ for 3h in a shaker at 250rpm in the absence of light.
7. Taking out the bacteria liquid after incubation for 3h in the dark in the steps 4 and 6, centrifuging 100ul of the bacteria liquid (10000g, 2min), removing the supernatant, then re-suspending the bacteria with 100ul of 100mM sterile phosphate buffer (pH 7.4), washing twice, and re-suspending the bacteria with 100ul of 100mM sterile phosphate buffer (pH 7.4).
8. After step 7, diluting the obtained bacterial liquid with 100mM sterile phosphate buffer (pH 7.4) according to gradient of 10 times each time, wherein the dilution gradient is 10 and 102、103、104、105And 4 mul of bacteria are dropped on a LB solid culture medium six-square plate at each dilution, the plate is placed in a constant temperature incubator at 37 ℃ for 12 hours, the death condition of the bacteria is observed, the colony count is carried out, and the survival rate of the staphylococcus aureus after being treated is calculated, and the result is shown in figure 1.
9. Repeating the steps 5, 6, 7 and 8 by using tobramycin with different concentrations respectively, wherein the concentration of the tobramycin is 50 mug/ml and 100 mug/ml; and taking the average value of the number of the surviving colonies of the three groups of experiments, calculating the survival rate of the staphylococcus aureus after being treated, and drawing a tobramycin concentration-number of surviving colonies curve. The results are shown in FIGS. 2 and 3.
TABLE 1 comparison of the efficacy of the adjuvants in increasing the killing of Staphylococcus aureus by tobramycin
Note: relative bactericidal efficiency (survival rate with adjuvant or without tobramycin) multiplied by survival rate with tobramycin/survival rate with adjuvant or with tobramycin
The result shows that the tobramycin is added into the bacterial liquid for 3 hours, and the tobramycin has the sterilization efficiency of 1 order of magnitude. And adding the auxiliary agent (indole and derivatives thereof) and tobramycin into the bacterial liquid for treatment for 3 hours, wherein the death rate of staphylococcus aureus can be improved by 6 orders of magnitude by the 4mM auxiliary agent compared with a group without the auxiliary agent, and the staphylococcus aureus in the plateau phase is killed to the lower limit of detection. The method has concentration-dependent relationship between the sterilization efficiency and the antibiotic concentration.
EXAMPLE 2 adjuvant combination with streptomycin, gentamicin, kanamycin kills the plateau staphylococcus aureus
1. Activating bacteria: the method comprises the following steps of (1) sucking 1 mu l of 20% glycerol bacterial liquid of staphylococcus aureus (S.aureus ATCC 25923) stored in a refrigerator at the temperature of-80 ℃, adding the glycerol bacterial liquid into 1ml of LB liquid culture medium, culturing the glycerol bacterial liquid to the stage by a shaking table (250rpm) at the temperature of 37 ℃, diluting the obtained bacterial liquid by 1000 times, inoculating the diluted bacterial liquid into 30ml of LB liquid culture medium, and culturing the glycerol bacterial liquid to the stage by the shaking table (250rpm) at the temperature of 37 ℃ for 24 hours to obtain a staphylococcus aureus culture solution.
2. The mother liquor of indole, 2-methylindole and 5-methylindole is prepared by dimethyl sulfoxide (DMSO) with the concentration of 1M, the mother liquor is diluted by DMSO to prepare the working solution with the concentration of 200mM, and the whole preparation process is protected from light.
3. The additive is matched with streptomycin to treat staphylococcus aureus in the platform stage: taking 7.5ml of the platform-stage staphylococcus aureus culture solution obtained in the step 1, and subpackaging the culture solution into 15 sterile glass test tubes, wherein each tube is subpackaged with 500 mu l of bacterial solution; the 15 glass tubes containing the bacterial liquid are randomly divided into 5 groups, and each group comprises 3 glass test tubes containing the bacterial liquid. Respectively an untreated group, a streptomycin treated group, an indole-streptomycin group, a 2-methylindole-streptomycin group and a 5-methylindole-streptomycin group.
Untreated group: adding neither adjuvant nor streptomycin into the bacterial liquid, and 3 test tubes (marked as C1, C2 and C3) containing the bacterial liquid;
streptomycin treatment group: adding streptomycin into the glass tube in the group, wherein the concentration of the streptomycin in the bacterial liquid is set to be 2000 mug/ml; 3 tubes were designated S-2000-1, S-2000-2 and S-2000-3(S means streptomycin addition, 2000 means streptomycin addition concentration of 2000. mu.g/ml);
indole-streptomycin group: adding indole and streptomycin into the glass tube in the group, wherein the concentration of the indole in the bacterial liquid is set to be 4 mM; the concentration of streptomycin in the bacterial liquid is set to be 2000 mug/ml; a set of 3 glass test tubes, 3 test tubes were designated I + S-2000-1, I + S-2000-2 and I + S-2000-3(I means indole addition, S means streptomycin addition, 2000 means streptomycin addition at a concentration of 2000. mu.g/ml);
2-methylindole-streptomycin complex group: adding 2-methylindole and streptomycin into the glass tube in the group, wherein the concentration of the 2-methylindole in the bacterial liquid is set to be 4 mM; the concentration of streptomycin in the bacterial liquid is set to be 2000 mug/ml; a set of 3 glass tubes, 3 tubes were designated 2MI + S-2000-1, 2MI + S-2000-2 and 2MI + S-2000-3(2MI for 2-methylindole addition, S for streptomycin addition, 2000 for streptomycin addition at 2000. mu.g/ml);
5-methylindole-streptomycin complex group: adding 5-methylindole and streptomycin into the glass tube in the group, wherein the concentration of the 5-methylindole in the bacterial liquid is set to be 4 mM; the concentration of streptomycin in the bacterial liquid is set to be 2000 mug/ml; a set of 3 glass tubes was identified as 5MI + S-2000-1, 5MI + S-2000-2 and 5MI + S-2000-3(5MI for 5-methylindole addition, S for streptomycin addition, 2000 for streptomycin addition) respectively.
4. The additive is matched with the gentamicin to treat staphylococcus aureus in the platform stage: taking 7.5ml of the platform-stage staphylococcus aureus culture solution obtained in the step 1, and subpackaging the culture solution into 15 sterile glass test tubes, wherein each tube is subpackaged with 500 mu l of bacterial solution; the 15 glass tubes containing the bacterial liquid are randomly divided into 5 groups, and each group comprises 3 glass test tubes containing the bacterial liquid. Respectively an untreated group, a gentamicin treated group, an indole-gentamicin matched group, a 2-methylindole-gentamicin matched group and a 5-methylindole-gentamicin matched group.
Untreated group: adding neither adjuvant nor gentamicin into the bacterial liquid, and 3 test tubes (marked as D1, D2 and D3) containing the bacterial liquid;
gentamicin treatment group: adding gentamicin into the glass tube in the group, wherein the concentration of the gentamicin in the bacterial liquid is set to be 500 mug/ml; marking 3 test tubes as G-500-1, G-500-2 and G-500-3(G represents adding gentamicin, and 500 represents adding gentamicin with the concentration of 500 mug/ml);
indole-gentamicin complex group: adding indole and gentamicin into the glass tube in the group, wherein the content of the indole in the bacterial liquid is set to be 4 mM; the concentration of the gentamicin in the bacterial liquid is set to be 500 mug/ml; a group of 3 glass test tubes, wherein 3 test tubes are respectively marked as I + G-500-1, I + G-500-2 and I + G-500-3(I represents adding indole, G represents adding gentamicin, and 500 represents adding gentamicin with the concentration of 500 mug/ml);
2-methylindole-gentamicin complex group: adding 2-methylindole and gentamicin into the glass tube in the group, wherein the concentration of the 2-methylindole in the bacterial liquid is set to be 4 mM; the concentration of the gentamicin in the bacterial liquid is set to be 500 mug/ml; a group of 3 glass test tubes, 3 test tubes were respectively marked as 2MI + G-500-1, 2MI + G-500-2 and 2MI + G-500-3(2MI indicates the addition of 2-methylindole, G indicates the addition of gentamicin, and 500 indicates the addition of gentamicin at a concentration of 500. mu.g/ml);
5-methylindole-gentamicin complex: adding 5-methylindole and gentamicin into the glass tube in the group, wherein the concentration of the 5-methylindole in the bacterial liquid is set to be 4 mM; the concentration of the gentamicin in the bacterial liquid is set to be 500 mug/ml; a set of 3 glass tubes was scored as 5MI + G-500-1, 5MI + G-500-2, and 5MI + G-500-3(5MI for 5-methylindole addition, G for gentamicin addition, and 500 for gentamicin addition at a concentration of 500. mu.g/ml).
5. The additive is matched with the staphylococcus aureus in the kanamycin treatment platform stage: taking 7.5ml of the platform-stage staphylococcus aureus culture solution obtained in the step 1, and subpackaging the culture solution into 15 sterile glass test tubes, wherein each tube is subpackaged with 1000 mu l of bacterial solution; the 15 glass tubes containing the bacterial liquid are randomly divided into 5 groups, and each group comprises 3 glass test tubes containing the bacterial liquid. The groups are respectively an untreated group, a kanamycin treated group, an indole kanamycin-matched group, a 2-methylindole kanamycin-matched group and a 5-methylindole kanamycin-matched group.
Untreated group: adding neither adjuvant nor kanamycin into the bacterial liquid, and 3 test tubes (marked as E1, E2 and E3) filled with the bacterial liquid;
kanamycin treatment group: adding kanamycin into the glass tubes in the group, wherein the concentration of the kanamycin in the bacterial liquid is set to be 1000 mu g/ml; 3 tubes were designated as K-1000-1, K-1000-2 and K-1000-3(K indicates kanamycin addition, 1000 indicates kanamycin addition at a concentration of 1000. mu.g/ml);
indole-kanamycin complex group: adding indole and kanamycin into the glass tube in the group, wherein the concentration of the indole in the bacterial liquid is set to be 4 mM; the concentration of kanamycin in the bacterial liquid is set to be 1000 mug/ml; a set of 3 glass tubes, 3 tubes were designated I + K-1000-1, I + K-1000-2 and I + K-1000-3(I for indole addition, K for kanamycin addition, 1000 for kanamycin addition at 1000. mu.g/ml);
2-methylindole with kanamycin group: adding 2-methylindole and kanamycin into the glass tube in the group, wherein the concentration of the 2-methylindole in the bacterial liquid is set to be 4 mM; the concentration of kanamycin in the bacterial liquid is set to be 1000 mug/ml; a set of 3 glass tubes, 3 tubes were designated as 2MI + K-1000-1, 2MI + K-1000-2 and 2MI + K-1000-3(2MI for 2-methylindole addition, K for kanamycin addition and 1000 for kanamycin addition at 1000. mu.g/ml);
5-methylindole with kanamycin group: adding 5-methylindole and kanamycin into the glass tube in the group, wherein the concentration of the 5-methylindole in the bacterial liquid is set to be 4 mM; the concentration of kanamycin in the bacterial liquid is set to be 1000 mug/ml; a set of 3 glass tubes was designated as 5MI + K-1000-1, 5MI + K-1000-2 and 5MI + K-1000-3(5MI for 5-methylindole addition, K for kanamycin addition and 1000 for kanamycin addition at 1000. mu.g/ml).
6. The test tubes of the experimental group of the step 3, the step 4 and the step 5 are placed in a shaker at 37 ℃ and 250rpm and incubated for 3 hours in the dark.
7. Taking out the bacteria liquid after incubation for 3h in the dark in the steps 3, 4 and 5, respectively taking 100ul of the bacteria liquid to centrifuge (10000g, 2min), removing the supernatant, then re-suspending the bacteria with 100ul of 100mM sterile phosphate buffer (pH 7.4), washing twice, and re-suspending the bacteria with 100ul of 100mM sterile phosphate buffer (pH 7.4).
8. After step 7, diluting the obtained bacterial liquid with 100mM sterile phosphate buffer (pH 7.4) according to gradient of 10 times each time, wherein the dilution gradient is 10 and 102、103、104、105After 4. mu.l of each dilution was dropped on a six-square plate of LB solid medium and cultured in a 37 ℃ incubator for 12 hours, bacterial death was observed and colony counting was carried out, the results are shown in FIG. 4.
The result shows that streptomycin is added into the bacterial liquid of the staphylococcus aureus in the plateau stage for treatment for 3 hours, and the streptavidin has the sterilization efficiency of 1 order of magnitude. The auxiliary agent (indole and derivatives thereof) and streptomycin are added into the bacterial liquid for treatment for 3 hours, compared with a group without the auxiliary agent, 4mM indole can improve the death rate of staphylococcus aureus by 3 orders of magnitude, 4mM 2-methylindole can improve the death rate of staphylococcus aureus by 4 orders of magnitude, and 4mM 5-methylindole can improve the death rate of staphylococcus aureus by 5 orders of magnitude. Wherein the auxiliary agent 5-methylindole has better sterilization promoting effect than 2-methylindole and indole.
Gentamicin is added into the bacterial liquid of the staphylococcus aureus in the plateau stage for treatment for 3 hours, and the gentamicin has the sterilization efficiency of 2 orders of magnitude. The auxiliary agent (indole and derivatives thereof) is matched with gentamicin and added into the bacterial liquid for treatment for 3 hours, and compared with a group without the auxiliary agent, the death rate of staphylococcus aureus can be improved by 5 orders of magnitude by the aid of 4mM of the auxiliary agent.
Kanamycin is added into the bacterium liquid of the staphylococcus aureus in the plateau phase for treatment for 3 hours, and the kanamycin has the sterilization efficiency of 1 magnitude order. And adding an auxiliary agent (indole and derivatives thereof) and kanamycin into the bacterial liquid for treatment for 3 hours, wherein 4mM of indole can improve the death rate of staphylococcus aureus by 1 order of magnitude, 4mM of 2-methylindole can improve the death rate of staphylococcus aureus by 3 orders of magnitude, and 4mM of 5-methylindole can improve the death rate of staphylococcus aureus by 4 orders of magnitude compared with a group without the auxiliary agent. Wherein the adjuvant has the following sterilization promoting effect: 5-methylindole > 2-methylindole > indole.
Example 3 adjuvant in combination with streptomycin killing plateau MRSA
1. Activating bacteria: the method comprises the steps of (1) sucking 1 mu l of 20% glycerol bacterial liquid of MRSA ATCC43300 stored in a refrigerator at-80 ℃, adding the glycerol bacterial liquid into 1ml of LB liquid culture medium, culturing the glycerol bacterial liquid to a platform stage by a shaker at 37 ℃ (250rpm), diluting the obtained bacterial liquid by 1000 times, inoculating the diluted bacterial liquid into 30ml of LB liquid culture medium, and culturing the diluted bacterial liquid to the platform stage (24h) by the shaker at 37 ℃ (250rpm) to obtain the MRSA culture solution.
2. The mother liquor of indole, 2-methylindole and 5-methylindole is prepared by dimethyl sulfoxide (DMSO) with the concentration of 1M, the mother liquor is diluted by DMSO to prepare the working solution with the concentration of 200mM, and the whole preparation process is protected from light.
3. Control group: taking 6ml of the platform-phase MRSA culture solution obtained in the step 1, and subpackaging the 6ml of the platform-phase MRSA culture solution into 12 sterile glass test tubes, wherein each tube is subpackaged with 500 mu l of bacterial solution; the 12 glass test tubes containing the bacterial liquid were randomly divided into 4 groups, i.e., adjuvant-free group, indole group, 2-methylindole group, and 5-methylindole group.
No auxiliary agent is added in 3 test-tube bacterial liquids of the adjuvant-free group, and the test-tube bacterial liquids are respectively marked as C-1, C-2 and C-3;
adding indole into each test tube in the indole group, wherein the concentration of the indole in a bacterial solution is set to be 4 mM; 3 tubes were designated I-1, I-2 and I-3, respectively (I indicates the addition of indole);
2-methylindole is added into each test tube in the 2-methylindole group, and the concentration of the 2-methylindole in a bacterial solution is set to be 4 mM; 3 tubes were scored as 2MI-1, 2MI-2 and 2MI-3(2MI for 2-methylindole addition);
5-methylindole is added into each test tube in the 5-methylindole group, and the concentration of the 5-methylindole in the bacterial liquid is set to be 4 mM; 3 tubes were scored as 5MI-1, 5MI-2 and 5MI-3(5MI for 5-methylindole addition);
4. the control test tubes of step 3 were incubated at 37 ℃ in a shaker at 250rpm for 3h in the dark.
5. Experimental groups: taking 7.5ml of the platform-phase MRSA culture solution obtained in the step 1, and subpackaging the 7.5ml of the platform-phase MRSA culture solution into 15 sterile glass test tubes, wherein each tube is subpackaged with 500 mu l of bacterial solution; the 15 glass tubes containing the bacterial liquid are randomly divided into 5 groups, and each group comprises 3 glass test tubes containing the bacterial liquid. Respectively an untreated group, a streptomycin treated group, an indole-streptomycin group, a 2-methylindole-streptomycin group and a 5-methylindole-streptomycin group.
Untreated group: neither adjuvant nor streptomycin was added to the bacterial solution, and the 3 tubes in this group were labeled F1, F2, and F3, respectively;
streptomycin treatment group: adding streptomycin into the glass tube in the group, wherein the concentration of the streptomycin in the bacterial liquid is set to be 1000 mug/ml; 3 tubes were designated S-1000-1, S-1000-2 and S-1000-3(S means streptomycin addition, 1000 means streptomycin concentration 1000. mu.g/ml);
indole-streptomycin group: adding indole and streptomycin into the glass tube in the group, wherein the concentration of the indole in the bacterial liquid is set to be 4 mM; the concentration of streptomycin in the bacterial liquid is set to be 1000 mug/ml; the 3 tubes in this group were labeled I + S-1000-1, I + S-1000-2 and I + S-1000-3, respectively (I indicates addition of indole, T indicates addition of streptomycin, and 1000 indicates addition of streptomycin at a concentration of 1000. mu.g/ml);
2-methylindole-streptomycin complex group: adding 2-methylindole and streptomycin into the glass tube in the group, wherein the concentration of the 2-methylindole in the bacterial liquid is set to be 4 mM; the concentration of streptomycin in the bacterial liquid is set to be 1000 mug/ml; the 3 tubes in this group were scored as 2MI + S-1000-1, 2MI + S-1000-2 and 2MI + S-1000-3(2MI for 2-methylindole addition, S for streptomycin addition, and 1000 for streptomycin addition at a concentration of 1000. mu.g/ml);
5-methylindole-streptomycin complex group: adding 5-methylindole and streptomycin into the glass tube in the group, wherein the concentration of the 5-methylindole in the bacterial liquid is set to be 4 mM; the concentration of streptomycin in the bacterial liquid is set to be 1000 mug/ml; the 3 tubes in this group were scored as 5MI + S-1000-1, 5MI + S-1000-2 and 5MI + S-1000-3(5MI for the addition of 5-methylindole, S for the addition of streptomycin, and 1000 for the addition of streptomycin at a concentration of 1000. mu.g/ml);
6. the test tubes of the experimental group of step 5 were incubated at 37 ℃ for 3h in a shaker at 250rpm in the absence of light.
7. Taking out the bacteria solution after incubation for 3h in the dark in the steps 4 and 6, centrifuging 100ul of the bacteria solution (10000g, 2min), removing the supernatant, then re-suspending the bacteria with 100ul of 100mM sterile phosphate buffer (pH 7.4), washing twice, and re-suspending the bacteria with 100ul of 100mM sterile phosphate buffer (pH 7.4).
8. After step 7, the obtained bacterial liquid is treated with 100mM sterile phosphate buffer solution according to a gradient of 10 times each time(pH 7.4) dilution with a dilution gradient of 10, 102、103、104、105And 4 mul of bacteria are dropped on a LB solid culture medium six-square plate at each dilution, and after the bacteria are cultured for 12 hours in a constant temperature incubator at 37 ℃, the death condition of the bacteria is observed, colony counting is carried out, and the survival rate of the MRSA after treatment is calculated, and the result is shown in figure 5.
9. Repeating the steps 5, 6, 7 and 8 by using streptomycin with different concentrations respectively, wherein the concentration of the streptomycin is 250 mug/ml and 500 mug/ml; the survival rate of the MRSA treated colonies was calculated by averaging the number of surviving colonies from the three groups of experiments, and a streptomycin concentration-number of surviving colonies curve was plotted, with the results shown in FIGS. 6 and 7.
TABLE 2 comparison of adjuvant to increase efficiency of streptomycin killing plateau MRSA
Note: relative bactericidal efficiency (survival rate with adjuvant and no streptomycin. times. survival rate with streptomycin/survival rate with adjuvant and streptomycin)
The result shows that streptomycin is added into the bacterial liquid, and the streptomycin has almost no sterilization efficiency after 3 hours of treatment. The 4mM adjuvant alone hardly killed the plateau MRSA. The auxiliary agent (indole and derivatives thereof) is added into the MRSA bacterial liquid to be treated for 3 hours, compared with the group without the auxiliary agent, the sterilization efficiency of the streptomycin is respectively improved by adding 4mM auxiliary agent: indole can increase the mortality of the platform stage MRSA by 1 order of magnitude, 2-methylindole can increase the mortality of the platform stage MRSA by 3 orders of magnitude, and 5-methylindole can increase the mortality of the platform stage MRSA by 2 orders of magnitude. After the treatment by the method, the bactericidal efficiency of the adjuvant matched with streptomycin for sterilization and the concentration of the streptomycin are in a concentration dependence relationship.
Claims (9)
1. The method for improving the sterilization efficiency of aminoglycoside antibiotics is characterized in that: adding an auxiliary agent and aminoglycoside antibiotics into the bacterial liquid containing bacteria to be killed to obtain a bacterial liquid treatment liquid, wherein the auxiliary agent is indole or indole derivatives.
2. The method of claim 1, wherein the method comprises: and carrying out shake cultivation on the bacteria liquid treatment solution.
3. The method for improving the bactericidal efficiency of an aminoglycoside antibiotic according to claim 2, wherein: the temperature of shaking table culture is 37 ℃, and the time is 1-5 hours.
4. The method of claim 1, wherein the method comprises: the indole derivative is 2-methylindole or 5-methylindole.
5. The method of claim 1, wherein the method comprises: the final concentration of the auxiliary agent in the bacterial liquid treatment liquid is 1mM-4 mM.
6. The method of claim 1, wherein the method comprises: the bacteria is staphylococcus aureus, and the aminoglycoside antibiotic is tobramycin.
7. The method of claim 6, wherein the method comprises: the final concentration of the tobramycin in the bacterial liquid treatment liquid is 50-500 mg/mL.
8. The method of claim 1, wherein the method comprises: the bacteria is methicillin-resistant staphylococcus aureus, and the aminoglycoside antibiotic is streptomycin.
9. The method of claim 8, wherein the method comprises: the final concentration of the streptomycin in the bacterial liquid treatment liquid is 250-1000 mg/mL.
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