CN108272809B - Method for improving sterilization efficiency of aminoglycoside antibiotics - Google Patents
Method for improving sterilization efficiency of aminoglycoside antibiotics Download PDFInfo
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- CN108272809B CN108272809B CN201810074813.3A CN201810074813A CN108272809B CN 108272809 B CN108272809 B CN 108272809B CN 201810074813 A CN201810074813 A CN 201810074813A CN 108272809 B CN108272809 B CN 108272809B
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- 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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Abstract
The invention discloses a method for improving the sterilization efficiency of aminoglycoside antibiotics, which comprises the step of freezing a gram-negative bacteria culture solution containing aminoglycoside antibiotics for a short time (10 s-30 s), wherein the freezing temperature is-80 ℃ to-196 ℃, and compared with the normal-temperature treatment, the bacterial death rate is increased by 2-4 orders of magnitude. The aminoglycoside antibiotics include Kanamycin (Kanamycin), Streptomycin (Streptomycin), Tobramycin (Tobramycin) and Gentamicin (Gentamicin), and the gram-negative bacteria are Escherichia coli (E.coli)E.coli) Or Pseudomonas aeruginosaP.aeruginosa). The method can greatly improve the sterilization efficiency of the aminoglycoside antibiotics, effectively reduce the risk of drug resistance of pathogenic bacteria, and simultaneously 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 field of antibiotics, in particular to a method for improving the sterilization efficiency of aminoglycoside antibiotics (aminoglycosides).
Background
Bacterial resistance is a major public health problem facing the world. In china, the problem of bacterial resistance is more pressing due to clinical antibiotic abuse and the abuse of aquaculture antibiotics. Improving the sterilization efficiency of the existing antibiotics and quickly and efficiently killing pathogenic bacteria are important means for reducing the drug resistance risk of bacteria.
Coli is an important bacterium parasitic to the human intestinal tract, and causes diseases in special cases, such as blood infection. Meanwhile, Escherichia coli is used as a common standard strain for biological research and is widely used for researching the drug resistance mechanism of bacteria. Pseudomonas aeruginosa is a pathogenic bacterium, and is also a common strain for researching the drug resistance mechanism of bacteria. Coli and pseudomonas aeruginosa belong to gram-negative bacteria.
Aminoglycoside antibiotics are important drugs for treating serious infection of aerobic gram-negative bacilli at present and belong to bactericidal antibiotics. The molecular structure of the compound has one amino cyclitol and one or more amino sugar molecules, and the compound is named after glycoside formed by connecting glycosidic bonds. Aminoglycoside antibiotics bind to the small 30S subunit of the bacterial ribosome, causing the bacteria to synthesize the wrong protein, producing harmful protein aggregates, and ultimately killing the bacteria. Such antibiotics have been subject to numerous limitations in clinical use, mainly due to the increasingly severe phenomenon of bacterial resistance and the nephrotoxicity and ototoxicity of such antibiotics. The aminoglycoside antibiotics currently used in clinical medicine or breeding industry mainly comprise: tobramycin, kanamycin, streptomycin, gentamicin, neomycin, amikacin, apramycin, dalbenamycin, netilmicin, sisomicin, and the like.
If the sterilization efficiency of the aminoglycoside antibiotics can be greatly improved, the risk of drug resistance generation of pathogenic bacteria can be effectively reduced, and meanwhile, on the premise of achieving the same treatment effect, the dosage and the administration time are reduced, so that the side effect is reduced.
Disclosure of Invention
The invention provides a method for improving the sterilization efficiency of aminoglycoside antibiotics, thereby achieving the purposes of reducing the drug resistance risk of aminoglycoside antibiotics and reducing the side effects of aminoglycoside antibiotics.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for improving the bactericidal efficiency of aminoglycoside antibiotics uses aminoglycoside antibiotics to kill gram-negative bacteria under freezing conditions. Specifically, the gram-negative bacteria culture solution containing aminoglycoside antibiotics is frozen at the temperature of-80 ℃ to-196 ℃.
Further, the gram-negative bacteria is escherichia coli, and the aminoglycoside antibiotic is tobramycin, kanamycin, streptomycin or gentamicin.
Further, the gram-negative bacteria are pseudomonas aeruginosa, and the aminoglycoside antibiotic is tobramycin, streptomycin or gentamicin.
Further, the gram-negative bacteria is escherichia coli or pseudomonas aeruginosa, and the aminoglycoside antibiotics include but are not limited to neomycin, amikacin, apramycin, dalbenamycin, netilmicin, and sisomicin.
Further, the freezing temperature is-80 deg.C, and gram-negative bacteria culture solution containing aminoglycoside antibiotics is frozen in alcohol pre-cooled in refrigerator at-80 deg.C for 25-35 s.
Preferably, the freezing treatment is carried out at-196 deg.C, and the gram-negative bacteria culture solution containing aminoglycoside antibiotics is frozen in liquid nitrogen at-196 deg.C for 8-12 seconds.
By adopting the technical scheme, the method can strengthen the killing of gram-negative bacteria by the aminoglycoside antibiotics, and the bacterial death rate can be increased by 2-4 orders of magnitude compared with the bacterial liquid containing the aminoglycoside antibiotics after the bacterial liquid is subjected to transient freezing treatment (-196 ℃ or-80 ℃) at normal temperature.
Taking tobramycin as an example, the sterilization efficiency of the tobramycin is respectively enhanced by 9720 times (-196 ℃ frozen) and 5667 times (-80 ℃ frozen) against escherichia coli, and the sterilization efficiency of the tobramycin is respectively enhanced by 19542 times (-196 ℃ frozen) and 24111 times (-80 ℃ frozen) against pseudomonas aeruginosa. The sterilization efficiency of streptomycin against Escherichia coli is respectively improved by 10125 times (-196 ℃ freezing) and 8500 times (-80 ℃ freezing), and the sterilization efficiency against Pseudomonas aeruginosa is respectively improved by 97 times (-196 ℃ freezing) and 115 times (-80 ℃ freezing). The sterilization efficiency of gentamicin against Escherichia coli is respectively increased by 10974 times (-196 ℃ frozen) and 6611 times (-80 ℃ frozen), and the sterilization efficiency against Pseudomonas aeruginosa is respectively increased by 503 times (-196 ℃ frozen) and 323 times (-80 ℃ frozen). The efficiency of killing Escherichia coli by kanamycin is improved by 17743 times (196 ℃ freezing) and 12219 times (80 ℃ freezing), and the specific results are shown in tables 1 and 2.
The method can greatly improve the sterilization efficiency of the aminoglycoside antibiotics, effectively reduce the risk of drug resistance of pathogenic bacteria, and simultaneously reduce the dosage and the administration time on the premise of achieving the same treatment effect, thereby reducing the side effect.
Detailed Description
Example 1
Freezing enhanced efficiency of aminoglycoside antibiotics in killing escherichia coli
1) Standard strain of activated escherichia coli (e.coli BW 25113). Sucking 1 μ L of the bacterial solution stored in a refrigerator at-80 deg.C with 20% glycerol, adding 1ml LB liquid medium (1L of the medium containing tryptone 10g, yeast extract 5g, NaCl 10g, and water in balance; sterilizing with high pressure steam), culturing at 37 deg.C with shaker (250rpm) to a plateau stage, diluting 1000 times, inoculating to 2ml of LB liquid medium, and culturing at 37 deg.C with shaker (250rpm) to a logarithmic phase (OD)600＝0.8)。
2) 15 sterilized EP tubes were taken and 100. mu.l of the bacterial suspension was dispensed into each tube. Wherein 12 EP tubes are respectively added with kanamycin, tobramycin, gentamicin or streptomycin to the final concentrations of 30 mug/ml, 25 mug/ml and 100 mug/ml, and are mixed evenly. The same antibiotic-treated bacteria liquid was used in three portions, one portion was used for normal temperature bath (as a control), and the other 2 portions were used for freezing treatment at-196 deg.C and-80 deg.C, respectively. Finally, 3 parts of the bacterial liquid are used as a blank control without adding antibiotics, 1 part of the bacterial liquid is placed at the normal temperature, and the other two parts are frozen at the temperature of-196 ℃ or-80 ℃.
3) Putting the bacterial liquid for freezing treatment in liquid nitrogen (-196 ℃), and taking out after 10 s; or placing in alcohol pre-cooled in refrigerator at-80 deg.C, and taking out after 30 s. And (5) placing the frozen bacterial liquid in a normal-temperature water bath for thawing for 2 minutes.
4) All the bacteria were centrifuged (10000g, 1min), the supernatant was removed, the cells were resuspended in 100. mu.l of 20mM sterile phosphate buffer, washed twice and finally resuspended in 100. mu.l of 20mM phosphate buffer.
5) Diluting the bacterial liquid with 20mM phosphate buffer solution sequentially according to gradient of 10 times each time, dripping 5 μ l of bacterial liquid at each dilution on a LB solid culture medium six-square grid plate, culturing in an incubator at 37 ℃ for 8-10 hours, checking bacterial death, counting bacterial colonies, and calculating survival rate of treated escherichia coli. The results of the experiment are shown in table 1.
TABLE 1 comparison of the efficiency of aminoglycoside antibiotics in killing E.coli at ambient temperature and under freezing conditions
aAnd (3) survival rate calculation: the number of Escherichia coli colonies surviving under the treatment conditions/the number of Escherichia coli colonies subjected to antibiotic-free normal-temperature warm bathbTaking 196 ℃ below zero as an example, the sterilization efficiency is improved by a factor of-196 ℃ survival rate without antibiotics multiplied by normal temperature survival rate with antibiotics multiplied by-196 ℃ survival rate with antibiotics multiplied by 100
As is clear from Table 1, the bactericidal efficiency of tobramycin against E.coli was increased 9720 times (-196 ℃ C. freezing) and 5667 times (-80 ℃ C. freezing), respectively. The efficiency of killing Escherichia coli by kanamycin is improved by 17743 times (196 ℃ freezing) and 12219 times (80 ℃ freezing), respectively. The sterilization efficiency of streptomycin against escherichia coli is respectively improved by 10125 times (-196 ℃ freezing) and 8500 times (-80 ℃ freezing). The sterilization efficiency of the gentamicin against the escherichia coli is respectively increased by 10974 times (-196 ℃ frozen) and 6611 times (-80 ℃ frozen).
From the above results, it is clear that the method of the present invention can enhance the killing of gram-negative bacteria by aminoglycoside antibiotics, thereby reducing the risk of drug resistance of aminoglycoside antibiotics.
Example 2
Freezing enhanced efficacy of aminoglycoside antibiotics in killing pseudomonas aeruginosa
1) The laboratory Pseudomonas aeruginosa strain (P. aeruginosa PAO1) was activated from 20% glycerol stock in a-80 ℃ refrigerator and cultured in LB liquid medium at 37 ℃ with a shaker (250rpm) to a plateau (about 16 hours).
2) 12 sterilized EP tubes were taken and 100. mu.l of the bacterial suspension was dispensed into each tube. Of these, 9 EP tubes were separately charged with gentamicin, tobramycin or streptomycin (note: Pseudomonas aeruginosa itself tolerates kanamycin and therefore the antibiotic was not tested) to final concentrations of 25. mu.g/ml, 25. mu.g/ml and 100. mu.g/ml, respectively, and mixed well. The same antibiotic-treated bacteria liquid was used in three portions, one portion was incubated at room temperature for 3min (as a control), and the other 2 portions were frozen at-196 deg.C and-80 deg.C, respectively. Finally, 3 parts of the bacterial liquid are used as a control without adding antibiotics, 1 part of the bacterial liquid is placed at the normal temperature, and the other two parts are frozen at the temperature of-196 ℃ or-80 ℃.
3) Putting the bacterial liquid for freezing treatment in liquid nitrogen (-196 ℃), and taking out after 10 s; or placing in alcohol pre-cooled in refrigerator at-80 deg.C, and taking out after 30 s. And (5) placing the frozen bacterial liquid in a normal-temperature water bath for thawing for 2 minutes.
4) The cells were centrifuged (10000g, 1min), the supernatant was removed, the cells were resuspended in 100. mu.l of 20mM sterile phosphate buffer, washed twice and finally resuspended in 100. mu.l of 20mM phosphate buffer.
5) And sequentially diluting the bacterial liquid with 20mM phosphate buffer solution according to the gradient of 10 times each time, dripping 5 mu l of bacterial liquid of each dilution on an LB solid culture medium six-square grid plate, placing the plate on a 37 ℃ incubator for culturing for 8-10 hours, checking bacterial death, counting bacterial colonies, and calculating the survival rate of the pseudomonas aeruginosa after treatment. The results of the experiment are shown in table 2.
TABLE 2 comparison of the efficacy of aminoglycoside antibiotics in killing Pseudomonas aeruginosa at ambient temperature and freezing conditions
aAnd (3) survival rate calculation: the number of the colonies of the pseudomonas aeruginosa surviving under the treatment condition/the number of the colonies of the pseudomonas aeruginosa after antibiotic-free normal-temperature warm bath
bWith-1For example, at 96 ℃, the bactericidal efficiency is increased by the factor of the survival rate without antibiotic at that temperature x the survival rate with antibiotic at room temperature/the survival rate with antibiotic at that temperature/100.
As can be seen from Table 2, the bactericidal efficiency of tobramycin against P.aeruginosa was increased 19542 times (-196 ℃ C. freezing) and 24111 times (-80 ℃ C. freezing), respectively. The sterilization efficiency of the streptomycin for the pseudomonas aeruginosa is respectively improved by 97 times (-196 ℃ freezing) and 115 times (-80 ℃ freezing). The sterilizing efficiency of the gentamicin against the pseudomonas aeruginosa is respectively improved by 503 times (-196 ℃ frozen) and 323 times (-80 ℃ frozen).
From the above results, it is clear that the method of the present invention can enhance the killing of gram-negative bacteria by aminoglycoside antibiotics, thereby reducing the risk of drug resistance of aminoglycoside antibiotics.
The foregoing is a detailed description of the invention with reference to specific preferred embodiments, and numerous derivations or substitutions may be made by those skilled in the art without departing from the spirit of the invention, which should be construed to be within the scope of the invention as defined in the appended claims.
Claims (4)
1. A method for improving the sterilization efficiency of aminoglycoside antibiotics is characterized in that: freezing the gram-negative bacteria by using the aminoglycoside antibiotics, wherein the freezing treatment is to freeze the gram-negative bacteria culture solution containing the aminoglycoside antibiotics in liquid nitrogen at the temperature of-196 ℃ for 8-12 seconds, or to freeze the gram-negative bacteria culture solution containing the aminoglycoside antibiotics in alcohol precooled by a refrigerator at the temperature of-80 ℃ for 25-35 seconds.
2. The method of claim 1, wherein the method comprises: the gram-negative bacteria are escherichia coli, and the aminoglycoside antibiotics are tobramycin, kanamycin, streptomycin or gentamicin.
3. The method of claim 1, wherein the method comprises: the gram-negative bacteria are pseudomonas aeruginosa, and the aminoglycoside antibiotics are tobramycin, streptomycin or gentamicin.
4. The method of claim 1, wherein the method comprises: the gram-negative bacteria are escherichia coli or pseudomonas aeruginosa, and the aminoglycoside antibiotics are neomycin, amikacin, apramycin, dalbenamycin, netilmicin or sisomicin.
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