A method of killing bacteria for non-therapeutic purposes

Abstract

The application discloses a method for killing bacteria for non-treatment purposes, which comprises the following steps: adding an auxiliary agent into a bacterial liquid containing bacteria to be killed to obtain a bacterial liquid treatment liquid, wherein the auxiliary agent is indole, 2-methylindole or 5-methylindole; and then performing a shaking table culture on the bacterial liquid treatment liquid. The method can kill a plurality of bacteria, effectively reduces the risk of drug resistance of the bacteria, and the sterilization effect is better with the prolongation of the treatment time.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for killing bacteria for non-therapeutic purposes. Background Technology

[0002] Antimicrobial resistance is a major global public health problem and a leading cause of chronic infections and post-treatment relapses. It threatens human life and health, is a significant factor in property damage, and poses a major challenge to veterinary public health, particularly in animal-derived bacteria. For over 70 years since the discovery of penicillin, the first antibiotic, antibiotics have been key drugs in clinical treatment of bacterial infections. However, long-term antibiotic use induces bacterial resistance, and antibiotic overuse has exacerbated this resistance, leading to the emergence of resistant strains and even "superbugs." The susceptibility of bacteria to multiple antibiotics has resulted in a decreasing number of drugs available to treat resistant bacterial infections. The bactericidal efficacy of existing antibiotics has decreased, and the development of new antibiotics is difficult and time-consuming; the number of new antibiotics approved by the FDA in the past 30 years has decreased by 90%. Therefore, screening existing compounds for drugs with broad-spectrum antibacterial activity is crucial.

[0003] With the widespread use of antibiotics globally, the incidence of drug-resistant bacterial infections due to antibiotic abuse is increasing year by year. In recent years, there have been reports of increasing nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Streptococcus pneumoniae. Staphylococcus aureus can cause chronic and recurrent infections, including osteomyelitis, endocarditis, and recurrent abscesses. Besides Staphylococcus aureus, many Gram-positive bacteria are pathogenic, such as Enterococcus faecalis, Streptococcus pyogenes, and Staphylococcus epidermidis. Among Gram-negative bacteria, Escherichia coli is an opportunistic pathogen, and Mycobacterium tuberculosis can infect host cells to form tuberculous nodules. Klebsiella pneumoniae commonly causes respiratory tract infections, urinary tract infections, liver / biliary tract infections, sepsis, meningitis, and peritonitis. Multidrug-resistant Pseudomonas aeruginosa is resistant not only to β-lactam antibiotics but also to carbapenems, quinolones, and aminoglycosides. This multidrug-resistant *Pseudomonas aeruginosa* readily infects the bloodstream and severely compromises the immune system, particularly in patients with weakened immune systems, such as those who have undergone bone marrow or organ transplants, ultimately leading to sepsis and pneumonia that are difficult to cure. Furthermore, *Shigella* is one of the most common pathogens causing intestinal diseases in humans, leading to diarrhea and a variety of other illnesses. Therefore, it is essential to find novel antibacterial agents with broad-spectrum bactericidal activity to eradicate these pathogens.

[0004] Indole is an aromatic heterocyclic compound whose structure consists of a benzene ring and a pyrrole ring. It serves as both an intermediate in chemical synthesis and a widely used additive in pharmaceuticals, pesticides, fragrances, and dyes. As a structural framework for chemical drugs, indole has been used to develop numerous highly active compounds, such as antibacterial, antiviral, analgesic, and antitumor agents. Daptomycin, launched in 2003, is an anti-infective drug developed using indole as its core, capable of treating complicated skin and skin structure infections, as well as bloodstream infections caused by Staphylococcus aureus. Therefore, other indole compounds synthesized using indole as a core may also possess bactericidal activity. Screening other indole compounds and testing their antibacterial activity is one approach to finding novel antibacterial agents. Summary of the Invention

[0005] The purpose of this invention is to provide a method for killing bacteria for non-therapeutic purposes.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for killing bacteria for non-therapeutic purposes involves adding an adjuvant to a bacterial suspension containing bacteria to be killed to obtain a bacterial suspension treatment solution, wherein the adjuvant is indole, 2-methylindole, or 5-methylindole, and then culturing the bacterial suspension treatment solution in a shaker.

[0008] The shaking incubator is incubated at 37°C for 1-5 hours.

[0009] The final concentration of the auxiliary agent in the bacterial solution is 1 mM-8 mM.

[0010] The bacteria include Gram-positive and Gram-negative bacteria. The Gram-positive bacteria are one of Staphylococcus aureus, MRSA, Staphylococcus epidermidis, Enterococcus faecalis, and Streptococcus pyogenes. The Gram-negative bacteria are one of Escherichia coli, Pseudomonas aeruginosa, Shigella, Klebsiella pneumoniae, and Mycobacterium tuberculosis.

[0011] Experiments have shown that the method of this invention can kill a variety of bacteria. For Staphylococcus aureus, the bactericidal efficiencies of 5 mM 2-methylindole and 5 mM 5-methylindole are three orders of magnitude higher; the bactericidal efficiencies of 7 mM 2-methylindole and 6 mM 5-methylindole are six orders of magnitude higher. For MRSA, both 8 mM 2-methylindole and 7 mM 5-methylindole achieve bactericidal efficiencies of three orders of magnitude higher. Other types of bacteria, especially those treated with 5-methylindole, show varying degrees of killing effects. This demonstrates that the method of this invention can kill a variety of bacteria, and that 5-methylindole possesses broad-spectrum bactericidal activity. Attached Figure Description

[0012] Figure 1The relationship between the concentration of indole and its derivatives and the number of surviving colonies. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, instruments, etc. used in the following embodiments can be obtained commercially. In the quantitative experiments in the following embodiments, three replicate experiments are set up, and the results are averaged.

[0014] The Staphylococcus aureus (ATCC25923), MRSA (ATCC43300), Staphylococcus epidermidis (CMCC26069), Enterococcus faecalis (ATCC29212), and Streptococcus pyogenes (ATCC19615) in the following examples; and Gram-negative bacteria: Escherichia coli (BW25113), Pseudomonas aeruginosa (PAO1), Shigella (24T7T), Klebsiella pneumoniae (KP-D367), and Mycobacterium tuberculosis (ATCC25177) are all biomaterials that are available to the public from the applicant. These biomaterials are only for repeating the relevant experiments of this invention and shall not be used for any other purpose.

[0015] Example 1: Indole, 2-methylindole, and 5-methylindole were used as bactericides to kill different types of bacteria.

[0016] 1. Activation of bacteria: Take 1 µl of 20% glycerol bacterial suspension of Staphylococcus aureus ATCC 25923 (S. aureus ATCC 25923) stored at -80℃ and add it to 1 ml of LB liquid medium. Incubate at 37℃ on a shaker (250 rpm) until the plateau phase. Dilute the obtained bacterial suspension 1000 times and inoculate it into 50 ml of LB liquid medium. Incubate at 37℃ on a shaker (250 rpm) until the plateau phase (24 h) to obtain Staphylococcus aureus culture.

[0017] 2. Prepare stock solutions of indole, 2-methylindole and 5-methylindole with dimethyl sulfoxide (DMSO) at a concentration of 1M. Then dilute the stock solutions with DMSO to prepare working solutions with a concentration of 200mM. The entire preparation process should be carried out in the dark.

[0018] 3. Take 37.5 ml of the plateau phase Staphylococcus aureus culture obtained in step 1 and dispense it into 75 sterile glass tubes, each containing 500 µl of bacterial culture;

[0019] The 75 glass tubes containing bacterial solutions were randomly divided into 4 groups: blank group, indole group, 2-methylindole group, and 5-methylindole group.

[0020] The three glass tubes containing bacterial solutions in the blank group are labeled A1, A2, and A3.

[0021] Indole was added to each glass tube in the indole group. The concentration of indole in the bacterial culture was set at 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, and 8 mM. Three glass tubes were set up for each concentration, and the three glass tubes were labeled as IX-1, IX-2, and IX-3, respectively (X is the corresponding indole concentration. For example, three parallel experiments of 1 mM indole were labeled as I-1-1, I-1-2, and I-1-3, respectively).

[0022] Add 2-methylindole to each glass tube in the 2-methylindole group. The concentration of 2-methylindole in the bacterial culture was set to 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, and 8 mM. Three glass tubes were used for each concentration, and the three glass tubes were labeled as 2MI-X-1, 2MI-X-2, and 2MI-X-3, respectively (X is the corresponding indole concentration, such as three parallel experiments of 1 mM 2-methylindole, which are labeled as 2MI-1-1, 2MI-1-2, and 2MI-1-3, respectively).

[0023] Add 5-methylindole to each glass tube in the 5-methylindole group. The concentration of 5-methylindole in the bacterial culture was set to 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, and 8 mM. Three glass tubes were used for each concentration, and the three centrifuge tubes were labeled as 5MI-X-1, 5MI-X-2, and 5MI-X-3, respectively (X is the corresponding indole concentration. For example, three parallel experiments with 1 mM 5-methylindole were labeled as 5MI-1-1, 5MI-1-2, and 5MI-1-3, respectively).

[0024] 4. Place the glass tube from step 3 in a shaker at 37°C and 250 rpm and incubate in the dark for 3 hours.

[0025] 5. After incubating the bacterial culture in the dark for 3 hours, take out 100 μL of the bacterial culture and centrifuge (10000 g, 2 min) to remove the supernatant. Then resuspend the bacterial cells in 100 µL of 100 mM sterile phosphate buffer (pH 7.4), wash twice, and then resuspend the bacterial cells in 100 µL of 100 mM sterile phosphate buffer (pH 7.4).

[0026] 6. After completing step 5, dilute the obtained bacterial solution with 100 mM sterile phosphate buffer (pH 7.4) in a 10-fold gradient, with each dilution being 10, ... 2 103 10 4 10 5 For each dilution, 4 µl of bacterial suspension was dropped onto a LB solid medium hexagonal plate and incubated at 37°C for 12 hours. Afterward, bacterial death was observed, colony counts were performed, the survival rate of Staphylococcus aureus after treatment was calculated, and the curve of adjuvant concentration versus surviving colony count was plotted.

[0027] 7. Repeat steps 3, 4, 5, and 6 three times, take the average number of surviving colonies in the three groups of experiments, calculate the survival rate of Staphylococcus aureus after treatment, and plot the additive concentration-surviving colony count curve.

[0028] 8. The treatment steps for other types of bacteria are the same as above: except for Enterococcus faecalis (ATCC29212), which uses MRS medium, the other 9 types of bacteria use LB medium. Gram-positive bacteria using LB medium are: Staphylococcus aureus (ATCC25923), MRSA (ATCC43300), Staphylococcus epidermidis (CMCC26069), and Streptococcus pyogenes (ATCC19615); Gram-negative bacteria using LB medium are: Escherichia coli (BW25113), Pseudomonas aeruginosa (PAO1), Shigella (24T7T), Klebsiella pneumoniae (KP-D367), and Mycobacterium tuberculosis (ATCC25177).

[0029] The results are as follows Figure 1 As shown in Tables 1 and 2.

[0030] Table 1. Concentration of additives used to kill 99.9% of bacteria

[0031]

[0032] Table 2 Concentrations of additives used to kill bacteria to the limit of detection

[0033]

[0034] Note: Sterilization efficiency = 1 - number of surviving colonies after treatment with fungicide / number of surviving colonies without treatment with fungicide

[0035] The sterilization concentration used to achieve a sterilization efficiency of 99.9% is the concentration of the bactericide that corresponds to a sterilization efficiency of just greater than 99.9%.

[0036] The concentration of the bactericide used to kill bacteria to the lower limit of detection is the bactericide concentration corresponding to a sterilization efficiency of 99.9999%.

[0037] The results showed that adding adjuvants (indole and its derivatives) to the bacterial culture for 3 hours increased the mortality rate of Staphylococcus aureus by three orders of magnitude compared to treatment without adjuvants. Further increasing the adjuvant concentration, 7 mM 2-methylindole and 6 mM 5-methylindole killed Staphylococcus aureus in the plateau phase to the limit of detection. The table above shows that different bacterial species have varying sensitivities to indole and its derivatives, requiring different adjuvant concentrations to achieve three orders of magnitude reductions. Overall, the bactericidal efficiency of the three adjuvants was 5-methylindole > 2-methylindole, and 2-methylindole > indole, i.e., 5-methylindole > 2-methylindole > indole.

Claims

1. A method for killing bacteria for non-therapeutic purposes, characterized in that: A bactericide is added to a bacterial culture containing bacteria to be sterilized to obtain a bacterial culture treatment solution, which is then cultured on a shaker. The bactericide is 2-methylindole or 5-methylindole. When the bacteria are Gram-positive bacteria, and specifically MRSA and Staphylococcus epidermidis, the final concentration of 2-methylindole in the bacterial suspension treatment solution is 7-8 mM, and the final concentration of 5-methylindole in the bacterial suspension treatment solution is 7-8 mM. When the bacteria are Gram-positive and Enterococcus faecalis, the final concentration of 2-methylindole in the bacterial solution is 5-8 mM, and the final concentration of 5-methylindole in the bacterial solution is 5-7 mM. When the bacteria are Gram-negative bacteria, and are one of Escherichia coli, Shigella, and Klebsiella pneumoniae, the final concentration of 2-methylindole in the bacterial suspension treatment solution is 5-8 mM, and the final concentration of 5-methylindole in the bacterial suspension treatment solution is 5-7 mM.

2. The method for killing bacteria for non-therapeutic purposes according to claim 1, characterized in that: The shaking incubator is incubated at 37°C for 1-5 hours.

3. A method for killing bacteria for non-therapeutic purposes, characterized in that: A bactericide is added to a bacterial culture containing bacteria to be sterilized to obtain a bacterial culture treatment solution, which is then cultured in a shaker. The bactericide is 2-methylindole or 5-methylindole, wherein the final concentration of 2-methylindole in the bacterial culture treatment solution is 5 mM, and the final concentration of 5-methylindole in the bacterial culture treatment solution is 3-4 mM. The bacteria is Pseudomonas aeruginosa.

4. The method for killing bacteria for non-therapeutic purposes according to claim 3, characterized in that: The shaking incubator is incubated at 37°C for 1-5 hours.

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