Combined antibacterial activity of streptozotocin and bacitracin

Abstract

The present invention discloses the application of streptozotocin for antibacterial and combined antibacterial activity. The present invention discloses that streptozotocin has broad-spectrum antibacterial activity against both drug-resistant Gram-positive and Gram-negative bacteria. Furthermore, when used in combination with other antibacterial drugs, streptozotocin exhibits additive or synergistic antibacterial activity against drug-resistant bacteria. This can address the current shortage of clinical anti-resistant bacteria drugs, improve efficacy, and reduce the spread of drug-resistant bacteria.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202210132500.5 filed on February 14, 2022, and invention name “Antibacterial and combined antibacterial activity of a nitrosourea compound”. Technical Field

[0002] The present invention relates to the biological field, and in particular to the application of streptozotocin in antibacterial and combined antibacterial activities. Background Art

[0003] Gram-negative (G - The drug resistance of multidrug-resistant bacteria is becoming increasingly serious, among which the most prominent are Enterobacteriaceae (including Escherichia coli and Klebsiella pneumoniae) that produce extended-spectrum β-lactamases (ESBLs) and are resistant to carbapenems. There is a lack of effective drugs, and clinical treatment is difficult. Polymyxins, which were basically abandoned many years ago due to their excessive nephrotoxicity and neurotoxicity, have to take on the current role in combating multidrug-resistant G - The mission of the last line of defense against bacteria is to be the first line of defense against bacteria. However, polymyxin-resistant strains have gradually emerged, especially the emergence of the plasmid-mediated polymyxin resistance gene mcr-1, which has further aggravated the current situation of bacterial resistance. What is more serious is that in the past 50 years, the only new structural antibacterial drugs launched globally are Gram-positive (G + ) bacteria, oxazolidinones, lipopeptides, pleuromutilins, and anti-tuberculosis diarylquinolines, none of which targets G - There are currently very few new backbone compounds in clinical and preclinical research. Finding effective antibacterial drugs and combined antibacterial regimens is an important way to combat the current difficult clinical G - Important strategies for combating drug-resistant bacterial infections.

[0004] Streptozotocin (CAS No. 18883-66-4), isolated in 1956 from the fermentation broth of Streptomyces achromogenes, is a small molecule (molecular weight 265.22) with a nitrosourea structure. It was first discovered for its antibacterial activity, with two publications in 1959 and 1960 reporting its in vitro and in vivo antibacterial activity. However, due to the age of these publications, the results are no longer traceable. However, since 1960, streptozotocin's antitumor activity as a DNA alkylating agent has gradually become apparent. Furthermore, antitumor research has revealed that streptozotocin can damage pancreatic β cells and subsequently induce type 2 diabetes. Since 1960, streptozotocin has primarily been used for antitumor therapy and the development of animal models for type 2 diabetes, while research on its antibacterial activity has been almost completely discontinued. In particular, no reports have been published on the antibacterial activity of streptozotocin in combination with other antibacterial agents. Summary of the Invention

[0005] Based on this, it is necessary to discover and explore the applications of streptozotocin's antibacterial and combined antibacterial activities.

[0006] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0007] Based on the current popular drug-resistant strains, antibacterial activity studies were conducted. Test strains include but are not limited to: G + Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) in bacteria, G - ESBL-producing, carbapenem-resistant, and polymyxin-resistant strains of bacteria were detected. The in vitro combined antimicrobial activity of streptozotocin and nine antimicrobial drugs was studied.

[0008] The agar dilution method was used to test the effects of streptozotocin on 18 species (G - 14 species of bacteria and G + The minimum inhibitory concentration (MIC) of the drug combination was determined for 37 bacterial strains (including 4 species of Escherichia coli) to clarify its antimicrobial spectrum. The combined antimicrobial susceptibility of streptozotocin and bacitracin against 15 bacterial strains from 9 species was tested using a KB disk test, as was the combined antimicrobial susceptibility of streptozotocin and 8 other antimicrobial agents against Escherichia coli (including resistant strains). The combined antimicrobial efficacy and combination index of the streptozotocin and bacitracin combination against extended-spectrum β-lactamase (ESBL)-producing, carbapenem-resistant, and polymyxin-resistant Escherichia coli were quantitatively determined using a checkerboard in vitro combined antimicrobial susceptibility test.

[0009] First, the present invention provides the use of streptozotocin in resisting Gram-positive bacteria and Gram-negative bacteria.

[0010] Preferably, the Gram-positive bacteria include Staphylococcus epidermidis, Staphylococcus aureus, and Enterococcus.

[0011] Preferably, the Staphylococcus aureus includes methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0012] Preferably, the enterococci include vancomycin-sensitive enterococci and vancomycin-resistant enterococci.

[0013] Preferably, the Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella aerogenes, Serratia marcescens, Citrobacter freundii, Pantoea agglomerans, Proteus vulgaris, Salmonella typhi, Morganella morganii, Stenotrophomonas maltophilia, and Shigella flexneri.

[0014] Preferably, the Escherichia coli includes extended-spectrum β-lactamase-resistant Escherichia coli, non-extended-spectrum β-lactamase-producing Escherichia coli, carbapenem-resistant Escherichia coli, polymyxin-resistant Escherichia coli, and New Delhi metallo-β-lactamase 1-producing Escherichia coli.

[0015] Preferably, the Klebsiella pneumoniae includes extended-spectrum β-lactamase-resistant Klebsiella pneumoniae, non-extended-spectrum β-lactamase-producing Klebsiella pneumoniae, carbapenem-resistant Klebsiella pneumoniae, polymyxin-resistant Klebsiella pneumoniae, and New Delhi metallo-β-lactamase 1-producing Klebsiella pneumoniae.

[0016] Furthermore, the invention also provides the combined use of streptozotocin and bacitracin in combating Gram-positive bacteria and Gram-negative bacteria.

[0017] Preferably, the Gram-negative bacteria include extended-spectrum β-lactamase-resistant strains, carbapenem-resistant strains, and polymyxin-resistant strains.

[0018] Furthermore, the invention also provides a combined use of streptozotocin and antibacterial drugs in antibacterial treatment, wherein the antibacterial drugs include roxithromycin, telithromycin, norfloxacin, vancomycin and novobiocin.

[0019] Based on the above technical solution, the present invention has the following beneficial effects:

[0020] Streptozotocin has broad-spectrum antibacterial activity, and when used in combination with other antibacterial drugs, it also has additive or synergistic antibacterial activity, effectively filling the gap in the application of antibacterial compounds, especially providing effective antibacterial solutions for drug-resistant bacteria, improving efficacy, and reducing the spread of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of the combined antibacterial test of streptozotocin and bacitracin using the KB paper disc method;

[0022] Figure 2 These are the results of the combined antibacterial experiment using streptozotocin and antibiotics using the KB disk method. DETAILED DESCRIPTION

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents in the following examples can be obtained from commercial sources.

[0026] Example 1 Agar dilution method for detecting the antibacterial MIC value of streptozotocin

[0027] The test strain was inoculated into an agar plate and incubated overnight in a 37°C constant temperature incubator. The next day, at least three well-separated colonies with the same morphology were picked from the agar plate and inoculated into an appropriate broth medium. The culture was cultured at 35°C for 2 to 6 hours. The concentration of the bacterial solution was adjusted to 0.5 McFarland turbidity (1 to 2 × 10 8 CFU / mL) for use. Dilute the stock solution of streptozotocin with the corresponding diluent to the required concentrations, add it to a 90mm sterile plate, and then add the corresponding volume of MH agar with a graduated pipette. After the drug solution and culture medium are fully mixed, a drug-containing agar plate with a final concentration of streptozotocin ranging from 0.06 to 128μg / mL is prepared for use. Dilute the 0.5 McFarland turbidity bacterial solution 10 times (about 10 7 CFU / mL), mix well and take 0.3mL to add to the inoculation plate in sequence. Use a multi-point inoculator to inoculate the above bacterial solution onto a series of plates containing different concentrations of drugs. The amount of inoculation bacteria at each inoculation point is about 10 4 CFU. Place the inoculated plate with the lid half-open in a constant temperature room. Once the inoculation point is completely dry, invert the plate and incubate it in a 35±2°C incubator or constant temperature room for 16–20 hours (20–24 hours for Acinetobacter). Arrange a series of plates in ascending order of drug concentration against a black, non-reflective background, and read the plate containing the lowest drug concentration. Observe bacterial growth visually; the lowest concentration at which bacterial growth is completely inhibited is the MIC.

[0028] Streptozotocin is effective against 18 species (G - 14 species of bacteria and G + The minimum inhibitory concentration (MIC) values of 37 bacterial strains (including 4 species of bacteria) are shown in Table 1. The results showed that streptozotocin has a broad-spectrum antibacterial activity against G + The minimum inhibitory concentration of bacteria (including MRSA and VRE) is 2-8 μg / mL. - The minimum inhibitory concentration (MIC) of streptozotocin against ESBL-producing, carbapenem-resistant, and polymyxin-resistant strains ranges from 2 to >128 μg / mL. The MIC values of streptozotocin against Staphylococcus aureus (including MRSA) range from 4 to 8 μg / mL, against Escherichia coli (including ESBL-producing, carbapenem-resistant, and polymyxin-resistant strains) from 4 to >128 μg / mL, and against Klebsiella pneumoniae (including ESBL-producing, carbapenem-resistant, and polymyxin-resistant strains) from 128 to >128 μg / mL. The MIC values for both susceptible and resistant Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter cloacae are all >128 μg / mL. MIC values of streptozotocin against other bacterial species are shown in Table 1.

[0029] Table 1 Streptozotocin MIC value determination results

[0030]

[0031] Note: MSSE, methicillin-susceptible Staphylococcus epidermidis; MSSA, methicillin-susceptible Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; VSE, vancomycin-susceptible enterococci; VRE, vancomycin-resistant enterococci; ESBL(+), producing extended-spectrum β-lactamases; ESBL(-), not producing extended-spectrum β-lactamases; mcr-1(+), carrying the polymyxin resistance gene mcr-1;

[0032] NDM-1(+), New Delhi metallo-β-lactamase 1-producing; CRPA, carbapenem-resistant Pseudomonas aeruginosa; CRAB, carbapenem-resistant Acinetobacter baumannii.

[0033] Example 2 K-B disk method combined with drug sensitivity test

[0034] The test strain was inoculated into an agar plate and incubated overnight in a 37°C constant temperature incubator. The next day, at least three well-separated colonies with the same morphology were picked from the agar plate and inoculated into an appropriate broth medium. The culture was cultured at 35°C for 2 to 6 hours. The concentration of the bacterial solution was adjusted to 0.5 McFarland turbidity (1 to 2 × 10 8 CFU / mL). Use a sterile cotton swab to smear the bacterial solution, gently squeeze to remove excess moisture, and evenly spread the solution on MH agar. Allow to air dry before use. The center-to-center distance between the two paper strips is adjusted based on drug sensitivity: 24 mm if both are sensitive, 15 mm if one is resistant and the other is sensitive, and 10 mm if both are resistant. A maximum of four strips can be placed on a 90 mm diameter MH plate. Incubate at 35°C for 24 hours, then examine the plate. The inhibition ring morphology is used to categorize the effects as synergistic, additive, unrelated, or antagonistic.

[0035] like Figure 1As shown, the combined use of streptozotocin (32 μg / tablet) and bacitracin (10 IU / tablet) exhibits synergistic or additive antibacterial activity against Enterobacteriaceae, including Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Shigella flexneri (S. flexneri). Among the nine Enterobacteriaceae tested, the streptozotocin inhibition zone shifted significantly toward the bacitracin susceptibility zone (black box). These included an ESBL-producing strain (E. coli ATCC 35218), a carbapenem-resistant strain (E. coli ATCC BAA-2469 and K. pneumoniae ATCC BAA-2146), and a polymyxin-resistant strain (E. coli 08-85). Furthermore, the combination of the two agents demonstrated a moderate degree of synergistic antibacterial activity against Staphylococcus aureus (S. aureus ATCC 29213) (black box). However, the combined use of the two has no obvious synergistic antibacterial effect against Acinetobacter baumannii (A.baumannii), Pseudomonas aeruginosa (P.aeruginosa), Enterobacter cloacae (E.coloacae), Serratia marcescens (S.marcescens), and Staphylococcus epidermidis (S.epidermidis).

[0036] like Figure 2 As shown in the figure, the antibacterial activity of streptozotocin in combination with eight antimicrobial agents, including linezolid, vancomycin, daptomycin, roxithromycin, telithromycin, novobiocin, norfloxacin, and co-trimoxazole, was investigated using the KB disk combined susceptibility test method with susceptible E. coli strains E. coli ATCC 25922 and ESBL-producing resistant E. coli ATCC 35218. The results showed that the combination of streptozotocin with roxithromycin, telithromycin, norfloxacin, vancomycin, and novobiocin exhibited a certain degree of synergistic or additive antibacterial activity against the tested E. coli.

[0037] Example 3 Chessboard method combined with drug sensitivity test

[0038] The test strain was inoculated into an agar plate and incubated overnight in a 37°C constant temperature incubator. The next day, at least three well-separated colonies with the same morphology were picked from the agar plate and inoculated into an appropriate broth medium. The culture was cultured at 35°C for 2 to 6 hours. The concentration of the bacterial solution was adjusted to 0.5 McFarland turbidity (1 to 2 × 10 8CFU / mL) and then diluted 20-fold for later use. The two compounds in the combination were diluted two-fold horizontally (Drug A) and two-fold vertically (Drug B) in two round-bottom 96-well plates, with the diluted drug concentrations being 2 times the final experimental concentration. After the last concentration gradient of each drug dilution, blank CAMH broth containing no drug was added. The two diluted drugs were combined at varying concentrations and added to the 96-well plates in a 1:1 ratio (50 μL each) so that each well contained 100 μL of drug-containing CAMH broth. 10 μL of the diluted bacterial solution was pipetted into the wells containing the drug-containing CAMH broth. The 96-well plates were sealed with sealing film and incubated at 37°C for 16–18 hours. When reading the results, the 96-well plates were placed in a well-lit area, and the broth turbidity and presence of bacterial colonies at the bottom of the wells were observed. The MIC values of the two drugs alone and the MIC values of the combination without bacterial growth were recorded in the 96-well plates. Calculation and judgment criteria of FIC index (FICI): FICI = MIC 甲药联用 / MIC 甲药单用 +MIC 乙药联用 / MIC 乙药单用 FICI ≤ 0.5, synergistic effect; FICI > 0.5-1, additive effect; FICI > 1-2, no effect; FICI > 2, antagonistic effect.

[0039] The chessboard method was used to quantitatively verify the synergistic or additive effects suggested by the KB paper strip combined drug sensitivity test. - The FICI for the combination of the two drugs (including resistant strains) ranged from 0.312 to 0.750, reducing the MICs of the two drugs used alone by 2- to 16-fold and 8- to 16-fold, respectively. The two drugs combined exhibited synergistic antibacterial activity against all tested Escherichia coli, including E. coli ATCC 25922 (FICI = 0.312), ESBL-producing E. coli ATCC 35218 (FICI = 0.375), carbapenem-resistant E. coli ATCC 2469 (FICI = 0.312), and polymyxin-resistant E. coli 08-85 (FICI = 0.375).

[0040] Table 2 Results of chessboard combined drug sensitivity test

[0041]

[0042] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. The combined use of streptozotocin and bacitracin in the preparation of drugs against drug-resistant Gram-negative bacteria, characterized in that: The resistant Gram-negative bacteria are resistant Escherichia coli or resistant Klebsiella pneumoniae, the resistant Escherichia coli is polymyxin-resistant Escherichia coli E. coli 08-85, carbapenem-resistant Escherichia coli E. coli ATCC BAA-2469 or extended-spectrum β-lactamase-producing Escherichia coli E. coli ATCC 35218, and the resistant Klebsiella pneumoniae is carbapenem-resistant Klebsiella pneumoniae K. pnuemoniae ATCC BAA-2146.

Images