Combined antibacterial activity of streptozotocin and antibacterial drugs

Abstract

The present invention discloses the application of streptozotocin in antibacterial and combined antibacterial activities. The present invention discloses that streptozotocin has broad-spectrum antibacterial activity, has antibacterial activity against drug-resistant Gram-positive bacteria and Gram-negative bacteria, and at the same time, when streptozotocin is used in combination with other antibacterial drugs, it has additive or synergistic antibacterial activity against drug-resistant bacteria, which can fill the shortage of current clinical anti-drug-resistant bacteria drug varieties, improve the curative effect, and reduce the spread of drug-resistant bacteria.

Description

[0001] This application is a divisional application of a Chinese patent application with the application number 202210132500.5 and the invention title "Antibacterial and combined antibacterial activities of a nitrosourea compound", which was filed on February 14, 2022. Technical Field

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

[0003] The resistance of Gram-negative (G-) bacteria is becoming increasingly serious. Among them, extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Enterobacteriaceae bacteria (including Escherichia coli, Klebsiella pneumoniae, etc.) are the most prominent. There is a lack of effective drugs and clinical treatment is difficult. As a result, polymyxins, which were basically abandoned many years ago due to excessive nephrotoxicity and neurotoxicity, have to undertake the task of being the last line of defense against multi-drug resistant G- bacteria. However, resistant strains of polymyxins are gradually emerging. In particular, the emergence of the plasmid-mediated polymyxin resistance gene mcr-1 has further exacerbated the current situation of bacterial resistance. Even more seriously, in the past 50 years, the newly-structured antibacterial drugs launched globally have only included oxazolidinones, lipopeptides, and pleuromutilins against Gram-positive (G + ) bacteria, and diarylquinolines against tuberculosis. None of them target G- resistant bacteria, and the new skeleton compounds currently in clinical and preclinical research are also very limited. Finding effective antibacterial drugs and combined antibacterial treatment regimens is an important strategy for combating the currently intractable G- resistant bacterial infections in clinical practice.

[0004] Streptozotocin (CAS No. 18883-66-4) was isolated from the fermentation broth of Streptomyces achromogenes in 1956 and belongs to a small molecule active substance with a nitrosourea structure type (molecular weight 265.22). It was first discovered due to its antibacterial activity, and two papers reported its antibacterial activity in vivo and in vitro in 1959-1960. However, due to the long time since the literature was published, the research results cannot be traced. Since 1960, streptozotocin has gradually shown its antitumor activity as a DNA alkylating agent. In addition, during the antitumor research process, it was found that streptozotocin has the activity of destroying pancreatic islet β cells and then triggering type II diabetes. Since 1960, streptozotocin has been mainly used for antitumor treatment and the construction of type II diabetes animal models, and its antibacterial activity research has almost completely stopped. In particular, there has been no report on the antibacterial activity of streptozotocin in combination with other antibacterial drugs. Summary of the Invention

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

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

[0007] Based on currently prevalent drug-resistant strains, antibacterial activity research was carried out. The test strains include, but are not limited to: methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) among G + bacteria, ESBL-producing drug-resistant strains, carbapenem-resistant strains, and polymyxin-resistant strains among G- bacteria. In vitro combined antibacterial activity research of streptozotocin and 9 antibacterial drugs was carried out.

[0008] The agar dilution method was used to detect the minimum inhibitory concentration (MIC) of streptozotocin against a total of 37 bacteria of 18 genera (14 genera of G- bacteria and 4 genera of G + bacteria), and clarify its antibacterial spectrum. The K-B disc diffusion combined susceptibility test was used to detect the combined susceptibility of the streptozotocin and bacitracin drug combination against a total of 15 bacteria of 9 genera, and the combined susceptibility of the streptozotocin and 8 other antibacterial drug combinations against Escherichia coli (including drug-resistant strains). The checkerboard method of in vitro combined susceptibility test was used to quantitatively detect the combined antibacterial effect and combined drug index of the streptozotocin and bacitracin drug combination against extended-spectrum β-lactamase (ESBL)-producing, carbapenem-resistant, and polymyxin-resistant Escherichia coli.

[0009] First of all, the present invention provides the application of streptozotocin in anti-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 Enterococcus includes vancomycin-sensitive Enterococcus and vancomycin-resistant Enterococcus.

[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-producing drug-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-producing drug-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 combined use of streptozotocin and bacitracin in the treatment of Gram-positive and Gram-negative bacteria is also provided.

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

[0018] Furthermore, the combined use of streptozotocin and antibacterial drugs in antibacterial treatment is also provided, and the antibacterial drugs include roxithromycin, telithromycin, norfloxacin, vancomycin, and novobiocin.

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

[0020] Streptozotocin has broad-spectrum antibacterial activity, and when combined with other antibacterial drugs, it also has additive or synergistic antibacterial activity, effectively filling the application gap of antibacterial compounds. In particular, it provides an effective antibacterial solution for drug-resistant bacteria, improves the curative effect, and reduces the spread of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Results of the combined antibacterial experiment of streptozotocin and bacitracin by the K-B disc diffusion method;

[0022] Figure 2 Results of the combined antibacterial experiment of streptozotocin and antibacterial drugs by the K-B disc diffusion method. DETAILED DESCRIPTION OF THE EMBODIMENTS

[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] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0025] All materials, reagents, etc. in the following examples can be obtained from commercial sources unless otherwise specified.

[0026] Example 1 Detection of the antibacterial MIC value of streptozotocin by the agar dilution method

[0027] Inoculate the test strain by streaking it on an agar culture dish and incubate it statically overnight in a 37°C constant temperature incubator. The next day, pick at least 3 well-separated and morphologically identical colonies from the agar plate and inoculate them into an appropriate broth medium. Incubate at 35°C for 2 - 6 hours, and use a turbidimeter to adjust the bacterial suspension concentration to 0.5 McFarland turbidity (1 - 2×10 8 CFU / mL) for standby. Dilute the streptozotocin drug stock solution successively with the corresponding diluent to various required concentrations, add it to a 90 mm sterile petri dish, and then add the corresponding volume of MH agar using a graduated pipette. After thoroughly mixing the drug solution and the medium, prepare a drug-containing agar plate with a final streptozotocin concentration range of 0.06 - 128 μg / mL for standby. Dilute the 0.5 McFarland turbidity bacterial suspension by another 10 times (about 10 7 CFU / mL), mix well, and take 0.3 mL and add it to the inoculation plate in sequence. Use a multi-point inoculator to inoculate the above bacterial suspension onto a series of petri dishes containing different concentrations of the drug, and the inoculum size at each inoculation point is about 10 4 CFU. Place the inoculated petri dishes with half-open lids in a constant temperature room. After the inoculation points are completely dry, invert the petri dishes and incubate them in a 35 ± 2°C incubator or constant temperature room for 16 - 20 hours (Acinetobacter spp. are incubated for 20 - 24 hours). Arrange a series of petri dishes in ascending order of drug concentration and place them against a black non-reflective background. Start reading from the petri dish containing the lowest concentration of the drug. Observe the bacterial growth with the naked eye, and the lowest concentration at which bacterial growth is completely inhibited is the MIC value.

[0028] The minimum inhibitory concentration (MIC) values of streptozotocin against a total of 37 strains of bacteria belonging to 18 species (14 species of G - bacteria and 4 species of G + bacteria) are shown in Table 1. The research results show that streptozotocin has broad-spectrum antibacterial activity. Its minimum inhibitory concentration against G + bacteria (including MRSA and VRE) is 2 - 8 μg / mL, and its minimum inhibitory concentration against G - bacteria (including ESBL-producing strains, carbapenem-resistant strains, polymyxin-resistant strains) is 2 - >128 μg / mL. Among them, the MIC value range of streptozotocin against Staphylococcus aureus (including MRSA) is 4 - 8 μg / mL, the MIC value range against Escherichia coli (including ESBL-producing, carbapenem-resistant, polymyxin-resistant strains) is 4 - >128 μg / mL, the MIC value range against Klebsiella pneumoniae (including ESBL-producing, carbapenem-resistant, polymyxin-resistant strains) is 128 - >128 μg / mL, and the MIC values against sensitive and resistant Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter cloacae are all >128 μg / mL. The MIC values of streptozotocin against other species of bacteria are shown in detail in Table 1.

[0029] Table 1 Results of determination of streptozotocin MIC values

[0030]

[0031] Note: MSSE, methicillin-sensitive Staphylococcus epidermidis; MSSA, methicillin-sensitive Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; VSE, vancomycin-sensitive Enterococcus; VRE, vancomycin-resistant Enterococcus; ESBL(+), producing extended-spectrum β-lactamase; ESBL(-), not producing extended-spectrum β-lactamase; mcr-1(+), carrying the polymyxin resistance gene mcr-1; NDM-1(+), producing New Delhi metallo-β-lactamase 1; CRPA, carbapenem-resistant Pseudomonas aeruginosa; CRAB, carbapenem-resistant Acinetobacter baumannii.

[0032] Example 2 K-B disk diffusion combined susceptibility test

[0033] The test strains were streaked on agar plates and incubated overnight in a 37°C incubator. The next day, at least 3 well-isolated and morphologically identical colonies were picked from the agar plates and inoculated into an appropriate broth medium. Incubate at 35°C for 2 - 6 hours, and use a turbidimeter to adjust the bacterial suspension concentration to 0.5 McFarland turbidity (1 - 2×10 8 CFU / mL). Use a sterile cotton swab to dip the bacterial suspension, gently squeeze out the moisture, and then evenly spread it on the MH agar medium. Let the surface moisture dry and set aside. The distance between the centers of the two disks is adjusted according to drug sensitivity. If both drugs are sensitive, the distance is 24 mm; if one drug is resistant and the other is sensitive, the distance is 15 mm; if both drugs are resistant, the distance is 10 mm. A maximum of 4 disks can be applied on a 90-mm-diameter MH plate. Then incubate at 35°C for 24 hours and take out to observe the results. According to the morphology of the inhibition zone, it is divided into synergistic, additive, irrelevant, and antagonistic effects.

[0034] Such as Figure 1As shown, the combined use of streptozotocin (32 μg / tablet) and bacitracin (10 IU / tablet) has certain synergistic or additive antibacterial activities against Enterobacteriaceae bacteria, including Escherichia coli (E. coli), Klebsiella pneumoniae (K. pnuemoniae), and Shigella flexneri (S. Flexneri). Among the 9 tested Enterobacteriaceae bacteria, the inhibition zones of streptozotocin in 6 strains significantly shifted towards the bacitracin susceptibility test paper (marked with black frames), including ESBL-producing strains (E. coli ATCC 35218), carbapenem-resistant strains (E. coli ATCC BAA-2469 and K. pneumoniae ATCC BAA-2146), and polymyxin-resistant strains (E. coli 08-85). Additionally, it was found that the combined use of the two also had a certain degree of synergistic antibacterial effect on Staphylococcus aureus (S. aureus ATCC 29213) (marked with black frames). However, the combined use of the two had no obvious synergistic antibacterial effect on Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), Enterobacter cloacae (E. coloacae), Serratia marcescens (S. marcescens), and Staphylococcus epidermidis (S. epidermidis).

[0035] As Figure 2 shown, using the K-B disk combination susceptibility method with the E. coli sensitive strain E. coli ATCC 25922 and the ESBL-producing resistant strain E. coli ATCC 35218 as the tested strains, the antibacterial activities of the combined use of streptozotocin with 8 antibacterial drugs, namely linezolid, vancomycin, daptomycin, roxithromycin, telithromycin, novobiocin, norfloxacin, and co-trimoxazole, were studied. The experimental results showed that the combined use of streptozotocin with roxithromycin, telithromycin, norfloxacin, vancomycin, and novobiocin had certain degrees of synergistic or additive antibacterial activities against the tested E. coli.

[0036] Example 3 Checkerboard combination susceptibility test

[0037] Inoculate the tested strains by streaking on an agar culture dish and incubate statically overnight in a 37°C constant temperature incubator. The next day, pick at least 3 well-separated and morphologically identical colonies from the agar plate and inoculate them into an appropriate broth medium. Incubate at 35°C for 2 - 6 hours, and use a turbidimeter to adjust the bacterial suspension concentration to 0.5 McFarland turbidity (1 - 2×10 8(CFU / mL), and then dilute it 20 times for standby. The two compounds in the combination drug are respectively subjected to horizontal two-fold dilution (Drug A) and vertical two-fold dilution (Drug B) in two round-bottom 96-well plates. The diluted drug concentrations are both 2 times the final experimental concentration. Among them, a blank CAMH broth medium without drug is added after the last concentration gradient of each drug dilution. The two diluted drugs are completely combined at different concentrations and added to the 96-well plate according to a volume ratio of 1:1 (50 μL each), so that the drug-containing CAMH broth medium in each well is 100 μL. Pipette 10 μL of the diluted bacterial solution into the above-mentioned drug-containing CAMH broth medium micro-wells, seal the 96-well plate with a sealing film, and incubate it statically at 37 °C for 16-18 hours. When reading the results, place the 96-well plate in a well-lit place, observe the turbidity of the broth in the wells and whether there is colony deposition at the bottom of the wells, and record the MIC values of the two drugs used alone and the MIC values without bacterial growth when using the combination drug. Calculation and judgment criteria of the fractional inhibitory concentration 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.

[0038] The checkerboard method combined drug susceptibility test was used to quantitatively verify a part of the synergistic or additive effects indicated in the K-B paper combined drug susceptibility. Table 2 shows that the combined drug index (FICI) of streptozotocin and bacitracin against G - bacteria (including drug-resistant bacteria) ranges from 0.312 to 0.750, and the MIC values of the two drugs used alone can be reduced by 2 - 16 times and 8 - 16 times respectively. The combination of the two drugs shows synergistic antibacterial activity against the tested Escherichia coli, including E. coli ATCC 25922 (FICI = 0.312), ESBL-producing strain E. coli ATCC 35218 (FICI = 0.375), carbapenem-resistant strain E. coli ATCC2469 (FICI = 0.312), and polymyxin-resistant strain E. coli 08-85 (FICI = 0.375).

[0039] Table 2 Results of checkerboard method combined drug susceptibility test

[0040]

[0041] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. The combined use of streptozotocin and antibacterial drugs in the preparation of drugs against drug-resistant Gram-negative bacteria, characterized in that, The drug-resistant Gram-negative bacterium is the drug-resistant strain E. coli ATCC 35218 that produces ESBL, and the antibacterial drugs are roxithromycin, telithromycin or norfloxacin.

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