Application of Creatmycin in Inhibiting Pathogenic Bacteria in Digestive Tract

Abstract

The present invention relates to the application of creatmycin in inhibiting gastrointestinal tract pathogenic bacteria, and the gastrointestinal tract pathogenic bacteria include but are not limited to: Helicobacter pylori, Clostridium difficile, Bacteroides fragilis, Escherichia fergusonii, Clostridium sporogenes.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology. Specifically, it relates to the application of creatmycin in inhibiting gastrointestinal pathogens. Background Art

[0002] There are more than 10 trillion microbial cells in the human digestive tract, especially in the intestine, which is equivalent to the number of human cells. The number of microbial cells per gram of feces is about 4.3×10 10 to 3.1×10 11 , and the genes of the gut microbiome are at least two orders of magnitude more than the human genome. [1] . The gut microbiota is a complex microecosystem containing a variety of microorganisms, including bacteria, archaea, eukaryotes, and viruses (or phages), etc. It plays an important role in the absorption of human nutrients, energy metabolism, and human immunity. The gut microbiota can prevent the colonization of potential pathogenic bacteria in the intestine and is an important biological barrier to protect the body from external damage. Diet, lifestyle, antibiotics and other drugs, hygiene, and the genetic and immune status of the host determine the composition of the microbiota and have an impact on the physiological and pathological states of the host. More and more studies have shown that changes in the composition of the gut microbiome (i.e., gut microbiota dysbiosis) can lead to the abnormal proliferation of some pathogenic bacteria, which is closely related to the incidence of infections, inflammations, allergic diseases, and some malignant tumors, etc. [2,3] .

[0003] Helicobacter pylori (abbreviated as H. pylori or Hp) is a Gram-negative microaerophilic bacillus colonized on the gastric mucosa. It infects more than half of the world's population and is the most common pathogenic factor causing chronic gastritis, peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Since the discovery of H. pylori in 1983, a variety of drugs have been successively used to treat gastric diseases caused by H. pylori. Currently, the clinical treatment of Hp infection mostly adopts the quadruple bismuth therapy (1 proton pump inhibitor + 1 bismuth agent + 2 antibiotics), and the antibiotics include different combinations of amoxicillin, furazolidone, tetracycline, levofloxacin, metronidazole, and clarithromycin. [4,5] , although it has certain effects, but the long-term application of antibiotics to patients during the treatment process is likely to cause the problem of Hp drug resistance in patients. Currently, the drug resistance problem of H. pylori has become more and more serious. [6] , and in 2017, the World Health Organization (WHO) listed Helicobacter pylori as one of the 20 pathogens that pose the most serious threats to human health due to its drug resistance. [7] .

[0004] Escherichia fergusonii belongs to the genus Escherichia in the family Enterobacteriaceae. It is mostly found in the environment and the intestines of humans and animals. It is a rod-shaped, sporeless, facultatively anaerobic Gram-negative bacterium. [8] Escherichia fergusonii is a zoonotic opportunistic pathogen closely related to Escherichia coli in the same genus. It can cause various opportunistic infections in humans and animals, including wound infections, urinary tract infections, bacteremia, diarrhea, and pleurisy. Since its discovery and naming in 1985, many studies have reported that Escherichia fergusonii has multiple drug resistances to antibacterial drugs for treating intestinal bacterial infections, such as resistance to cephalosporins, quinolones, colistin, tigecycline, etc. It may be an important reservoir of neglected drug resistance genes and should attract wide attention.

[0005] Clostridioides difficile (CD) is a Gram-positive anaerobic spore-forming bacillus that is widely present in the environment and the intestines of humans and animals. It is the main pathogen causing antibiotic-associated diarrhea, hospital infection-associated diarrhea, and colitis. Severe Clostridioides difficile infection (CDI) can be fatal. It is listed as one of the urgent public health threats related to antibiotics (https: / / www.cdc.gov / DrugResistance / Biggest-Threats.html). CDI is usually caused by antibiotics disrupting the normal intestinal flora, resulting in a dysbiosis dominated by the proliferation of CD. Currently, it is considered that the abuse of antibiotics is the most important influencing factor for the increasing incidence of CDI, and the drug resistance of Clostridioides difficile is also gradually increasing. CDI has become a global problem. Metronidazole, vancomycin, and fidaxomicin are currently the first-line drugs for treating CDI, but resistance has also started to appear.

[0006] With the widespread use and abuse of broad-spectrum antibiotics, many strains have developed drug resistances. The three gastrointestinal pathogens mentioned above are currently also facing severe antibiotic resistance problems. The development of new anti-drug-resistant bacteria drugs is extremely urgent. Chuangxinmycin (CM for short, the chemical structure is shown in Figure 1 ) was born in 1964. It is the first antibiotic with a completely new structure discovered in China and is produced by fermentation of a strain of Actinoplanes tsinanensis CPCC 200056 isolated from the soil in Jinan, Shandong, China. [9,10] It has an inhibitory effect on Staphylococcus aureus, Shigella dysenteriae, Escherichia coli, and Haemophilus influenzae in vitro.

[11] This is achieved by selectively inhibiting the activity of tryptophanyl-tRNA synthetase.

[12] , there is currently no clinically used antibiotic targeting this target, which has no cross-resistance with commonly used antibiotics, low toxicity, and few side effects. Preliminary clinical trials were conducted in the 1970s, and the results showed that it had a unique effect on infections caused by Escherichia coli, especially sepsis. [9,13] , but at that time, due to the high fermentation cost, narrow antibacterial spectrum, and relatively low antibacterial activity of creatmycin, its widespread use in clinical practice was affected. Based on the completely new skeleton structure and novel action target of creatmycin, in the current situation where the problem of antibiotic resistance is becoming increasingly serious, the development of new uses for creatmycin has new significance.

[0007]

References

[0008] [1].Vandeputte D, Kathagen G, D'Hoe K, et al. Quantitative Microbiome Profiling Links Gut Community Variation to Microbial Load[J]. Nature, 2017, 551(7681): 507-511.

[0009] [2]. Sommer F, Backhed F. The Gut Microbiota--Masters of Host Development and Physiology[J]. Nat Rev Microbiol, 2013, 11(4): 227-238.

[0010] [3]. Gilbert JA, Blaser MJ, Caporaso JG, et al. Current Understanding of the Human Microbiome[J]. Nat Med, 2018, 24(4): 392-400.

[0011] [4]. Liu WZ, Xie Y, Lu H, et al. Fifth Chinese National Consensus Report on the Management of Helicobacter Pylori Infection[J]. Helicobacter, 2018, 23(2): e12475.

[0012] [5]. Matsumoto H, Shiotani A, Graham DY. Current and Future Treatment of Helicobacter Pylori Infections[J]. Adv Exp Med Biol, 2019, 1149: 211 - 225.

[0013] [6]. Savoldi A, Carrara E, Graham DY, et al. Prevalence of Antibiotic Resistance in Helicobacter Pylori: A Systematic Review and Meta - Analysis in World Health Organization Regions[J]. Gastroenterology, 2018, 155(5): 1372 - 1382e1317.

[0014] [7]. Tacconelli E, Carrara E, Savoldi A, et al. Discovery, Research, and Development of New Antibiotics: The Who Priority List of Antibiotic - Resistant Bacteria and Tuberculosis[J]. Lancet Infect Dis, 2018, 18(3): 318 - 327.

[0015] [8]. Chen Yifei, Yang Hua, Xia Xiaodong, et al. Research Progress on the Prevalence and Drug Resistance of Escherichia fergusonii[J]. Microbiology China, 2020, (06): 1973 - 1981.

[0016] [9]. Report on the Identification Meeting of Chuangxinmycin[J]. Pharmaceutical Industry, 1972, (01): 24.

[0017]

[10] . Research Group of Chuangxinmycin, Institute of Materia Medica, Chinese Academy of Medical Sciences. Research on a New Antibiotic - Chuangxinmycin[J]. Science in China, 1976, (3): 295 - 300.

[0018]

[11] . Yan Guihua, Wang Chengde, Lin Futian, et al. Antibacterial Effects in Vivo and in Vitro and Distribution in Vivo of Chuangxinmycin[J]. Chinese Journal of Antibiotics, 1980, (05): 41 - 46.

[0019]

[12] . Qi Tianqing, Liu Xiu, Yang Yufen. Inhibition of Enzyme Synthesis in the Tryptophan Pathway of Escherichia coli by Chuangxinmycin [J]. Acta Academiae Medicinae Sinicae, 1980, (01): 32 - 37.

[0020]

[13] . Clinical Trial of Chuangxinmycin [J]. Bulletin of Medical Research, 1972, (01): 16 - 17.

[0021]

[14] . CLSI, Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria. 3th Ed. CLSI Standard M45. 2015, Wayne, PA: Clinical and Laboratory Standards Institute.

[0022]

[15] . CLSI, Methods for Antimicrobial Susceptibiluty Testing of Anaerobic Bacteria. 8th Ed. CLSI Standard M11. 2012, Wayne, PA: Clinical and Laboratory Standards Institute. Summary of the Invention

[0023] The present invention first relates to an application of chuangxinmycin in inhibiting digestive tract pathogenic bacteria, and the digestive tract pathogenic bacteria include but are not limited to: Helicobacter pylori, Clostridium difficile, Bacteroides fragilis, Escherichia fergusonii, Clostridium sporogenes.

[0024] The present invention also relates to the following applications of chuangxinmycin and its derivatives:

[0025] (1) Preparing a drug for inhibiting Helicobacter pylori, Clostridium difficile, Bacteroides fragilis, Escherichia fergusonii, Clostridium sporogenes;

[0026] (2) Preparing a drug for repairing intestinal flora imbalance, where the intestinal flora imbalance is: intestinal flora imbalance caused by the over - proliferation of Clostridium difficile, Bacteroides fragilis, Escherichia fergusonii, Clostridium sporogenes;

[0027] (3) Preparing a drug for treating diarrhea and intestinal inflammation, where the diarrhea and intestinal inflammation are caused by the over - proliferation of Clostridium difficile, Bacteroides fragilis, Escherichia fergusonii, Clostridium sporogenes;

[0028] (4) Prepare a drug for treating gastric diseases caused by Helicobacter pylori.

[0029] The innovative mycin derivatives mentioned above are 3-demethyl-innovative mycin (DCM) and 3-methyl-innovative mycin (MCM), and their structures are shown in the following formula:

[0030]

[0031] The beneficial effects of the present invention are as follows.

[0032] 1. We determined the antibacterial activities of innovative mycin and its derivatives against a variety of bacteria, including the above-mentioned 3 digestive tract pathogenic bacteria with serious drug resistance problems. The results showed that innovative mycin had good inhibitory activities against Gram-negative Helicobacter pylori (Hp) and Escherichia fergusonii;

[0033] 2. Similarly, innovative mycin and its derivatives had certain inhibitory activities against Gram-positive Clostridioides difficile (CD);

[0034] 3. Based on the research results of the present invention, it can be seen that the C-3 methyl group is very important for the above-mentioned functional activities of innovative mycin, and it also lays a foundation for further structural optimization of innovative mycin to discover compounds with better activities;

[0035] The above results suggest that innovative mycin has great potential in the treatment of digestive tract bacterial infections. Description of the Drawings

[0036] Figure 1 The chemical structures of innovative mycin and its derivatives Detailed Embodiments

[0037] Materials and Reagents

[0038] Innovative mycin (CM), 3-demethyl-innovative mycin (DCM), and 3-methyl-innovative mycin (MCM) were prepared in our laboratory. The preparation method can be referred to CN202110601165.4.

[0039] 1. Test Strains

[0040] Helicobacter pylori (Helicobacter pylori ATCC 43504), Clostridioides difficile (Clostridioides difficile ATCC 700057), and Bacteroides fragilis (Bacteroides fragilis ATCC 25285) were all purchased from the American Type Culture Collection (ATCC);

[0041] Escherichia fergusonii CICC 24137 and Clostridium sporogenes CICC 10409 were purchased from China Center of Industrial Culture Collection (CICC).

[0042] The strains were activated on solid medium for three generations for activity measurement.

[0043] 2. Culture Medium:

[0044] Defibrinated sheep blood was purchased from Solarbio (Cat#TX0030).

[0045] Anticoagulated horse blood was purchased from Solarbio (Cat#KN0040).

[0046] Mueller-Hinton Blood Agar Plate: 38.0 g of Mueller-Hinton Agar (purchased from oxoid, Cat#CM0337) was weighed and dissolved in 1000 ml of distilled water, autoclaved at 121 °C for 15 minutes. 5% defibrinated sheep blood was added before pouring the plates.

[0047] Enriched Brucella Liquid Medium: 28.0 g of Brucella Broth (purchased from BD, Cat#211088) was weighed and dissolved in 900 ml of distilled water, hemin (5 mg / L) was added, vitamin K1 (0.01 ml / L) was added, and the volume was made up to 1 L after dissolution, then autoclaved at 121 °C for 15 minutes. 5% anticoagulated horse blood was added before use.

[0048] Enriched Brucella Solid Blood Agar Plate: 43.0 g of Brucella Agar (purchased from BD, Cat#211086) was weighed and dissolved in 900 ml of distilled water, hemin (5 mg / L) was added, vitamin K1 (0.01 ml / L) was added, and the volume was made up to 1 L after dissolution, then autoclaved at 121 °C for 15 minutes. 5% defibrinated sheep blood was added before pouring the plates.

[0049] 3. Compounds (Positive Control Drugs)

[0050] Clarithromycin was purchased from J&K Scientific (Cat#261815),

[0051] Levofloxacin was purchased from J&K Scientific (Cat#129219),

[0052] Metronidazole was purchased from sigma (Cat#M3761),

[0053] Chloramphenicol was purchased from J&K Scientific Ltd. (Cat#444411).

[0054] Example 1. Detection of the antibacterial activity of chuangxinmycin and its derivatives against bacteria

[0055] The activities of chuangxinmycin and its derivatives were detected using Helicobacter pylori (Helicobacter pylori ATCC 43504), Escherichia fergusonii (Escherichia fergusonii CICC 24137), Clostridioides difficile (Clostridioides difficile ATCC700057), Clostridium sporogenes (Clostridium sporogenes CICC 10409) and Bacteroides fragilis (Bacteroides fragilis ATCC 25285).

[0056] 1. Determination of the activity of chuangxinmycin and its derivatives against Helicobacter pylori

[0057] Referring to the Clinical and Laboratory Standards Institute (CLSI) guidelines for antimicrobial susceptibility testing of microaerophilic bacteria

[14] , clarithromycin and levofloxacin were used as positive control drugs, and the minimum inhibitory concentration (MIC) of the drugs against Helicobacter pylori was detected by the agar dilution method.

[0058] Agar dilution method:

[0059] (1) Prepare Mueller-Hinton blood plates containing the drugs (900 μl Mueller-Hinton blood agar medium + 90 μl sterile water + 10 μl drug dissolved in 100× DMSO, mix well and add to a 24-well plate), so that the final concentrations of the drugs to be tested in each well are 32, 16, 8, 4, 2, 1 μg / ml respectively, and the final concentrations of the positive drugs clarithromycin and levofloxacin are 2, 1, 0.5, 0.25, 0.125, 0.0625 μg / ml respectively.

[0060] (2) The strain was activated on Mueller-Hinton blood plates for three generations, and each generation was cultured at 37 °C under microaerophilic conditions (5% O2–10% CO2–85% N2) for 72 hours. The colonies from the third to fifth generations were scraped into sterile water, and the bacterial concentration was adjusted to 1×10 8 CFU / ml. 5 μl of the bacterial solution was spotted on the drug-containing plate (final concentration approximately 5×10 5 CFU / spot). After complete absorption, it was inverted and cultured at 37 °C under microaerophilic conditions for 72 hours, and then the results were read. Three biological replicates were performed.

[0061] 2. Determine the activities of creatmycin and its derivatives against Escherichia fergusonii, Bacteroides fragilis, and Clostridium sporogenes

[0062] Refer to the Clinical and Laboratory Standards Institute (CLSI) guidelines for antimicrobial susceptibility testing of anaerobic bacteria

[15] , using metronidazole and chloramphenicol as positive control drugs, and detecting the minimum inhibitory concentration (MIC) of the drugs against Escherichia fergusonii, Bacteroides fragilis, and Clostridium sporogenes by the broth microdilution method

[0063] (1) Broth microdilution method: Prepare reinforced Brucella broth medium. Take a sterile 96-well plate, add 100 μl of reinforced Brucella broth medium to the first well of each row as a blank control; add 100 μl of reinforced Brucella broth medium to wells 3 - 12; add 200 μl of reinforced Brucella broth medium and 1 μl of the test compound stock solution (final DMSO concentration < 0.5%, final drug concentration 64 μg / ml) to the second well. Perform serial dilutions in wells 2 - 11 so that the final drug concentrations in each well are 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml respectively. The 12th well contains no drug and only the bacterial suspension is added as a positive growth control

[0064] (2) The strains are activated on reinforced Brucella solid blood plates for three generations. Take a monoclonal colony and inoculate it into reinforced Brucella broth medium. Culture it anaerobically (10% CO2–10% H2–80% N2) at 37°C for 16 hours. Adjust the bacterial concentration to 1×10 7 CFU / ml. Add 10 μl of the bacterial suspension to wells 2 - 12, and at the same time set a solvent control well. After incubating each drug sensitivity plate anaerobically at 37°C for 48 hours, read the results. Conduct three biological replicates. Escherichia fergusonii is also cultured under aerobic conditions at 37°C for 16 hours

[0065] 3. Determine the activities of creatmycin and its derivatives against Clostridium difficile

[0066] Refer to the Clinical and Laboratory Standards Institute (CLSI) guidelines for antimicrobial susceptibility testing of anaerobic bacteria

[15] , using metronidazole as a positive control drug, and detecting the minimum inhibitory concentration (MIC) of the drugs against Clostridium difficile by the agar dilution method

[0067] (1) Solid agar dilution method: Prepare fortified Brucella solid blood agar plates containing the drug (900 μl fortified Brucella solid blood agar medium + 90 μl sterile water + 10 μl drug dissolved in 100× DMSO, mix well and add to a 24-well plate), so that the final concentrations of the drugs to be tested in each petri dish are 64, 32, 16, 8, 4 μg / ml, and the final concentration of the positive drug metronidazole is 2, 1, 0.5, 0.25, 0.125 μg / ml.

[0068] (2) The strain is activated for three generations on fortified Brucella solid blood agar plates, and each generation is cultured anaerobically (10% CO2–10% H2–80% N2) at 37 °C for 48 hours. Scrape the colonies into sterile water and adjust the bacterial concentration to 1×10 7 CFU / ml. Take 10 μl of the bacterial solution and spot it on the drug-containing plate (final concentration about 1×10 5 CFU / spot). After complete absorption, invert and culture anaerobically at 37 °C for 48 hours and then read the results. Conduct three biological replicates.

[0069] The minimum inhibitory concentrations (MICs) measured under aerobic, anaerobic, and microaerophilic conditions are shown in Table 1

[0070] Table 1. Activities of chuangxinmycin and its derivatives against some gastrointestinal pathogens

[0071]

[0072] LEV: Levofloxacin; CCR: Clarithromycin; Cm: Chloramphenicol; MTR: Metronidazole

[0073] The results show that:

[0074] (1) Chuangxinmycin (CM) has good inhibitory activity against Helicobacter pylori and Escherichia fergusonii (anaerobic culture), weak inhibitory activity against Clostridium difficile, and weak inhibitory activity against Escherichia fergusonii, Clostridium sporogenes, and Bacteroides fragilis in aerobic culture;

[0075] (2) 3-Desmethylchuangxinmycin (DCM) and 3-methylchuangxinmycin (MCM) have only slight inhibitory activity against the tested gastrointestinal bacteria, indicating that the C-3 methyl group is very important for activity. The above results suggest that chuangxinmycin has great potential in the treatment of intestinal bacterial infections.

[0076] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the protection scope of the present invention.

Claims

1. The following applications of creatmycin: (1) Preparation of a medicament for inhibiting Helicobacter pylori; (2) Preparation of a medicament for treating gastric diseases caused by Helicobacter pylori.

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