阅读说明：本技术 西塞卡那用于抑制革兰阳性细菌生物活性的用途 (Use of cisacana for inhibiting biological activity of gram-positive bacteria ) 是由 余治健 王占文 熊焱鹏 邓启文 于 2020-08-14 设计创作，主要内容包括：本发明公开了西塞卡那用于抑制革兰阳性细菌生物活性的用途,所述西塞卡那用于抑制革兰阳性细菌生长的活性。本发明的技术方案公开了西塞卡那的一个新的用途,其可以抑制多种革兰阳性细菌的浮游菌生长的生物活性,尤其是西塞卡那对粪肠球菌具有抑菌和清除生物被膜的活性的作用。在有粪肠球菌生物被膜感染的环境中,西塞卡那可兼具抑菌活性以及清除生物膜的活性的作用。而且,西塞卡那与其他抗生素联用与单药相比更具抗菌活性,能明显降低单药抗生素的用量,并降低其耐药选择压力。(The invention discloses application of sisalan in inhibiting biological activity of gram-positive bacteria, and the sisalan is used for inhibiting growth activity of the gram-positive bacteria. The technical scheme of the invention discloses a new application of the sisalan, which can inhibit the biological activity of planktonic bacteria growth of various gram-positive bacteria, in particular the effect of the sisalan on inhibiting bacteria and removing biofilm activity of enterococcus faecalis. In the environment infected by enterococcus faecalis biofilm, the sitecana has the functions of bacteriostasis activity and biomembrane eliminating activity. Moreover, the combination of the sitecana and other antibiotics has better antibacterial activity compared with single drug, can obviously reduce the dosage of the single drug antibiotics and reduce the drug-resistant selection pressure.)

1. Use of cisacana for inhibiting the biological activity of gram-positive bacteria, characterized in that: the cisacana is used for inhibiting the biological activity of the growth of gram-positive bacteria.

2. Use of cisaccania according to claim 1 for inhibiting the biological activity of gram-positive bacteria, characterized in that: the MIC of the sisalana for inhibiting gram-positive bacteria is not more than 12.5 mu g/mL.

3. Use of celecoxib for combating enterococcus faecalis biofilm activity, characterized in that: the celecoxib has activity of clearing enterococcus faecalis biofilm at a concentration of 8 × MIC.

4. The application of the sisalan in preparing the medicine for resisting gram-positive bacterial infection is characterized in that: the drug against gram-positive bacterial infection includes celecoxib.

5. Use of cisocara according to claim 4 in the manufacture of a medicament against gram-positive bacterial infection, characterized in that: the drug for resisting gram-positive bacterial infection comprises at least one of ampicillin and linezolid.

6. Use of cisacana in the preparation of a coating for the surface of a medical device, characterized in that: the coating for the surface of the medical device comprises cisaca.

7. Use of cisocara according to claim 6 in the preparation of a coating for the surface of a medical device, wherein: in the coating on the surface of the medical device, the concentration of the sisalan is not less than 6.25 mu g/mL.

8. A coating for a medical device surface, characterized by: including cisacana.

9. The application of the sisalan in preparing the disinfectant for resisting gram-positive bacteria is characterized in that: the gram-positive bacteria-resistant disinfectant comprises sisalan.

10. A disinfectant against gram-positive bacteria, characterized by: including cisacana.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of cisacana in inhibiting bioactivity of gram-positive bacteria.

Background

Enterococcus faecalis is a common nosocomial infection, a common bacterium of the human gastrointestinal tract, can cause a variety of nosocomial infections, is also a recognized opportunistic pathogen, and can form biofilms, highly correlated with urinary infections, wound infections, abdominal and pelvic infections, and bloodstream infections. Enterococcus faecalis is the most common one of enterococcus and accounts for 85% -90% of human enterococcus infections. According to previous reports, the biomembrane is considered to be the main cause of nosocomial infection, and accounts for more than 80% of microbial infection, and most of enterococcus faecalis can form the biomembrane. Enterococci in biofilms are more resistant to antibacterial agents than planktonic bacteria. It has now been found that enterococci are increasingly resistant to oxazolidinones, glycopeptides, aminoglycosides, β -lactams and macrolides. In clinical treatment, the unreasonable use of antibiotics improves the sensitivity of enterococcus faecalis to most commonly used antibiotics, and the continuous emergence of drug-resistant strains such as tetracycline-resistant enterococcus faecalis and doxycycline-insensitive enterococcus faecalis a threat in the field of medical care, so that an alternative treatment method for antibacterial drug resistance is urgently needed.

Ciacacet (Cinacalciet) is a new class of compounds used to treat hyperthyroidism, also a calcimimetic, with potential advantages for the treatment of secondary hyperparathyroidism by decreasing the synthesis and secretion of parathyroid hormone (PTH) through increasing the sensitivity of the calcium-sensitive receptor of the parathyroid gland (CaR) to extracellular calcium ions. At present, no report about the antibacterial activity and the effect on the biological membrane of the medicine is found.

Disclosure of Invention

The research shows that the sisalan has better bacteriostatic and bactericidal effects on various gram-positive bacteria such as staphylococcus aureus, enterococcus faecalis, streptococcus agalactiae and the like, can remarkably inhibit the formation of a gram-positive bacterial biofilm, particularly can remarkably inhibit the formation of an enterococcus faecalis biofilm, and can be combined with other antibiotics to effectively remove the formed enterococcus faecalis biofilm.

In contrast, the technical scheme adopted by the invention is as follows:

use of celecoxib for inhibiting the biological activity of gram-positive bacteria for inhibiting the biological activity of the growth of gram-positive bacteria. Wherein, the gram-positive bacteria are staphylococcus aureus, enterococcus faecalis, streptococcus agalactiae and the like.

Further, the sisalana is used for inhibiting the biological activity of planktonic growth of enterococcus faecalis.

Cisacan hydrochloride is a "calcium mimetic". The main indications are the reduction of secondary hyperparathyroidism and the reduction of parathyroid hormone levels in patients with end-stage renal disease. In chronic kidney disease, patients are often high in phosphorus, variable in calcium, and elevated in parathyroid hormone levels (> 300 pg/mL), in which case cisecan acts to lower serum parathyroid hormone levels, preventing bone destruction, this intervention restores calcium levels within the target range defined by the renal disease outcome quality initiative (K-DOQI) for disease management, and reduces the chance of secondary hyperparathyroidism requiring parathyroidectomy.

The research of the invention preliminarily discovers that the sisalan has good effect of inhibiting the bioactivity of gram-positive bacteria, can remove biofilm, and has antibacterial activity and a low MIC (minimal inhibitory concentration) value in various antibiotics related to bacterial infection caused by various gram-positive bacteria (such as staphylococcus aureus, enterococcus faecalis, streptococcus agalactiae and the like).

In particular, the siteca has good bacteriostatic activity on enterococcus faecalis, can effectively inhibit the growth of the enterococcus faecalis, and can effectively remove mature biofilm of the enterococcus faecalis, which provides a new direction for treating related infection caused by the enterococcus faecalis.

As a further improvement of the invention, the MIC of the sisalan inhibiting gram-positive bacteria is not more than 12.5 mu g/mL. For staphylococcus aureus, the MIC values of the sisalan are all less than or equal to 12.5 mu g/mL; for enterococcus faecalis, the MIC values of the siseca are all less than or equal to 12.5 mu g/mL; for Streptococcus agalactiae, the MIC values of the sisalana are less than or equal to 6.25 mu g/mL.

As a further improvement of the invention, the concentration of the sisalan is not less than 6.25 mug/mL. Further, the concentration of the sisalana is not less than 12.5 mu g/mL.

The invention discloses an application of cetacaran for resisting the activity of enterococcus faecalis biofilms, and the cetacaran has the activity of clearing the enterococcus faecalis biofilms at the concentration of 8 × MIC.

The invention discloses application of sisalan in preparing a medicament for resisting gram-positive bacterial infection, wherein the medicament for resisting gram-positive bacterial infection comprises the sisalan. Wherein, the gram-positive bacteria are staphylococcus aureus, enterococcus faecalis, streptococcus agalactiae and the like. Preferably, the concentration of the sisalan is not less than 6.25 μ g/mL. Preferably, the concentration of the sisalan is not less than 12.5 μ g/mL.

As a further improvement of the invention, the medicament against gram-positive bacterial infection comprises other medicaments, such as other antibiotics. Further, the drug for inhibiting gram-positive bacterial infection comprises at least one of ampicillin and linezolid. The combination of the sisekalan, the ampicillin and the linezolid has better effect of resisting gram-positive bacteria, particularly enterococcus faecalis infection.

The main clinical treatments for enterococcus faecalis infection are to select gram-positive cocci-resistant drugs such as ampicillin and linezolid. The research shows that although the single-medicine antibacterial effect of the sisekalan is not as good as that of the ampicillin and the linezolid, compared with the single-medicine, the combination of the sisekalan and the ampicillin or the linezolid has better antibacterial activity, the dosage of the single-medicine antibiotic can be obviously reduced, and the drug-resistant selective pressure of the antibiotic can be reduced. A treatment regimen using celecoxib in combination with traditional antibiotics may be a more rational way of combating celecoxib.

As a further improvement of the invention, in the medicine for resisting gram-positive bacterial infection, the concentration of ampicillin and/or linezolid is not less than 0.5 mu g/mL.

As a further improvement of the invention, the sisalan is used as a related antibiotic for various bacterial infections caused by gram-positive bacteria (Staphylococcus aureus, enterococcus faecalis, Streptococcus agalactiae, etc.).

The invention discloses application of sisalan in preparing a coating for the surface of a medical device, wherein the coating for the surface of the medical device comprises the sisalan, and the sisalan is used for inhibiting biofilm formation and adhesion of gram-positive bacteria.

As a further improvement of the invention, in the coating for the surface of the medical device, the concentration of the sisalane is not less than 6.25 mu g/mL.

The invention discloses a coating for the surface of a medical device, which comprises sisecane. Preferably, the concentration of the sisalan is not less than 6.25 μ g/mL. Further preferably, the concentration of the sisalan is not less than 12.5 μ g/mL.

The invention discloses application of sisalan in preparing a disinfectant for resisting gram-positive bacteria, which comprises the sisalan.

As a further improvement of the invention, in the disinfectant against gram-positive bacteria, the concentration of the sisalana is not less than 6.25 mug/mL. Further, the concentration of the sisalana is not less than 12.5 mu g/mL.

The invention discloses a disinfectant for resisting gram-positive bacteria, which comprises sisalan. Preferably, the concentration of the sisalan is not less than 6.25 μ g/mL. Further preferably, the concentration of the sisalan is not less than 12.5 μ g/mL.

Compared with the prior art, the invention has the beneficial effects that:

the technical scheme of the invention discloses a new application of the siteca, which is used for inhibiting the biological activity of enterococcus faecalis, and the siteca has the functions of inhibiting bacteria and removing the activity of a biofilm on the enterococcus faecalis. In the environment with enterococcus faecalis biofilm infection, the sitecana has the functions of bacteriostasis activity and biofilm clearing activity, so that the adverse prognosis problems of repeated chronic infection, delayed recovery and the like caused by the enterococcus faecalis biofilm infection can be reduced. In addition, the combination of the sitecana and other antibiotics has better antibacterial activity compared with single medicine, can obviously reduce the dosage of the single medicine antibiotics and reduce the selection pressure of drug resistance.

Drawings

FIG. 1 is a graph showing the results of the inhibition of various enterococcus faecalis by different concentrations of Sesacena (A5) according to the present invention; wherein a) is 16C102 enterococcus faecalis, b) is 16C152 enterococcus faecalis, C) is 16C170 enterococcus faecalis, and d) is 16C201 enterococcus faecalis.

FIG. 2 is a graph showing the analysis of the results of the bacteriostatic activity of the combination of Xisaikana (A5) and ampicillin and linezolid of the present invention against various enterococcus faecalis; wherein a) is 16C102 enterococcus faecalis, b) is 16C152 enterococcus faecalis, C) is 16C170 enterococcus faecalis, and d) is 16C201 enterococcus faecalis.

FIG. 3 is a graph showing the results of experiments on the activity of Sesacena of the present invention in removing mature biofilm of enterococcus faecalis; wherein, a) is against enterococcus faecalis 16C3, 16C9, 16C25, 16C 109; b) is against enterococcus faecalis 16C124, 16C137, 16C139, 16C 170.PIs less than 0.05, and has significant difference.

Detailed Description

Preferred embodiments of the present invention are described in further detail below.

Enterococcus faecalis is a common bacterium in the human gastrointestinal tract and can cause a variety of nosocomial infections. They not only have various inherent antibiotic resistance, but also are capable of acquiring mutated and/or new resistance genes. In clinical treatment, the unreasonable use of antibiotics improves the sensitivity of enterococcus faecalis to most commonly used antibiotics, and the continuous emergence of drug-resistant strains such as tetracycline-resistant enterococcus faecalis and doxycycline-insensitive enterococcus faecalis a threat in the field of medical care, so that an alternative treatment method for antibacterial drug resistance is urgently needed.

The invention finds a new application of the sisalan in inhibiting the biological activity of enterococcus faecalis, has the functions of inhibiting the bacteria and removing the activity of a biological membrane, and comprises the following experimental processes:

1.1 strains

The test strain was selected from enterococcus faecalis which was clinically isolated from my hospital and was biofilm-positive. After the strain is recovered, the strain is subjected to primary identification and analysis by using a Phoenix 100 automatic analysis identifier of American BD company, and is identified again by using a flight mass spectrometer (IVD MALDI Biotyper, Germany) after being inoculated and cultured for two generations. The drug sensitive quality control strain is enterococcus faecalis ATCC 29213. Staphylococcus aureus, enterococcus faecalis and Streptococcus agalactiae clinical strains were all collected from the clinical microbiology laboratory of Shenzhen Hospital, Kyowa university of science and technology.

1.2 growth Curve determination experiment

Enterococcus faecalis strain 1:200 was inoculated into 4mL of TSB medium, cultured at 37 ℃ and at 220rpm for 12 h (once activated). The activated culture of 12 h of bacterial liquid 1:200 was inoculated into 4mL of TSB medium, cultured at 37 ℃ and at 220rpm for 12 h. The growth curve of the bacteria was examined using a finland Bioscreen fully automated growth curve analyzer: adding bacterial liquid containing different concentrations of Sesacena into a special pore plate (provided with 3 multiple pores) in an amount of 200 mul/pore, placing the pore plate into a growth curve analyzer, measuring OD600 once every 1h, continuously measuring OD600 absorbance for 16h, and drawing a growth curve of each strain according to the measured value.

1.3 Combined bacteriostasis experiment of sitecana and clinical common antibiotic medicine

Enterococcus faecalis strain was inoculated at 1:200 into 4mL of TSB medium, cultured at 37 ℃ at 220rpm for 12 h (once activated). The activated culture of 12 h of bacterial liquid 1:200 was inoculated into 4mL of TSB medium, cultured at 37 ℃ and at 220rpm for 12 h. The growth curve of the bacteria was examined using a finland Bioscreen fully automated growth curve analyzer: adding bacterial liquid containing ampicillin and linezolid with corresponding concentrations into a specially-made 96-well plate (3 multiple wells) in an amount of 200 mul/well, placing the plate into a growth curve analyzer, measuring OD600 once every 1h, continuously measuring OD600 absorbance for 24h, and drawing a growth curve of each strain according to the measured value.

1.4 biofilm removal experiments

Inoculating enterococcus faecalis strain into tryptone soybean culture medium (TSB culture medium) at 37 ℃, shaking the strain overnight, diluting the strain with TSBG culture medium (TSB contains 0.25% glucose) at 1:200, adding the diluted strain into a 96-well culture plate (Costar 3599 plate, 200 mu L/well), setting 3 wells, standing and culturing at 37 ℃ for 24 hours (forming a mature biofilm), discarding the culture supernatant, washing with sterile 0.9% NaCl for 3 times, adding fresh TSBG culture medium containing corresponding concentration of Sesacena, continuing to culture for 48 hours, finally discarding the culture supernatant, washing for 3 times, staining with 1% crystal violet for 20min, slowly washing with dddH2O to remove unbound crystal violet, and determining the quantity of the biofilm by measuring the absorbance at 570nm (OD 570 nm) with a microplate reader.

1.5 statistical analysis

Statistical analysis was performed using SPSS software (version 19.0) and GraphPad Prism software (version 5.0). Results are expressed as mean ± standard deviation. Multiple comparisons were performed using one-way analysis of variance (ANOVA) and post-HOC-Dunnett test.PLess than 0.05 is statistically significant.

Through the above experiments, the experimental results after statistical analysis are as follows:

(1) bacteriostatic action of Xisaikana on enterococcus faecalis

To verify the bacteriostatic activity of the sisalana on enterococcus faecalis, the MIC value of 60 clinical strains of enterococcus faecalis was tested, and the MIC value was determined₅₀/MIC₉₀The concentrations were 12.5/12.5. mu.g/mL, respectively. The effect of different concentrations of sisalan on the growth curve of the experimental strains was further investigated, and the results are shown in FIG. 1, where the MIC values were all 12.5. mu.g/mL. The result shows that the concentration of the S-type S-.

50 clinically isolated staphylococcus aureus, 60 feces and 60 agalactia streptococcus strains were collected from the clinical microbial community of synechiae Shenzhen hospital, university of science and technology in Huazhong, and the cemacana MIC value was determined: the Sisaciana MIC values of 60 staphylococcus aureus are all less than or equal to 12.5 mu g/mL; the Sissekana MIC values of 60 enterococcus faecalis are respectively less than or equal to 12.5 mu g/mL, and the Sissekana MIC values of 60 streptococcus agalactiae are respectively less than or equal to 6.25 mu g/mL.

(2) Sisacana enhances the bacteriostatic activity of ampicillin and linezolid on enterococcus faecalis

In order to investigate whether the antibiotic of the combination of the Xisaikana and the ampicillin and the linezolid for treating the enterococcus faecalis can enhance the bacteriostatic action of the single drug. The growth conditions of enterococcus faecalis under the combined action of the sisomica, the ampicillin and the linezolid are observed, the result is shown in figure 2, it can be seen that 1/2 XMIC of the sisomica has a lower antibacterial effect than 1/4 XMIC of the ampicillin and the linezolid, and MIC of the sisomica is 2 mug/mL; compared with single medicine, after the drug is combined with 1/2 times MIC cetaccaine, the antibacterial activity of the ampicillin and the linezolid is obviously improved.

(3) The obtained Xisaikana has effect in removing enterococcus faecalis biofilm

In order to investigate whether the sisalan has a clearing effect on mature biofilms of enterococcus faecalis, 8 clinical separated strains with positive biofilm are screened, a biofilm model is constructed, the biofilm clearing activity of the sisalan is verified by a microplate crystal violet staining method, and the result is shown in figure 3, so that the 8 XMIC of the sisalan can effectively clear the mature biofilms of the enterococcus faecalis, and the MICs of the strains used in the experiment are all 12.5 mu g/mL.

The experiment shows that 12.5 mu g/mL of the sitecana has good bacteriostatic activity on the enterococcus faecalis and can effectively remove mature biofilm of the enterococcus faecalis. The characteristics of wide distribution tissue, good absorption effect and the like are expected to become another alternative for antibiotic therapy. Although the single drug of the sisalan has a lower antibacterial effect than that of the ampicillin and the linezolid, compared with the single drug, the combination of the sisalan and the ampicillin or the linezolid has better antibacterial activity, so that the dosage of the single drug antibiotic can be obviously reduced, and the drug resistance selection pressure of the antibiotic can be reduced. Thus, a treatment regimen using cisaccaine in combination with traditional antibiotics may be a more rational way for cisaccaine to resist infection.

The embodiment of the invention also discloses application of the sisalan in preparing a medicament for resisting gram-positive bacterial infection, wherein the medicament for resisting gram-positive bacterial infection comprises the sisalan. Wherein, the gram-positive bacteria are staphylococcus aureus, enterococcus faecalis, streptococcus agalactiae and the like. Preferably, the concentration of the sisalan is not less than 6.25 μ g/mL. Preferably, the concentration of the sisalan is not less than 12.5 μ g/mL.

Preferably, the drug for inhibiting gram-positive bacterial infection comprises at least one of ampicillin and linezolid. The concentration of the ampicillin and/or linezolid is not less than 0.5 mu g/mL. The combination of the sisekalan, the ampicillin and the linezolid has better effect of resisting gram-positive bacteria, particularly enterococcus faecalis infection.

The embodiment of the invention discloses application of sisalan in preparing a coating for the surface of a medical device, wherein the coating for the surface of the medical device comprises the sisalan, and the sisalan is used for inhibiting the formation of a biofilm of gram-positive bacteria and the adhesion of the gram-positive bacteria. Preferably, in the coating for the surface of a medical device, the concentration of the sisalan is not less than 6.25 μ g/mL.

The embodiment of the invention discloses a coating for the surface of a medical device, which comprises sisalan. Preferably, the concentration of the sisalan is not less than 6.25 μ g/mL. Further preferably, the concentration of the sisalan is not less than 12.5 μ g/mL.

The embodiment of the invention discloses application of sisalan in preparing a disinfectant for resisting gram-positive bacteria, wherein the disinfectant for resisting gram-positive bacteria comprises the sisalan. Preferably, the concentration of the sisalan in the disinfectant against gram-positive bacteria is not less than 6.25 μ g/mL. Further preferably, the concentration of the sisalan is not less than 12.5 μ g/mL.

The embodiment of the invention discloses a disinfectant for resisting gram-positive bacteria, which comprises sisalan. Preferably, the concentration of the sisalan is not less than 6.25 μ g/mL. Further preferably, the concentration of the sisalan is not less than 12.5 μ g/mL.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.