CN111265568A - Application of phlegm-heat clearing or combination of phlegm-heat clearing and antibiotics in inhibition of multidrug-resistant pseudomonas aeruginosa - Google Patents
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Abstract
The invention relates to an application of phlegm heat clearing in inhibiting multiple drug-resistant pseudomonas aeruginosa; also relates to the application of the combination of the phlegm-heat-clearing drug and the antibiotic in inhibiting the multidrug-resistant pseudomonas aeruginosa; in particular to a method for inhibiting flagella movement of multiple drug-resistant pseudomonas aeruginosa by phlegm heat clearing, reducing surface hydrophobicity of the multiple drug-resistant pseudomonas aeruginosa, inhibiting an efflux pump of the multiple drug-resistant pseudomonas aeruginosa, inhibiting a quorum sensing system of the multiple drug-resistant pseudomonas aeruginosa and reducing virulence of the multiple drug-resistant pseudomonas aeruginosa. The invention has great value for the antibiosis of multiple drug-resistant pseudomonas aeruginosa and also has great value for public health service.
Description
Technical Field
The invention relates to application of phlegm-heat clearing or combination of phlegm-heat clearing and antibiotics in inhibition of multidrug-resistant pseudomonas aeruginosa.
Background
Pseudomonas aeruginosa can be resistant to 3 and above medicaments in 5 types of antibacterial medicaments, namely multiple-resistant strains, including cephalosporins (such as ceftazidime or cefapizone), carbapenems (such as imipenem), β -lactamase inhibitors (such as cefoperazone/sulbactam), fluoroquinalones (such as ciprofloxacin) and aminoglycosides (such as amikacin), if the antibacterial medicaments are resistant to all the medicaments, including cefapizone palate, ceftazidime, imipenem, meropenem, piperacillin/tazobactam, ciprofloxacin and levofloxacin, the medicaments are named as drug-resistant Pseudomonas aeruginosa (PDR-PA), Pseudomonas aeruginosa is suitable for growing in a humid environment, and oxygen humidification bottles, bath heads, medical instruments and the like cause pollution, and is often the main cause of nosocomial infection in hospitals.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides an application of phlegm-heat clearing or the combination of the phlegm-heat clearing and antibiotics in inhibiting multiple drug-resistant pseudomonas aeruginosa.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, the use of phlegm-heat clearing for inhibiting multidrug-resistant pseudomonas aeruginosa is provided.
Preferably, the phlegm fever reducing agent inhibits flagellar motility of the multidrug-resistant pseudomonas aeruginosa, reduces surface hydrophobicity of the multidrug-resistant pseudomonas aeruginosa, inhibits the multidrug-resistant pseudomonas aeruginosa efflux pump, inhibits the multidrug-resistant pseudomonas aeruginosa quorum sensing system, and reduces virulence of the multidrug-resistant pseudomonas aeruginosa.
In a second aspect, the use of a combination of phlegm-heat clearing and an antibiotic for inhibiting multidrug-resistant pseudomonas aeruginosa is provided.
Preferably, the antibiotic is one of tyloxapol and ceftazidime.
Preferably, the combination of the phlegm fever reducing agent and the antibiotic inhibits flagellar movement of the multidrug-resistant pseudomonas aeruginosa, reduces surface hydrophobicity of the multidrug-resistant pseudomonas aeruginosa, inhibits efflux pumps of the multidrug-resistant pseudomonas aeruginosa, inhibits the quorum sensing system of the multidrug-resistant pseudomonas aeruginosa, and reduces virulence of the multidrug-resistant pseudomonas aeruginosa.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the combination of the phlegm-heat clearing agent or the phlegm-heat clearing agent and the antibiotic has obvious inhibition effect on the multidrug-resistant pseudomonas aeruginosa; the drug-resistant pseudomonas aeruginosa protein has the effects of inhibiting flagella movement of multiple-drug-resistant pseudomonas aeruginosa, reducing the surface hydrophobicity of the multiple-drug-resistant pseudomonas aeruginosa, inhibiting an efflux pump of the multiple-drug-resistant pseudomonas aeruginosa, inhibiting a quorum sensing system of the multiple-drug-resistant pseudomonas aeruginosa, reducing the toxicity of the multiple-drug-resistant pseudomonas aeruginosa, and has great value for the antibiosis of the multiple-drug-resistant pseudomonas aeruginosa and great value for public health service.
Drawings
FIG. 1 shows the diameter of a colony of Pseudomonas aeruginosa after the sputum heat clearing culture in example 1 of the present invention;
FIG. 2 is a graph showing the adhesion rate of Pseudomonas aeruginosa group after the phlegm-heat clearing action in example 2 of the present invention;
FIG. 3 is a graph showing the adhesion rate of multiple drug-resistant P.aeruginosa groups after the phlegm-heat clearing action in example 2 of the present invention;
FIG. 4 shows the diameters of the zones of inhibition of each antibiotic in example 3 of the present invention (LB medium);
FIG. 5 shows the diameters of the inhibition zones of the antibiotics (LB medium containing PA β N) in example 3 of the present invention;
FIG. 6 shows the diameter of the zone of inhibition of each antibiotic in example 3 of the present invention (LB medium with phlegm-heat-clearing);
FIG. 7 is a graph showing the inhibition of phlegm-heat in 1508032 and 1708116 efflux pumps in example 3 of the present invention;
FIG. 8 shows that the combination of phlegm-heat-clearing medicine and ceftazidime, cefoperazone and sulbactam in example 3 of the present invention can increase the curative effect of the medicine;
FIG. 9 shows that the combination of phlegm-heat-clearing drug with ceftazidime, cefoperazone and sulbactam in example 3 of the present invention increases the drug efficacy;
FIG. 10 shows the effect of phlegm-heat-clearing on bacterial growth and the inhibition of PA01 pyocin release in example 4 of the present invention;
FIG. 11 shows the inhibition of the expression of las (Panel A), the expression of rhIR (Panel B) and the expression of mvf (Panel C) in the PA01 quorum sensing system by phlegm-heat-clearing in example 4 of the present invention;
FIG. 12 shows the inhibition of las expression (panel A), rhIR expression (panel B) and mvf expression (panel C) in the PA01 quorum sensing system by the five components contained in TanreReqing according to example 4 of the present invention;
FIG. 13 shows the contribution of five components contained in TanreReqing in example 4 of the present invention to inhibition of PA01 quorum sensing system;
FIG. 14 shows the survival rates of different concentrations of phlegm-heat-clearing nematode in example 5 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Example 1
The influence of phlegm-heat clearing on the flagellum movement trend of PA and MDR-PA is observed:
preparing a solid culture medium plate with the swimming motility detection function, dipping bacterial liquid on the culture medium by using an inoculating needle, culturing for 12-14h at 30 ℃, and observing the movement track formed on the culture medium by the movement of the bacteria depending on flagella.
As shown in FIG. 1, there was no significant change in the PA group, while the colony diameters of the fifth and sixth passages after subculture of the expectorant-PA group were significantly reduced compared with the control group, indicating that the expectorant-PA group was able to inhibit the flagellar motility of MDR-PA.
Example 2
Influence of pseudomonas aeruginosa on surface hydrophobicity due to phlegm-heat clearing:
phlegm-heat clearing action the LB culture medium for pseudomonas aeruginosa and the phlegm-heat clearing agent are mixed according to a ratio of 1: 1 dilution log phase of PA and MDR-PA, incubation in a shaker (100rpm) at 37 ℃ for 5 hours, collecting 4ml samples every 30 minutes, centrifuging at 4700rpm for 5 minutes, discarding the supernatant, washing twice with PBS, and resuspending the cells in 3ml of PBS.
Before measurement, 1ml of PA bacterial liquid, 1ml of MDR-PA bacterial liquid, 1ml of phlegm-heat-clearing effect PA bacterial liquid and 1ml of phlegm-heat-clearing effect MDR-PA bacterial liquid are taken from the cetane OD value, and the absorbance at 540nm is measured.
And adding 0.8mL of n-hexadecane into the washed cell suspension after the measured cetane OD value, swirling for 30s, separating the mixture into a water phase and an n-hexadecane phase, taking 1mL of n-hexadecane phase PA bacterial liquid and 1mL of n-hexadecane phase MDR-PA bacterial liquid, 1mL of n-hexadecane phase phlegm heat clearing effect PA bacterial liquid, and 1mL of n-hexadecane phase phlegm heat clearing effect MDR-PA bacterial liquid in a cuvette, and measuring the absorbance of the mixture at 540 nm.
The adhesion ratio Percentage adherence [100 (OD540 pre-hexane-OD 540 post-hexane) ]/OD540 pre-hexane) was calculated by using the formula
The results are shown in fig. 2-3, the adhesion rate of the MDR-PA group is reduced overall after the phlegm-heat clearing action, especially the adhesion rate after 270min is obviously reduced compared with the MDR-PA control group, which indicates that the surface hydrophobicity of the MDR-PA group can be reduced after the phlegm-heat clearing action, the cohesive force is weakened, the local colonization is not facilitated, and the pathogenic force is weakened, which may be due to the phlegm-heat clearing blocking of cell wall adhesion mediators or the damage of cell wall surface components.
Example 3
Whether the phlegm-heat clearing has an inhibiting effect on the pseudomonas aeruginosa efflux pump is researched:
preparing an LB plate added with PA β N (the final concentration is 50 mug/mL), detecting the diameters of the bacteriostatic rings of clinical MDR-PA1508032 on azlocillin, meropenem, aztreonam, ceftazidime, cefoperazone and Shupu deep drug sensitive paper sheets by using a paper diffusion method (KB method), taking the LB plate without PA β N as a control, preparing an LB group containing phlegm-heat clearing (the final concentration is 3mg/mL) and comparing the LB group with the LB group, and observing whether the traditional Chinese medicine has an inhibition effect on an efflux pump of pseudomonas aeruginosa.
The results are shown in fig. 4-6, and the LB plate control group can show that after the bacteria are cultured by the phlegm-heat-clearing, the inhibition zones of five antibiotics except for meropenem are slightly increased, which indicates that the combined use of the phlegm-heat-clearing and the antibiotics can reduce the dosage of the antibiotics.
Two clinically-separated multidrug-resistant pseudomonas aeruginosa (serial numbers 1508032 and 1708116) with high expression of an efflux pump are selected, firstly, the diameters of the bacteriostatic rings of two MDR-PA strains on the phlegm-heat clearing, ceftazidime, cefoperazone, sulpu, aztreonam, meropenem and azlocillin drug sensitive paper sheets are detected by a classical paper sheet diffusion method, and an LB (Luria-Beta) flat plate without the phlegm-heat clearing injection is used as a control. The results show that TRQ has inhibitory effect on both efflux pumps 1508032, 1708116, and the results are shown in fig. 7.
Continuously culturing two clinically-separated multidrug-resistant pseudomonas aeruginosa (serial numbers 1508032 and 1708116) with sub-MIC concentration TRQ for six generations, and then respectively detecting the diameters of the bacteriostatic rings of the two MDR-PA continuously-cultured six generations by using a classical paper diffusion method on the phlegmapyrexia, ceftazidime, cefoperazone, Shupu-shen, aztreonam, meropenem and azlocillin drug sensitive paper, and taking an LB plate without phlegmatic pyrexia injection as a control. The result shows that the PA1508032 continuously cultured for six generations by the phlegm heat clearing can increase the inhibition zone of the ceftazidime and the aztreonam by 2cm and the inhibition zone of the cefoperazone and the sulbactam by 1 cm; through continuous culture of the phlegm-heat-clearing agent for six generations, the PA1708116 can increase the inhibition zone of ceftazidime by 3cm, the inhibition zone of sulbactam by 2cm and the inhibition zones of cefoperazone and meropenem by 1cm, thus prompting that the phlegm-heat-clearing agent can be combined with the ceftazidime, the cefoperazone and the sulbactam to improve the curative effect of the medicine. The results are shown in FIGS. 8 to 9.
The effect of TRQ on the expression of efflux pump-related genes (mex-AB, mex-EF and mex-XY) was observed by RT-PCR. The result shows that compared with the control group, the phlegm-heat clearing MDR-PA can reduce the expression quantity of the bacterial efflux pump gene, and the effect is consistent with that of the antibiotic cefoperazone. When the combination of the phlegm-heat clearing drug and the antibiotic cefoperazone is used, the expression quantity of mex-AB, mex-EF and mex-XY genes of the bacterial efflux pumps can be reduced; when the combination of the phlegm-heat-clearing and the antibiotic Shupu is used, the expression quantity of mex-EF and mex-XY genes of the bacterial efflux pump can be reduced, the indication that the combination of the phlegm-heat-clearing and the cefoperazone and the Shupu is used is provided, the drug resistance of bacteria is weakened by changing the expression level of the genes of the bacterial efflux pump, and the clinical use dosage of the antibiotic is reduced, so that the bacteriostatic action is achieved. The differences in efflux pump gene expression levels between treatment groups were statistically significant (P <0.05) and the results are shown in the following table.
TABLE 1
Example 4
The effect of TRQ on the PAO1 quorum sensing system was observed using a different virulence factor fluorescent marker P.a:
construction of separate population intervention systems the bacteria should be labeled P.a, as shown in the following table.
Table 2 Strains and plasmids used in this study.
The influence of TRQ and the components thereof on the quorum sensing system and the virulence of the PAO1 is reflected by observing the change of the TRQ and the five components thereof on the fluorescence value of the PAO1 bacteria within 24 h.
The results are shown in fig. 10, which indicates that TRQ has little effect on bacterial growth within 24h, but TRQ at concentrations of 1/4-1/16 can significantly inhibit the release of PAO1 pyocin.
As shown in FIG. 11, TRQ at concentrations of 1/4-1/8 significantly inhibited las expression (panel A), rhlR expression (panel B), and mvf expression (panel C) in the PAO1 quorum sensing system.
As shown in FIG. 12, the five components contained in TRQ have different inhibiting effects on las expression (panel A), rhlR expression (panel B) and mvf expression (panel C) in PAO1 quorum sensing system, wherein the scutellaria baicalensis effect is particularly remarkable. The pseudomonas aeruginosa is not clinically pathogenic bacteria of the main condition of the respiratory system, the quorum sensing system plays an extremely important role in the clinical pathogenic process of the pseudomonas aeruginosa, and the inhibition of TRQ on the PAO1 quorum sensing system also indicates the rationality of the application of the TRQ in the treatment process of the respiratory system diseases.
As shown in fig. 13, the five components have different contribution degrees to inhibition of the PAO1 quorum sensing system, but the inhibition effects of the single components do not reach the full formula effect, which indicates the rationality of the full formula compatibility of the TRQ.
Example 5
The RNA-Seq technology is utilized to research PAO1 in a stationary phase and a propagation phase respectively so as to determine the influence of TRQ on a PAO1 quorum sensing system and virulence related genes. The results indicate that TRQ can down-regulate or up-regulate various links of amino acid biosynthesis and metabolism, carbon compound catabolism, intermediate product metabolism, and energy metabolism of PAO1 bacteria.
Classification analysis of regulatory genes revealed that TRQ affected 514 and 367 genes, respectively, in the stationary phase and the reproductive phase of the bacteria, which were not 193 and 161 genes, respectively, associated with quorum sensing, again suggesting that TRQ limited inhibitory effects on quorum sensing.
The effect of pyrexia on the virulence of pseudomonas aeruginosa bacteria was demonstrated from an in vivo experimental perspective by observing changes in survival rates of N2 nematodes infected with pyrexia intervention P.a. As shown in FIG. 14, the results indicate that the life cycle of normal N2 nematodes was 2-3 weeks, whereas nematodes infected with PAO1 all died within 10 days. The survival rate of the infected nematodes after TRQ treatment is remarkably improved, the 1/8 concentration group has better treatment effect than other concentration groups 5 days before statistics, and the statistical analysis also has significant difference compared with other concentration groups. The TRQ has treatment effect on the nematodes infected with the PAO1, and the TRQ effect is optimal at 1/8 concentration.
Example 6
The effect of combined medication of phlegm-heat clearing and four antibiotics (tylan, ceftazidime, temporax and ciprofloxacin) on inhibiting PA and MDR-PA is researched:
through the in-vitro combined anti-pseudomonas aeruginosa activity detection of the phlegm-heat clearing and the four antibiotics, the application of a chessboard method in the in-vitro combined antibacterial drug screening research is utilized to research the inhibiting effect of the combined application of the phlegm-heat clearing and the four antibiotics on PA and MDR-PA. The activity of the phlegm-heat clearing medicament and the four medicaments on the pseudomonas aeruginosa is observed in a combined manner, the antibacterial combined index is calculated, and the FIC index is not more than 0.5 as a synergistic effect according to the FIC index interpretation standard; 0.5 FIC < 1 is additive effect; 1< FIC < 2 is irrelevant; FIC >2 is antagonistic.
The results are shown in the following table, with no significant change in the PA group, and FICI values for the MDR-PA group: the tylene and the ceftazidime have synergistic action. The test result is consistent with the previous MIC value test result, namely the combination of the phlegm heat clearing drug and the antibiotic has the inhibition effect on the multidrug-resistant pseudomonas aeruginosa.
TABLE 3 inhibition of MDR-PA by combined use of phlegm-heat-clearing and four antibiotics
In conclusion, the combination of the phlegm-heat clearing drug or the phlegm-heat clearing drug and the ceftazidime has an obvious inhibiting effect on multiple drug-resistant pseudomonas aeruginosa; the drug-resistant pseudomonas aeruginosa protein has the effects of inhibiting flagella movement of multiple-drug-resistant pseudomonas aeruginosa, reducing the surface hydrophobicity of the multiple-drug-resistant pseudomonas aeruginosa, inhibiting an efflux pump of the multiple-drug-resistant pseudomonas aeruginosa, inhibiting a quorum sensing system of the multiple-drug-resistant pseudomonas aeruginosa, reducing the toxicity of the multiple-drug-resistant pseudomonas aeruginosa, and has great value for the antibiosis of the multiple-drug-resistant pseudomonas aeruginosa and great value for public health service.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (5)
1. The application of the phlegm heat clearing medicine in inhibiting multiple drug-resistant pseudomonas aeruginosa.
2. The use of the phlegm-heat-clearing agent in inhibiting multiple-drug-resistant pseudomonas aeruginosa according to claim 1, wherein the phlegm-heat-clearing agent inhibits flagellar motility of the multiple-drug-resistant pseudomonas aeruginosa, reduces surface hydrophobicity of the multiple-drug-resistant pseudomonas aeruginosa, inhibits efflux pumps of the multiple-drug-resistant pseudomonas aeruginosa, inhibits quorum sensing system of the multiple-drug-resistant pseudomonas aeruginosa, and reduces virulence of the multiple-drug-resistant pseudomonas aeruginosa.
3. The application of the combination of the phlegm-heat-clearing medicament and the antibiotic in inhibiting the multidrug-resistant pseudomonas aeruginosa.
4. The use of the combination of phlegm fever reducing and antibiotic in inhibiting multidrug-resistant pseudomonas aeruginosa according to claim 3, wherein the antibiotic is one of tyloxapol and ceftazidime.
5. The use of the combination of phlegm heat clearing and antibiotics of claim 3, wherein the combination of phlegm heat clearing and antibiotics inhibits flagellar motility of the multidrug-resistant pseudomonas aeruginosa, reduces surface hydrophobicity of the multidrug-resistant pseudomonas aeruginosa, inhibits efflux pump of the multidrug-resistant pseudomonas aeruginosa, inhibits quorum sensing system of the multidrug-resistant pseudomonas aeruginosa, and reduces virulence of the multidrug-resistant pseudomonas aeruginosa.
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