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Application of epsilon-polylysine or hydrochloride thereof in preparation of medicine for inhibiting cronobacter spp or intervening cronobacter spp biofilm

A technology of Cronobacter and polylysine, applied in the field of biomedicine, achieving good application prospects, no toxic and side effects, and widening the application field

Pending Publication Date: 2021-12-10
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report of ε-polylysine or its hydrochloride inhibiting Cronobacter and its biofilm

Method used

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  • Application of epsilon-polylysine or hydrochloride thereof in preparation of medicine for inhibiting cronobacter spp or intervening cronobacter spp biofilm
  • Application of epsilon-polylysine or hydrochloride thereof in preparation of medicine for inhibiting cronobacter spp or intervening cronobacter spp biofilm
  • Application of epsilon-polylysine or hydrochloride thereof in preparation of medicine for inhibiting cronobacter spp or intervening cronobacter spp biofilm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Example 1: ε-polylysine detects the minimum inhibitory concentration (minimuminhibitory concentration, MIC) and minimum bactericidal concentration (minimum bactericidal concentration, MBC) of Cronobacter planktonic bacteria

[0037] The inhibitory effect of ε-polylysine on Cronobacter was determined by broth microdilution method.

[0038] ε-polylysine was dissolved in sterile water, sterilized by filtration through a 0.22 μm filter membrane, and prepared as a 64 mg / mL stock solution. Streak inoculate the strains in Table 1 frozen at -20°C on TSA medium, and culture them statically at 37°C for 16-18 hours. Then pick a single colony and inoculate it in TSB medium, shake it at 37°C and cultivate it to the logarithmic growth phase, collect the bacterial pellet by centrifugation and resuspend the bacterial cell in fresh TSB medium, adjust OD600nm=0.5, and the total number of bacteria obtained is about 10 8 CFU / mL bacterial suspension, again using TSB medium to dilute the ba...

Embodiment 2

[0042] Example 2: Effect of ε-polylysine on the Microscopic Morphology of Cronobacter

[0043] Cronobacter sakazakii ATCC29544 was used as the experimental strain, and the preparation of the bacterial solution was the same as in Example 1. The bacterial solution (OD600nm=0.5) added with different concentrations of ε-polylysine (0, MIC and MBC) was placed in a 37°C incubator and cultured for 2 h, and the bacterial precipitate was collected by centrifugation, and the bacterial cell was washed 3 times with PBS. The cells were resuspended in 2.5% glutaraldehyde solution and fixed at room temperature for 1-2 hours, then transferred to 4°C for overnight fixation, and then washed three times with phosphate buffer (PH=7.2) for 10 minutes each time, and then used different concentrations ( 25%, 50%, 70%, 80%, 90% and 95%) in ethanol solution for dehydration step by step, 15min each time, remove 95% ethanol solution, and treat the sample twice with absolute ethanol, 15min each time. Fi...

Embodiment 3

[0045] Example 3: Effect of sub-inhibitory concentration ε-polylysine on Cronobacter biofilm formation

[0046] Inoculate XZCRO43, a strong biofilm-producing strain frozen at -20°C, into LB broth, culture on a shaking table at 37°C until logarithmic growth phase, adjust OD600nm=0.5, and obtain a total of about 10 bacteria 8 CFU / mL bacterial solution, set aside. Use LB broth to dilute ε-polylysine to equal times, add 100 μL each to a 96-well culture plate, and then add an equal volume of LB broth to dilute to 1.0×10 6 CFU / mL bacterial suspension. At this time, the final concentration of ε-polylysine was 1 / 4MIC, 1 / 8MIC, 1 / 16MIC, 1 / 32MIC, 1 / 64MIC. The same volume of LB broth was added to the bacterial growth control group. Three repetitions were made for each concentration. After mixing, culture the plate at 37°C for 24 hours, and then carry out crystal violet staining: remove the supernatant, wash the plate with sterile distilled water 3 times to remove planktonic bacteria. ...

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Abstract

The invention discloses application of epsilon-polylysine or hydrochloride thereof in preparation of a medicine for inhibiting cronobacter spp or intervening a cronobacter spp biofilm. Experimental results show that epsilon-polylysine has the effects of inhibiting formation of the cronobacter spp and the biofilm thereof and removing the mature biofilm of the cronobacter spp. According to the invention, the bacteriostatic activity of the epsilon-polylysine to the cronobacter spp is determined by adopting a standard trace broth dilution method, the MIC value is 0.125-0.25 mg / mL, and the MBC value is 0.125-0.50 mg / mL; and by a crystal violet staining method and scanning electron microscope observation, the invention finds that the epsilon-polylysine with sub-inhibitory concentration has a remarkable inhibition effect on formation of the cronobacter spp biofilm, and the epsilon-polylysine with high concentration can destroy the mature biofilm. The invention provides experimental basis for clinical application of the epsilon-polylysine in the aspect of preparation of drugs for inhibiting the cronobacter spp or intervening the biofilm of the cronobacter spp, and the epsilon-polylysine has good application prospects and huge potential value.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the application of ε-polylysine or its hydrochloride in the preparation of drugs for inhibiting Cronobacter or intervening Cronobacter biofilm. Background technique [0002] Cronobacter spp. is a facultative anaerobic, peri-flagellated, motile Gram-negative bacillus that can cause infection in people of all ages, especially in immunocompromised neonates and infants The threat is greater, capable of causing severe necrotizing enterocolitis, sepsis, and meningitis. Although its infection rate is low, the mortality rate of infants and young children infected with this bacteria is as high as 40% to 80%. In addition, even if the Cronobacter infectious disease is cured, it will be accompanied by severe neurological sequelae, including brain abscess, neurodevelopmental delay, hydrocephalus, and quadriplegia. Cronobacter contamination sources are very extensive, and the bacteria ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/785A61K31/198A61P31/04A01N37/46A01P1/00
CPCA61K31/785A61K31/198A61P31/04A01N37/46
Inventor 李远宏向月卲继红姜华周丽任香梅
Owner XUZHOU MEDICAL UNIV
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