| Pseudomonas aeruginosa is opportunistic pathogen that easily infects host cells with impaired mucosal barriers,and it is prone to inflammation of the livestock and human lung diseases with high mortality.P.aeruginosa which get multi-drug resistance easily is widely found in water,air and feces.It is one of the multi-drug resistant bacteria currently published worldwide.P.aeruginosa can obtain drug resistance through a variety of ways,including the expression of a variety of antibiotic-modified enzymes,antibiotic efflux pumps,chromosomal mutations,low membrane permeability and the acquisition of other organism resistance genes.Drug resistance is easily produced during the antibiotic treatment of P.aeruginosa infection during poultry farming.At the same time,P.aeruginosa is easy to form biofilm in the environment,as a model strain for studying biofilms,which brings greater threat to the its control.The study consists of three parts:the molecular modification of the natural antibacterial peptide Sakacin P and its antibacterial mechanism against P.aeruginosa,the influencing factors of P.aeruginosa biofilm formation and the control of biofilm,the study of cross-resistance and collateral sensitivity of P.aeruginosa.The main experimental results are summarized as follows:1.Molecular design of antimicrobial peptide Sakacin P and its antibacterial mechanism against.aeruginosaThe α-helix sequence in Sakacin P was truncated and the amino acid at a specific position was replaced with arginine and lysine to increase the hydrophobicity the amount of positive charge.After molecular designed,three antibacterial peptides were obtained,including Sakacin P16-R6,Sakacin P16-R8 and Sakacin P16-R6.After modification,the antibacterial activity of the antibacterial peptide is obviously improved and the inhibition spectrum is broadened.The results of bactericidal kinetics of antimicrobial peptides showed that the bactericidal effect was mainly concentration-dependent.Sakacin P16-R8 has the highest activity in combination with various factors.The stability and safety of Sakacin P16-R8 were investigated and it was found that salt ions would have an effect on their activity but would not be inactivated.In a mixture of mouse macrophages and P.aeruginosa,Sakacin P16-R8 can kill P.aeruginosa and has a cell viability of more than 75%at 4×MIC.The results showed that Sakacin 16-R8 has high security.By measuring the content of nucleic acid and protein in the P.aeruginosa 96846 supernatant treated by Sakacin P16-R8 and flow cytometry experiments,the results showed that this antimicrobial peptide was inhibited P.aeruginosa by destroying the cell membrane.2.Influence of environmental factors on the P.aeruginosa biofilm formation and the control of biofilmThe effects of environmental factors on the P.aeruginosa biofilm formation were studied under laboratory conditions,including culture time,temperature,medium type,sodium chloride content,glucose content and pH.And the concentration of the P.aeruginosa suspension under the corresponding conditions was determined.The amount of biofilm formation was measured by crystal violet staining.The results showed that P.aeruginosa have Strong biofilm formation ability at 25℃-30℃,36 h,TSB medium,1%-2%sodium chloride content,0.2%-0.4%glucose content and pH 6-7,respectively.The P.aeruginosa biofilm formation can be effectively inhibited at low temperature,high or low pH and high glucose concentration.In addition,biofilm formation ability is not completely proportional to the growth of P.aeruginosa.The P.aeruginosa biofilm was treated with 84 disinfectant(concentration of 84 disinfectant conversion by effective chlorine),alcohol and detergent,and the damage of common disinfectant to P.aeruginosa biofilm was observed.The results showed that removal effect was not satisfactory,among which 84 disinfectant had the best removal effect.The effective chlorine content in 256 μg/ml could clear about 20%of the biofilm.At the same time,the control of the of P.aeruginosa biofilm by three antimicrobial peptides obtained in the first part of the molecular transformation were studied,and it was found that the removal effect was better than that of the chemical disinfectant,but it was still not completely clear.At 4 × MIC concentration,about 50%of the biofilm can be removed.3.The rule of cross-resistance and collateral sensitivity of P.aeruginosaThe drug resistance of P.aeruginosa was determined by drug-sensitive plate method.Ten antibiotic-resistant were used for the single antibiotics induction of resistance aginst P.aeruginosa ATCC 27853 and Pseudomonas aeruginosa 02818 with no resisitance.The sensitivity of the strain to other antibiotics was determined after the induction.Both strains were found to be resistant to induced drugs in a relatively short period of time.In addition to peptide antibiotics,resistance to other antibiotics increased by 32-128 times.The MIC of peptide antibiotics are no longer improved after 2-fold increase.The drug-resisitance artificial induction experiment of Sakacin P16-R8 against P.aeruginosa ATCC 27853 showed that the evolution trend of drug resistance was similar to other peptide drugs,and it did not cause a large and rapid increase in drug resistance.Drug-resistant strains are susceptible get cross-resistance to the same type of antibiotics,and collateral sensitivity often occurs between different types of antibiotics.However,the specific results of cross-resistance or collateral sensitivity between the two strains are different.For the drug-resistant strains,the antibiotics with enhanced sensitivity were used for the second induction for 7 days,and then the antibiotic resistance test was carried out with the original antibiotics.It was found that 11 of the 20 resistant strains were sensitive.And the other 9 strains showed no significant changes,and the MIC value did not increase. |
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