Study On Antibacterial Activity Of The Trp-containing Antimicrobial Peptides On Multiple-drug Resistant Klebsiella Pneumoniae MRKP1203

Klebsiella pneumoniae is resistant to a variety of clinic commons antibiotics,and treatment of related diseases caused by it becomes a problem in clinical practice.It is known as the three most difficult to treat pathogens in the 21 st century intensive care unit with Acinetobacter baumannii and Pseudomonas aeruginosa.Therefore,we urgently need to find out novel drugs to treat the infection caused by multi-drug resistant Klebsiella pneumoniae.antimicrobial peptides have the characteristics of small molecular weight,strong thermal stability and low immunogenicity,and have strong bactericidal activity and are not easy to produce drug resistance.The unique bactericidal mechanism of antimicrobial peptides has made it a hot topic in recent years.Temporin-1CEb is a natural antimicrobial peptide isolated and purified from frog skin secretion of Rana chensinensis in our laboratory,and exhibits certain antibacterial activity.In previous study,a series of the Trp-containing antimicrobial peptides was designed and synthesized by substitution of lysine,leucine with tryptophan residues based on Temporin-1CEb.These Trp-containing antimicrobial peptides showed high bactericidal activity against Gram-negative and Gram-positive bacteria such as drug-resistant Pseudomonas aeruginosa and drug-resistant Staphylococcus epidermidis,and were able to disrupt bacterial cell membranes.In this paper,the bactericidal activity,mechanism of action of the Trp-containing antimicrobial peptides on the clinically isolated multidrug-resistant K.pneumoniae(MRKP1203)were studied.Furthermore,the therapeutic effect of the Trp-containing antimicrobial peptides on lung infection induced by MRKP1203 in mice was studied.The Trp-containing antimicrobial peptides showed good antibacterial activity against MRKP1203 strain with the MIC value was 12.5-50 ?M.Among them,I4 W and I1WL5 W showed strong antibacterial activity,and the MIC value was 12.5 ?M.The results of bactericidal kinetics showed that the bactericidal activity of the Trp-containing antimicrobial peptides on MRKP1203 was in a concentration-dependent and time-denpendent manner.The bacteria treated by I4 W,L12W and I1WL5 W were all died at 16×MIC,120 min,16×MIC,180 min and 8×MIC,180 min,respectively.The Post-Antibiotic Effects Experiment(PAE)investigated the time for bacteria to recover logarithmic growth phase after moving peptides.The results showed that PAE of MRKP1203 treated with the Trp-containing antimicrobial peptides was 2-5 h.The effects of of the Trp-containing antimicrobial peptides on themorphology of MRKP1203 cells were observed by scanning electron microscopy.The results showed that a partial cell surface disruption and leakage of the intracellular contents of MRKP1203 cells after treatment with 2×MIC of I4 W,L12W and I1WL5 W,respectively.Subsequently,the effects of the Trp-containing antimicrobial peptides on inner and outer membrane permeability of MRKP1203 strain were studied.The cell membrane depolarization result showed that the addition of I4 W,L12W and I1WL5 W was only 1 minute,and the fluorescence value of DiSC3-5 was significantly increased.When concentration of the three peptides increased to 4×MIC,the fluorescence value of DiSC3-5 increased by 68%,59.5 %,and 72.4% respectively.Triton X-100,a positive control,increased fluorescence value of DiSC3-5 by 90%,while the negative control vancomycin increased by only 27%.This results suggest that the Trp-containing antimicrobial peptides can induce cell membrane depolarization of MRKP1203 cells and increase the permeability of cell membrane.The tryptophan residue in the Trp-containing antimicrobial peptides shows specific fluorescence absorption at a wavelength of 340 nm.When side chain of tryptophan inserts into the cell membrane,the fluorescence spectrum of peptide is changes.The experimental results showed that the absorption peak of the tryptophan residue has different degrees of blue shift from 340 nm to 308 nm after the treatment with the three Trp-containing antimicrobial peptides on the MRKP1203 bacteria.I1WL5 W,I4W and L12 W made the absorption peak of the tryptophan residue blue shift 32 nm,30 nm and 26 nm,respectively.This result suggests that the Trp-containing antimicrobial peptides can disrupt the stability of the cell membrane by inserting the tryptophan residue into membrane phospholipid bilayer of MRKP1203 bacteria,thereby kill the bacteria.The effect of the Trp-containing antimicrobial peptides on the permeability of the artificially mimicked-cell membrane Klebsiella pneumoniae also was studied.The result showed that I4 W,L12W,and I1WL5 W increased the permeability of the bacterial cell membrane with a 100% release of calcein at concentrations of 6.25 ?M,12.5?M,and 6.25 ?M,respectively.Further,we used the hydrophobic fluorescent probe NPN to detect the effect of the Trp-containing antimicrobial peptides on the outer membrane permeability of MRKP1203.The results showed that the three antimicrobial peptides could increase the permeability of MRKP1203 outer membrane in a concentration-dependent manner.Next,we observed the effect of the Trp-containing antimicrobial peptides on the membrane integrity of MRKP1203 cells by using LIVE/DEAD Bacterial Activity Detection Kit and performed by a laser confocal microscopy.The results showed that the live bacteria stained by the green fluorescent stain SYTO 9 significantly reduced in MRKP1203 cells treated with the peptides and dead bacteria stained by the red-fluorescent stain PI increased,compared with the control group.And as the concentration of the peptides increased,the number of dead bacteria also increased.I4 W and I1WL5 W at a concentration of 25 ?M killedmore than 90% of bacteria This result suggests that the Trp-containing antimicrobial peptides can disrupt the integrity of the MRKP1203 cell membrane.The mouse lung MRKP1203 infection model was established.After a treatment with the Trp-containing antimicrobial peptide I1WL5 W,the number of CFU in the lung tissue and blood of mice,the levels of inflammatory factors TNF-? and IL-6,and the histochemistry of lung tissue were detected.The results showed that after a treatment with I1WL5 W at 2,4 and8 mg/kg for 3 days,the number of CFU of blood decreased by 10-25.39% in lung-infected mice compared with the model group;TNF-? and IL-6 decreased by 17.09-23.27% and7.42-10.35%,respectively.After 6-days,the number of CFU of blood decreased by42.72-84.98% compared with the model group;TNF-? and IL-6 decreased by 31.42-48.94%and 19.71-41.17%,respectively.After 9-days of treatment,the number of CFU of blood decreased by 54.58-92.24% compared with the model group;TNF-? and IL-6 decreased by33.52-89.71% and 51.58-87.45%,respectively.The lung histochemistry(H&E staining)showed that I1WL5 W restored the reticulated loose structure of MRKP1203-infected mouse lung tissue,and the alveolar cavity became clean and free of exudate,compared with the control group,suggesting that the Trp-containing antimicrobial peptide I1WL5 W can inhibit the expression of inflammatory factors and effectively treat lung infection in mice.