绿脓杆菌对鲎素耐受性特点及耐受性机制的研究
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摘要
【目的】为了探讨鲎素作为抗菌药物在临床使用中的安全性问题,通过鲎素连续增高浓度法对绿脓杆菌进行耐受性诱导,并对其耐受性机制进行初步研究,以期为鲎素的广泛应用提供理论依据。【方法】绿脓杆菌ATCC27853为试验菌株,通过连续增高浓度诱导法筛选抗药菌株,并通过抗药稳定性、交叉抗药性、抗药性代偿测定来探究其耐受性特点,通过对其胞外蛋白酶活性、生物膜形成、胞外多糖含量的变化来探讨其抗药性机制。【结果】通过连续增高鲎素浓度法对原始菌株进行30多代诱导后,绿脓杆菌ATCC27853对鲎素的MIC值逐渐增高,80多代时产生了明显抗药性。抗药菌株对丁胺卡那以及pexiganan、鲎素同源肽tachyplesin III、polyphemusin I均能产生不同程度的抗药性。在无药培养基中抗药菌株以更长的延滞期作为抗药性代偿,但在有药培养基中具有更短的延滞期和更大的生长速率。抗药菌株较原始菌株分泌的胞外蛋白酶活性增高,并能降低鲎素的抗菌活性。在同样条件下抗药菌株较原始菌株胞外多糖含量增高,更易形成生物膜。【结论】在长期选择压力下绿脓杆菌ATCC27853对鲎素能产生抗药性,其抗药性机制可能与生物膜形成、胞外蛋白酶失活鲎素有关。关于细菌对鲎素的抗药性机制,有待进一步研究。
[Objective] To explore the safety of tachyplesin I as an antibacterial drug for wide clinical application,we induced Pseudomonas aeruginosa resistance to tachyplesin I and studied the preliminary resistance mechanisms,which may provide theoretical basis for the widely application of tachyplesin-I.[Methods] First,we induced P.aeruginosa resistance to tachyplesin I using continuously increasing concentration selection pressure and monitored bacterial resistance.Second,we studied the stability,cross-resistance and cost of resistance of resistant strain.Last,we investigated the potential role of extracellular proteases,extracellular polysaccharide content and biofilm formation in the resistance mechanism.[Results] After more than 30 serial transfers in P.aeruginosa ATCC27853 under increasing concentrations of tachyplesin I selection,the MIC values for P.aeruginosa was gradually increased,whereas high resistance to tachyplesin I was produced until 80 serial transfers.P.aeruginosa ATCC27853 showed resistance to tachyplesin I under long-term,continuously increasing concentration selection pressure.Cross-resistances between tachyplesin I,amikacin and other antimicrobial peptides(pexiganan,tachyplesin III,and polyphemusin I) were observed in resistant mutant.Our resistant strain displayed a substantial cost of resistance mainly in the form of a much longer lag phase in the absence of tachyplesin I in P.aeruginosa,whereas in the presence of tachyplesin I,resistant strain had a shorter lag phase and greater growth rate.The resistant strain P.aeruginosa ATCC27853-88-2 exhibited increased levels of extracellular proteolytic activity and reduced the antimicrobial activity of tachyplesin I.Under the same conditions,extracellular polysaccharide content of the resistant strain was higher,more easily to form biofilm than the original strain.[Conclusion] We demonstrate that long-term continuous exposure to high concentrations of tachyplesin I can induce resistance in P.aeruginosa ATCC27853 and the potential involvement of extracellular protease and biofilm formation in mediating this resistance.
[Objective] To explore the safety of tachyplesin I as an antibacterial drug for wide clinical application,we induced Pseudomonas aeruginosa resistance to tachyplesin I and studied the preliminary resistance mechanisms,which may provide theoretical basis for the widely application of tachyplesin-I.[Methods] First,we induced P.aeruginosa resistance to tachyplesin I using continuously increasing concentration selection pressure and monitored bacterial resistance.Second,we studied the stability,cross-resistance and cost of resistance of resistant strain.Last,we investigated the potential role of extracellular proteases,extracellular polysaccharide content and biofilm formation in the resistance mechanism.[Results] After more than 30 serial transfers in P.aeruginosa ATCC27853 under increasing concentrations of tachyplesin I selection,the MIC values for P.aeruginosa was gradually increased,whereas high resistance to tachyplesin I was produced until 80 serial transfers.P.aeruginosa ATCC27853 showed resistance to tachyplesin I under long-term,continuously increasing concentration selection pressure.Cross-resistances between tachyplesin I,amikacin and other antimicrobial peptides(pexiganan,tachyplesin III,and polyphemusin I) were observed in resistant mutant.Our resistant strain displayed a substantial cost of resistance mainly in the form of a much longer lag phase in the absence of tachyplesin I in P.aeruginosa,whereas in the presence of tachyplesin I,resistant strain had a shorter lag phase and greater growth rate.The resistant strain P.aeruginosa ATCC27853-88-2 exhibited increased levels of extracellular proteolytic activity and reduced the antimicrobial activity of tachyplesin I.Under the same conditions,extracellular polysaccharide content of the resistant strain was higher,more easily to form biofilm than the original strain.[Conclusion] We demonstrate that long-term continuous exposure to high concentrations of tachyplesin I can induce resistance in P.aeruginosa ATCC27853 and the potential involvement of extracellular protease and biofilm formation in mediating this resistance.
引文
[1]Anaya-López JL,López-Meza JE,Ochoa-Zarzosa A.Bacterial resistance to cationic antimicrobial peptides.Critical Reviews in Microbiology,2013,39(2):180–195,doi:10.3109/1040841X.2012.699025.
[2]Cai Y,Chai D,Wang R,Liang BB,Bai N.Colistin resistance of Acinetobacter baumannii:clinical reports,mechanisms and antimicrobial strategies.Journal of Antimicrobial Chemotherapy,2012,67(7):1607–1615.
[3]Perron GG,Zasloff M,Bell G.Experimental evolution of resistance to an antimicrobial peptide.Proceedings of the Royal Society B:Biological Sciences,2006,273(1583):251–256.
[4]Peschel A,Sahl HG.The co-evolution of host cationic antimicrobial peptides and microbial resistance.Nature Reviews Microbiology,2006,4(7):529–536.
[5]Hong J,Guan WT,Jin G,Zhao HY,Jiang XH,Dai JG.Mechanism of tachyplesin I injury to bacterial membranes and intracellular enzymes,determined by laser confocal scanning microscopy and flow cytometry.Microbiological Research,2015,170:69–77.
[6]Dai JG,Xie HW,Jin G,Wang WG,Zhang Y,Guo Y.Preliminary study on high-level expression of tandem-arranged tachyplesin-encoding gene in Bacillus subtilis Wb800 and its antibacterial activity.Marine Biotechnology,2009,11(1):109–117.
[7]田颖.鲎素抗菌肽对海兰褐蛋鸡产蛋后期蛋品质及子宫Ca BP-D28k m RNA表达量影响的研究.吉林大学硕士学位论文,2016.
[8]Zhang Y,Feng X,Han D,Tie KY,Zhang QY,Yu X,Han WY.Therapeutic effect of genetic engineering tachyplesin I on Staphylococcus aureus infection in mice.Chinese Journal of Veterinary Science,2017,37(4):682–685,691.(in Chinese)张洋,冯新,韩东,铁鲲源,张楸杨,于曦,韩文瑜.基因工程鲎素对金黄色葡萄球菌小鼠感染模型治疗效果.中国兽医学报,2017,37(4):682–685,691.
[9]Gao Y,Zhao HL,Feng X,Zhai RD,Zhu S,Du CT,Sun CJ,Lei LC.Expression of recombinant human lysozyme-tachyplesin I(h LYZ-TP I)in Pichia pastoris and analysis of antibacterial activity.Biomedical and Environmental Sciences,2013,26(4):319–322.
[10]曹原.基因工程鲎素Tachyplesin I的工业化生产及其皮肤抗菌凝胶的研制.吉林大学硕士学位论文,2017.
[11]Hong J,Dai JG,Guan WT,Jin G,Huang ZL,Zhang LJ,Zhang Y.Tachyplesin I induce drug resistance in bacteria in vitro.Journal of Animal and Veterinary Advances,2012,11(7):939–945.
[12]Hong J,Hu JY,Ke F.Experimental induction of bacterial resistance to the antimicrobial peptide tachyplesin I and investigation of the resistance mechanisms.Antimicrobial Agents and Chemotherapy,2016,60(10):6067–6075.
[13]Li M,Lai YP,Villaruz AE,Cha DJ,Sturdevant DE,Otto M.Gram-positive three-component antimicrobial peptide-sensing system.Proceedings of the National Academy of Sciences of the United States of America,2007,104(22):9469–9474.
[14]Lai YP,Villaruz AE,Li M,Cha DJ,Sturdevant DE,Otto M.The human anionic antimicrobial peptide dermcidin induces proteolytic defence mechanisms in staphylococci.Molecular Microbiology,2007,63(2):497–506.
[15]Dean SN,Bishop BM,van Hoek ML.Susceptibility of Pseudomonas aeruginosa biofilm to alpha-helical peptides:D-enantiomer of LL-37.Frontiers in Microbiology,2011,2:128.
[16]曾祥萍.AL-1单用及联合用药对铜绿假单胞菌生物膜形成的抑制作用及其作用机理研究.暨南大学硕士学位论文,2011.
[17]Mehla J,Sood SK.Substantiation in Enterococcus faecalis of dose-dependent resistance and cross-resistance to pore-forming antimicrobial peptides by use of a polydiacetylene-based colorimetric assay.Applied and Environmental Microbiology,2011,77(3):786–793.
[18]Habets MGJL,Brockhurst MA.Therapeutic antimicrobial peptides may compromise natural immunity.Biology Letters,2012,8(3):416–418.
[19]Lofton H,Pr?nting M,Thulin E,Andersson DI.Mechanisms and fitness costs of resistance to antimicrobial peptides LL-37,CNY100HL and wheat germ histones.PLo S One,2013,8(7):e68875.
[20]Gruenheid S,Le Moual H.Resistance to antimicrobial peptides in Gram-negative bacteria.FEMS Microbiology Letters,2012,330(2):81–89.
[21]Nuri R,Shprung T,Shai Y.Defensive remodeling:How bacterial surface properties and biofilm formation promote resistance to antimicrobial peptides.Biochimica et Biophysica Acta(BBA)-Biomembranes,2015,1848(11):3089–3100.
[22]Sieprawska-Lupa M,Mydel P,Krawczyk K,Wójcik K,Puklo M,Lupa B,Suder P,Silberring J,Reed M,Pohl J,Shafer W,Mc Aleese F,Foster T,Travis J,Potempa J.Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus-derived proteinases.Antimicrobial Agents and Chemotherapy,2004,48(12):4673–4679.
[23]Fuente-Nú?ez CDL,Korolik V,Bains M,Nguyen U,Breidenstein EBM,Horsman S,Lewenza S,Burrows L,Hancock REW.Inhibition of bacterial biofilm formation and swarming motility by a small synthetic cationic peptide.Antimicrobial Agents and Chemotherapy,2012,56(5):2696–2704.
[2]Cai Y,Chai D,Wang R,Liang BB,Bai N.Colistin resistance of Acinetobacter baumannii:clinical reports,mechanisms and antimicrobial strategies.Journal of Antimicrobial Chemotherapy,2012,67(7):1607–1615.
[3]Perron GG,Zasloff M,Bell G.Experimental evolution of resistance to an antimicrobial peptide.Proceedings of the Royal Society B:Biological Sciences,2006,273(1583):251–256.
[4]Peschel A,Sahl HG.The co-evolution of host cationic antimicrobial peptides and microbial resistance.Nature Reviews Microbiology,2006,4(7):529–536.
[5]Hong J,Guan WT,Jin G,Zhao HY,Jiang XH,Dai JG.Mechanism of tachyplesin I injury to bacterial membranes and intracellular enzymes,determined by laser confocal scanning microscopy and flow cytometry.Microbiological Research,2015,170:69–77.
[6]Dai JG,Xie HW,Jin G,Wang WG,Zhang Y,Guo Y.Preliminary study on high-level expression of tandem-arranged tachyplesin-encoding gene in Bacillus subtilis Wb800 and its antibacterial activity.Marine Biotechnology,2009,11(1):109–117.
[7]田颖.鲎素抗菌肽对海兰褐蛋鸡产蛋后期蛋品质及子宫Ca BP-D28k m RNA表达量影响的研究.吉林大学硕士学位论文,2016.
[8]Zhang Y,Feng X,Han D,Tie KY,Zhang QY,Yu X,Han WY.Therapeutic effect of genetic engineering tachyplesin I on Staphylococcus aureus infection in mice.Chinese Journal of Veterinary Science,2017,37(4):682–685,691.(in Chinese)张洋,冯新,韩东,铁鲲源,张楸杨,于曦,韩文瑜.基因工程鲎素对金黄色葡萄球菌小鼠感染模型治疗效果.中国兽医学报,2017,37(4):682–685,691.
[9]Gao Y,Zhao HL,Feng X,Zhai RD,Zhu S,Du CT,Sun CJ,Lei LC.Expression of recombinant human lysozyme-tachyplesin I(h LYZ-TP I)in Pichia pastoris and analysis of antibacterial activity.Biomedical and Environmental Sciences,2013,26(4):319–322.
[10]曹原.基因工程鲎素Tachyplesin I的工业化生产及其皮肤抗菌凝胶的研制.吉林大学硕士学位论文,2017.
[11]Hong J,Dai JG,Guan WT,Jin G,Huang ZL,Zhang LJ,Zhang Y.Tachyplesin I induce drug resistance in bacteria in vitro.Journal of Animal and Veterinary Advances,2012,11(7):939–945.
[12]Hong J,Hu JY,Ke F.Experimental induction of bacterial resistance to the antimicrobial peptide tachyplesin I and investigation of the resistance mechanisms.Antimicrobial Agents and Chemotherapy,2016,60(10):6067–6075.
[13]Li M,Lai YP,Villaruz AE,Cha DJ,Sturdevant DE,Otto M.Gram-positive three-component antimicrobial peptide-sensing system.Proceedings of the National Academy of Sciences of the United States of America,2007,104(22):9469–9474.
[14]Lai YP,Villaruz AE,Li M,Cha DJ,Sturdevant DE,Otto M.The human anionic antimicrobial peptide dermcidin induces proteolytic defence mechanisms in staphylococci.Molecular Microbiology,2007,63(2):497–506.
[15]Dean SN,Bishop BM,van Hoek ML.Susceptibility of Pseudomonas aeruginosa biofilm to alpha-helical peptides:D-enantiomer of LL-37.Frontiers in Microbiology,2011,2:128.
[16]曾祥萍.AL-1单用及联合用药对铜绿假单胞菌生物膜形成的抑制作用及其作用机理研究.暨南大学硕士学位论文,2011.
[17]Mehla J,Sood SK.Substantiation in Enterococcus faecalis of dose-dependent resistance and cross-resistance to pore-forming antimicrobial peptides by use of a polydiacetylene-based colorimetric assay.Applied and Environmental Microbiology,2011,77(3):786–793.
[18]Habets MGJL,Brockhurst MA.Therapeutic antimicrobial peptides may compromise natural immunity.Biology Letters,2012,8(3):416–418.
[19]Lofton H,Pr?nting M,Thulin E,Andersson DI.Mechanisms and fitness costs of resistance to antimicrobial peptides LL-37,CNY100HL and wheat germ histones.PLo S One,2013,8(7):e68875.
[20]Gruenheid S,Le Moual H.Resistance to antimicrobial peptides in Gram-negative bacteria.FEMS Microbiology Letters,2012,330(2):81–89.
[21]Nuri R,Shprung T,Shai Y.Defensive remodeling:How bacterial surface properties and biofilm formation promote resistance to antimicrobial peptides.Biochimica et Biophysica Acta(BBA)-Biomembranes,2015,1848(11):3089–3100.
[22]Sieprawska-Lupa M,Mydel P,Krawczyk K,Wójcik K,Puklo M,Lupa B,Suder P,Silberring J,Reed M,Pohl J,Shafer W,Mc Aleese F,Foster T,Travis J,Potempa J.Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus-derived proteinases.Antimicrobial Agents and Chemotherapy,2004,48(12):4673–4679.
[23]Fuente-Nú?ez CDL,Korolik V,Bains M,Nguyen U,Breidenstein EBM,Horsman S,Lewenza S,Burrows L,Hancock REW.Inhibition of bacterial biofilm formation and swarming motility by a small synthetic cationic peptide.Antimicrobial Agents and Chemotherapy,2012,56(5):2696–2704.
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