Discovery Research Of Synergists For Polymyxin B Against Resistant Pseudomonas Aeruginosa DK2

Pseudomonas aeruginosa(P.aeruginosa)is a widespread opportunistic pathogen which could cause multiple hospital-acquired infections,mainly in immunocompromised and cystic fibrosis(CF).It mostly established chronic infection that accompanied with patients until the end of life,which caused a significant proportion of morbidity and mortality.Antibiotic treatment is one of the most essential methods for bacterial infections,due to the inherent and acquired resistance,the infections caused by P.aeruginosa are often difficult to treat.Polymyxin B(PB)is considered to be the last line of defense against life-threatening infections caused by Gram-negative bacteria.However,with the excessive abuse of agents,the CF clinical isolated strain P.aeruginosa DK2 has developed severely resistant to PB,MIC=512 ?g/mL.One of the most effective strategies to overcome the susceptibility decrease is combined with synergist to improve the activity of PB.In order to find good synergists of PB,we systematically screened 1348 approved drugs synergized with PB to against DK2,fortunately,we found fluoroquinolones and niclosamide showed significant synergistic activity that could restore the susceptibility of PB in DK2.In summary,we carried out two part of the work,the one is the development of fluoroquinolones in combination with PB against resistant DK2,and the other is design,synthesis,and synergistic activity of benzoylanilides in combination with PB against resistant DK2.In the first part,we determined the synergistic activity of 19 fluoroquinolones combined with PB in checkerboard assay,and 12 fluoroquinolones showed effective synergistic activity.Especially,combined with gemifloxacin,sparfloxacin,enrofloxacin,ciprofloxacin,sarafloxacin and moxifloxacin,PB exhibited high therapeutic potential with an obvious reduction in MIC below the clinical susceptible breakpoint 2 ?g/mL.Encouragingly,gemifloxacin achieved a 4096-fold reduction in MIC of PB from 512 ?g/mL to 0.125?g/mL.In addition,the synergistic activity was also observed in the combination of gemifloxacin and colistin(CL),the MIC of CL was reduced from 1024 ?g/mL to 4 ?g/mL.In order to investigate whether the synergy is conserved beyond the clinical isolated DK2,the time-killing kinetic assay of gemifloxacin was conducted on two wild-type P.aeruginosa strains PAO1 and MPAO1.The results showed that the combination of gemifloxacin and PB showed the weak synergistic effect in PAO1 within 8 h,and no synergy in MPAO1.Finally,the outer membrane permeability assay showed that gemifloxacin could increase the cell membranes permeability in DK2,which support the optimal synergistic activity in DK2 and could partially explain the mechanism of synergy.These results provide a reference for the clinical application of fluoroquinolones combined with PB in the treatment of resistant DK2 infections.In the second part,we found a class of benzoylanilides exhibit synergistic effect with PB to restore the susceptibility of DK2 without affect the growth.As the synergist,niclosamide still has disadvantages in weak synergistic activity,poor solubility,high cytotoxicity,and unknown in vivo activity.Therefore,we used niclosamide as the lead compound,designed and synthesized 38 compounds,and 26 of them showed effecetive synergistic activity with PB,19 compounds could reduce the MIC of PB below the resistant breakpoint 8 ?g/mL,7 compounds could reduced the MIC of PB below the susceptible breakpoint 2 ?g/mL Especially,the compound B4 achieved the highest synergistic effect with PB,reduced the MIC of PB from 512?g/mL to 1 ?g/mL in DK2,which had 1-fold increase of niclosamide.The synergistic effect of the compound B4 combined with CL was also improved,the MIC of CL was reduced from 1024 ?g/mL to 1?g/mL,which had 8-fold increase of niclosamide.The time-killing kinetic assay showed that the optimal compound B4 also behaved a significant synergistic activity on the sensitive strain P.aeruginosa PAO1,in 24h,the combination of compound B4 and PB declined the bacterial cell count by 5.7 log CFU/mL which showed significant synergistic activity in both logarithmic growth phase and the plateau phase of bacteria,this performance was not found in niclosamide.In addition,we first report the C.elegans treatment assay applied in evaluating the combination synergistic effect in vivo.The results of C.elegans liquid treatment assay showed that the combination of compound B4 and PB achieved extremely significant therapeutic effect on PAO1-infected C.elegans,and restore the survival curve of infected-C.elegans same to normal-C.elegans,which displayed an extremely therapeutic effect in vivo.The human kidney cells 293T cytotoxivity test showed compound B4 had 24.8-fold reduction in cytotoxity compared with niclosamide.Finally,the compound B4 showed obvious inhibitory effect on quorum sensing(QS)signal molecule C4-HSL of DK2 and PAO1,the inhibitory effect in DK2 was more siginificant indicated that compound B4 has inhibitory effect on QS and become a QS inhibitor of DK2.The results also support the optimal synergistic effect in DK2.These results support that compound B4 could be identified a potential polymyxin synergist for further research and development to overcome the emergence of polymyxin-resistance.