Study On Anti-Bacterial Biofilm Activity And Mechanism Of Novel Pyran Derivatives

ObjectiveMost of the bacteria cannot survive as single cells but exist in thin-layered colonies called bacterial biofilms,which are attached to abiotic or biological surfaces and are encapsulated by their own extracellular polymer and matrix networks.The National Institutes of Health reported that among all microbial and chronic infections,65%and 80%,respectively,were associated with bacterial biofilms,including osteomyelitis,infective endocarditis,wound infection and implant-associated infections,such as catheter and joint prosthesis.Moreover,approximately 17 million new biofilm-associated infections arise annually in the United States.Since biofilms can reduce the susceptibility of bacteria to antimicrobial agents and to immune defenses,they promote bacterial survival and multiplication in numerous microbial infection conditions.Consequently,the microbial colonies develop into a chronic stage-type of infection and lead to increased morbidity,mortality and duration of hospitalization.Staphylococcus aureus（S.aureus）and Pseudomonas aeruginosa（P.aeruginosa）are two major opportunistic pathogens that cause biofilm-associated infections.and there are few drugs available in clinical to against these two bacterial biofilms related infection,especially the emergence of pan drug resistance and multiple drug resistance.Meanwhile,the speed of new anti-bacterial drug development is not able to keep up with the growth rate of drug-resistant strains,which leads to the urgent necessity of developing new agents with biofilm-forming inhibitory ability.Recently,pyran derivatives are received more and more attention due to their wide biological activities,including antibacterial and antifungal activities,and many of them have been developed as commercial antibacterial and antifungal agents such as triadimefon,triadimenol,diniconazole,myclobutanil and bitertanol.Furthermore,pyran structural features are responsible for the anti-bacterial activity of these chemicals,such as the tetrahydropyran rings present in the antibiotics erythromycin and streptomycin.In the present study,we synthesized fourteen novel pyran derivatives and analyzed the in vitro and in vivo anti-bacterial biofilm activity against S.aureus and P.aeruginosa,then explored the possible mechanism of anti-bacterial biofilm of the pyran derivative.Methods1.Antibacterial activity of novel pyran derivatives in vitroIn S.aureus（USA 300）and P.aeruginosa（ATCC 27853）,the minimal inhibitory concentration（MIC）and the effect of pyran derivatives to the bacterial growth curve were determined by microbroth dilution.2.Anti-biofilm activity of novel pyran derivatives in vitro（1）The biofilm formation capacity of S.aureus（USA 300）and P.aeruginosa（ATCC27853）was verified by crystal violet staining.（2）Effect of 14 compounds on S.aureus（USA 300）and P.aeruginosa（ATCC 27853）biofilm formation were observed by crystal violet staining,fluorescence microscopy and scanning electron microscopy.（3）The minimal biofilm inhibitory concentration（MBIC）of compoundⅠc andⅣc against S.aureus（USA 300）and P.aeruginosa（ATCC 27853）were determined by microbroth dilution.3.Effect of compoundⅣc on extracellular polymeric substance of S.aureus（USA300）biofilm in vitro（1）The amount of residual biofilm after treatment with or without proteinase K（0.1mg/m L）,DNaseⅠ（0.1 mg/m L）or sodium metaperiodate（10μM）was measured at OD_630nm.（2）Main components of S.aureus（USA 300）biofilm were extracted and the extracellular protein concentration in the biofilm was determined by Bradford method.The content of e DNA was determined by micronucleic acid protein analyzer,and the exopolysaccharides in biofilm was measured by phenol-sulfuric acid method.4.Anti-biofilm activity of novel pyran derivatives in vivo（1）Using the subcutaneous tissue cage S.aureus biofilm infection mouse model,the colony forming unit（CFU）on implanted tissue cage was measured after administration of2.5 mg/kg,5 mg/kg or 10 mg/kg compoundⅣc for 7 days,the biofilm formation on the surface of the implanted tissue cage was observed under the scanning electron microscope.（2）Using the urinary tract catheters P.aeruginosa biofilm infection rat model,the bacterial CFU of kidneys,bladder and implanted catheter was measured after daily administration of compoundⅣc at 2.5 mg/kg and 5 mg/kg,and the effect of compoundⅣc to the P.aeruginosa biofilm was evaluated in vivo.5.Mechanism of anti-biofilm activity of novel pyran derivatives（1）Transcriptome sequencing（RNA-seq）was applied to analyze the difference in the gene expression of S.aureus（USA 300）biofilm after compoundⅣc treatment.（2）RT-PCR was used to detect the expression level of pyrimidine synthesis related genes（car A,car B,pyr B,pyr C,pyr E,pyr F,upp and pyr R）in P.aeruginosa（ATCC 27853）after compoundⅣc treatment.（3）Upon addition of UMP,Uracil and UTP at the concentration from 0.01 m M to 10m M,the biofilm formation of S.aureus（USA 300）and P.aeruginosa（ATCC 27853）was detected after compoundsⅣc treatment by crystal violet staining.（4）The expression level of biofilm related gene（sig B,sar A,ica R,ica A and Las I/Las R,Rhl I/Rhl R,Fap A,Fap B and Fap C）in S.aureus（USA 300）and P.aeruginosa（ATCC 27853）after compoundⅣc treatment was measured by RT-PCR.（5）The effect of compoundⅠc andⅣc on△agr S.aureus（USA 300）biofilm formation was detected by crystal violet staining,and the expression level of pyrimidine synthesis related genes（car B,pyr B,pyr C,pyr E,upp and pyr R）in S.aureus（USA 300）was measured by RT-PCR after silencing the expression of lux S gene.（6）The effect of compoundⅣc on△psmα/βS.aureus（USA 300）biofilm formation was measured by crystal violet staining.（7）The relative expression levels of psmα,psmβof S.aureus（USA 300）,and the expression levels of Fap A,Fap B,Fap C of P.aeruginosa（ATCC 27853）after treatment of100μg/m L compoundⅣc with or without addition of 1 m M UMP,Uracil and UTP were measured by RT-PCR.Results1.Anti-bacterial activity of novel pyran derivatives in vitro（1）The MICs of the 14 novel pyran derivatives were higher than 256μg/m L against S.aureus（USA 300）and P.aeruginosa（ATCC 27853）in vitro.（2）The 14 novel pyran derivatives had no effect to the growth curves of S.aureus（USA300）and P.aeruginosa（ATCC 27853）in vitro.2.Anti-bacterial biofilm activity of novel pyran derivatives in vitro（1）The tested compounds exhibited variations inhibitory potency to the biofilm formation of S.aureus（USA 300）and P.aeruginosa（ATCC 27853）,and ClassⅣcompounds exhibited the most potent activity against bacterial biofilm formation.（2）CompoundⅠc andⅣc showed strong inhibitory effect to the bacterial biofilm formation at the concentration of 200μg/m L during 24 h,while they showed significant inhibitory effect in 50μg/m L or 100μg/m L at the early stage of biofilm formation.The inhibitory effect gradually weakened at the later stage of maturity（16-24 h）.（3）The MBIC of compoundⅠc andⅣc to S.aureus（USA 300）and P.aeruginosa（ATCC 27853）were higher than 200 and 256μg/m L,indicating these two compounds had no effects on mature biofilm in vitro.3.Effect of compoundⅣc on extracellular polymeric substance of S.aureus（USA300）biofilm in vitro（1）In the extracellular polymeric substance of S.aureus（USA 300）biofilm,the proportion of protein and e DNA were more than exopolysaccharide.（2）CompoundⅣc decreased the EPS of the USA 300 biofilm at different inhibition concentration,reduced the content of e DNA in 50,100,and 200μg/m L,and inhibited the formation of extracellular polysaccharide or protein in 100μg/m L and 200μg/m L,respectively.4.Anti-biofilm activity of novel pyran derivatives in vivo（1）Compared with the control group,daily administration of compoundⅣc at 2.5,5and 10 mg/kg reduced the formation of S.aureus（USA 300）biofilm and the corresponding bacterial number in the tissue cage biofilm infection model mice significantly within 7 days.CompoundⅣc treatment inhibited the formation of S.aureus biofilm in the tissue cage on days 1 and 7,and the in vivo inhibitory activity was in a time and dose-dependent manner.（2）In the urinary tract catheters P.aeruginosa biofilm infection rat model,2.5 and 5mg/kg compoundⅣc treatment reduced the bacterial burden in bladder,kidney and implanted catheter significantly,suggesting that this compound could also inhibit the P.aeruginosa biofilm formation and spread in vivo.5.Mechanism of anti-biofilm formation of novel pyran derivatives（1）In 2682 genes,25 genes were expressed different significantly after compoundⅣc treatment comparing to the control,nine of them were up-regulated,and the sixteen genes were significantly down-regulated.Based on KGEE Pathway enrichment,seven significantly down-regulated expressed genes were related with pyrimidine biosynthesis pathway closely.（2）In P.aeruginosa（ATCC 27853）,0.1 mg/m L compoundⅣc reduced the expression of car B,pyr B,pyr E,pyr F,upp and pyr R significantly.（3）The addition of UMP,UTP and Uracil（lower than 10 m M）did not affect the growth of S.aureus（USA 300）and P.aeruginosa（ATCC 27853）biofilm,but reversed the inhibitory effect of 100μg/m L compoundⅣc treatment to f bacterial biofilm formation.（4）CompoundⅣc reduced the expression of sig B,ica A,psmα,psmβin S.aureus,and Rhl I,Rhl R,Fap A,Fap B,Fap C in P.aeruginosa（ATCC 27853）significantly.（5）CompoundⅣc could inhibit the△agr S.aureus（USA 300）biofilm formation at the 50,100 or 200μg/m L,and the expression of pyrimidine synthesis related genes after silencing the expression of lux S gene in S.aureus（USA 300）.（6）CompoundⅣc could not affect the△psmα/βS.aureus（USA 300）biofilm formation,indicating that PSM protein was involved in the anti-biofilm mechanism of compoundⅣc.（7）After additional supplement of 1 m M UMP,UTP or Uracil,100μg/m L compoundⅣc could not inhibit the expression of psm and Fap in S.aureus（USA 300）.Conclusions1.14 novel pyran compounds did not affect the growth of S.aureus（USA 300）and P. aeruginosa（ATCC 27853）,but inhibited the formation of bacterial biofilm in vitro.2.CompoundⅣc could inhibit the S.aureus（USA 300）biofilm formation in mice model of subcutaneous tissue cage,and P.aeruginosa（ATCC 27853）biofilm formation in rat model of urinary catheter in vivo.3.The novel pyran derivatives inhibited the expression of psm and Fap by affecting the pathway of pyrimidine synthesis.4.CompoundⅣc also could inhibit the production of PIA,reduced the expression of ica A,and inhibited the formation of biofilm by promoting the e DNA degradation in S. aureus（USA 300）.