Inhibition Of Naftifine Derivatives On Staphylococcal Biofilm Formation

Staphylococcus aureus is one of the most important foodborne pathogens.Clonal complex 75 lineage was newly defined as a novel species,Staphylococcus argenteus,naturally lacking one important pigment,staphyloxanthin.The staphyloxanthin biosynthesis strengthens the bacterium's stress resistance,meanwhile biofilm is an important way to enhance its resistance.Therefore,a hypothesis is put forward that there is correlation between the staphyloxanthin biosynthesis and biofilm formation.Naftifine and its derivatives can effectively inhibit staphyloxanthin biosynthesis by inhibiting the key catalytic enzyme CrtN.In this study,the effects of naftifine derivatives on staphyloxanthin biosynthesis and biofilm formation will help us to further understand their correlation.The main findings of this study are as follows:1.One hundred and ninety-three isolates of S.aureus and 6 isolates of S.argenteus were used in the species identification with both methods either recommended in GB 4789.10-2016 or PCR amplification of nuc1 and NRPS genes.It was unfortunate that S.argenteus isolates were all falsely detected as S.aureus with the method recommended in GB 4789.10-2016 and the nuc1-PCR method,however,the NRPS-PCR method established in this study could distinguish these two species.Therefore,a new protocol was established based on the combination of GB 4789.10-2016 and NRPS-PCR to identify and distinguish S.aureus and S.argenteus.2.The ability of staphyloxanthin synthesis was quantitatively evaluated by methanol extraction of the pigment.It was surprised that 31.6% of S.aureus isolates were non-pigmented.These non-pigmented isolates were detected for the integrity of crtM and crtN genes.There were different degrees of synonymous mutation,missense mutation,frameshift mutation and nonsense mutations.3.The ability of biofilm formation was quantitatively evaluated by crystal violet staining.Most of the S.aureus and 7 S.argenteus isolates were able to form biofilm,however,there was no direct correlation between staphyloxanthin biosynthesis and biofilm formation in phenotypes.The effect of naftifine derivatives on the biofilm producers showed that the biofilm formation of S.aureus isolates was significantly inhibited by naftifine derivatives(the strongest inhibitory effect was found in JX08806),which was also found in S.argenteus.It indicated that CrtN was not the only target for naftifine derivatives,and the regulatory network of staphyloxanthin biosynthesis and biofilm formation was also possibly targeted.4.The transcriptomic and proteomic correlative analysis was performed on inhibition of JX08806 on biofilm formation.A total of 674(616 up-regulated and 58 down-regulated)significantly differential genes were detected(fold change greater than 2 or less than 0.5);a total of 396(175 up-regulated and 220 down-regulated)significant differential proteins were screened(fold change greater than 1.2 or less than 0.83).Differentially expressed proteins in correlative analysis between transcriptomics and proteomics were primarily involved in cellular and metabolism process,cells and membranes,as well as catalytic activity and binding.The results of KEGG Pathway analysis showed that the expression of the biofilm-associated adhesion protein was down-regulated in S.aureus infection pathway;the expression of operon crtOPQMN,as well as key enzymes CrtM,CrtN,and CrtO were significantly down-regulated in carotenoid synthesis pathway.