Autoinducer-2 Of Streptococcus Mitis As A Target Molecule To Inhibit Pathogenic Multi-species Biofilm Formation In Vitro And In An Endotracheal Intubation Rat Model

PART ⅠAutoinducer-2 as a target molecule to inhibit pathogenic multi-species biofilm formation in vitro modelObjective:Streptococcus mitis(S.mitis)and Pseudomonas aeruginosa(PA)are typically found in the upper respiratory tract of infants.We previously found that PA and S.mitis were two of the most common bacteria in biofilms on newborns’ endotracheal tubes(ETTs)and that S.mitis was able to produce autoinducer-2(AI-2),whereas PA was not.Recently,we also found that exogenous AI-2 and S.mitis could influence the behaviors of PA.The aim to the study was to investigate whether inhibition of AI-2 could result in inhibition of these effects.Methods:To test this hypothesis,we selected PAO1 as a representative model strain of PA and evaluated the effect of S.mitis as well as an AI-2 analog(D-ribose)on mono-and co-culture biofilms in both in-vitro model.The changes of biofilm formation of each group was studied by scanning electron microscopic image(SEM),laser-scanning confocal microscopy(LSCM),crystal violet staining assay,q-PCR and colony counts to determine if AI-2 analogue could inhibit the biofilm formation of co-culture biofilm.Results:S.mitis could produce AI-2,while PAO1 couldn’t.S.mitis promoted PAO1 biofilm formation and pathogenicity.Dual-species(PAO1 and S.mitis)biofilms exhibited higher expression of quorum sensing genes than single-specie(PAO1)biofilms did.D-ribose could inhibit Dual-species(PAO1 and S.mitis)biofilms,decreasing the bacterial counts,the density,the structure complicacy and thickness of Dual-species(PAO1 and S.mitis)biofilms.Conclusions:S.mitis promoted PAO1 biofilm formation though secreted signal molecules,which was inhibited by D-ribose.Our results demonstrated that S.mitis AI-2 plays an important role in interspecies interactions with PAO1 and may be a target for inhibition of biofilm formation.PART Ⅱ AI-2 analogue interferes with dual-species biofilms infection in an endotracheal intubation rat modelObjective: S.mitis promoted PAO1 biofilm formation though secreted signal molecules,which was inhibited by D-ribose.In this part,we would discuss whether S.mitis could enhance the pathogenicity of PAO1 and D-ribose could reduce the burden of BF infection.Methods: The study was divided into 5 groups included:Intubations with PAO1(P group),PAO1&S.mitis(PS group),PAO1&S.mitis& D-ribose(PSD group),and S.mitis(S group)biofilm-covered tubes were chosen as the four infection groups,and intubation with sterile tubes served as the control.We observed the general condition,survivorship curve,total cell count of alveolar lavage,bacterial colony count and hematoxylin-eosin staining.Interleukin-4,Interleukin-6 and Interleukin-10 levels in BALF were detected by ELISA assay.Results:The rats in PS group were lower activity,higher death rate,more severe pathologic changes and higher bacterial load than P group in the endotracheal intubation rat model,all of which were inhibited by D-ribose.At the same time,the inflammatory markers and pathologic changes in C group and S group were much lower than other 3 group.Conclusions: It may be one reason for lower eliminate bacterial ability,higher death rate,more severe pathologic changes and higher bacterial load in PS group than P group that S.mitis could promoted PAO1 biofilm formation,resulting in immune escape.However AI-2 analogue(D –ribose)could reduce the extent of the infection.