Study Of The Surface Adhesion And Motility Mechanisms Of Pseudomonas Aeruginosa

Antibiotics therapy is one of the most effective way to treat the regular infectious diseases mediated by bacterial pathogens in current clinical cases.However,the prog-nosis of the antibiotics therapy are unsatisfactory when the therapy is used to treat the infections arising from biofilms or drug-resistant bacteria,in which the fatal cases are often related to the biofilms-associated infections.Therefore,the development of new therapies that can be used to treat those infections is urgent.Bacteria typically pro?duce massive extracellular polymeric substances(EPS)to enclose themselves during the biofilms formation.These EPS can protect the bacteria to resist to various external envi-ronmental stresses,including immune defenses,antibiotics,heavy metals,surfactants,resulting that the biofilms cannot be eradicated.It is well known that the initial surface adhesion and subsequently surface adaption of bacteria are essential to the biofilms for-mation.Therefore,understanding the mechanisms of bacterial surface adhesion as well as the surface adaption are important for the development of new methods to prevent biofilms formation.However,the methods that can be used to in situ characterize the bacterial surface adhesion and adaption are rather limited.In addition,various polymer materials are broadly used to produce biomedical devices.The investigation of bacte-rial adhesions and adaptions on those surfaces of various polymer materials is needed.Based on the above considerations,we first established a novel method,then we used the method to systematically investigate the surface adhesion and surface adaption of one of three major nosocomial pathogens Pseudomonas aeruginosa on the surfaces of various polymer materials.Adhesion mechanisms of P.aeruginosa on the surfaces of various polymer materials:Using a combination of microfluidics,microscopy,molecular biology and parti-cles tracking techniques,we first established a high-throughput method that can be used to characterize bacterial adhesion and quantify the adhesion strength in single-cell scale.Second,we investigated how adhesion factors to facilitate P.aeruginosa to attach on the surfaces of various polymer materials using different adhesion factors mutants.Thirdly,we quantified the adhesion strength and estimated the mechanical properties of extra-cellular polymeric substances.Our results indicated that:1)the difference of adhesion strength between single cells reach three orders of magnitudes;2)bacteria differently responded to mechanical washings,which can be sorted by three types,including the cells stuck on the surface despite the shear flow(stick-type),the cells kept sliding on the surface(slide-type)with an approximately constant velocity and the cells detached from the surface(detach-type);3)15%-25%bacteria persistently adhered to the glass surface despite the shear strength of 2000 Pa.Furthermore,we found that this small subpopulation cells can adhere to the surfaces of various polymer materials,including(PP,PDMS,PTFE)and termed this newly identified subpopulation of P.aeruginosa as strong shear flow persisters(SSP)cells.In addition,we observed that SSP cells can develop on different materials to form distinct biofilms that are tolerant to high doses of tobramycin.After screening the adhesion factors in P.aeruginosa,we found that SSP cells form due to the high expression of the outer-membrane protein CdrA.Most impor-tantly,we elucidated a general molecular mechanism underlying the high tolerance of SSP cells to mechanical washings on the surface of various materials:outer-membrane proteins can crosslink with the polysaccharides to form gel-like adhesion complexes in the bacterial wall,which can exert extremely strong adhesion strength(up to 50 N/mm2)on distinctive substrates.Understanding the general molecular mechanism underlying the high tolerance of SSP cells to mechanical washings on different materials can help ongoing research focused on preparing antifouling biomedical materials.Surface adaption mechanism of P.aeruginosa on polymer brush surfaces:To investigate whether P.aeruginosa cells can adapt to different polymer mate-rials possessing different viscoelasticity(softness),we prepared the surfaces that are grafted with a layer of thermally sensitive polymer chains PNIPAM,where the softness of brush-layer is tunable by applying a small temperature change.Using a high-speed microscope and our established two-point tracking algorithm,we found that P.aerugi-nosa cells would deploy their type IV pili(TFP)to slingshot more on soft surfaces at a shear-thinning condition.Furthermore,we clearly demonstrated that frequent slingshot on the long-brush surface can greatly reduce the surface viscosities,thus facilitating cells to crawl on surfaces by means of reducing the energy dissipation.This adaptive response suggests that P.aeruginosa cells may be able to sense the local viscoelasticity,and then deploy TFP to adapt to their physical surroundings.