Study Of The Effects Of Surface-deposited Dead Bacteria On The Three-dimensional Behaviors Of Planktonic Bacteria

Surface sterilization to inactivate pathogenic bacteria are commonly used in the related fields of medical care and industry.However,the dead bacteria after inactivation are usually unable to be completely removed from the surface,so that they might shows a long-term impact on the subsequent behaviors and adhesion of viable bacteria near the surface.The previous study of our group showed that the biofilm grown on dead Pseudomonas aeruginosa(PAO1)is softer and less populated.Nevertheless,the impact of dead bacteria on the planktonic bacteria before the formation of the biofilm attached to the surface and the related mechanism are still not clear.Herein,we used digital holographic microscopy(DHM)to track the three-dimensional behaviors and spatial distribution of PAO1 near a deposited layer of their dead siblings.Meanwhile,combined with RNA-sequencing of the planktonic bacteria and metabonomics analysis of dead bacteria,we reveal the corresponding molecular mechansim.The main contents of this thesis are as follows:(1)Based on DHM,the three-dimensional behaviors of planktonic viable PAO1 on the surface of either dead or viable siblings after ultraviolet(UV)inactivation were studied and compared.UV light was used to inactivate PAO1,and a uniformly deposited layer of either dead or viable bacteria surface was prepared.The 3D motions and accumulations of Pseudomonas aeruginosa upon these bacterial layers were observed by DHM in real time.It was found that the planktonic cells near the surface covered with dead siblings have a lower density and a reduced 3D velocity compared with those upon viable ones,accompanied by the increase of the frequency of bacteria “flick” behavior.This suggests that planktonic bacteria move away from the surface of dead siblings by adopting more frequent adaptive response behaviors.On the other hand,the near-surface hydrodynamics might be changed by the deposited dead bacteria,which further increases the degree of PAO1 aggregation on the surface of the viable bacteria.As a result,the accumulation of bacteria is inhibited near the surface of dead siblings and activate adaptive defensive responses of planktonic bacteria.(2)We performed RNA-seq measurements on the planktonic PAO1 swimming on the surface of dead siblings,and used LC-MS technology-based metabonomics to analyze the differential metabolites and metabolic pathways on the surface of viable and dead bacteria.The transcriptome data of RNA-seq revealed that the up-regulation of dgc E and dgc M genes of planktonic PAO1 is related with the inhibited movement near the surface upon dead siblings.Meanwhile,the presence of dead bacteria up-regulates genes hcp1,clp V1 and vgr G1,which are closely related to PAO1 virulence factors.In addition,the differential metabolic pathways of the significant enrichment of dead bacteria and viable bacteria indicate that the increase of succinic acid and the decrease of L-glutamate of dead bacteria promote the spatial diffusion of planktonic bacteria.In summary,utilizing 3D real-time tracking of planktonic bacteria near a layer of their dead siblings,combined with RNA-seq and metabolomics analysis techniques,we are able to investigate the impacts of dead bacteria on the 3D motional and adhesion behaviors of planktonic bacteria,and reveals the molecular mechanism behind them.Our results show that the accumulation of planktonic Pseudomonas aeruginosa near the surface of dead bacteria is weakened compared with those upon viable deposited bacteria.The reason is surface properties and extracellular metabolites of dead bacteria activate the adaptive response mechanism of viable planktonic PAO1 to adjust its motion and subsequent adhesion behaviors.These findings shed lights for the development of new antifouling coatings and antibacterial surfaces.