Application And Mechanism Of Negative Pressure Wound Therapy In P.Aeruginosa Infected Wounds

Background:Infected wound plays an important role in endangering public health and costs nearly ten billion RMB every year.Previous studies indicated that P.aeruginosa infected wound is a complicated procedure which is influenced by the interaction between bacteria and wound.Besides,biofilm,motility and virulence factors play important roles in P.aeruginosa infected wound.Negative pressure wound therapy(NPWT)is considered to be an effective method to treat and repair wound.Considerable research shows that NPWT could alleviate infection and promote wound healing when it is used for P.aeruginosa infected wound.However,the underlying mechanisms are still not clear.Our previous study indicated that NPWT could reduce the virulence factor and inhibit the expression of virulence regulated genes in early time.Further investigation of the underlying mechanisms is helpful for revealing the theory of infection control which could be used as theory foundation and treatment strategy for clinician.Objective(1)To observe the influence of negative pressure on range of bacteria motility,biofilm formation and virulence factor secretion in vitro;(2)To explore the effect of NPWT on biofilm morphology,key component of biofilm as well as the depth of bacteria invasion in vivo;(3)To analyze the effect of NPAWT on variation of pH values and immune response in vivo;(4)To quantitatively measure and compare epithelial and granulation gaps,new epithelial and granulation areas,angiogenesis and wound healing between NPWT and gauze treatment.Methods(1)P.aeruginosa proliferation model was built in vitro and,crystal violet staining and enzyme linked immunosorbent assay(ELISA)were used to measure the influence of negative pressure on bacteria motility,biofilm and virulence factor;(2)Scanning electron microscope(SEM),concanavalin A(ConA)staining and laser scanning confocal microscope(CLSM)were used to observe P.aeruginosa biofilm,measure biofilm components and bacteria invasion depth in vivo;(3)pH meter,real-time quantitative polymerase chain reaction(Real-Time PCR)and Hematoxylin and eosin(HE)staining were used to assess microenvironment and immune factors.(4)Histology analyses and Real-Time PCR were used to compare wound healing between NPWT and Gauze treatment.Results(1)Negative pressure(-125mmHg and-200mmHg)could inhibit P.aeruginosa motitility(swimming,swarming,and twitching),biofilm formation and virulence secretion in vitro.(2)Bacteria bioburden in soft tissue in NPWT group was decreased below 105 CFU/g tissue guideline earlier than the control.The mean bacteria counts in NPWT group on day 17 has decreased below 102 CFU/g tissue.Besides,biofilm in NPWT group was relatively decentralized and bacteria invasion depth was shallower than that in control.Invasion depth was 18.2±9.7μm in NPWT group and 40.00±12.3μn in the control on day 17(P<0.01).(3)Comparing with the control,NPWT could significantly decrease pH values,increase expression of IL-8 and,inhibit overexpression of IL-1β,IL-6,TNF-α and neutrophil recruitment in superficial layer.(4)NPWT leads to better histologic parameters including epithelial and granulation gaps,new epithelial and granulation areas,angiogenesis and wound healing.The mean healing time of NPWT and the control were 18.8±2.0 days and 23.5±2.7 days(P<0.01).Expression of VEGF and TGF-β1 in NPWT group were higher than that in the control.Conclusion(1)P.aeruginosa motility,biofilm formation and virulence factors secretion were all retarded under-125 mmHg pressure or lower in vitro,but no level of negative pressure tested in this study inhibited these completely.(2)NPWT could change biofilm structure and decrease biofilm components compared with the control in vivo which made bacteria more likely exposed to the host immune cells and therefore be more easily eliminated.(3)NPWT could promote inflammatory cells and cytokines recruitment in the early days,inhibit continuous excessive inflammation and reduce the pH values of wound which could improve microenvironment for infected wound.(4)NPWT was demonstrated to be an effective strategy to alleviate infection and promote wound healing compared with the gauze treatment.