| 【Background and Objective】The navy is an important military combat force in China,whose strategic importance is recognized and with the development of maritime activities such as transportation fisheries,the occurred rate of fracture in the marine environment is increasing.So the research on fracture seawater bacteria infraction become worthy of attention.It is still in the primitive stage and the research is relatively and remains compared with other branches of marine science.There are fewer domestic investigations and analysis of seawater bacterial community composition,and there are fewer related animal models of limb trauma combined with seawater bacterial infection.The"Illumina Mi Seq Sequencing+Bacterial Culture"method is based on a comprehensive analysis of seawater bacterial composition in the Southeastern sea of China,and a new model of rabbit tibia seawater bacterial infection is provided to provide basic data and experimental methods for marine science and clinical medicine.【Method】Experiment 1:Composition analysis of seawater bacterial communities in the southeastern coastal waters.On-site sampling and measurement of seawater environmental factors in the southeastern coastal waters:including Temperature,PH,Osmotic pressure,Na+,K+,Ca2+concentration.Using a suction filtration system to filter 500ml of seawater with a filter membrane（pore diameter0.22μm,diameter 50mm,water system MCE）,and randomly divide the seawater filter membrane into 4 groups:8 total bacterial community groups in seawater,3bacterial community groups in seawater,seawater 8 bacteria in the bacterial culture group and 10 in the experimental group.DNA and RNA extraction of 16S r DNA and16S r RNA genes（V3-V4 region）were performed by Illumina Miseq sequencing method in the seawater total bacterial community group and the seawater active bacterial community group,respectively,and then performed PCR.Amplification,sequencing,sequence processing,and finally statistical analysis.Seawater bacteria are complex,and 8 types of bacterial culture plates are used for cultivation,isolation,identification and counting according to different growth requirements.Experiment 2:Construction of a new rabbit tibia seawater bacterial infection model.14 New Zealand rabbits were randomly numbered 1-14 and divided into 2groups:10 in the experimental group（of which EG13 died during anesthesia）and 3 in the control group.Relevant indicators in vivo were measured after anesthesia in only experimental rabbits:including body temperature,blood biochemistry(p H,osmotic pressure,Na+,K+,Ca2+concentration).All animals were prepared with a left tibial bone defect model The experimental group put 10 pieces of seawater filter membranes into the bone marrow cavity of each animal,and the control group put the same sterile filter membranes for bone wax sealing and suture.Routine nursing after surgery,passed 1 week after surgery.General conditions,statistical infection rate and mortality,genetic sequencing of infectious bacteria in the bone marrow cavity,microbial culture and drug sensitivity tests,histopathology of the bone marrow cavity,and scanning electron microscope observation for comprehensive evaluation and analysis.【Result】Experiment 1:The sequencing results of seawater bacteria genes in the coastal waters of southeast China showed that the high-abundance bacteria at the door level of the total seawater bacterial community group and seawater active bacterial community group were Cyanobacteria,Proteobacteria,Bacteroidetes are the main species.Oxyphotobacteria,Alphaproteobacteria,and Gammaproteobacteria are the main species at the level of the genus.Synechococcus＿CC9902,Chloroplast,Cyanobium＿PCC-6307 mainly.Beta diversity analysis and species difference analysis show that there is a significant difference between the total bacterial community and the active bacterial community in the seawater（P<0.05）,mainly the difference in low-abundance bacteria.53 strains of bacteria:20 strains of Vibrio（37.74%）,22 strains of Enterobacter（41.51%）,and 11 strains of nonfermentative bacteria（20.75%）.This investigation is not only beneficial for the diagnosis and treatment of seawater bacterial infections,but also for seawater bacterial infections.It is important to prevent the spread of infectious diseases caused by seawater bacteria.Experiment 2:1 week after operation:1.The infection rate in the experimental group was 100%and the mortality rate was 20%.The infection rate and mortality rate in the control group were 0,and the difference was statistically significant（P<0.05）.2.The bone marrow cavity of the experimental group The results of the sequencing of infected active bacteria genes showed that:Firmicutes,Proteobacteria,Thermotogae were mainly at the level of Phylum.Clostridia、Gammaproteobacteria andThermotogaeweremainlyatthelevelof Class.Clostridium＿sensu＿stricto＿7,Haloimpatiens,Clostridium＿sensu＿stricto＿15 at the genus Level.Through sample hierarchical clustering analysis,PCo A analysis,ANOSIM analysis and significant difference test between groups It showed that the bacterial community composition of the experimental group and the seawater group,the experimental group and the control group were statistically different（P<0.05）.In addition,the environmental factors were related to the changes of the marine bacteria in the experimental group and the seawater group due to changes in the living environment.Analysis:Including ordinal regression analysis and correlation Heatmap plot analysis,the results show that most of the dominant bacteria in the seawater group are positively related to PH,Osmotic pressure,and Na+,K+,Ca2+concentrations,and negatively related to temperature.the experimental group is the opposite.The hypoxic environment of the bone marrow cavity can not be ignored,making Clostridium in the inferior flora in seawater the main pathogenic bacteria.3,10 experimental group animal infection specimens isolated 45 strains of 11types of bacteria,Among them,5 strains of Vibrio（11.11%）,21 strains of Enterobacter（46.67%）,10 strains of Non-fermentative bacteria（22.22%）,9 strains of Gram-positive cocci（20.00%）,and seawater group and control group The differences in bacterial culture results were statistically significant（P<0.05）.4.Drug resistance and sensitive antibiotics of infected bacteria were obtained through drug sensitivity experiments,providing scientific basis for the treatment of marine bacterial infections.5.Application of The average number of marine active bacteria loaded into the tibia of the rabbit by RT-PCR measurement was 1.6711x10 8copies/ml;the average number of active bacteria infected in the experimental group was1.609x107copies/ml.6.Histopathological evaluation:Sample treatment and After HE staining,the results of microscopic observation were all positive infections,which corresponded to the general observation,the genetic sequencing of the infected bacteria,and the results of microbiological evaluation,while the control group showed no signs of infection.7.Scanning electron microscope observation:A large number of bacteria were observed in the experimental group.On the surface and deep of the bone marrow cavity,the morphology is thick and short.The long rod is thin,spherical,spindle-shaped,spindle-shaped,etc.A large amount of secretions can be seen between bacteria.Therefore,we have comprehensively evaluated the rabbit tibia seawater bacterial infection model and verified the feasibility,scientificity and reliability of the model.Sex.【Conclusion】1.This experiment is the first to apply the"Illumina Mi Seq sequencing+bacterial culture"method to researches on the species and composition of seawater bacteria in the Southeast sea of China,and to compare the differences between marine total bacteria and active bacteria,on microbial communities in aquatic ecosystems providly basic information.2.This experiment is the first one that uses seawater bacteria filtration and enrichment to load rabbit tibia bone marrow cavity to successfully establish a new model of rabbit tibia seawater bacterial infection,which is based on the genetic detection of infected bacteria.Comprehensive evaluations are performed in four aspects,including bacterial culture,histopathology,and scanning electron microscopy.On the basis of quantitative research using RT-PCR method to complete the measurement of bacterial load,improve marine active bacteria in seawater and experimental animals,respectively.Changes in community species and composition in two different environments in the body,revealing the pathogenic environment of seawater pathogenic bacteria,analyzing the pathogenic conditions of Vibrio alginolyticus as conditioned pathogenic bacteria,and proposing the importance of anaerobic bacteria infection after seawater bacterial infection In the end,drug susceptibility tests are conducted on pathogenic bacteria,and the use of carbapenem antibiotics is recommended for combined,sufficient,and full use.It provides a certain scientific basis for marine science,basic medicine,and clinical diagnosis and treatment of related patients,and also for China The navy and fisheries provide a certain logistical support foundation. |
