| Multidrug-resistant Acinetobacter baumannii is a gram-negative bacillus,which is an significant pathogen of nosocomial infection mostly causing pneumonia,lower respiratory tract infection,urinary tract infections and meningitis.The epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains suggested that nosocomial infections rate was increasing year by year.Multidrug-resistant Acinetobacter baumannii is widely distributed in hospital and is capable of surviving for a long time,threatening inpatients seriously.Multiple drug-resistant Acinetobacter baumannii,sometimes gives rise to nosocomial infection outbreaks,especially in intensive care units,this situation has got widespread concern of doctors.The rapid rise of multidrug-resistant Acinetobacter baumannii has led to a lack of clinical treatment,most of the strains for the current clinical application of antibiotics are resistant.Initially,multidrug-resistant infections are limited to nosocomial patients,however,the current multidrug-resistant pathogens are often planted in vulnerable patients(including patients with underlying disease and immune hypofunction patients,etc.);What's worse is that multidrug resistant bacteria are prevalent in community currently;as a rusult Acinetobacter baumannii has recently been listed top six most dangerous opportunistic pathogens.Acinetobacter baumannii can survive long in different environments,mainly due to the formation of bacterial biofilm,which is also the major reason for Acinetobacter baumannii chronically colonization in human tissue and in medical devices.in the traditional sense,it is considered that the bacterial biofilm is the combination of bacteria,which is irreversibly adherent to the surface of medical devices or organisms;recently,some researchersnoticed that biofilm was not just a simple mixture of bacteria at different growth stages,but rather a special growth period of bacteria through proteomics analysis and transcription;which had their own unique protein phenotype,different from planktonic bacteria.The ability of multidrug-resistant Acinetobacter baumannii forming biofilm has become one of the major factors in assessing its pathogenicity.The biofilm of bacteria is a dynamic stable whole formed by the aggregation of multiple colonies.It is composed of multiple interconnected colonies embedded in a large number of extracellular matrix(including extracellular polysaccharides,extracellular genetic materials,proteins and lipids).The chronic colonization of bacteria,difficult clearance,chronic and repeated infections,depend on the formation of bacterial biofilm and shape maintenance.Under the continuous protection of biofilm,bacteria can be long-term survived,and achieve stronger resistance and pathogenicity as well;which is a serious clinical problem to be solved currently.We believe that the formation of bacterial biofilm as well as the maintenance of morphologies are regulated by specific pathways,signaling factors and transcription factors.In the state of planktonic bacteria,which are not expressed or activated.Therefore,in this study,we want to explore the mechanism of the biofilm state of Acinetobacter baumannii,compared with the floating state of the cell,which has a unique pathogenicity,leading to persistent chronic infection and recurrence;and we try to study the specific genetic expression of bacterial biofilm.Part one The multidrug-resistant strains of Acinetobacter baumannii in different states of the strain screeningObjective: Multidrug-resistant Acinetobacter baumannii is an important pathogen of nosocomial infection and often causes extensive nosocomial infections.The epidemiological survey of multidrug-resistant Acinetobacter baumannii strains found that the infection rate in nosocomial infections was increasing year by year.We investigated the epidemiological trend ofmultidrug-resistant Acinetobacter baumannii,and make the subtype analysis of the strain DNA to prepare the multidrug-resistant strain model under different growth conditions.Methods:1 Investigation on the infection trend of pathogenic bacteria with multiple drug-resistant strains: to investigate the infection trend of multiple drug-resistant strains in our hospital from 2011 to 2015.2 Screening of multidrug-resistant Acinetobacter baumannii by group,PCR analysis,subtypes analysis.3 Preparation of multidrug-resistant Acinetobacter baumannii strains of different growth condition,including rapid growth period and biofilm fixation period.Results:1 The epidemiological investigation of multidrug resistance pathogens in our hospital showed that from 2011 to 2015,both of multidrug-resistant strains and multidrug-resistant Acinetobacter baumannii increased year by year,increasing rates are1.1067 and 1.2287.2 This study selected 608 cases of multidrug-resistant Acinetobacter baumannii in our hospital from March 2014 to March 2015 which screened by bacterial chamber,and the DNA of all the collected strains was extracted and sorted by PCR.Among the 7 subtypes divided,the number of subtype 5strains was the most abundant,and further studied.3 Observed the biofilm growth of multidrug-resistant Acinetobacter baumannii strains.Conclusion: The epidemiological investigation of multidrug-resistant pathogens showed that multidrug-resistant strains were detected in our hospital,and the multidrug-resistant of Acinetobacter baumannii increased year by year.Multidrug resistant strains of infection was no longer limited to critically ill subjects,and showed a general trend of increasing.Part Two Analysis of High-throughput Sequencing of Multidrug-resistant Acinetobacter baumannii Strains in Different Growth Stages for biofilm formation gene expression changesObjective: Biofilm is not just a simple mixture of bacteria at different growth stages,but rather a special growth period in which they are located;they differ from planktonic bacterial states and have their own unique protein phenotype and gene expressed.We compared the same strain,under different growth conditions,the whole genome analysis,compared to its gene expression differences,revealed multidrug resistant strains in the biofilm stage unique phenotype,and biofilm for the strains to provide maintaining mechanism.Methods:1 The quality and quantity of the dominant Acinetobacter baumannii strains were detected by extracting the genetic material during the rapid growth period and the solid period of biofilm.2 In order to study gene expression,the high throughput sequencing of genetic material was carried out in the two states of rapid growth and biofilm fixation respectively.3 The gene sequences of the two samples from the study strains were compared with the common database for gene function annotation.4 The sequence expression of sequenced genes under different growth conditions of the same strain was studied.Results:1 In this study,the quality of mRNA data is tested and evaluated,including pollution assessment.The results showed that the data of this study is high in purity.The original sample sequencing sequence was not significantly contaminated with other species sequences.This sample points to Acinetobacter baumannii BJAB0868.2 Analysis of multiple drug resistant Acinetobacter baumannii strains of different growth periods of gene expression,found that two samples of the most common expression mainly amino acid transport and metabolism related genes,generation and transformation of energy related gene,inorganic iontransport and metabolism related genes,transport and metabolism of carbohydrate related genes,enzymes transport and metabolism related genes,bacteria cell wall formation related genes et al,all of these genes maintain bacteria activities and structure.3 The sequencing gene sequence and whole gene sequencing analysis of Acinetobacter baumannii BJAB0868 strain were significantly different in the two states.There were significant differences in the expression of 198 genes,among which 106 genes were up-regulated and 92 genes were down-regulated in the biofilm-like cells compared to the rapidly growing cells.In the 106 genes that were up-regulated,53 genes were significantly elevated in the biofilm state of the bacteria,but were rarely expressed or even completely inhibited in the planktonic bacterial state.These 53 genes were significantly different in expression,and the encoded proteins were mainly involved in bacterial biofilm formation and morphological maintenance.Conclusion: We sequenced the genome-wide sequencing of the multidrug-resistant Acinetobacter baumannii BJAB0868.We used the same strain but in different states of the bacteria,selected the most pathogenic two stages: rapid growth status and biofilm status to make the system of the whole genome comparison,found that there is a genetic expression difference between them.The biofilm is a unique form of bacteria that has its own unique characteristics.The bacteria under protection of biofilm,have its own unique genetic expression and protein phenotype,and also have its own unique pathogenicity.The part of expression significant differences in genes,mostly involved in the biofilm formation and morphology maintenance,revealing the biofilm formation and morphology maintenance mechanism.Part Three Studies on the gene knockout and pathogenicity of mutant strains of multidrug-resistant Acinetobacter baumannii BJA B0868Objective: Biofilm is a unique form of bacteria that has its own unique genetic expression.The genes that expressed significant different wereinhibited in the bacterial planktonic cells.The TetR family transctiptional regulator gene in the biofilm was significantly increased.We believed that target gene was involved in the formation and morphology of the biofilm.The target gene was screened for gene knockout,and the mutant strain was constructed to study the unique pathogenicity of biofilm.Furthermore,we further explore the changes in the pathogenicity of the acute and chronic infection of the lungs and the effect of biofilm formation on the pathogenicity of the bacteria.Methods:1 Make accurate measurement of Tet R family transctiptional regulator gene expression concentration once again for multidrug-resistant Acinetobacter baumannii BJAB0868 strain in different states.Real-time PCR was used to detect the expression level of target gene between the two samples.2 Knock out the Tet R family transctiptional regulator gene of multidrug-resistant Acinetobacter baumannii BJAB0868 strain.Amplify the upper and lower homologous recombination arms of the TetR family transctiptional regulator gene on the genome of the study strain by using the high-fidelity PCR enzyme.The target fragment of the TetR family transctiptional regulator gene knockout(upstream homologous arm-downstream homologous arm)was obtained by fusion PCR technique.Then we cloned the target fragment into the suicide vector pUC19 vector to obtain the pUC19 / TetR plasmid.The reverse gene was used to remove the target gene sequence from the pUC19 / TetR plasmid.The amplified sequence was digested and self-ligated to obtain the pUC19-?TetR gene plasmid.The resistance gene from the p KD4 plasmid was inserted at the upstream and downstream targeting arm attachment site of this plasmid to obtain the pUC19-?TetR::Kan plasmid.The target plasmid(upstream homologous arm-resistance gene-downstream homologous arm)was excised from pUC19-?TetR::Kan plasmid and subcloned into pCVD442 suicide plasmid to obtain the target plasmid pCVD442-?TetR::Kan.The target plasmidpCVD442-?TetR::Kan was transferred to E.coli ?21552 by electroporation,and the donor strain ?21552 / pCVD442-?TetR::Kan was obtained.(Fusion PCR product)was cloned with the donor strain ?21552 /pCVD442-?TetR::Kan and the recipient bacteria Acinetobacter baumannii BJAB0868.The cloned BJAB0868 strain was screened on the sterile LB plate and the genome was integrated into the target Sequence,known as BJAB0868/ pCVD442-?TetR.BJAB0868 / pCVD442-?TetR was inoculated on LB plates containing 10% sucrose and cultured to monoclonal.The clones of TetR gene were screened by PCR.The local sequence was confirmed by sequencing and named BJAB0868 / ?TetR.3 After PCR and sequencing confirmed,LB plate screening transformation mutant cloned strain can stabilize the genetic related genes,the upstream and downstream gene stability is also verified.4 To evaluate the ability of stable mutant strain BJAB0868 / ?TetR to produce biofilm and the resistance of strains to various antibiotics.5 The effects of wild strains and mutant strains on the pathogenicity of rats were studied by animal experiments.Results:1 The expression of the gene TetR family transctiptional regulator was significantly increased in the biofilm of the multidrug-resistant Acinetobacter baumannii strain,and the expression of the gene was inhibited in the rapid growth period of bacteria.2 Knock out the target gene,construct a stable mutant strain BJAB0868 /?TetR,and make gene sequencing to verify its upstream and downstream gene stability.In the mutant strain BJAB0868 / ?TetR,the ability to generate biofilm was significantly reduced and inhibited.The biofilm produced by the mutant strain BJAB0868 / ?TetR was inhibited and decreased significantly.3 After knocking out the Tet R family transctiptional regulator gene,the resistance of the mutant strain did not change significantly,and it was still resistant to multiple antibiotics.4 Animals were used to study the pathogenicity of wild strains and mutantstrains in vivo,quantitative culture of bacteria in lung lavage fluid showed that the quantitative culture of BJAB0868 / ?TetR group was significantly lower than that of the infected multidrug-resistant strains.The results showed that the number of bacteria in the BJAB0868 / ?TetR group was significantly lower than that in the control group.Acinetobacter baumannii BJAB0868.Pathological section of pulmonary infection rats showed that the infection of the mutant strain BJAB0868 / ?TetR was significantly lighter than that of the multidrug-resistant strain Acinetobacter baumannii BJAB0868.Conclusion: TetR family transctiptional regulator gene was significantly increased in the biofilm of the multidrug-resistant Acinetobacter baumannii strain,while the expression of the gene was inhibited during the rapid growth of bacteria.We believed that it participated in the formation and morphology of biofilm,which regulates biofilm formation and morphology maintenance.We knocked out the target gene to construct a stable mutant strain BJAB0868/ ?TetR,and the gene sequencing confirmed its upstream and downstream gene stability.The resistance of the mutant strain to variety antimicrobial agents did not change significantly,but the biofilm formation ability was signigicantly reduced.Mutant strains were more likely to be cleared in the face of host immunization attacks and antimicrobial killings.Infection of the mutant strains group was significantly lighter than the infected wild strains group.The pathogenicity of mutant strains decreased significantly.The increasing virulence was due to the continued strong protection of the biofilm.Because the bacteria in the biofilm state,the virulence is significantly increased,the biofilm protect the strain from host immunity and make sure its exsistence to be a continuous source of infection,leading the infection increasing and recurring. |
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