| Objective:1.To observe the changes of drug-resistance during 2013-2015 of Pseudomonas aeruginosa isolated from Clinical Laboratory of the First Affiliated Hospital of Guangxi Medical University.2.To understand the changes of whole cell proteins before and after the drug-resistance to cefoperazone sodium and sulbactam sodium of Pseudomonas aeruginosa,and discover different proteins between wide-type Pseudomonas aeruginosa PAO1 and its cefoperazone sodium and sulbactam sodium resistant strains,and to investigate the relationship between these different proteins and the resistance of Pseudomonas aeruginosa to cefoperazone sodium and sulbactam sodium.3.To understand the changes of genome before and after the drug-resistance to cefoperazone sodium and sulbactam sodium of Pseudomonas aeruginosa,and discover mutational sites of gene of drug-resistant strain of PAO1,and to investigate the relationship between these genetic mutations and the resistance of Pseudomonas aeruginosa to cefoperazone sodium and sulbactam sodium.Methods:1.Collect the information of Pseudomonas aeruginosa that isolated from Clinical Laboratory of the First Affiliated Hospital of Guangxi Medical University during 2013-2015,after to observe and analysis the drug sensitivity of Pseudomonas aeruginosa to cefoperazone sodium and sulbactam sodium in the 3 years,and a subgroup analysis was carried out in clinical departments which detect a large amount of Pseudomonas aeruginosa,to acquaint the changes of drug-resistance of Pseudomonas aeruginosa to cefoperazone sodium and sulbactam sodium in this clinical departments. 2.To induce PAO1 resistant to cefoperazone sodium and sulbactam sodium by the method of increase sub-inhibitory concentration (1/2 minimum inhibitory concentration) by degrees in vitro,and accompany a control-group of in-synchronism, after to process the whole cell protein solution of the experimental-group(3 strains) and the control-group(3 strains),and detect the whole cell protein solution by CM10 protein chip on Surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI),then analyze the informations of these whole cell proteins by Biomarker Wizard software and screen the different proteins between the two groups.3.We extract the genome DNA of the experimental-group(3 strains) and the control-group(3 strains),after to detect the nucleotide sequences of the genome DNA,then we discover the mutations by compare between the Basesequence of the two groups and the PAO1 standard strain gene sequence published by NCBI,at last,to explore the Bioinformatics information of the genes which have mutations.Results:1.In 2013,the drug-sensitivity test were carried out for 1306 strains of Pseudomonas aeruginosa isolated from Clinical Laboratory of the First Affiliated Hospital of Guangxi Medical University,and the drug sensitivity of Pseudomonas aeruginosa to cefoperazone sodium and sulbactam sodium were susceptible 52.4%(684/1306),intermediate 26.4%(345/1306) and resistance 21.2%(345/1306) respectively; in 2014,the drug-sensitivity test were carried out for 1149 strains of Pseudomonas aeruginosa isolated from Clinical Laboratory of the First Affiliated Hospital of Guangxi Medical University,and the drug sensitivity of Pseudomonas aeruginosa to cefoperazone sodium and sulbactam sodium were susceptible 54.0% (621/1149),intermediate 26.4% 24.6%(282/1149)and resistance 21.4%(246/1149)respectively;in 2015,the drug-sensitivity test were carried out for 1101 strains of Pseudomonas aeruginosa isolated from Clinical Laboratory of the First Affiliated Hospital of Guangxi Medical University,and the drug sensitivity of Pseudomonas aeruginosa to cefoperazone sodium and sulbactam sodium were susceptible 53.1%(585/1101),intermediate26.4%(345/1306)and resistance 21.2%(345 /1306)respectively.Subgroup analysis shows that ICU (intensive care unit),Traditional Chinese Medicine Department,Department of Burn and Department of respiratory medicine isolated a large number of Pseudomonas aeruginosa,and the drug-resistance rate of Pseudomonas aeruginosa isolated from ICU was the highest.2.Experimental-group compare with the control-group,26 different proteins were up-regulated (P<0.05),and their molecular weight are 1235Da,4439Da,4538Da,5145Da,6269Da,6569Da,8920Da, 8971 Da,9081 Da,9213Da,9297Da,9380Da,9861 Da,10126Da,10946Da,11037Da, 11138Da,11174Da,11255Da,11328Da,11522Da,11573Da,13520Da,13583Da,13 647Da and 13776Da respectively;15 different proteins were down-regulated (P<0.05),their molecular weight are 3790Da,3910Da,4439Da,5734 Da, 5787Da,5880Da,5946Da,6673Da,6906Da,7576Da,7788Da,8561Da,11731Da, 12276Da and 12352Da respectively.3.The Basesequence of experimental-group PAO1 compare with the PAO1 standard strain gene sequence published by NCBI,we found 792 mutations; while the Basesequence of control-group PAO1 compare with the PAO1 standard strain gene sequence published by NCBI,we found 141 mutations,when we exclude same gene mutation sites between the two groups,there are still 679 mutation sites only exist in the experimental-group. We analyze the 42 high quality mutations by compare with NCBI bioinformatics database, found those mutations exist in 35 genes, the expression products of these genes are enzymes, transcription factor, and so on, they are all involved in the metabolism of Pseudomonas aeruginosa.Conclusion:1.The resistance rate had gradually rising trend of Pseudomonas aeruginosa to cefoperazone sodium and sulbactam sodium during 2013-2015 in the First Affiliated Hospital of Guangxi Medical University,and the intermediate rate decreased year by year, the susceptible rate with slight fluctuations, the resistance rate of Pseudomonas aeruginosa to cefoperazone sodium and sulbactam sodium is not the same in different wards, the strains of Pseudomonas aeruginosa isolated from ICU had the highest resistance rate. 2. We can take advantage of the techniques of protein-chip and SELDI to detect different proteins between cefoperazone sodium and sulbactam sodium resistant strains and control-group strains,and different proteins were screened which may contribute to PAO1 resistant to cefoperazone sodium and sulbactam sodium. 3.There are a large number of mutations in genomic DNA of Pseudomonas aeruginosa after it resistant to cefoperazone sodium and sulbactam sodium;it’s a complicated process for PAO1 resistant to cefoperazone sodium and sulbactam sodium,it needed joint many genes and mechanisms to participate the process. |
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