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1、Characteristics of Escherichia coli biolm production, genetic typing ,drug resistance pattern and gene expression under aminoglycoside pressures,Escherichia coli 大肠杆菌 infection 感染 Aminoglycoside 氨基糖苷类 antibiotic 抗生素 Pseudomonas 假单胞菌 aeruginosa 聚合酶链反应技术 Crystal violet staining method 结晶紫染色法 phenotype
2、 表现型 drug resistance pattern耐药模式 Antimicrobial agent 抗菌药物 molecular biology reagent 分子生物学试剂 Broth microdilution method 肉汤稀释法 Susceptibility testing 药敏试验 Genotype 基因型 Real-time qRT-PCR 实时定量荧光PCR Streptomycin 链霉素 amikacin 丁胺卡那霉素 Gentamycin 庆大霉素 apramycin 安普霉素,1. New words,Background,In addition, under
3、 extensive and persistent pressure of antibiotics, in order to survive, the E. coli forms biolm to evade the immune clearance and results in multiple antibiotics resistance. Hoffman et al. (2005) found that subinhibitory levels of the aminoglycoside antibiotics could induce E. coli and Pseudomonas a
4、eruginosa biolm formation. Some other studies also revealed that biolm formation by E. coli in vitro correlated with the virulence factors (Naves et al., 2008; Rijavec et al., 2008).,Abstract,In this study, qualitative (scanning electron microscope) and semi-quantitative (modied crystal violet stain
5、ing method) methods had been used to evaluate Escherichia coli biolm-forming ability. Broth microdilution method and enterobacterial repetitive intergenic consensus-based PCR (ERIC-PCR) were performed to study E. coli drug resistance pattern and genetic typing. Based on the results above, we studied
6、 the correlation between biolm-forming ability phenotype, drug resistance pattern and genetic typing in E. coli. Real-time qPCR (qRT-PCR) was used to reveal mRNA expression level of E. coli biolm related multiple antibiotics resistance genes (acrA, agn43, csgA, csgD, ompF and pgaA) under different c
7、oncentrations of four aminoglycoside pressures.,Contents,1. Objective,The primary aims of this study were to investigate: (i) The E.coli biolm-forming ability based on the qualitative (scanning electronmicroscope) and quantitative (modied crystal violet staining method) methods. (ii) The correlation
8、s between E. coli genotype,drug resistance pattern and biolm-forming ability phenotype. (iii)Changes of mRNA level of acrA, agn43, csgA, csgD, ompF and pgaA under persistent and extensive antibiotic pressures using qRT-PCR method.,2. Materials and methods,37 C,64 E. coli strains,tryptic soy brot 胰蛋白
9、酶大豆肉汤,2.1. Bacterial strains,2.2.Antimicrobial agents and molecular biology reagents,10 antimicrobial agents,TRIzol reagent Kit,SYBR Prime Script RT-PCR Kit,Prime Script RT reagent Kit,other molecular biology reagents for qRT-PCR and universal PCR,2.3. Study design and experimental approach,2.3.1. S
10、emi-quantitative biolm formation experiment,Semi-quantitative 半定量的,Spectrophotometer Reader 分光光度计,2.3.2. Scanning electron microscope (SEM),SEM 扫描电子显微镜,2.3.3. Correlation between drug resistance pattern and biolm-forming ability,Broth microdilution method was used for antimicrobial drug susceptibili
11、ty testing of 10 antimicrobial agents to 64 clinical isolates and the experimental data was statistically analyzed by WHONET5.3 software. E. coli ATCC25922 was used as reference strain for quality controls Based on the results of semi-quantitative biolm formation experiment in Section 2.3.1, we focu
12、sed on whether biolm formation was related to drug resistance patterns (Moskowitz et al., 2004; Frank et al., 2007; Suman et al., 2007).,2.3.4. Correlation between genotype and biolm-forming ability,2.3.5. RNA extraction and real-time qRT-PCR,3.1. Biolm-forming ability of 64 E. coli,3.Results,3.2. S
13、EM photograph of strain E53,3.3. Drug resistance pattern and biolm-forming ability,3.4. ERIC-PCR and biolm-forming ability,3.5. mRNA levels of acrA, agn43, csgA, csgD, ompF and pgaA under four aminoglycoside pressures,3.5. mRNA levels of acrA, agn43, csgA, csgD, ompF and pgaA under four aminoglycosi
14、de pressures,3.5. mRNA levels of acrA, agn43, csgA, csgD, ompF and pgaA under four aminoglycoside pressures,4.Conclusions,Form above results,we can draw these conclusions :,(iii) qRT-PCR revealed mRNA expression of acrA, agn43, csgA, csgD, ompF and pgaA genes changed accordingly by stimulation of di
15、fferent concentrations of four aminoglycosides. Design Inc.,(ii) ERIC-PCR showed that there was signicant correlation between biolm-forming ability and genotype; while there was weak correlation between biolm-forming ability and drug resistance patterns based upon the results of semi-quantitative method and antibiotics susceptibility test; Design Inc.,(i) forty-nine out of 64 strains of E. coli (76.56%) showed signicant production of biolm and most of them performed weak biolm forming ability,Thank You !,
