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,Several types of
3、infection caused by Escherichia coli (e.g., urethral catheter, biliary tract prosthesis, biliary tract infection, lithangiuria, urinary tract infection andmany other diseases) are associated with biolm formation,which leads to an inability to eradicate the infection due to its intrinsic nature to re
4、sist high levels of antibiotics (Fontaine and Smith, 2006; Melchior et al., 2006),,Background,In addition, under 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.
5、 (2005) found that subinhibitory levels of the aminoglycoside antibiotics could induce E. coli and Pseudomonas aeruginosa 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).,,Abst
6、ract,In this study, qualitative (scanning electron microscope) and semi-quantitative (modied crystal violet staining method) methods had been used to evaluate Escherichia coli biolm-forming ability. Broth microdilution method and enterobacterial repetitive intergenic consensus-based PCR (ERIC-PCR) w
7、ere performed to study E. coli drug resistance pattern and genetic typing. Based on the results above, we studied 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. co
8、li biolm related multiple antibiotics resistance genes (acrA, agn43, csgA, csgD, ompF and pgaA) under different concentrations 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 (
9、scanning electronmicroscope) and quantitative (modied crystal violet staining method) methods. (ii) The correlations 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 extensi
10、ve antibiotic pressures using qRT-PCR method.,,2. Materials and methods,37 C,64 E. coli strains,tryptic soy brot 胰蛋白酶大豆肉汤,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 Ki
11、t,other molecular biology reagents for qRT-PCR and universal PCR,,2.3. Study design and experimental approach,2.3.1. Semi-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
12、drug resistance pattern and biolm-forming ability,Broth microdilution method was used for antimicrobial drug susceptibility 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 s
13、train for quality controls Based on the results of semi-quantitative biolm formation experiment in Section 2.3.1, we focused 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 b
14、iolm-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. SEM 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
15、 and pgaA under four aminoglycoside pressures,,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 aminoglycoside pressures,,4.Conclusions,Form above results,we can draw these conclusion
16、s :,(iii) qRT-PCR revealed mRNA expression of acrA, agn43, csgA, csgD, ompF and pgaA genes changed accordingly by stimulation of different concentrations of four aminoglycosides. Design Inc.,(ii) ERIC-PCR showed that there was signicant correlation between biolm-forming ability and genotype; while t
17、here 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 !,
