高渗对多重耐药大肠杆菌外排泵和外膜孔道蛋白基因表达及其生长的影响

Effect of Hypertonicity on Gene Expression of Efflux Pump and Outer Membrane Protein in Multidrug Resistant Escherichia coli and Its Growth

为了探究高渗对多重耐药菌外排泵和外膜孔道蛋白基因表达及其生长的影响,试验应用生长曲线和相对适应性测定,对比考察不同盐胁迫条件对多重耐药大肠杆菌QL15和敏感大肠杆菌ATCC 25922的生长差异,采用RT-PCR方法考察2种盐胁迫条件下外排泵与外膜孔道蛋白基因表达差异。结果表明,大肠杆菌QL15为高水平多重耐药菌,其中对大观霉素和恩诺沙星耐药最为严重,可达耐药标准MIC的32倍;独立培养时大肠杆菌QL15对NaCl胁迫的敏感性大于大肠杆菌ATCC 25922,并且在6.0%NaCl胁迫下,大肠杆菌QL15在10 h内生长缓慢,但是在24 h细菌峰值浓度更高;混合培养时大肠杆菌QL15在6.0%NaCl胁迫时具有更高的适应性,在体外培养中更具竞争优势。大肠杆菌QL15共携带5大类、15种耐药相关基因,其中含8种外排泵基因和3种外膜孔道蛋白基因;大肠杆菌ATCC 25922在6.0%NaCl浓度下的5种基因表达量较3.5%NaCl均出现约50%的显著下调(P<0.05),大肠杆菌QL15在6.0%NaCl浓度下除acrB、ompF基因下调外,其余3种外排泵与外膜孔道蛋白基因的表达量则显著上调(P<0.05)。推测大肠杆菌QL15对盐胁迫具有更高的适应性的原因可能与细胞膜相关蛋白的表达相关。

In order to explore the effects of hypertonic stress on gene expression and growth of efflux pump and outer membrane protein of multidrug-resistant bacteria,growth curve and relative adaptability were used to compare the effects of different salt stress conditions on resistant Escherichia coli QL15 and sensitive Escherichia coli ATCC 25922.RT-PCR was used to investigate the difference of gene expression of efflux pump and outer membrane protein under salt stress.The results showed that Escherichia coli QL15 was a high-level multidrug-resistant bacterium,and the most serious resistance to spectinomycin and enrofloxacin was 32 times of the standard MIC.When cultured independently,Escherichia coli QL15 was more sensitive to salt stress than Escherichia coli ATCC 25922,and under 6.0%NaCl stress,Escherichia coli QL15 grew slowly within 10 h,but its concentration was higher at 24 h.When cultured in mixture,Escherichia coli QL15 had higher adaptability to 6.0%NaCl stress,and had more competitive advantage in vitro culture.Escherichia coli QL15 carried 15 drug resistance related genes in 5 categories,including 8 efflux pump genes and 3 outer membrane protein genes.The expression levels of 5 genes in Escherichia coli ATCC 25922 at 6.0%NaCl concentration were about 50%lower than that in 3.5%NaCl concentration(P<0.05).However,the expression of Escherichia coli QL15 were significantly up-regulated at 6.0%NaCl concentration except acrB and ompF genes(P<0.05).It was speculated that the higher adaptability of Escherichia coli QL15 to salt stress might be related to the expression of membrane associated proteins.