生物膜铜绿假单胞菌MexAB-OprM及MexR的表达
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摘要
目的研究铜绿假单胞菌在浮游态、生物膜态及经亚胺培南、环丙沙星诱导下,外排泵MexAB-OprM及其负性阻遏蛋白MexR表达情况。
方法用铜绿假单胞菌PA01在eppendorf管内建立生物膜,通过结晶紫快速染色法证实生物膜的形成。用1/2MIC的亚胺培南、环丙沙星对铜绿假单胞菌浮游菌诱导4小时;4MIC的亚胺培南、2MIC的环丙沙星对铜绿假单胞菌生物膜菌诱导12小时,分别于生物膜形成的第3天及第5天,用实时荧光定量PCR的方法,测定铜绿假单胞菌外排泵MexAB-OprM及MexR的基因表达。
结果铜绿假单胞菌由浮游菌变为生物膜菌后(第3天和第5天),其外排泵MexAB-OprM的表达水平明显降低,其相对表达量最高相差近10倍,其差别存在统计学意义(P<0.05),而生物膜菌第3天和第5天相比,尽管MexAB-OprM相对表达量相差近3倍,但差别无统计学意义(P>0.05)。铜绿假单胞菌由浮游菌变为生物膜菌后(第3天和第5天),阻遏蛋白MexR表达差别无统计学意义(P>0.05)。生物膜菌第3天和第5天,其MexAB-OprM和MexR表达差别无统计学意义(P>0.05)。在亚胺培南及环丙沙星诱导后,浮游菌和生物膜菌其基因MexAB-OprM、MexR表达差别均无统计学意义(P>0.05)。
结论铜绿假单胞菌由浮游菌转变为生物膜菌后,外排泵MexAB-OprM表达明显减少,MexR基因表达无影响。生物膜菌的第3天与第5天,其MexAB-OprM、MexR基因表达无差别。亚胺培南、环丙沙星对浮游菌及生物膜菌的MexAB-OprM、MexR均无诱导作用。
Objective To study the expression of MexAB-OprM and its repressor protein-MexR in planktonic and biofilm bacteria of Pseudomonas aeruginosa when exposed to imipenem and ciprofloxacin.
Methods The P.aeruginosa strain PAO1 was used in this study.In vitro models of Pseudomonas aeruginosa bacterial biofilm were built up in eppendorf tube and a rapid staining procedure of crystal violet was used to verify them.The test was developed under two groups,planktonic and biofilm bacteria of Pseudomonas aeruginosa.The planktonic were exposed to 1/2MIC imipenem and ciprofloxacin for 4 hours;The biofilms were exposed to 4 MIC imipenem and 2 MIC ciprofloxacin for 12 hours,the expression of multiple drugs efflux pumps MexAB-OprM and its repressor protein MexR were detected by using the method of Real-time quantitative PCR at the days of 3 and 5 after biofilms forming.
Results When the P.aeruginosa strain PAO1 transferred its growth mode from planktonic to biofilms(days of 3 and 5),the efflux pump MexAB-OprM gene expression level was significantly decreased(P<0.05),the highest relative quantity level was nearly 10 folds decreased compared to the lowest.The difference was significant between planktonic and biofilms.As the biofilms on the days of 3 and 5,though the relative quantity was 3 folds lower,but it was not different.The expression level of repressor protein MexR had no difference between planktonic and bioiflms(days of 3 and 5)(P>0.05).The expression of MexAB-OprM and MexR of biofilms on the days of 3 and 5 had no statistical significance(P>0.05).The expression level of MexAB-OprM and MexR,none was different after imipenem and ciprofloxacin induced both in planktonic and biofilms.
Conclusion When pseudomonas aeruginosa PAO1 transferred its growth mode from planktonic to biofilms,the expression level of efflux pumps MexAB-OprM was greatly decreased,the MexR had no change.There had no difference of MexAB-OprM and MexR between 3days and 5days after biofilm forming.Imipenem and ciprofloxacin had no effect on the expression of MexAB-OprM and MexR both in planktonic and biofilms.
方法用铜绿假单胞菌PA01在eppendorf管内建立生物膜,通过结晶紫快速染色法证实生物膜的形成。用1/2MIC的亚胺培南、环丙沙星对铜绿假单胞菌浮游菌诱导4小时;4MIC的亚胺培南、2MIC的环丙沙星对铜绿假单胞菌生物膜菌诱导12小时,分别于生物膜形成的第3天及第5天,用实时荧光定量PCR的方法,测定铜绿假单胞菌外排泵MexAB-OprM及MexR的基因表达。
结果铜绿假单胞菌由浮游菌变为生物膜菌后(第3天和第5天),其外排泵MexAB-OprM的表达水平明显降低,其相对表达量最高相差近10倍,其差别存在统计学意义(P<0.05),而生物膜菌第3天和第5天相比,尽管MexAB-OprM相对表达量相差近3倍,但差别无统计学意义(P>0.05)。铜绿假单胞菌由浮游菌变为生物膜菌后(第3天和第5天),阻遏蛋白MexR表达差别无统计学意义(P>0.05)。生物膜菌第3天和第5天,其MexAB-OprM和MexR表达差别无统计学意义(P>0.05)。在亚胺培南及环丙沙星诱导后,浮游菌和生物膜菌其基因MexAB-OprM、MexR表达差别均无统计学意义(P>0.05)。
结论铜绿假单胞菌由浮游菌转变为生物膜菌后,外排泵MexAB-OprM表达明显减少,MexR基因表达无影响。生物膜菌的第3天与第5天,其MexAB-OprM、MexR基因表达无差别。亚胺培南、环丙沙星对浮游菌及生物膜菌的MexAB-OprM、MexR均无诱导作用。
Objective To study the expression of MexAB-OprM and its repressor protein-MexR in planktonic and biofilm bacteria of Pseudomonas aeruginosa when exposed to imipenem and ciprofloxacin.
Methods The P.aeruginosa strain PAO1 was used in this study.In vitro models of Pseudomonas aeruginosa bacterial biofilm were built up in eppendorf tube and a rapid staining procedure of crystal violet was used to verify them.The test was developed under two groups,planktonic and biofilm bacteria of Pseudomonas aeruginosa.The planktonic were exposed to 1/2MIC imipenem and ciprofloxacin for 4 hours;The biofilms were exposed to 4 MIC imipenem and 2 MIC ciprofloxacin for 12 hours,the expression of multiple drugs efflux pumps MexAB-OprM and its repressor protein MexR were detected by using the method of Real-time quantitative PCR at the days of 3 and 5 after biofilms forming.
Results When the P.aeruginosa strain PAO1 transferred its growth mode from planktonic to biofilms(days of 3 and 5),the efflux pump MexAB-OprM gene expression level was significantly decreased(P<0.05),the highest relative quantity level was nearly 10 folds decreased compared to the lowest.The difference was significant between planktonic and biofilms.As the biofilms on the days of 3 and 5,though the relative quantity was 3 folds lower,but it was not different.The expression level of repressor protein MexR had no difference between planktonic and bioiflms(days of 3 and 5)(P>0.05).The expression of MexAB-OprM and MexR of biofilms on the days of 3 and 5 had no statistical significance(P>0.05).The expression level of MexAB-OprM and MexR,none was different after imipenem and ciprofloxacin induced both in planktonic and biofilms.
Conclusion When pseudomonas aeruginosa PAO1 transferred its growth mode from planktonic to biofilms,the expression level of efflux pumps MexAB-OprM was greatly decreased,the MexR had no change.There had no difference of MexAB-OprM and MexR between 3days and 5days after biofilm forming.Imipenem and ciprofloxacin had no effect on the expression of MexAB-OprM and MexR both in planktonic and biofilms.
引文
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