| This study is divided into two parts:Part1:The role of las quorum sensing system in the formation of Pseudomonas aeruginosa biofilm. The differences of the expression of AmpC gene in Pseudomonas aeruginosa induced by antibiotics at planktonic and biofilm phases.Part2:The role of las quorum sensing system in antimicrobial induced ampC expression in Pseudomonas aeruginosa.The role of las quorum sensing system in the formation of Pseudomonas aeruginosa biofilm. The differences of the expression of AmpC gene in Pseudomonas aeruginosa induced by antibiotics at planktonic and biofilm phases.Objective:1.To study the differences in the shape of biofilms between pseudomonas aeruginosa standard strains PAO-1and the mutants in quorum sensing systems at different growth stages.2.To study the ampC gene expression of PAO1planktonic bacteria before and after antibiotics induction.3. To study the differences in the ampC gene expression between PAO1 planktonic bacteria and biofilm bacteria at different growth stages induced by antibiotics.Methods:Biofilm model was established by improved piping way. Biofilm was established on the nutrient solution infusion pump tube. Biofilm generation was identified by silver staining method,8h,1d,3d,6d,12d biofilm were observed by optical microscopy morphology. we characterized five stages of biofilm development: planktonic cells from the chemostat;8-h biofilm, reversible attachment;1-day biofilm, irreversible attachment;3-day biofilm, maturation-1;6-day biofilm, maturation-2; and12-day biofilm, dispersion. The MIC of planktonic bacteria and biofilm bacteria to imipenem and ceftazidime were measured by automatic susceptibility analyzer. First the concentrations of imipenem and ceftazidime that can induce planktonic bacteria and established8h,1d,3d,6d,12d biofilm bacteria producing the largest AmpC activity was measured. To induce AmpC gene expression, planktonic bacteria and established8h,1d,3d,6d,12d biofilm bacteria were cultured in medium containing the above concentrations of antibiotics. The ampC gene expression of PAO1planktonic bacteria before and after antibiotics induction was measured by real-time quantitative PCR. The ampC gene expression between PAO1planktonic bacteria and biofilm bacteria at different growth stages induced by antibiotics was measured.Results:There was short rod bacteria gathered piles each other and no black dye cotton wool material in the vision of PAO1initial attachment stage and irreversible attachment silver-stained biofilm. Mature biofilm could be seen at maturation-1stage and maturation-2stage, the cotton wool、unevenly distributed biofilm was dyed dark brown. PAO1silver stained biofilm photo of dispersion stage, The black dyed cotton wool biofilm subsided in dispersion stage. Strong black dyed floccules biofilm could be seen at maturation-2stage mature biofilm of mutant strains PDO100. Dark dyed biofilm was less and unevenly distributed can be seen at maturation-2stage biofilm of mutant strain PAO-JP1and PAO-MW1. The MIC of imipenem and ceftazidime to both Planktonic bacteria and various stages of biofilm bacteria were4mg/L, and the MIC of these two drugs to biofilm bacteria measured after ultrasonic vibration tuomo was not change. The concentration of imipenem inducing maximum AmpC activity of planktonic bacteria and8h、1d、3d、6d、12d biofilm bacteria was respectively2mg/L(1/2MIC),2mg/L(1/2MIC)、8mg/L(2MIC)、16mg/L(4MIC)、16mg/L(4MIC)、8mg/L(2MIC). The concentration of ceftazidime inducing maximum AmpC activity of planktonic bacteria and8h、1d、3d、6d、12d biofilm bacteria was respectively2mg/L(1/2MIC)、8mg/L(2MIC)、24mg/L(6MIC)、40mg/L (10MIC)、48mg/L (12MIC)、24mg/L (6MIC). The ampC gene expression levels without antibiotics induction in both PAO1planktonic bacteria and biofilm bacteria were low, there was no statistically significant difference between the two. Induced by antibiotics which concentration can induce the maximum ampC enzyme activity,the ampC gene expression level of PAO1planktonic bacteria were significantly elevated. The ampC gene expression levels of planktonic bacteria induced by imipenem were higher than that of ceftazidime,there was highly statistically significant difference between the two. Induced by imipenem which concentration can produce maximum AmpC enzyme activity,the ampC gene expression levels of various stages of biofilm PAO1bacteria except dispersion stage biofilm bacteria were higher than that of planktonic bacteria.With biofilm gradually maturing and thickening,the ampC gene expression levels induced by imipenem were increasing, the ampC gene expression level of maturation-2stage was the highest. In the dispersion stage,the ampC gene expression induced by imipenem decreased. The difference of ampC gene expression between dispersion stage and planktonic bacteria、initial attachment stage、irreversible attachment stage was not statistically significant.Induced by ceftazidime which concentration can produce maximum AmpC enzyme activity,the ampC gene expression levels of various stages of biofilm PAO1bacteria were higher than that of planktonic bacteria. With biofilm gradually maturing and thickening,the ampC gene expression levels induced by ceftazidime were increasing, the ampC gene expression level of maturation-2stage was the highest. In the dispersion stage,the ampC gene expression induced by ceftazidime decreased. The difference of ampC gene expression between dispersion stage and initial attachment stage was not statistically significant. AmpC gene expression levels of various stages PAO1biofilm bacteria induced by imipenem were higher than that of ceftazidime, the differences were statistically significant.Conclusion:1、The PA biofilm formation was related to the role played by the las Q.S system regulation through direct way or indirect way, rhl QS system was less or not involved in biofilm differentiation, while it was not excluding the unknown signal system involved.2, Induced by antibiotics,the ampC gene expression levels of various stages of biofilm PAO1bacteria except dispersion stage biofilm bacteria were higher than that of planktonic bacteria, imipenem induced the expression of ampC much more potently than ceftazidime.The role of las quorum sensing system in antimicrobial induced ampC expression in Pseudomonas aeruginosa.Objective:1、To study the effect of furanone C-30on PA1ampC gene expression levels induced by antibiotic.2、The comparison of ampC gene expression between PA1and mutants in quorum sensing systems induced by antibiotics.3、To study the effects of Las quorum sensing systems (QS) in the expression of AmpC gene induced by antibiotics in PA.Methods:In order to study the influence of Furanone C-30in PAO1AmpC gene expression induced by antibiotic, furanone C-30were added to the medium with antibiotics at the same time in the induction process. The concentration of furanone C-30in the medium were2.5μg/ml and5μg/ml。To study the differences in the ampC gene expression between pseudomonas aeruginosa standard strains PAO-1and the mutants in quorum sensing systems at different growth stages induced by antibiotics was measured by real-time quantitative PCR.The induced biofilm bacteria was tuomoed by ultrasonic oscillation (100W×15min).Results:The decrease of ampC gene expression level in maturation-2stage biofilm bacteria with2.5μg/ml and5μg/ml furanone C-30induced by antibiotics were more obvious than that of planktonic bacteria with2.5μg/ml and5μg/ml furanone C-30respectively. The decrease of ampC gene expression level in maturation-1stage biofilm bacteria with5μg/ml furanone C-30induced by imipenem was more obvious than that of planktonic bacteria with5μg/ml furanone C-30. The decrease of ampC gene expression level in maturation-2stage biofilm bacteria with2.5μg/ml furanone C-30induced by antibiotics was more obvious than that of initial attachment stage biofilm、dispersion stage biofilm with2.5μg/ml furanone C-30respectively. The decrease of ampC gene expression level in maturation-2stage and maturation-1stage biofilm bacteria with5μg/ml furanone C-30induced by imipenem were more obvious than that of initial attachment stage biofilm、dispersion stage biofilm with5μg/ml furanone C-30respectively. The decrease of ampC gene expression level in maturation-2stage biofilm bacteria with5μg/ml furanone C-30induced by ceftazidime was more obvious than that of initial attachment stage biofilm、 dispersion stage biofilm with5μg/ml furanone C-30respectively, no significant difference in ampC gene expression levels between PDO100and PAO1at planktonic stage and various biofilm stages induced by antibiotics. No significant difference in ampC gene expression levels between PAO-MW1and PAO-JP1at planktonic stage and the various biofilm stages induced by antibiotics. The ampC gene expression level of PAO-MW1and PAO-JP1were lower than that of PAO1and PDO100respectively at various biofilm stages. The above results were considered partly relating to the changes of biofilm structure and biofilm bacteria survival status.Conclusion:The ampc expression level of maturation-2stage biofilm bacteria was higher than that of planktonic bacteria and other stages of biofilm bacteria relating to different regulatory role played by las QS system in planktonic state and various biofilm states.The drop of ampC gene expression in mutant strain PAO-MW1and PAO-JP1 induced by antibiotic at biofilm stage partly relevant to the missing regulation of las QS system. |
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