| Part I Intervention of tea polyphenols on drug-resistant Acinetobacter baumannii biofilm in vitroObjective To study the destructive effect and mechanism of tea polyphenols(TP)on planktonic drug-resistant Acinetobacter baumannii(A.baumannii)and A.baumannii biofilm(BF).Methods The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of TP against planktonic drug-resistant A.baumannii were determined by broth dilution method and dilution coating plate method respectively.Meanwhile,the growth inhibition curve was determined.The destructive effect of TP on A.baumannii was detected by fluorescence microscopy,and the structure of bacteria was observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The growth curve of A.baumannii BF was drawn by ultraviolet spectrometry.The influence of TP on the formation of biofilm was measured by Crystal violet(CV)staining method.In addition,the effect of TP on the number of viable bacteria in biofilm was examined by MTT assay.The effects of TP on total extracellular polysaccharides and protein content of A.baumannii BF were evaluated by phenol-sulfuric acid method and Coomassie brilliant blue method separately.The relative m RNA expression of aba I,Bap,Csu E and Omp A related to BF formation in A.baumannii was detected by Q-PCR.Results The MIC and MBC of TP against planktonic drug-resistant A.baumannii were 750 ?g/m L and 3000 ?g/m L respectively.The growth of A.baumannii was significantly inhibited by TP at MIC,and the inhibition was gradually weakened at 1/2 MIC.It could be seen clearly under fluorescence microscope that the number of planktonic drug-resistant A.baumannii was significantly reduced by TP administration.SEM and TEM indicated that the cell membrane structure of bacteria was destroyed by TP with the contents of cells flowing out,etc.Plate culture method was used to establish biofilm model of A.baumannii in vitro.Initial BF and mature BF formed after 9 and 48 hours of culture respectively.TP treatment with 1/8 MIC for 12 h inhibited the formation of initial BF(p<0.05),and TP treatment with MIC for 24 h decreased the number of viable bacteria in initial BF(p<0.05).The inhibitory effect of TP treatment with 1/4 MIC on mature biofilm was demonstrated after 6 hours.Moreover,TP treatment with 1/2 MIC for 6 h reduced the number of viable bacteria in mature BF.Total extracellular polysaccharides in TP group and control group were 16.8982 ± 1.4178 VS 22.1154 ± 3.7127,respectively.The total extracellular polysaccharide content of TP group decreased(F=323.3,P<0.01).The content of extracellular protein in TP group and control group were 15.1456 ± 1.818 VS 22.3139 ± 1.2210),respectively.The content of extracellular protein in TP group decreased(F=11.18,p<0.01).In TP group,the m RNA expression of genes associated with BF formation including aba I,Bap,Csu E and Omp A was 0.3928 ± 0.0560(F=255.8,p<0.01),0.1477 ± 0.0051(F=1549,p<0.01),0.4517 ± 0.0806(F=38.57,p<0.01)and 0.4616 ± 0.0494(F=126.9,p<0.01),respectively.Compared with control group,the m RNA expression of aba I,Bap,Csu E and Omp A in TP group decreased significantly.Conclusion TP could inhibit the growth of planktonic drug-resistant A.baumannii and destroy the structure of bacteria.In addition,TP not only inhibited the formation of initial BF in A.baumannii,destroyed the structure of mature BF,but also reduced the number of live bacteria in BF.In conclusion,TP was able to destroy A.baumannii BF by reducing the content of total extracellular polysaccharides and proteins as well as inhibiting the expression of aba I,Bap,Csu E and Omp A genes related to BF formation.Part II Intervention of tea polyphenols on catheter-related drug-resistant Acinetobacter baumannii biofilm infection in vivoObjective To establish a model of infection induced by BF of tracheal intubation-related drug resistance A.baumannii in SD rats and investigate the effect of TP on A.baumanni BF and pulmonary infection in vivo.Methods SPF Sprague Dawley rats were divided into four groups randomly with eight in each group: the Normal group,rats with sterile tube intubation;the Control group,rats with biofilm-covered tube intubation as model;the TP group,rats with biofilm-covered tube intubation were given tea polyphenols;the SCF group,rats with biofilm-covered tube intubation were given SCF.Surgical models were established by invasive tracheal intubation.The rats in TP group and SCF group were administered for 7 days respectively,and their weight changes were recorded at a fixed time every day.All rats were sacrificed after 8 days.The catheters were removed for living bacterial counting in BF by using dilution coating plate method.Furthermore,the biofilm structure of catheters was observed by SEM.The morphological changes of lung and trachea were observed,and the number of bacteria in lung tissue was detected.The pathological changes of lung tissue was evaluated by Hematoxylin-eosin staining(HE)staining.The levels of TNF-?,IL-1? and IL-6 in serum was detected by Enzyme linked immunosorbent assay(ELISA).Fluorescence quantitative polymerase chain reaction(Q-PCR)was used to detect the relative m RNA expression of TNF-?,IL-1?,IL-6,My D88,p65 and TGF-?,which related to inflammation.Relative expression of inflammatory proteins in lung tissues such as My D88 and p65 was measured by performing Immunohistochemistry(IHC)and Western blotting(WB).Results During the whole experiment period,the weight of rats in the control group increased most slowly.By comparison,the weight of rats in the TP group increased significantly,and the weight of rats in the Normal group increased fastest.The number of bacteria in BF of TP group and Control group were 8.8266 ± 0.8724 lg CFU VS 11.0234 ± 0.6487 lg CFU),respectively.By contrast,the number of bacteria in TP group decreased significantly(F=71.57,p<0.05).SEM results showed that BF on the catheter of control group was complete and compact,with a large amount of mucous matrix.That of TP group was obviously broken and reduced.The appearance of lung and trachea in Normal group was normal.In control group,necrosis and bleeding were found in the lungs,and there were obvious abscess in the trachea at the intubation site.There was no lung necrosis in TP group with the significant reduction of bleeding,and the intubation site recovered well.The number of bacteria in lung tissue of TP group and Control group were 5.3821 ± 0.537 lg CFU VS 6.0289 ± 0.6487 lg CFU,respectively.The number of bacteria in TP group decreased significantly(F=16.51,p<0.05).HE results revealed that the lung tissue structure of Normal group was normal without inflammatory cells,and alveolar cavity together with alveolar wall were normal.By contrast,the lung tissue structure of Control group exhibited obvious destruction,inflammatory cell infiltration,alveolar cavity rupture,dilation,alveolar wall thinning.In TP group,the lung tissue structure recovered significantly,inflammatory cell infiltration decreased markedly,alveolar cavity and alveolar wall tended to be normal.ELISA results indicated that the levels of TNF-? in the Control and TP group were VS 199.9334 ± 51.33335 pg/m L VS 65.6 ± 17 pg/m L,respectively.The contents of IL-1? were 651.06 ± 102 pg/m L VS 410.06 ± 101 pg/m L,respectively.The contents of IL-6 were 809.6 ± 245 pg/m L VS 182 ± 58.6 pg/m L.Compared with the control group,TP could significantly reduce the serum levels of TNF-?(F=10.02,P<0.05),IL-1?(F=5.628,P<0.05)and IL-6(F=8.037,P<0.05).The results of Q-PCR demonstrated that the relative m RNA expression of TNF-? in TP and Control group were 2.6225 ± 0.5376 VS 5.8288 ± 1.4819,respectively.The relative expression of IL-1? were 3.3794 ± 1.6140 VS 8.2196±2.4838,respectively.The relative expression of IL-6 were 1.3779 ± 0.1804 VS 2.4171 ± 0.4310,respectively.The relative expression of My D88 were 1.5195 ± 0.1827 VS 2.5807 ± 0.2674,respectively.The relative expression of p65 were 1.8729 ± 0.1202 VS 2.7811 ± 0.1110.The relative expression of TGF-? were 0.9036 ± 0.2158 VS 2.3340 ± 0.4937. Compared with the control group,TP evidently suppressed the relative expression of TNF-?(F=13.28,p<0.01),IL-1?(F=7.206,p< 0.05),IL-6(F=11.28,p<0.01),My D88(F=22.08,p<0.01),p65(F=23.18,p<0.01)and TGF-?(F=5.852,p<0.01).IHC results showed that the expression of My D88 and p65 in TP group was significantly lower than that in Control group,while the expression of My D88 and p65 in Normal group was the lowest.WB results were consistent with IHC results.Conclusion The BF infection model of SD rats with tracheal intubation-related drug resistance A.baumannii was successfully established.TP was provided with the functions to destroy the structure of BF on tracheal intubation catheter in rats,remove BF and reduce the number of bacteria in BF.TP could improve pulmonary inflammation in BF-infected rats with the mechanism that it could not only decrease the levels of TNF-?,IL-1? and IL-6 in serum,but also inhibit the expression of TNF-?,IL-1?,IL-6,My D88,p65 and TGF-? in lung tissues. |
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