| 1.Object To characterize N-acylhomoserine lactone and study the role in bacterial drug resistance in Acinetobacter Baumanii Ab S.2.Materials and methods The study is divided into three parts:(1)To characterize N-acylhomoserine lactones(AHLs)in Acinetobacter Baumanii.First,we incubated Agrobacterium tumefaciens KYC55 with AHLs extract from culture supernatant of Ab S which incubated at 4,8,16,24,32,40,48 H,and then the AHLs activity curve was built by ?-galactosidase activity detetion;at last,we characterized the AHLs molecule structure and validated with AHLs standard by use of high performance liquid chromatography-mass spectrometry(HPLC-MS).(2)To construct the AHLs mutant strain of Acinetobacter Baumanii Ab S--Ab S-M.First,we transfect the plasmid p KNG101.aba I::Km into E.coli SM10 to form E.coli SM10/p KNG101.aba I::Km;then,the plasmid p KNG101.aba I::Km was introduced into Ab S by filter mating with E.coli SM10/p KNG101.aba I::Km to form Ab S-M;at last,detect the difference between Ab S and Ab S-M with PCR electrophoresis,AHLs activity detecting and laser scanning confocal microscopy.(3)To observe the role of AHLs in Ab S drug resistance.First,test the minimal inhibitory concentration(MIC)of meropenem,piperacillin,ceftazidime,ciprofloxacin,trimethoprim sulfamethoxazole and minocycline to Ab S?Ab S-M?Ab S-M+AHLs;then,compare the gene OXA-51,Amp C,Ade A,Ade B,OXA-23,IMP-4,VIM-2,Ade C expression of Ab S?Ab S-M?Ab S-M+AHLs induced by meropenem with real time PCR.The SPSS 19.0 software was used to analyze the data with t test and variance analysis3.Result(1)We proved that Ab S can produce AHLs with blue display by parallel line incubation;the AHLs activity of Ab S was low(5.00±1.00 Miller Units)in 4H,reached the peak with 279.33±27.59 Miller Units in 8H,then descended gradually(16H:28.67±4.16 Miller Units)over time.But OD600 value of Ab S reached the peak with 0.90±0.01 in 24 H incubation,then entered the stationary phase.And Ab S can produced only one kind of AHLs,N-3-OH-C12-HSL confirmed with HPLC-MS.(2)Suceeded in constructing the AHLs mutant strain of Acinetobacter Baumanii,Ab S--Ab S-M.And verified the mutant strain with PCR electrophoresis and AHLs activity detecting.In PCR electrophoresis,a belt can be observed in 400 bp position in Ab S which was not found in Ab S-M.The mutant strain Ab S-M AHLs activity(12.67±1.53 Miller units)was lower than wide type strain Ab S(255.67±16.01 Miller units)(P<0.01).With laser confocal microscopy,Ab S was easy to gather into mass and Ab S-M was likely to be suspension.The biofilm thickness of the Ab S-M(5.96±0.56?m)was thinner than Ab S biofilm thickness(7.75±0.48?m)(P<0.05).(3)The MIC of meropenem(0.25?g/ml),piperacillin(1?g/ml)to mutant strain Ab S-M is lower than MIC to wide type strain Ab S(meropenem,0.5?g/ml;piperacillin,2?g/ml),and the MIC of meropenem(0.5 ? g/ml)and piperacillin(2 ? g/ml)to Ab S-M was restored after the culture medium was added AHLs.There was no difference of MIC of ceftazidime,ciprofloxacin,trimethoprim/ sulfamethoxazole and minocycline between Ab S,Ab S-M and Ab S-M+AHLs.Gene OXA-51?Amp C?Ade A?Ade B expression was detected in Ab S group,Ab S-M group and Ab S-M+AHLs group.The gene expression in mutant strain Ab S-M(OXA-51,0.68±0.04;Amp C,0.60±0.04;Ade A,0.59±0.08;Ade B,0.51±0.09)was much lower than wide type strain Ab S(OXA-51,1.09±0.13;Amp C,0.94±0.11;Ade A,1.17±0.17;Ade B,1.08±0.16),and the expression in Ab S-M+AHLs(OXA-51,1.74±0.04;Amp C,1.55±0.04;Ade A,1.66±0.25;Ade B,1.31±0.11)was restored.Gene OXA-23?IMP-4?VIM-2?Ade C expression was not detected in Ab S group,Ab S-M group and Ab S-M+AHLs group.4.Conclusion We proved that Ab S only produce one kind of AHL,N-3-OH-C12 HSL.N-3-OH-C12 HSL can regulate the gene OXA-51?Amp C?Ade A?Ade B expression to affect drug resistance : low N-3-OH-C12 HSL activity can inhibit the gene expression and high N-3-OH-C12 HSL activity can promote the gene expression.And Ab S may become pan resistant strain through gene Ade A?Ade B expression promoted by N-3-OH-C12 HSL with meropenem inducing. |
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