| The bacteria biofilm is a cell community with a kind of solid structure, it is formed by the polymer which secreted by itself, and packed after the bacterial adhesion to abiotic or biological surface. The main component of the extracellular polymer which forms the bacteria biofilm involves polysaccharides and dissociative DNA, RNA, protein, fat and other substances. Escherichia Coli are important pathogenic bacteria in hospital infection, and it is also the common pathogen to biofilm infection. Escherichia Coli which have formed the biofilm shows highly resistant to escape the immune system attacks, the infection of Escherichia Coli is easily to be chronic and difficult to control. The mechanism of biofilm drug-resistant mainly involves penetration obstacles, special physical condition of intramembranous bacteria not being sensitive to antimicrobial agents and high expression from part of the drug-resistant gene. During the process of bacterial biofilm formation, through the QS (Quorum Sensing) Regulation,the bacteria lyses and releases great amount of dissociative DNA,which forms the skeletal structure of bacterial biofilm, and maintains the biofilm space conformation. High concentrations of bacteria cells in biofilm are in close contacting state, plus the high concentrations of dissociative DNA, which provides favorable conditions for passing on genes in bacterial biofilm.Previously, with a specific resistance gene as the goal, through the gene distribution in different bacterial species in different periods, we concluded that between different bacterial species, it is possible for the occurrence of gene transinformation in genome. Because of the low incidence,there ware no direct observation to prove that. So far, transfer phenomena observation of the genes transmission in the bacterial biofilm often adopts the plasmid with characteristics of obvious selection, the bacteria which has been reported includes Alcaligenes Eutrophus, Pseudomonas Aeruginosa, Lactococcus Lactis, Acinetobacter Sp. and Streptococcus Mutans, etc. Among them, the Alcaligenes Eutrophus, Pseudomonas Aeruginosa, Lactococcus Lactis is able to occur transfer bonding, The Acinetobacter and Streptococcus Mutans is able to be formed to competence under natural conditions and transform; under natural conditions, even if that does not form the competence Escherichia Coli,it could also observed plasmid transmission during the biofilm formation process. Compared with the planktonic cells, the plasmid transmission in biofilm showed the characteristics of high efficiency, up to tens of thousands of times. In bacterial biofilm, the occurrence of this gene transmission may also be the main way for bacteria to gain the resistance gene, and as the biofilm dissociation, bacterial strain which gained the resistance gene would cause the dissemination of the drug-resistance, which brings more damage. At the same time, these studies indicates that the biofilm formation is the important guarantee for gene transmission between bacteria, therefore, the regulation factor which can affect the biofilm formation is bound to occur regulation to the gene delivery in the biofilm. The study of its influencing factors and mechanisms can contribute to prove the knowledge of the drug-resistant genes spreading in the biofilm, but it is rarely reported.This study is aimed to Escherichia Coli,and is to research the two important regulatory genes LuxS and Pga operon in biofilm formation, they are encoded on the signaling molecules synthase of quorum sensing system in escherichia coli and theΒ-1-6-N-Acetyl - dextran amines ynthase, The major components of polysaccharide in biofilm, which plays an important regulatory role during the process of initial formation adhesion and maturation of the bacterial biofilm. In this study, the wild type of Escherichia Coli from clinical separation is provided by the first hospital of third military medical university,its drug-resistance, the capability of the biofilm formation and influencing factors to the biofilm formation from different medium was analyzed; we studied the drug-resistant plasmid transmission frequency from the clinical isolates strains and the standard Escherichia Coli bacteria in biofilm formation; through the fluorescence quantitative PCR, we analyzed the influence to the expression of main regulation gene LuxS the Pga from the LB medium with sugar content of 0.5%;construct the quorum sensing signal synthase gene LuxS knockout strain of Escherichia Coli CAG18439, study the changes of drug-resistant plasmid transmission in the gene luxS knockout strain, and investigate its mechanism.The Study Is Divided Into 4 Steps:1. The study of drug-resistance and the capability of biofilm formation in Escherichia Coli from clinical separation;2. The influence to the drug-resistant plasmid transmission in Escherichia Coli biofilm from different culture mediums;3. The biofilm formation of Escherichia Coli from clinical separation and the expression of the main regulation gene Lux and Pga;4. The influence to drug-resistant plasmid transmission in Escherichia Coli biofilm and possible mechanism from knocking out the gene luxS.Material And Method1.MeterialThere are 127 strains of Escherichia Coli from clinical separation (s1,s2,s3……S127),which are all separated in the first hospital of third military medical university, from inpatient clinical specimens, from july, 2005 to december, 2007; Escherichia Coli ATCC25922,DH5Α(Pgreentir-) (F-,Φ80d Lacz△M15,△(Laczya-Argf)U169, Deor, Reca1, Enda1, Hsdr17(Rk-,Mk+ ) and MG1655 was provided from national drug clinical research base of chong qing southwest hospital;Escherichia Coli CAG18439(F, Lacz118(Oc), Laci3042::Tn10, LAM- Rph-1) was presented by professor xu qishou from the military academy of medical sciences;DH5Α(Pgreentir+),which contains the nuclear green fluorescent expressing the mating type plasmid, was presented by professor li guo from viruses institute in wu han university.2.Method2.1 The study of drug-resistance and the capability of biofilm formation in Escherichia Coli from clinical separation.2.1.1 The MIC evaluation of 10 kinds of commonly used drugs to the 127 strains of Escherichia Coli from clinical separationUse Ceftazidime, Chloramphenicol, Kanamycin, Tetracycline, Aztreonam, Cefoperazone, Left Oxygen Sand Stars, Ampicillin, Ampicillin / Clavulanic Acid and Imipenem to do the sensitivity experiments on Escherichia Coli From Clinical Separation, adopt the agaragar multiple proportions method;2.1.2 in 127 strains of Escherichia Coli from clinical separation, do the PCR detection onΒ-Lactamase drug-resistant strains carrying commonΒ-Lactamase gene.According to the result from the drug sensitive test of 127 strains of the Escherichia Coli, select ceftazidime and cefoperazone resistant strains as well as the Ampicillin / Clavulanic Acid with concentration of 256ΜG/Ml, which can reverse the Ampicillin-Resistant strains. Based on the above criteria, select the drug-resistant Escherichia Coli, detect TEM and the seven kinds ofΒ-Lactamase genes, use PCR amplification, verify agarose gel electrophoresis, select a positive product for sequence; the homology analysis of theΒ-Lactamase Gene in Escherichia Coli from clinical separation was contrasted in the genebank sequence by using the PCR sequencing products.2.1.3 the study of biofilm formation from 127 strains of Escherichia Coli from clinical separationThe biofilm formation detection of Escherichia Coli in LB Medium, using 96-well plates crystal violet staining method; the correlation analysis of the capability of biofilm formation in Escherichia Coli and bacterial drug-resistance, using Non-Paired T Test;2.2 the study of drug-resistant plasmid transmission in biofilm of Escherichia Coli CAG184392.2.1 the study of uptake of exogenous drug-resistant plasmid pgreentir in Escherichia Coli CAG18439The extraction of the pgreentir plasmid adopted the alkaline lysis method ;methodologically investigate about the GFP labeling method for detecting bacterial plasmid uptake in biofilm and the resistance screening method by use of 96-well plates,to determine the observation methods; comparison reported in the literature by adding the exogenous plasmid concentration with equivalent content of biofilm DNA, contrast the plasmid ingestion frequency of Escherichia Coli ATCC25922, MG1655, the low resistant strain of s17 from clinical separation (sensitive to ampicillin), DH5ΑAnd CAG18439 after 24h of the inoculation. Observe the different periods (4h,8h,12h,16h,24h,36h and 48h) after the inoculation of Escherichia Coli CAG18439 and the plasmid ingestion frequency after 24h of the inoculation in different exogenous plasmid concentration (0.5ΜG/Ml, 1ΜG/Ml, 2ΜG/Ml, 4ΜG/Ml And 8ΜG/Ml);comparison reported in the literature by adding the exogenous plasmid concentration with equivalent content of biofilm DNA,determine the plasmid ingestion frequency of the biofilm bacteria and planktonic bacteria after 24h and 48h of the inoculation.2.2.2 the study of plasmid transmission between bacteria during the process of symbiosis biofilm formation by Escherichia Coli CAG18439 and DH5Α(Pgreentir+) in different culture mediums.2.2.2.1 Determine the best observation period for symbiosis culturing drug-resistant plasmid, use the common LB medium (Ph 7.4) for the drug-resistant plasmid transmission during the symbiosis biofilm formation by culturing DH5Α(Pgreentir+) and CAG18439, detect the drug-resistant plasmid transmission frequency after 4h,8h,12h,16h,24h,36h and 48h of inoculation by use of the 96-well plates two-resistance screening.2.2.2.2 The influence to the plasmid transmission between bacteria during the process of symbiosis biofilm formation from Escherichia Coli CAG18439 and DH5Α(Pgreentir+) from different culture mediums. Use the common LB medium (Ph7.4) As contrast, determine the period according to 2.2.2.1, detect bacteria by use of 96-well plates two-resistance screening which inoculated in different mediums:containing 0.5% glucose or 100mmcacl2 in common LB and TSB medium (Ph 7.4), symbiosis culturing the Escherichia Coli CAG18439 and DH5Α(Pgreentir+) for biofilm formation from in partial acid (Ph6) and partial alkaline (Ph9)LB medium and detect the plasmid ingestion frequency;detect the escherichia coli growth curve under the same condition.2.3 The influence to the biofilm formation and the expression of the main regulation gene luxS and Pga from common LB medium which contains 0.5% glucose.2.3.1 The PCR detection of the biofilm formation and the expression of the main regulation gene luxS and Pga in127 strains of Escherichia Coli from clinical separation. The detection of gene Pga and luxS, which related to Escherichia Coli biofilm formation, use PCR to amplify, and verify by using agarose gel electrophoresis.2.3.2 The influence to the strain of Escherichia Coli in biofilm formation and the expression of the main regulation gene luxS and Pga from common LB medium which contains 0.5% glucose. 127 strains from clinical separation and 4 strains of laboratory bacteria (ATCC25922, DH5Α, MG1655 And CAG18439),the LB medium which contains 0.5% glucose and the formation capability of bacterial biofilm in LB medium, aodpt 96-well plates crystal violet staining method, and the Paired T Test analysis; select out the most obvious bacterial strains which increases and decreases in biofilm formation, and detect the expression of Pga and luxS by use of fluorescence quantitative PCR method.2.4 The influence to the biofilm formation and its drug-resistant plasmid transmission from the gene luxS removing in Escherichia Coli2.4.1The construction of Escherichia Coli CAG18439 with gene luxS knockout the constructure of pat2-kan plasmid,amplify kan gene sequences by PCR, connect the pat2 carrier after the a tail added to the product; the constructure of pat2-△luxS carrier used PCR amplification for the upstream and downstream sequence of the Escherichia Coli CAG18439 luxS gene sequence respectively, connect the pat2 carrier after the a tail added to the product,after the correct sequencing, use bamh i and nde i double digestion respectively to pat2-kanplasmid and pat2 plasmid containing the upstream sequences, after the recycling of gel, connect the upstream sequence to pat2-kanplasmid, with the same method, connect the downstream sequence to the pat2-kanplasmid which has already connected to the upstream sequence; obtain the fragment of the homologous recombination, transform the pat2-kanplasmid into DH5Αcompetent cells, extract the plasmid after enrichment, and obtain the fragment of the homologous recombination by gel recycling after xhoi and bamh i double digestion;the obtaining of the CAG18439 with luxS gene knockout , use electrotransformation on the fragment of the homologous recombination ; the evaluation of the Escherichia Coli CAG18439 with luxS gene knockout,used the PCR sequencing method for amplification.2.4.2 the influence to the biofilm formation and its drug-resistant plasmid transmission from luxS gene knockout in Escherichia ColiAfter 24h of inoculation on the Escherichia Coli CAG18439△luxS,determine the biofilm formation and its drug-resistant plasmid transmission separately by using the 96-well plates crystal violet staining method and resistance screening,the quantification of the dissociated DNA in the biofilm used the ethanol precipitation medium method.the expression of 16h Pgac mrna In CAG18439△luxS bacterial strain after inoculation,used the method of RT-PCR.3. Major Result3.1 The study of the drug-resistance and formation capability of biofilm in Escherichia Coli from clinical separationThe MIC detection showed that,in addition to 100% sensitive to imipenem,the drug-resistant rate of 127 strains of Escherichia Coli from clinical separation to Ceftazidime, Chloramphenicol, Kanamycin, Tetracycline, Aztreonam, Cefoperazone, Left Oxygen Sand Stars, Ampicillin, Ampicillin / Clavulanic Acid and Imipenem is 74.8%(95/127), 63.0%(80/127), 95.3(121/127), 48.8%(62/127), 59.8%(76/127), 40.9%(52/127), 37.8%(48/127), 74.0%(94/127) and 49.6%(63/127). In the 57 resistant strains ofΒ- Lactamase, the proportion of positive amplification in Bla-PER, Bla-TEM, Bla-PSE, Bla-OXA, Bla-CTX, Bla-SHV and Bla-VEB Is 0%(0/59), 85.96%(49/57), 66.67%(38/57), 14.04%(8/57), 82.46%(47/57), 22.81%(13/57) and 19.30% (11/57);homology analysis the corresponding genes PUBMED,the 5 kinds ofΒ- Lactamase homology of PCR products (Bla-TEM, Bla-OXA, Bla-CTX, Bla-SHV and Bla-VEB) are all above and beyond 98%.The Escherichia Coli from clinical separation is generally able to form biofilm, there are no relativities between the capability of bacterial biofilm formation and bacterial drug-resistance.3.2 The study of plasmid transmission in the biofilm of Escherichia Coli CAG184393.2.1 The study of ingesting exogenous drug-resistant plasmid pgreentir in Escherichia Coli CAG18439In the GFP labeling method, the occasional factors showed huge influence to the plasmid ingestion rate, so we determined to use the 96-well plates screening for the experiments;when the plasmid concertration is 2ΜG/Ml,the ingestion frequency after 24h of inoculation in escherichia colibacterial strain of ATCC25922, MG1655, DH5Αand CAG18439 Is 0.67×10-8, 0.33×10-8, 1.40×10-7 and 1.71×10-7, and the S17 showed no plasmid ingestion in bacterial biofilm;after 4h, 8h, 12h, 16h, 24h, 36h and 48h of inoculation , the ingestion frequency of drug-resistant plasmid in Escherichia Coli CAG18439 Is 0.22×10-7, 0.36×10-7, 0.68×10-7, 1.22×10-7, 1.57×10-7 and 1.93×10-7;When The plasmid concentration is in 0.5mg/ml,1 mg/ml,2 mg/ml,4 mg/ml and 8 mg/ml,The plasmid ingestion frequency after 24h of inoculation is 0.53×10-7, 1.13×10-7, 1.68×10-7, 1.84×10-7 and 2.40×10-7;after 24h and 48h of the inoculation of biofilm bacteria and planktonic bacteria,the plasmid ingestion frequency is 1.72×10-7 and 2.31×10-7,3.16×10-10 and 8.62×10-10.3.2.2 Common LB medium (Ph7.4) symbiosis culturing DH5Α(Pgreentir+) and CAG18439 for biofilm formation, after 4h, 8h, 12h, 16h, 24h, 36h and 48h of inoculation, the transmission frequency of drug-resistan plasmid is 0,1.78×10-8, 6.89×0-8, 1.13×10-7, 2.07×10-7 and 3.10×10-7; common LB and TSB medium(Ph7.4) which contains 0.5% glucose or 100mm Cacl2, partial acid(Ph6) and partial alkaline(Ph9) LB Medium symbiosis culturing DH5Α(Pgreentir+) and CAG18439 for biofilm formation, after 24h of inoculation, the frequency of plasmid transmission is 2.20×10-7, 2.43×10-7, 2.40×10-7, 2.13×10-7, 1.80×10-7 and 1.73×10-7,use one way ANOVA statistics analytical method to analyze the differences of plasmid transmission in different mediums and LB medium (Ph7.4),P>0.05,with no obvious differnece; the determination result of growth curve showed that different mediums had no obvious influence to bacterial growing.3.3 The influence to the biofilm formation and it's main regulation gene luxS and Pga from LB medium which contains glucose (0.5%)3.3.1 127 strains of bacteria all contains gene Pga and luxS.3.3.2 the influence to the biofilm formation in Escherichia Coli and the expression of gene luxS and Pga from LB medium which contains glucose (0.5%). paired T test discovered that 29 (29/131) strains showing obvious increase in biofilm formation capability, 42 (42/131) strains showing obvious decrease;the most obvious increase in biofilm formation is S59,the most obvious decrease in biofilm formation is S100. The expression of Pga and luxS in S59 bacterial strain was higher than bacteria in common LB medium;the expression of Pga in S100 decreased,but the expression of luxS increased.3.4 The influence to the biofilm formation and it's drug-resistant plasmid transmission from Escherichia Coli with luxS gene knockout3.4.1 The constructure of luxS gene knockout strain in Escherichia Coli CAG18439 sequencing of PCR amplification products proved to be successful in building a luxS knockout strain.3.4.2 The influence to the biofilm formation and its drug-resistant plasmid transmission from Escherichia Coli with luxS gene knockoutAfter 24h of inoculation, the study showed that the capability of biofilm formation decreased in Escherichia Coli cag18439△luxS,the contents of the dissociated DNA in biofilm decreased,plasmid transmission frequenct decreased. after 16h of inoculation, the expression level decreased in pga mrna.4. Conclusion4.1 The drug-resistant rate is quiet high in Escherichia Coli from clinical separation, and there is condition of a few ofΒ-Lactamases being carried by one strain of bacteria, and aΒ-Lactamase being located in a number of bacteria,under the normal culturing condition, almost all the bacterial strains were able to form biofilm,but the capabilities of biofilm formation were quiet different,there were no obvious differences between drug-resistance and the capability of biofilm formation in bacterial strain.4.2 The drug-resistant plasmid transmission in bacterial biofilm had bacterial strain specificity,the incidence rate was highest during the incipient period of biofilm formation,and this transformation frequency increased as plasmid concentration increased,but showed no linearity relations;the existence of different mediums and calcium showed no obvious influence to drug-resistant plasmid transmission in biofilm, which indicated that this drug-resistant plasmid transmission mechanism was totally different from the common transformation ways.4.3 127 strains of escherichia coli were all contains pgaA, pgaB, pgaC, pgaD and luxS gene,which indicated that the Pga and luxS gene was quiet high conservative;LB medium which contains 0.5% glucose in different bacterial strains could showed increase and decrease of the bacterial biofilm formation capability,the expression of bacterial pga gene had the same effect;but whether the biofilm formation increase or decrease in bacterial strains,the expression of the luxS gene all increased,which indicated that as the upstream regulatory gene for biofilm formation, the luxS is responsive to the changes in the external environment,but the Pga was just the effectors for biofilm regulation,the expression intensity is in accordant with biofilm formation intensity. on bacterial strain which Pga gene with inherent low expression, even if environmental factors had led to increased expression of luxS gene, biofilm formation would still not enhanced.4.4 Escherichia Coli CAG18439 with luxS gene deletion showed decrease of the biofilm formation capability, the transmission frequency of drug-resistant plasmid in biofilm reduced,the mechanism may be caused by the luxS mutation which reduced expression of Pga,the decrease of formation capability in biofilm, and the reduction of the release of dissociative bacterial DNA from schizolysis at the same time.
|
PDF Full Text Request