Effects Of Sub-Minimum Inhibitory Concentrations Antibiotics On Conjugation Between Escherichia Coli And Pseudomonas Aeruginosa

ObjectivePseudomonas aeruginosa is an important opportunistic pathogen, a facultative anaerobic non-fermenting Gram-negative bacteria. Widespread use of antibiotics have brought about the serious increase of bacterial resistance in Clinical Epidemiology. PA resistance can be separated into natural resistance and acquired resistance. Natural resistance is mediated by chromosome, which is usually a vertical transmission to the offspring of the bacteria, little or no horizontal transmission. Natural resistance determines the phenotype of a wild species, Acquired resistance contribute more on the change of clinical epidemiology. There are three main mechanisms of acquired resistance:First, lower permeability or efflux systems are adjusting to reach the target due to reduced antibiotic dose; Second, target modification; Third, inactivation of antimicrobial agents. Horizontal gene transfer (horizontal gene transfer, HGT) is to exchange genes between bacteria which can produce resistance. conjugation is an important way of horizontal gene transfer.There are many ways of communication between bacteria:previous study have learned self-induced behavior of bacteria-the bacterial quorum sensing system (Quorum Sensing System, QS) or quorum sensing systems that regulate the whole:Many types of bacteria monitoring the surroundings can change their number depending on the concentration of bacteria or other specific signaling molecules, the signal molecules can enter the cell receptor binding and intracellular expression of genes when they start to adapt to changes in the environment. The acyl homoserine lactone (acylated homoserine lactones, HSL) signal molecules play an important role in the conjugation of the Ti plasmid of Agrobacterium tumefaciens. After preliminary experiments, we found that in Pseudomonas aeruginosa HSL reactions play an important role:HSL-related genes regulate the activity of a plurality of conjugation and intervention the process.antibiotics is also a signaling molecule for bacterial like HSL. A review of 《Nature》studied the use of antibiotic in the last century, said antibiotic was a good detector,which can help us to understand the complexity of microorganisms. There is relatively clearer understanding of the targets of the antibiotics.However HSL belongs to global regulatory systems, It is difficult to clarify the complex specific mechanism. On the other hand, antibiotics are also secondary metabolites which evolved from bacteria. The study found that the use of antibiotics is closely related to drug resistance of bacteria. With the increasing use of antibiotics, resistance to antibiotics of the bacteria have increased. This is not limited to the PA and E. coli and other gram-negative bacteria, Gram-positive cocci is also of the same way, The less of erythromycin,The less of the number of antibiotic-resistant strains of type A streptococcus.Bacteriostatic effect is not just that simple.SOS system or microorganism called LexA regulon is inducible DNA repair system, lexA protein regulatory network system helps maintain the integrity of the bacterial DNA. It has been proved that inhibitory concentration of antibiotics can activate the SOS response, such as the role of antibiotics Pseudomonas aeruginosa associated with activation of the SOS response. What is the relationship between SOS response and the conjugation of bacteria? This physiological behavior of bacterial bonding involves many bacterial regulatory system, will it have a very significant correlation with SOS regulatory network? We have a lot of evidence, but for Pseudomonas aeruginosa, the exact mechanism is unknown.This paper intends to study the sub-inhibitory concentrations of antibiotics used in the clinic of Pseudomonas aeruginosa,the role it played in the conjugation reaction of P. aeruginosa.As we all know, antibiotics can inhibit even kill the bacteria, and how it can promote the horizontal transfer of resistance genes in effect? In fact, it is not contradictory, because antibiotics play its role only when it achieve effective drug concentrations and enough time of periods. Antibiotics play their role dependenting on the concentrations and the time.,. In order to maintain the antibiotic concentration that is greater than the minimum inhibitory concentration (minimum inhibitory concentration, MIC). the clinical application of antibiotics need regular dosing based on pharmacokinetic parameters, so as to inhibit or kill bacteria.However, there are actually a variety of factors affect that antibiotics can reached only "sub MIC"(sub-MIC) levels in the site of infection, Antibiotic abuse is an important reason; Second, the bacteria can form a special structure, such as PA resistant to antibiotics for the formation of biofilms.Third, the special structure of human organs, such as the blood-brain barrier may prevent some drugs into the ventricle; Fourth, the distribution of drugs in different tissues of different organs and tissues is different or infected by poor blood supply etc.; Fifth, the body’s immune system is low and the bacteria is not being killed. although the bacteria is engulfed by phagocytic cells of the body that later become a shelter. for the bacteria. sub-MIC levels of antibiotics can hardly kill the bacteria.This paper intends to optimize the conjugation model between donor bacteria E.coli and receptor bacteria PA created in the previous project, making it suitable for antibiotic studies.Then compare the effects of different antibiotic to treat the donor or the receptor,we found the donor bacteria E.coli more important and sub-MIC concentrations of antibiotics can promote conjugation reaction efficiency,the result of quantitative PCR can prove the related changes in the expression of genes at different time points. We analyze the gene expression of PA under the treatment of sub-MIC antibiotics by transcriptome sequencing, In order to fully clarify the effects of sub-MIC antibiotics on bacterial conjugation reactions, We also screen positive genes from those data to analyze gene function in the conjugation reaction. Methods1. An optimized model for conjugation①Donor strain and Recipient strainDonor strain E. coliSM10λpir (pUCP24T), Recipient strain PAO1is provided by a kind professor.And the number of donor and recipient strains:the need to maintain a fixed ratio,0.5MCF is commonly used in the standard quantitation of microrganism.②filter hybridization compared with the hybridization of liquidIt is of the same for the steps before hybridization.What is the difference is after mixing the donor and recipient strain of bacteria after quantitative mixing:place them under a liquid environment or on the membrane for2h.③the time of the conjugationTo select the time of conjugation, factors must be considered influencing the experimental results within the whole experimental cycle:pre-treatment、washing the membrance、antibiotic selection and the counting of the conjugates on the agar.We have tried1h、2h、4h.④results of the fluorescence detection of the reporter gene and the method of counting the number of the colonyFluorescence detection choose the double plasmids that one can prohibit the expression of the reporter gene of the other plasmid.After the conjugation,the plasmid which have the function of suppression can be lost.Then the fluorescence detection can be made of the conjugates.The method of counting the number of the conjugates use the antibiotics to repress the growth of the donor and recipient strain.⑤conjugation related factorsThe main consideration are the three factors:number of bacteria on the filter、the time of washing the membrane、dilution. We make a two-level、three-factors orthogonal design.2. antibiotic selection and the treatmentAccording to the recommendation of the A、B class clinical use of drugs by clinical guidelines of 《CLSI》 of Enterobacteriaceae and Pseudomonas aeruginosa, also depending on the the standard antibiotics available in the market:we choose ticarcillin、levofloxacin、 ciprofloxacn、tobramycin、meropenem、cefepime these six antibiotics.Because the main components about conjugation involved in the donor strain, we chose the donor strain E.coliSM10λpir (pUCP24T) for antibiotic treatment after verification testing. To avoid the synergistic role of the antibiotic resistance selection and gentamicin which is the maintenance of antibiotic plasmid pUCP24T, we do not use the gentamicin to maintain the continuing resistance of the plasmids when treating with the donor bacteria. 3. Observation of the effects of antibiotics on the role of donor strain and the conjugation reaction,explore the optimal sub-MIC concentration and time pointThe sub-inhibitory concentrations is determined by macrodilution technique and microdilution method. Then draw the growing curves of the donor bacteria treated with the antibiotic of sub-inhibitory concentrations.We treat the donor strain with antibiotics for1h,2h,10h, to see if there are changes in the conjugation efficiency.4. Transcriptome sequencingFunctional analysis of the genome of the E.coliSM10λpir (pUCP24T)treated with the antibiotics of sub-inhibitory concentrations:the effect of the antibiotic of Sub-mic、the regulons、Signal Transduction and the effect to the host.5. qPCR to detect the expression of related genes of E.coli conjugationWe keep the specimens after antibiotic treatment for6h,8h,10h and RNA was extracted to reverse transcribed into cDNA. Select reference gene araC and detect two genes related to the conjugation traI, traG by qPCR.Results1. The best conditions are:the main factor-dilution of the bacteria when washing the membrane is240times;then the number of bacteria on the filter is thirty μL;third,the time of washing the membrane is two minutes. The time of the conjugation is two hours.colony counting still have good sensitivity in this condition:conjugation efficiency can be quantitated with calculated significant results.2.the treatment of antibiotics:We treat the donor strain with ticarcillin, levofloxacin, ciprofloxacin,tobramycin, meropenem, cefepime and keep the samples to do the experiment of conjugation by the standard that the five relatively stable decreasing sub-inhibitory concentrations of1/2below the lowest inhibitory concentration.Finally through statistical calculations, ciprofloxacin and levofloxacin show a regular changes of conjugation efficiency with the changes of sub-inhibitory concentrations. Then we also try to treat antibiotic with1h、2h, the bacterial growth time is fixed at10h, found1h,2h failed to show regular changes.3.We chose to focus on ciprofloxacin and levofloxacin to treat the donor strain, and the processing time for the growth of bacteria and antibiotics in longitudinal6h、8h、10h to do the experiments to see the changes of conjugation efficiency. Final results are:with the change of sub-inhibitory concentrations the differences in efficiency are not reflected in6h, but has a clear difference in8h:with the increase of sub-inhibitory concentrations, conjugation efficiency is also increased, the control group has the lowest efficiency.8h and10h has the consistency of the trend of efficiency.4.Results of transcriptome sequencing:the RP4plasmid of E.coli SM10λpir(pUCP24T) treated With ciprofloxacin and levofloxacin can be clustered into ten group. Some genes express in the same mode that they go down with the lower of of the Sub-mic.We pay more attention to the screening of the genes related to the type IV secretion system (T4SS).5.With ciprofloxacin and levofloxacin treating sample for6h、8h、10h, the results of quantitative PCR show obvious changes in the expression of these two genes tral, traG.The expression of genes related to conjugaton with sub-inhibitory concentrations rise in6h,8h has a more obvious rise in expression.the statistics are of significance.Conclusion:1.A successful model to optimize the conjugation.2.Conclusions can be drawn that sub-inhibitory concentrations of the antibiotic ciprofloxacin and levofloxacin can promote the conjugation efficiency.3.The results of transcriptome sequencing show sub-inhibitory concentrations of antibiotics has important implications on conjugation,mainly on the effects of RP4plasmids.4.Quantitative PCR results also prove sub-inhibitory concentrations of antibiotics up-regulate the conjugation related genes traI、traG, which is consistent with the quantitation of the transcriptome sequencing.