Horizontal Transformation And Spread Of Antibiotic Resistance Genes Under Chlorination

To date,antibiotic resistance bacteria（ARB）and antibiotic resistance genes（ARGs）,as new type of environmental pollutants,is becoming an increasing worldwide concern.Natural water environment is considered as a huge ARGs pool in which hundreds of various ARB and ARGs could preserve their resistance genes for long time and deliver the mobile gene elements through horizontal gene transfer（HGT）,which pose a great threat to human health and environmental safety.It is widely believed that HGT is mediated by three mainly different mechanisms:transduction,conjugation and transformation.Transduction is a specific process mediated by temperate phages that could insert themselves into chromosomes of hosts without causing cell lysis.Conjugation requires direct contact of donors and recipients and involves mating-pair formation and DNA exchange mediated by so-called“conjugative bridge”---F-pilus.Transformation refers to the uptake of free DNA by the competent bacterial cell and its stable integration into the bacterial genome.There are few relative documents reported on natural transformation in water environment,however,as more and more free antibiotic resistance genes and plasmids have been discovered,the problems of environmental pollution caused by ARGs through HGT have been unnegligible.Sodium hypochlorite has been widely used on industrial production because of its high efficiency,broad spectrum,safety and stability,which has become the most common treatment applied in drinking water disinfection.Many reports showed that incomplete disinfection of drinking water could generate sub-lethal bacteria（injured bacteria via disinfection）.The bacteria with physical defect,could hardly be detected by the conventional methods due to their great repair ability to restore normal activity under some certain conditions,bringing huge threat to drinking water safety.Currently,few studies are devoted on water-disinfection injured bacteria.The main opinions accepted by the majority of researchers is that,disinfectants damage cell walls because of their strong oxidation,changing membrane permeability and leading to unbalance of metabolism,finally causing cell injuries.Thus,illustrating the properties and exploring formation laws of injured bacteria could help to further deepen research on disinfection mechanism as well as facilitate prevention and control the pollution of drinking water.Natural transformation involves both competent of recipients and free plasmids or DNA fragmentations from donors.There are some studies reported that disinfection could not eliminate free ARGs,on the contrary,the presence of ARB and ARGs increased.The main reasons may be that abundant ARB in water systems releasing their ARGs to potential natural recipients which are injured bacteria under competent state after disposing by disinfection,what’s worse,resulting to horrible ARGs contamination through dissemination mediated by natural transformation.This study dealt with the problems from the following four aspects:（1）The construction method of detecting disinfect-caused injured bacteria.（2）Influences on ARB and ARGs by chlorination disinfection.（3）The research on properties of disinfection injured bacteria.（4）Conditions and factors effecting the transformation of ARGs and injured bacteria.The main conclusions are as follows:（1）Successful evaluation of disinfect-caused injured bacteria.The repair medium TSYA was effectively used to better detect injured bacteria compared with conventional methods such as multi-tube fermentation,membrane filter and enzyme substrate tests.It could successfully detect the injured bacteria with the detection rate of 100%.MTF could only detect injured wild strains of E.coli completely,while EST method could detect wild types and only parts of GE E.coli,however,MF methods could detect neither of them.Therefore,TSYA is the most suitable approach to detect and evaluate injured bacteria.（2）Demonstrating the inaction principles of ARB by sodium hypochlorite and formation principles of injured bacteria caused by disinfection.Our study took E.coli HB101（p UC19）as research object.Taking use of first order kinetic model of chlorine attenuation,we analysed the degenerative law of free chlorine.According to the enumeration results on selective medium（Endo）and non-selective medium（TSYA）of culturable bacteria after chlorination,we fit EFH models and obtained Ct values for 2-,3-,4-and 5-log of E.coli HB101（p UC19）inactivation at different p H and temperature,respectively.Therefore,We found that appropriate temperature and reducing p H values could help improve the efficiency of disinfection.When the killing log value reached 5-lg and p H=7.2,the Ct value decreased from 10.28mg/L·min to 7.67mg/L·min at36℃,and even reduced to 6.68mg/L·min at p H=6.0,suggesting that the change of p H could pose a larger influence on disinfection efficiency compared to temperature.Meanwhile,the comparison results of resistance among different bacteria showed that the strongest resistance of Na Cl O was E.faecalis,the following were P.aeruginosa,S.aberdeen and E.coli.we also found that when the Ct values reached 10.88～23.73mg/L·min,it could disinfect all the E.coli while E.faecalis were still left with concentration of more than 10²cfu/ml in water,additionally,the free plasmids remained transformatively active,which created favorable conditions for natural transformation.（3）Analysis the effect of chlorine disinfection on antibiotic resistance plasmid and ARGs.We found that the transformation function and integrity of antibiotic resistance plasmid had not been destroyed due to bacteria lysis by full length sequence PCR amplification and plasmid transformation function tests and discovered that Ct values of the injury were affected by treat conditions.Under our research conditions,the integrity damage Ct values of antibiotic resistance plasmids p UC19 ranged from 3.59 to 75.57mg/L·min,and the Ct values of ARGs were within 194.62～4809.20mg/L·min.Besides,results showed simultaneous damage of length integrity and conversion function.Consequently,we identified that large amounts of antibiotic resistance plasmids and genes still existed in drinking water under the normal disinfection treatment except under ultrahigh dose.（4）Demonstrating that disinfection-injured bacteria were kinds of competent form as well as conducting researches on bacterial biological characteristics including antioxidase systems,membrane permeability and morphology.Our results showed that the SOD,CAT and GSH-PX enzyme activities of E.coli,P.aeruginosa,S.aberdeen and E.faecalis had improved 3.03～4.78,1.48～16.08and 4.11～12.32 folds,respectively.While the MDA content was below the detection limit.As a result,we believed that disinfection-injured bacteria could resist chlorine oxidation by eliminating oxygen radicals.After disinfection,membrane permeability was increased 2.5～3.1 times by measuring the decomposition rates of ONPG.We also observed the change of cell shapes of four types of bacteria using TEM,and discovered that the main changes of injured bacteria was plasmolysis which could increase membrane permeability and finally caused the functional alterations of cell walls.Transformation by heat shock methods verified that disinfection-injured bacteria were in forms of competent cell.（5）Study of the natural transformation conditions between free antibiotic resistance plasmid and injured bacteria under chlorination and the relative effect factors.The results obviously showed that,chlorination disinfection significantly promoted both free plasmid transformations and uptake of free DNA in disinfect-injured bacteria.The longer incubation time would promote the transformation frequency of RP4 plasmid.1m M concentration of plasmids showed a high transformation frequency.A higher concentration of injured bacteria could increase the effect of transformation.During the study of water factors affecting transformation,we found that neutral p H value would remarkably enhance the frequency of transformation,while water temperature has little influences on the transformation.Metal ions such as Ca²⁺,Mg²⁺,K⁺could promote the conversion with the highest transformation frequency of0.1m M,approximately equals the concentrations in practical water.The strong-to-weak rank of facilitation effect were Ca²⁺>Mg²⁺>K⁺,respectively.Ca²⁺could promote injured bacteria transformation for 3～8 times,while RP4 was6～36;Mg²⁺and K⁺showed both 2～8\2～5 times higher than healthy cells transformed by p UC19,and RP4 were 6～34\3～19times.While Mn²⁺had inhibit effects to a certain extent.The study found that disinfection will surely promote natural transformation of ARGs by horizontal transfer,not only by facilitating the free plasmids into cell but also promoting the uptake of extracellular DNA into injured bacteria.In this study,we simulated chlorination treatment of water to explore the interactions between free antibiotic resistance plasmids and injured bacteria.We have found that,chlorination in drinking water could release free antibiotic resistance plasmids and produced disinfection-injured bacteria which were in competence forms.In the same water systems,the two factors might cause natural transformation and spread of ARGs eventually.In a word,the results will contribute to improve drinking water disinfection treatment technology,as well as provide a strong theoretical foundation on ARGs horizontal transfer and spread in water.