Effects Of Organic Matter Polluted Water And Constructed Wetlands On Transfer Efficiency Of Antibiotic Resistance Genes

Tetracyclines are the most widely used antibiotics,in livestock production in recent decades.However,there are few reports on the natural factors affecting the spread of such antibiotic resistance genes.This study used the doxycycline resistance gene（Dox）as the research objective.（1）The drug susceptibility test of 7 bacterial pathogens（6 of which are clinically isolated pathogens with unclear genetic background,1 of which is a conditional pathogenic Klebsiella pneumoniae mutant TR-M30-1 with mTn5gusA-pgfp21 insertion mutation and Kan^R（kanamycin resistance）,preserved in the laboratory）,was performed using 18 commonly used antibiotics to obtain the drug resistance spectrum of these 7strains.Candidate-donors with phenotype of Dox^RX^S and candidate-recipients with phenotype of Dox^SX^R were selected（X represents any of the 18 antibiotics tested）.（2）After the bacterial conjugation of donor and recipient bacteria,the zygotes with phenotype of Dox^RX^R was screened,and the target donor and recipient bacteria were determined.（3）The target donor and recipient bacteria were identified by morphological,physiological,biochemical,molecular methods and BIOLOG method.（4）The tetracycline resistance genes and mobile genetic elements of target donor and recipient were identified.（5）The effect of the survival rate of bacteria and transfer efficiency of Dox gene in different sections of sewage channel of pig farm and different concentrations of leachate from organic fertilizer was detected.（6）The downflow constructed wetlands were constructed using Myriophyllum spicatum L,Eichharnia crassipes,Myriophyllum sp.+Eichharnia crassipes and no plant（control）to detect the survival rate of bacteria and transfer efficiency of Dox gene.（7）The correlation between the transfer efficiency of Dox gene from donor to recipient and pH value,chemical oxygen demand（COD）,the type and content of amino acid,total sugar,reducing sugar,Kjeldahl nitrogen,total phosphorus（TP）and available phosphorus was analyzed in water of sewage channel of pig farm and constructed wetlands.（8）The effect of constructed wetlands on COD removal was examined.The results are as follows.（1）Both of the seven pathogens could tolerate multiple antibiotics（up to 15antibiotics）.The drug resistance rates of streptomycin（aminoglycosides）,nalidixic acid（quinolones）and sulfamethazine（sulfonamides）were 100%.However,the drug resistance rates of gentamicin（aminoglycosides）,norfloxacin（quinolones）and ciprofloxacin（quinolone）were 0.The phenotype of target donor is Dox^RKan^S,the phenotype of target recipient is Dox^SKan^R,the phenotype of zygote（named as Con-II）is Dox^RKan^R.（2）The target donor with phenotype of Dox^RKan^S was identified as Escherichia coli O157:H7（E.coli O157:H7）.The result of BIOLOG identification for the target recipient TR-M30-1（Dox^SKan^R）（K.acidophilus）is consistent with that of 16S rDNA identification by Yu.（3）E.coli O157:H7（Dox^RKan^S）and Con-II（Dox^RKan^R）carry tetB resistance gene.However,TR-M30-1（Dox^SKan^R）don’t carry tetB.E.coli O157:H7（Dox^RKan^S）contains F plasmid,class I integron and class I integron variable region.TR-M30-1（Dox^SKan^R）contains F plasmid.Con-II（Dox^RKan^R）contains F plasmid and class I integron.Dox^R of Con-II（Dox^RKan^R）is not transferred from E.coli O157:H7（Dox^RKan^S）to TR-M30-1（Dox^SKan^R）by F plasmid.（4）Lasted 9 days,Determination on 3rd,6th and 9th day,E.coli O157:H7（Dox^RKan^S）and TR-M30-1（Dox^SKan^R）in complete medium（LB）,different sections of sewage channel of pig farm,different concentrations of leachate from organic fertilizer and constructed wetland system,the survival rates gradually decreased compared with the original concentration（0 d）and the decline rate of Experience group was higher than control group（LB medium）.In LB medium,determine from（Average±standard deviation）,there was no difference in survival rate between the two Bacterias on the 3rd day,but on the 6th and 9th day,the survival rate of TR-M30-1（Dox^SKan^R）was higher than E.coli O157:H7（Dox^RKan^S）.The survival rate of organic matter polluted water（pig farm sewage and organic fertilizer leachate）was higher than constructed wetland water.In time dimension（0-3 d,3-6 d,6-9 d）,survival rates presented:（1）Subtraction-subtraction-increase:3 sections of the pig discharge channel（upper,middle and lower reaches）sewage,150 g organic fertilizer+1 L distilled water of E.coli O157:H7（Dox^RKan^S）;pig farm drainage channel（upper and lower）,（150,100,50） g organic fertilizer+1 L distilled water and Myriophyllum spicatum constructed wetland of TR-M30-1（Dox^SKan^R）;（2）Subtraction-increase-subtraction:maximum water holding capacity state organic fertilizer（water content 57%）,（100,50）g organic fertilizer+1 L distilled water,plant-free constructed wetland water of E.coli O157:H7（Dox^RKan^S）;200 g organic fertilizer+1 L distilled water of TR-M30-1（Dox^SKan^R）.（3）Subtraction-subtraction-subtraction:other test conditions other than（1）and（2）,such as （200,10）g organic fertilizer+1 L distilled water,（Eichharnia crassipes,Myriophyllum sp.,Eichharnia crassipes+Myriophyllum sp.）constructed wetland,LB medium of E.coli O157:H7（Dox^RKan^S）;pig farm sewage drain（middle）,maximum water holding state organic fertilizer（water content 57%）,10g organic fertilizer+1 L distilled water,（Eichharnia crassipes,Eichharnia crassipes+myriophyllum,no plant）constructed wetland system and LB medium of TR-M30-1（Dox^SKan^R）.（5）The order of conjugation efficiency between E.coli O157:H7（Dox^RKan^S）and TR-M30-1（Dox^SKan^R）for 3 d in different systems is as follow:LB medium(3.25×10^-6)>upstream of sewer of pig farm(1.124×10^-6)>midstream of sewer of pig farm(9.03×10^-7)>organic fertilizer with maximum water content（57%）(6.23×10^-9)>constructed wetland system（0）.（6）Pearson correlation analysis of conjugation efficiency between E.coli O157:H7（Dox^RKan^S）and TR-M30-1（Dox^SKan^R）for 3 d co-culture showed that pH were significantly negative correlated with conjugation efficiency（p<0.05）,chemical oxygen demand（COD）were significantly positive correlated with conjugation efficiency（p<0.05）,amino acid content was very significantly positive correlated with conjugation efficiency（p<0.01）,but conjugation efficiency was not significantly correlated with total sugar,reducing sugar,Kjeldahl nitrogen,total phosphorus（TP）,available phosphorus.（7）The average removal rates of CODcr in the constructed wetlands of Myriophyllum spicatum,Eichharnia crassipes,Myriophyllum sp.+Eichharnia crassipes and non-plant control group were 38.82%,34.1%,32.37%and 40.75%,respectively.