The Effect And Mechanism Of Quaternary Ammonium Compound On The Distribution Of Antibiotic Resistant Genes

With the discovery and wide use of antibiotics,Antibiotic Resistance Genes(ARGs)have drawn more and more attention as an emerging environmental pollutant.Quaternary Ammonium Compound(QAC)is a common disinfectant and detergent,it has many similar drug-resistance mechanisms to antibiotics and may play a part in promoting the spread and diffusion of ARGs.In this study,we detected one QAC resistance gene(qacE?1),one integron gene(intI1)and 12 kinds of antibiotic resistance genes(sul1,sul2,cfr,cml,fexA,tetA,tetG,tetQ,tetX,ermB,blaTEM and dfrA1)in the watersheds of Ningbo,Wenzhou and Huzhou.Using SPSS analysis software to analyse the correlation between QAC resistance genes and antibiotic resistance genes.Bacterium were continuously cultured for 30 generations under QAC and antibiotic environmental pressure.Then withdrawed the environmental pressure and carried on culturing for another 30 generations.The sensitibilities of bacteria to tetracycline(Tc),ciprofloxacin(Cip)and chloramphenicol(Chl)were tested every 5 generations,in order to explore whether the environmental pressure of QAC can promote the bacterial resistance to antibiotics.In addition,five QACs were selected to study the effect of QAC on the efficiency of conjugational transfer of RP4 antibiotic resistant plasmids between donor bacteria and recipient bacteria.Finally,we used Annexin V/PI double dye kit and DCFH-DA fluorescent probe to detect the change of bacterial cell membrane and the production of ROS respectively in the presence of QAC,for the purpose of exploring the mechanism of QAC in promoting the spread of antibiotic resistant.The results show:(1)QAC resistance gene is ubiquitous in the environment and it has significant correlation with class ? integrons gene(r = 0.999,p<0.01).Among the 12 ARGs,there are 7 ARGs were significantly associated with QAC resistance gene.QAC resistance gene had the strongest correlation with ARGs which equipped the resistance mechanism of inhibiting enzyme activity,followed by ARGs with ribosomal protection protein resistance mechanism,and the correlation is relatively weak with the class of efflux pump.(2)The sensitivity of bacteria cultured continuously under the environmental pressure of QAC and antibiotics both decreased to a certain extent.Among the three selected QACs,the bacteria cultured in didodecyldimethylammonium chloride(DDDAC)had the maximum decrease in the sensitivity to ciprofloxacin.After 30 generations of continuous culture,the bacteriostatic circle diameter decreased from 25 mm to 19 mm.Bacteria cultured in dodecyltrimethylammonium chloride(DTAC)showed the greatest reduction in chloramphenicol sensitivity,with the diameter of bacteriostatic circle reduced from 19 mm to 13 mm.The bacteria cultured in benzyldimethyldodecylammonium chloride(DDBAC)had he fasted reduced of tetracycline sensitivity,and the diameter of bacteriostatic circle completely disappeared when they were cultured continuously till the 20th generation.When QAC environmental pressure was withdrawed,the sensitivity of the bacteria to antibiotics increased.(3)The five QACs selected in this experiment all can promote the conjugational transfer of RP4 resistant plasmid,although their promoting effects were not exactly the same.The rules of their promoting effect on conjugational transfer efficiency are as follows:Benzyldimethyldodecylammonium chloride(DDBAC)>Benzyldimethy-ltetradecylammonium chloride(TDBAC)>Dodecyltrimethylammonium chloride(DTAC)>Didodecyldimethylammonium chloride(DDDAC)>Benzyldimethy-lhexadecylammonium chloride(HDBAC)(4)After adding QAC,the cell membranes of donor and recipient bacteria were damaged to a certain extent,and the apoptosis phenomenon appeared.That is,the ratio of donor bacteria with cell membrane damage increased from 2.49%to 95%and the ratio of recipient bacteria increased from 3.81%to 97.52%.The amount of ROS produced by donor bacteria increased from 0.31%to 16.72%,and the amount of ROS produced by recipient bacteria increased from 0.01%to 6.23%.QAC can damage the cell membrane of bacteria to improve the membrane permeability of bacterial cell,and as a result stimulate bacteria to produce ROS.The alter in the bacterial cell membrane can promote the transfer of antibiotic resistant plasmids between bacterium.