Diversity Of Disinfectant Resistance Bacteria And Pollution Of Disinfectant Resistance Genes In Haihe River Basin,Tianjin

BackgroundThe disinfectants play an important role in controlling the spread of pathogenic microorganism,which have been widely employed in daily life,hospitals,packinghouse,and so on.The problem that the bacteria have the capability of resistance to disinfectants has received more attention.If the bacteria have the ability to resist the disinfectants,they will cause the infection which becomes difficult to control and cure.The disinfectant resistance bacteria have become the dominant causation of nosocimial infection in hospital.The definition of disinfectant resistance bacteria is the bacteria which can not be inactivated or inhibited by the disinfectant on the concentration,while the great mass of bacteria can be inactivated or inhibited.The ability to resist the disinfectants of the experiment bacteria can be judged by comparing the MIC and MBC value against the experiment bacteria with those against the standard bacterium strains.The dominant mechanism of which the bacteria resist to disinfectant includes innate resistance and acquired resistance.The innate resistance has a correlation with the bacteria’s structure or characteristic.The bacterial endospores are generally the least susceptible to disinfectants because the outer spore coat(s)can prevent the disinfectants from penetrating.The gram-negative bacteria can more strongly resist to disinfectants than the gram-positive bacteria due to the differences in the structure of cell wall.The acquired resistance has a correlation with the disinfectant resistance genes in environment.The disinfectant resistance genes can be classified as plasmid-encoded resistance genes or chromosome-encoded resistance genes.The QAC genes family is widely studied.There are eight kinds of QAC genes encoded by plasmids including qacA/B,qacE,qacE△1,qacF,qacG,qacH,qac I and sugE(p),which can transmit horizintally and vertically between bacteria.The bacteria taking the genes of sugE(c),emr E,ydgE/ydgF and mdfA which are encoded by chromosome can resist the disinfectant QAC too.ObjectivesThe objectives of this paper were three-fold.The first of these aims was to estimate the ability of bacteria resistance to sodium hypochlorite,iodine and benzalkonium bromide in Haihe River Basin,Tianjin.The second purpose was to analyze the diversity of disinfectant resistance bacteria in samples.The final point was to know the pollution of the disinfectant resistance genes in Haihe River Basin,Tianjin.MethodsThe water samples were sampled on the different sites according to the location along the Haihe River Basin in Tianjin.The ability of bacteria resistant to disinfectant in samples was evaluated by the detection of MIC and MBC value.Sodium hypochlorite,iodine and benzalkonium bromide were chosen as the experiment disinfectants.Escherichia coil ATCC 25922 was chosen as the standard bacterium strain in the experiments.The disinfectant resistance genes were studied by using Polymerase chain reaction(PCR)in the samples against which the MIC value of sodium hypochlorite,iodine and benzalkonium bromide were respectively higher than those of the ATCC25922.The diversity of the bacteria in samples against which the MIC and MBC value were higher than those of the standard bacteria strains was analyzed by using 16 S rRNA amplicon sequencing.The bacteria in the samples were concentrated by using positive pressure filtration method.The genomic DNA in the samples was extracted by using TIANamp Bacteria DNA Kit.The disinfectant resistance genes were detected by using real-time quantitative PCR with SYBR GREEN.Results1.The capability of disinfectant resistance of bacteria in samples from the Haihe River Basin in Tianjin.There were eight samples coming from the upstream,midstream and downstream location along the Haihe River Basin in Tianjin.The MIC and MBC value of sodium hypochlorite against all samples were between 850mg/L to 1350mg/L,which were higher than those against ATCC 25922.The MIC and MBC value of iodine against all samples were higher than those against ATCC25922,which were between 120mg/L to 270mg/L.The MIC and MBC value of benzalkonium bromide against all samples besides the sample from the South Canal were between 90mg/L to 270mg/L,which were higher than those against ATCC25922.In the 24 samples against which the MIC value was higher than those against ATCC 25922,the presence ratio of qac E△1,qacE,emrE,sugE(p),sugE(c)and ydgE/F were 10/24,3/24,3/24,2/24,6/24 and 7/24,respective.2.The diversity of the bacteria in the MIC and MBC samples.There were 38 samples against which the MIC and MBC value were higher than those against the standard bacteria strains.The diversity of the bacteria in 38 samples was different.The differences were among 11 phyla.Proteobacteria and Frimicutes were the dominant phyla presenting in most samples.Cyanobacteria and Actinomycetes were presenting at high level in the MIC sample of iodine from the Southern Canal,and Actinomycetes was observed in the MIC sample of benzalkonium bromide from the Diamond Bridge.In the samples against which the MIC and MBC value of sodium hypochlorite were higher than those against ATCC 25922,the relative frequency of Bacillus,Pseudomonas,Jeotgalibacillus,Virgibacillus and Lysinibacillus were higher.While in the samples against which the MIC and MBC value of iodine were higher than those against ATCC 25922,Pseudomonas,Bacillus,Aeromonas,Synechocossus,and Lactobacillus were presenting in high frequency.And Pseudomonas,Aeromonas,Providence,Morgan and Bacillus were presenting frequently in the samples of benzalkonium bromide.3.Quantitative analysis of disinfectant resistance genes in samples from the Haihe River Basin in Tianjin.The qacE,qacE△1,qacG of the QAC gene family were existent in the samples from the Haihe River Basin in Tianjin,while the qacA/B gene was nonexistent.The genes of ydgE and ydgF were detectable in the samples from the upstream and the midstream of the Haihe River Basin in Tianjin.The sugE(c),sugE(p)and emrE genes were existent in the samples from the sampling location of the South Canal,the Jingang Bridge,the Daguangming Bridge and the Outside loop.The concentration of qacE,qacE△1 were from 102 to 104 copies/ m L.The concentration of qacG was from 102 to 103 copies/ m L.The concentration of the sugE(c),sugE(p)and emrE gene encoded by chromosome ranged from 100 to 103 copies/mL.The copy number of the ydgE and ydgF gene were from 101 to 102/mL.ConclusionThere were some bacteria which could resist to sodium hypochlorite,iodine and benzalkonium bromide in the Haihe River Basin in Tianjin.The phyla of Proteobacteria,Firmicutes,Cyanobacteria and Actinomycetes were presenting frequently.In addition,Aeromonas,Providencia and Morgan which were the opportunity pathogens could resist to the disinfectants in the samples.The bacteria in the Haihe River Basin in Tianjin carried a certain copies number of the disinfectant resistance genes.The potential endangerment of the disinfectant resistance bacteria and the disinfectant resistance genes should be attracted in environment.