Bacterial Antibiotic Resistance Shift And Its Molecular Ecology Mechanism During Drinking Water Chlorination

Extensive use and abuse of antibiotics stimulates the spread of antibiotic resistant bacteria(ARB)and resistance genes(ARGs)in aquatic environments,which has conferred enormous and complicated impacts on human health and environmental safety.Disinfection is a crucial step in drinking water treatment plants for controlling the spread of pathogenic bacteria,but recent studies indicate that chlorination promotes the antibiotic resistance of residual bacteria.However,chlorination effects on the ARGs diversity and abundance and the underlying molecular mechanism were rarely reported.Polymerase chain reaction(PCR)and quantitative real time PCR(qRT-PCR)have been widely applied to detect environmental ARGs,but limited primers are available for ARGs,and bias may exist during the amplification for environmental samples.Application of hybridization-based techniques in environmental ARGs detection often encounters difficult technical problems,such as low detection limit and complex sample pretreatment.In this study,a large-scale waterworks in Jiangsu Province was investigated and a series of experimental methods including high-throughput sequencing techniques-based metagenomic approaches,qRT-PCR and antibiotic sensitivity analysis were applied to investigate the shift of antibiotic resisitome,mobile genetic elements(MGEs)and bacterial community and their potential correlations.This thesis also explored the underlying molecular ecological mechanisms regarding chlorination effects on bacterial antibiotic resistance,theoretically helping to develop control techniques for ARGs and antibiotic resistant pathogens in drinking water.Overall,the main research conclusions are listed as belows.(1)Chlorination effects on bacterial antibiotic resistance in drinking waterBacterial strains were screened from filtered water,chlorine-disinfected water and tap water using MacConKey nutrient agar for taxonomic identification.The results showed that the chlorination and pipeline distribution induced an obvious change in the composition of cultivable bacteria.At genus level,the bacterial community was mainly composed of Escherichia,Enterobacteriaceae and Klebsiella before chlorination,while the abundance of Pseudomonas greatly increased after chlorination.Molecular cloning and 454 pyrosequencing also indicated great changes in bacterial community composition during chlorination and pipeline transport,and chlorination reduced the bacterial diversity.The Proteobacteria phylum dominated in the drinking water system,and its proportion gradually increased during chlorination and pipeline transportation.Antibiotic sensitivity analysis revealed that the percentage of chloramphenicol,trimethoprim and cephalothin resistant bacteria was enhanced by 4.1,1.5,and 3.0 folds,respectively,but the relative abundance of ampC?bla-TEM-1?tet(B)?tet(C)and aphA2 in isolated bacteria decreased,while the relative abundance of tet(A)and sulI increased.Compared with the filtered water,resistance to most antibiotics of ARB increased after pipeline transportation and the abundance of ARB resistant to ampicillin,tetracycline,trimethoprim and cefalotin were increased by 1.5,2.8,4.7,3.3 and 4.8 folds,respectively,as well as the relative abundance of blaTEM-1,tet(A),tet(B),and aphA2 gene in isolated bacteria.qRT-PCR results based on the total DNA of drinking water samples showed that chlorination significantly increased the abundances of 7 ARGs(ampC,aphA2,blaTEM-1,tet(A),tet(G),ermA and ermB)in drinking water(p<0.05)while the relative abundances of the 8 ARGs detected after pipeline transport were all significantly decreased(p<0.05).(2)Characteristics effects on diversity and abundance of ARGs and MGEs in drinking waterSource water,effluent of sedimentation tank,filtered water,chlorine-disinfected water and tap water were collected continuously,and total DNA of these samples were extracted.A total of 151 ARGs within 15 types were detectable in all the drinking water samples by using high-throughput sequencing and metagenomics technologies,and multidrug,bacitracin and sulfonamide resistance genes were the dominant types,accounting for 75.44-94.60%of the total abundance of ARGs,while the relative abundance of Resistance-Nodulation-Cell Division(RND)efflux pumps ARGs accounted for 99.33-100%of the total relative abundance of multidrug resistance genes.Chlorination significantly reduced the diversity of ARGs(p<0.05)while significantly increased their total relative abundance(p<0.05),especially the abundance of RND-related ARGs and bacA(p<0.05),significantly increasing to 71.10 ± 4.81 ppm and 15.45± 2.62 ppm,respectively,which mainly contributed to the increase of total relative abundance of ARGs.In addition,22 persistent ARGs(most were multidrug resistance genes)were detected.Although the diversity of persistent ARGs was low,their relative abundance accounted for 81.80-95.00%of the total ARGs relative abundance in the drinking water.Among the persistent ARGs,the relative abundance of bacA gene and RND-related genes significantly increased after chlorination(p<0.05).Principal coordinate analysis and Adonis assay showed significant changes in the antibiotic resistome after chlorination(p<0.01)and residual chlorine was identified as the key contributing factor driving the resistome alteration during drinking water treatment and pipeline distribution.At the same time,the relative abundances of MGEs including integrons,plasmids and IS were significantly increased after chlorination with the highest abundances of 84.23 ±10.20 ppm for integrons,3869.65 ± 1246.01 ppm for plasmids and 182.46 ± 87.38 ppm for IS in the chlorine-disinfected water.The relative abundances of MGEs and ARGs were significantly correlated,and the correlation between the relative abundances of integrons and ARGs was the highest(R2=0.85),followed by the insertion sequence(R2=0.66)and plasmid(R2=0.62).(3)The change of bacterial community structure and potential pathogens during drinking water chlorinationThe changes of bacterial community structure were investigated during chlorination and pipeline transportation based on the pyrosequencing of 16S rRNA gene.The results indicated that bacterial diversity significant decreased(p<0.05)and bacterial community significantly changed(p<0.01)after chlorination.The percentage of Proteobacteria had gradually increased along chlorination and distribution system,with the highest proportion(94.22 ± 0.24%)in tap water sample B,but the proportion of Actinomycetes gradually decreased.At genus level,the relative abundances of residual genera,including Pseudomonas,Acidovorax,Sphingomonas,Pleomonas and Undibacterium seemed to greatly increase after chlorination,and some of them were significantly and positively correlated with bacA and RND-related genes(p<0.02).Host analysis and redundancy analysis consistently revealed that Pseudomonas and Acidovorax were the main hosts of bacA and multi drug-resistant ARGs.Enrichment of chlorine-resistant bacteria(e.g.Pseudomonas and Acidovorax)significantly changed the bacterial community,and residual chlorine was the key contributing factor shaping the bacterial community(p<0.05).In addition,13 potential pathogens,mainly including Pseudomonas alcaligenes,Pseudomonas aeruginosa,Acinetobacter junii and Aeromonas hydrophila were detected in all the drinking water samples using MetaPhlAn2.Precipitation and sand filtration reduced the relative abundances of potential pathogens which dropped from 18.76%in source water to 0.45%in chlorinated water,while the relative abundances of potential pathogens increased to 21.39%and the species number increased to 10 after chlorination,posing potential human health risks after bacterial regrowth.(4)The underlying molecular mechanisms of antibiotic resistome change after drinking water chlorinationThe Mantel test revealed a significant correlation between bacterial communities and ARGs profiles(p<0.001).Bacterial community shift contributed to 57.22%of the resistome variation,which was much higher than MGEs alterations based on result of variation partitioning analysis.It indicated that bacterial community shift was the major driver shaping the antibiotic resistome in drinking water,which may be the potential mechanism of bacterial antibiotic resistance alteration by drinking water chlorination.Meanwhile,the total abundance of antibiotic resistance open reading frames(AR-ORFs)increased significantly from 121.13×/Gb to 818.84 ×/Gb after chlorination(p<0.01)based on metagenomics assembly combined with Resfams annotation.AR-ORFs within RND and ABC antibiotic efflux were identified as the core resistome in drinking water,their abundances accounted for 41.94-74.81%of the total abundance of AR-ORFs and both increased significantly after chlorination(p<0.01).Chlorination significantly increased the average abundance of MGE-like ORFs and promoted co-occurrence of various ARGs and the co-existence of ARGs and MGEs in drinking water.After chlorination,the obvious increase in abundance of the hosts(mainly Sphingomonas,Polaromonas,Bdellovibrio,Hyphomicrobium,Pseudomonas and Acidovorax)carrying RND and ABC efflux genes verified the results based on statistical methods,that is,the enrichment of chlorine-resistant bacteria carrying RND and ABC-related ARGs and the increase in the abundance of MGEs(including plasmids and transposons)mainly contributed to the increase of total relative abundance of ARGs in drinking water after chlorination,greatly promoting the microbial antibiotic resistance in drinking water after chlorination.