Occurrence of antibiotic resistance genes in urban water environment in hanoi

VIETNAM NATIONAL UNIVERSITY VIETNAM JAPAN UNIVERSITY s NGUYEN THI NGA OCCURRENCE OF ANTIBIOTIC RESISTANCE GENES IN WATER ENVIRONMENT IN HANOI MASTER THESIS Environmental Engineering Hanoi, 2019 VIETNAM NATIONAL UNIVERSITY VIETNAM JAPAN UNIVERSITY NGUYEN THI NGA OCCURRENCE OF ANTIBIOTIC RESISTANCE GENES IN WATER ENVIRONMENT IN HANOI MASTER’S PROGRAM: ENVIRONMENTAL ENGINEERING STUDENT ID: 17110042 SUPERVISORS: ASSOC PROF IKURO KASUGA PROF HIROYUKI KATAYAMA Hanoi, 2019 ACKNOWLEDGEMENT Master thesis was completed perfectly in the Vietnam Japan University (VJU) which is a great educational environment for research activities I would like to send my special thanks and gratefulness to my supervisors, Assoc Prof Ikuro Kasuga and Prof Katayama Hiroyuki who despite of being extraordinarily busy, took time out to hear, guide me many valuable lessons even the basic knowledge Their advice and encouragement had significantly enriched my quality of master thesis Without their guidance, it is impossible for me to finish effectively the thesis Many thanks go to the other professors in Master's program of Environmental Engineering at Vietnam Japan University, who kindly give necessary advices during my research progress I am using this opportunity to express my deepest gratitude to NIHE-NAGASAKI Friendship Laboratory that supported us having opportunity to the most important experiment via their facilities (Real-time PCR system) Last but not least, I would like to thank The Japan International Cooperation Agency (JICA) for supporting VJU students having a great opportunity for the study at VJU and internship in Japan I perceive as this opportunity as a big milestone in my career development and I hope to continue cooperation with all of you in the future Sincerely thank CONTENTS INTRODUCTION LITERATURE REVIEW 2.1 Antibiotic resistance mechanism and spreadi 2.1.1 Gene m 2.1.2 Horizon 2.2 Wastewater treatment plant as the hot-spot 2.2.1 Wastewater treatment plant - meeting place for selection of antibiot resistance 2.2.2 Removal efficiency of antibiotic, ARB and ARGs in WWTP 2.3 Antibiotic resistance in water environment 2.3.1 Two approaches antibiotic resistance in waster environments including ARB and ARGs 2.3.2 General review for ARB and ARGs in water environments 2.3.3 Antibiotic, ARB, ARGs in different water environments in Vietnam 2.3.4 Emergence of colistin resistance genes in water environments METHODS 3.1 Sampling 3.1.1 Sampling sites in Japan 3.1.2 Sampling sites in Hanoi 3.2 Persistence of ARGs in lab-scale experiment 3.3 Water quality measurement 3.3.1 Temperature and Electrical conductivity 3.3.2 Ammonium 3.3.3 E coli and total coliform 3.3.4 Flow cytometry 3.4 Molecular biological analysis 3.4.1 DNA extraction 3.4.2 PCR and gel agarose electrophoresis for presence of colistin resistan gene (mcr-1) 3.4.3 Real time PCR (qPCR) 34 PREVALENCE OF ANTIBIOTIC RESISTANCE GENES (ARGS) IN DIFFERENT WATER ENVIRONMENTS IN JAPAN 39 4.1 Water quality 39 4.1.1 Water quality in Tama river 39 4.1.2 Water quality in Lake Kasumigaura 40 4.1.3 Correlation among water quality parameters 40 4.2 ARGs in Tama River 41 4.3 ARGs in Lake Kasumigaura 44 PREVALENCE OF ANTIBIOTIC RESISTANCE GENES (ARGs) IN DIFFERENT WATER ENVIRONMENTS IN HANOI 48 5.1 Water quality 48 5.1.1 Water quality in all water environments 48 5.1.2 Correlation among water quality parameters 49 5.2 ARGs abundance in different water environments 49 5.2.1 ARGs abundance in various water environments 49 5.2.2 Presence of colistin resistance gene (mcr-1) in different water environments 52 5.3 Seasonal variations of removal efficiency of ARGs in wastewater treatment plant 55 5.3.1 Seasonal variations of ARGs abundances 55 5.3.2 Seasonal variation of removal rates of ARGs in wastewater treatment plant 58 5.4 Persistency of ARGs in lab-scale experiment 61 5.4.1 Water quality 61 5.4.2 Persistence of ARGs in lab-scale experiment 63 5.4.3 Presence of colistin resistance gene (mcr-1) in lab-scale experiment 65 COMPARISON OF ANTIBIOTIC RESISTANGE GENES (ARGs) BETWEEN JAPAN AND HANOI SAMPLES 66 6.1 Water quality 66 6.2 ARGs abundance 66 6.3 Wastewater treatment plant removal efficiency 69 CONCLUSION 71 REFERENCES APPENDIX LIST OF TABLES Table 2.1 Removal rates of ARGs in WWTPs in China 11 Table 2.2 Culture-dependent method and culture-independent method .14 Table 3.1 Description of sampling points in Japan 23 Table 3.2 Seasonal sampling points in Hanoi 25 Table 3.3 Extended sampling points in Hanoi 27 Table 3.4 FastDNA Spin Kit for Soil Components 32 Table 3.5 The reaction mixture for PCR 34 Table 3.6 Table of sequence primer 36 Table 3.7 The reaction mixture for qPCR 36 Table 4.1 Water quality of samples in Tama River 39 Table 4.2 Water quality of samples in Lake Kasumigaura 40 Table 5.1 Presence of Colistin resistance gene (mcr-1 ˷ 330 base pairs) 53 LIST OF FIGURES Figure 2.1 Antibiotic resistance mechanism - Gene mutations Figure 2.2 Transfer of ARGs in microorganism community Figure 2.3 Integron is involved in HGT (Gillings et al 2015) Figure 2.4 Antibiotic consumption in Vietnam reference (Gelband et al 2015) 17 Figure 3.1 Sampling sites in Tama River, Tokyo 21 Figure 3.2 Sampling sites in Lake Kasumigaura, Ibaraki prefecture 22 Figure 3.3 Kim Nguu river and Yen So WWTP 24 Figure 3.4 Extended sampling sites in Hanoi 26 Figure 3.5 Schematic of lab-scale pilot 28 Figure 4.1 Correlation between ammonium concentration and log E coli 41 Figure 4.2 Absolute and relative abundances of ARGs in Tama River and Kanda River 42 Figure 4.3 E coli concentration in Tama River and Kanda River 43 Figure 4.4 Absolute abundance of 16S rRNA genes and Total cell counts in Tama River and Kanda River 44 Figure 4.5 Absolute abundance of ARGs and IntI1 in Lake Kasumigaura 45 Figure 4.6 Relative abundance of ARGs and IntI1 in Lake Kasumigaura 46 Figure 4.7 Relative ratio of ARGs to those at KS1 47 Figure 5.1 Correlation between log E coli and ammonium concentration 49 Figure 5.2 Abundance of ARGs in different water environments 50 Figure 5.3 Relative abundance of ARGs in different water environments 51 Figure 5.4 Correlation between each ARGs and IntI1 (MGEs) 52 Figure 5.5 Image of electrophoresis of mcr-1 genes 54 Figure 5.6 Presence of mcr-1 in various categorizes 55 Figure 5.7 Absolute abundance of ereA, tetA, tetW, sul in seasonal samples .56 Figure 5.8 Absolute abundance of qnrD, blaTEM, 16S rRNA genes and IntI1 in seasonal samples 57 Figure 5.9 Log removal rate of E coli in WWTP 59 Figure 5.10 Log removal rate of ammonium in WWTP 59 Figure 5.11 Log removal values of each ARGs and 16S rRNA genes, IntI1 .60 Figure 5.12 Ammonium concentration in lab-scale pilot 61 Figure 5.13 Relative change in E coli in lab-scale pilot 62 Figure 5.14 Relative change in Total coliform in lab-scale pilot 62 Figure 5.15 Log change of ARGs, 16S rRNA genes, IntI1 in INF-aeration (a); INFNo aeration (b); EFF-aeration (c); EFF-No aeration (d) 64 Figure 6.1 Comparison the water quality of Hanoi and Japan samples .66 Figure 6.2 Relative abundance of ARGs to the level at TM1 (Upstream of Tama River) for all samples, circle symbols for Japan samples, and triangle symbols for Hanoi samples 67 Figure 6.3 Log removal values of each ARGs and 16S rRNA genes, IntI1 in Japan 69 LIST OF ABBREVIATIONS Acronyms Original words 16S rRNA 16S ribosomal RNA A/O Anaerobic oxic ARB Antibiotic resistant bacteria ARGs Antibiotic resistance genes CAS Conventional activated sludge CASS Cyclic activated sludge system CDDEP Center for Disease Dynamics, Economics & Policy CFU Colony forming unit CLSI Clinical & Laboratory Standards Institute E coli Escherichia coli EC Electric conductivity EFF Effluent ESBL Extended spectrum beta-lactamase HGT Horizontal gene transfer INF Influent MGEs Mobile gene elements MICs Minimum inhibitory concentrations MLSB Macrolide-lincosamide-streptogramin B NGS Next generation sequencing PCR Polymerase Chain Reaction qPCR Quantitative polymerase chain reaction WWTP Wastewater treatment plant Stange, C et al 2019 “Distribution of Clinically Relevant Antibiotic Resistance Genes in Lake Tai, China.” Science of the Total Environment 655: 337–46 Sutcliffe, Joyce, Thorsten Grebe, Amelia Tait-kamradt, and Lillian Wondrack 1996 “Detection of Erythromycin-Resistant Determinants by PCR.” 40(11): 2562–66 Takasu, Hiroyuki, Satoru Suzuki, Alissara Reungsang, and Pham Hung Viet 2011 “Fluoroquinolone (FQ) Contamination Does Not Correlate with Occurrence of FQ-Resistant Bacteria in Aquatic Environments of Vietnam and Thailand.” Microbes and Environments 26(2): 135–43 Thi, Do et al 2014 “Antibiotic Sales in Rural and Urban Pharmacies in Northern Vietnam : An Observational Study.” BMC Pharmacology and Toxicology 15(6): 1–10 Thu, Truong Anh et al 2012 “Antibiotic Use in Vietnamese Hospitals: A Multicenter Point-Prevalence Study.” American Journal of Infection Control 40(9): 840–44 Tuo, Hongmei et al 2018 “The Prevalence of Colistin Resistant Strains and Antibiotic Resistance Gene Profiles in Funan River, China.” Frontiers in Microbiology 9(December): 1–10 Urase, Taro, and Takaya Sato 2016 “Quantitative Monitoring of Resistance in Escherichia Coli to Clinically Important Antimicrobials in an Urban Watershed.” Journal of Water and Environment Technology 14(5): 341–49 Van, Thi Thu Hao, George Moutafis, Linh Thuoc Tran, and Peter J Coloe 2007 “Antibiotic Resistance in Food-Borne Bacterial Contaminants in Vietnam.” Applied and Environmental Microbiology 73(24): 7906–11 Wang, Ruobing et al 2018 “The Global Distribution and Spread of the Mobilized Colistin Resistance Gene Mcr-1.” Nature communications 9(1): 1179 Watanabe, Kazuya, Yumiko Kodama, and Shigeaki Harayama 2001 “Design and Evaluation of PCR Primers to Amplify Bacterial 16S Ribosomal DNA Fragments Used for Community Fingerprinting.” Journal of Microbiological 76 Methods 44(3): 253–62 Wen, Qinxue et al 2018 “Insight into Effects of Antibiotics on Reactor Performance and Evolutions of Antibiotic Resistance Genes and Microbial Community in a Membrane Reactor.” Chemosphere 197: 420–29 Xu, Jian et al 2015 “Occurrence of Antibiotics and Antibiotic Resistance Genes in a Sewage Treatment Plant and Its Effluent-Receiving River.” Chemosphere 119: 1379–85 Xu, Yan Bin et al 2017 “Distribution of Tetracycline Resistance Genes and AmpC β-Lactamase Genes in Representative Non-Urban Sewage Plants and Correlations with Treatment Processes and Heavy Metals.” Chemosphere 170(100): 274–81 Yamamoto, Yoshimasa et al 2019 “Wide Dissemination of Colistin-Resistant Escherichia Coli with the Mobile Resistance Gene Mcr in Healthy Residents in Vietnam.” Journal of Antimicrobial Chemotherapy 74(2): 523–24 Yamashita, Naoyuki, Yohei Katakawa, and Hiroaki Tanaka 2017 “Occurrence of Antimicrobial Resistance Bacteria in the Yodo River Basin, Japan and Determination of Beta-Lactamases Producing Bacteria.” Ecotoxicology and Environmental Safety 143(April): 38–45 Yang, Dong et al 2017 “The Occurrence of the Colistin Resistance Gene Mcr-1 in the Haihe River (China).” International Journal of Environmental Research and Public Health 14(6): 1–10 Yang, Yuyi et al 2018 “Antibiotics and Antibiotic Resistance Genes in Global Lakes: A Review and Meta-Analysis.” Environment International 116(April): 60–73 Zhou, Hong-Wei et al 2017 “ Occurrence of Plasmid- and Chromosome-Carried Mcr-1 in Waterborne Enterobacteriaceae in China ” Antimicrobial Agents and Chemotherapy 61(8) 77 Zhou, Zhen Chao et al 2018 “Prevalence and Transmission of Antibiotic Resistance and Microbiota between Humans and Water Environments.” Environment International 121(July 2018): 1155–61 Zurfuh, Katrin et al 2016 “Occurrence of the Plasmid-Borne Mcr-1 Colistin Resistance Gene in Extended-Spectrum-Lactamase-Producing Enterobacteriaceae in River Water and Imported Vegetable Samples in Switzerland.” Antimicrobial Agents and Chemotherapy 60(4): 2594–95 78 APPENDIX Table A1 Water quality of Hanoi samples Sampling time 17/09/2018 Sampling sites Kim Nguu river Kim Nguu river Kim Nguu river Kim Nguu river Yen So lake Yen So lake Duckpond Kim Nguu river 25/12/2018 Kim Nguu river Kim Nguu river Yen So lake Yen So lake Kim Nguu river 17/01/2019 Kim Nguu river Yen So lake Yen So lake Next to Yen So lake 18/02/2019 Van Quan lake Sampling time Sampling sites Truc Back lake West lake Duong river 19/02/2019 Hoan Kiem lake Bay Mau lake Bay Mau lake Set river Lu river 20/02/2019 Nhue river Nhue river To Lich river To Lich river 21/02/2019 Kim Nguu river Kim Nguu river Yen So lake Yen So lake Yen So lake Linh Dam lake Filtered Kim Nguu river sample Filtered Kim Nguu river sample Sampling time Sampling sites Filtered sample Filtered sample 3/3/2019 Groundwater Filtered groundwater Day river 26/03/2019 Kim Nguu river Kim Nguu river Yen So lake Yen So lake Next to Yen So lake Nearby duckpond 10/4/2019 Hung Yen Hang Chi Hang Thuong 15/04/2019 Kim Nguu river Kim Nguu river Yen So lake Yen So Yen So Sampling time Sampling sites Yen So lake Next to Yen So lake Nearby duckpond Table A2 Water quality of Japan samples Sampling Sampling Time 5/10/2018 9/10/2018 19/10/2018 22/10/2019 sites Ara river Edo river Kanda river Tama river Tama river Tama river Tama river Tama river Tama river WWTP influent WWTP effluent Lake Kasumigaura 24/10/2019 7/11/2019 Code AR ED KD TM1 TM2 TM3 TM4 TM5 TM6 SIF SEF KS1 Lake Kasumigaura Lake Kasumigaura Lake Kasumigaura Lake Kasumigaura Sakura river WWTP influent WWTP effluent KS2 KS3 KS4 KS5 RS KSI KSE Figure A1 Relative abundance of ARGs in different water environments in Hanoi Jan.2019 Feb.2019 Mar.2019 Apr.2019 2.50 2.00 1.50 1.00 0.50 0.00 tetA tetW sul1 qnrD blaTEM 16S IntI1 rRNA Figure A2 Log removal value of ARGs w/o UV disinfection in WWTP Jan.2019 Feb.2019 Mar.2019 Apr.2019 Dec.2018 3.00 log removal of ARGs,16S rRNA, IntI1 log removal of ARGs,16S rRNA, IntI1 3.00 2.50 2.00 1.50 1.00 0.50 0.00 tetA tetW sul1 qnrD blaTEM 16S IntI1 rRNA Figure A3 Log removal value of ARGs with UV disinfection in WWTP log Average ARGs (copies/L) 12 10 r = 0.975 p < 0.01 024681012 log absolute abundance of IntI1 (copies/L) Figure A4 Correlation between log average of ARGs including tetA, ereA, sul1, blaTEM and log IntI1 for all Japan and Hanoi samples ... prevalence of ARGs in water environments in Hanoi, especially the impacts of WWTP in transmission of ARGs remains unknown 2.3.4 Emergence of colistin resistance genes in water environments Colistin is... review for ARB and ARGs in water environments 2.3.3 Antibiotic, ARB, ARGs in different water environments in Vietnam 2.3.4 Emergence of colistin resistance genes in water environments METHODS... THI NGA OCCURRENCE OF ANTIBIOTIC RESISTANCE GENES IN WATER ENVIRONMENT IN HANOI MASTER’S PROGRAM: ENVIRONMENTAL ENGINEERING STUDENT ID: 17110042 SUPERVISORS: ASSOC PROF IKURO KASUGA PROF HIROYUKI