Behavior of plasmid mediated colistin resistance gene in urban water environment

VIETNAM NATIONAL UNIVERSITY, HANOI uu VIETNAM JAPAN UNIVERSITY VU THI MY HANH BEHAVIOR OF PLASMID-MEDIATED COLISTIN RESISTANCE GENE IN URBAN WATER ENVIRONMENT AND FOOD CHAIN MASTER'S THESIS VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY VU THI MY HANH BEHAVIOR OF PLASMID-MEDIATED COLISTIN RESISTANCE GENE IN URBAN WATER ENVIRONMENT AND FOOD CHAIN MAJOR: ENVIRONMENTAL ENGINEERING CODE: 8520320.01 RESEARCH SUPERVISOR: Associate Prof Dr KASUGA IKURO Prof Dr KATAYAMA HIROYUKI Hanoi, 2020 CODE: ………………… ACKNOWLEDGMENT Foremost, I would like to express my sincere gratitude to Vietnam Japan University for creating a wonderful international educational environment for research activities I would like to acknowledge the Japan International Cooperation Agency (JICA) for financial support and the University of Tokyo for giving me internship opportunity My deepest thanks to my supervisors, Assoc Prof Dr Kasuga Ikuro and Prof Dr Katayama Hiroyuki who have always supported and assisted me throughout the preparation and during my research time They did teach me the way of thinking thoroughly and encourage me to express my opinions They have given me many opportunities to expand my international friends and working relationship I would like to give gratitude to other professors in the Master's program in Environmental Engineering for kindly guide and help me in the past time Many thanks to Prof Takemura and lab assistants in NIHE-Nagasaki Friendship Laboratory for supporting me during the experiment time I would like to thank the program assistant Ms Hang, lab assistants Ms Huong, Ms Xuyen as well as my classmates for accompanying me and helping me a lot Last but not least, I could not complete this two-year master course without the supporting from my family and friends during this time I am extremely grateful to them Sincerely thank i TABLE OF CONTENTS ACKNOWLEDGMENT i TABLE OF CONTENTS ii LIST OF TABLES v LIST OF FIGURES .vi LIST OF ABBREVIATIONS viii INTRODUCTION CHAPTER LITERATURE REVIEW 1.1 Antimicrobial resistance (AMR) 1.1.1 AMR definition and mechanism 1.1.2 One Health approach: Human-Animal-Environment interfaces in AMR 1.1.3 Antibiotic uses and resistance situation 1.2 Colistin resistance 13 1.2.1 Colistin mechanism 13 1.2.2 Colistin resistance mechanism 13 1.3 Plasmid-mediated colistin resistance gene (mcr-1) 16 1.3.1 Global dissemination of mcr-1 16 1.3.2 Correlation of mcr-1 with other genes 21 1.4 Research gaps 22 CHAPTER METHODOLOGY 23 2.1 Water sampling 23 2.1.1 Sampling in Hanoi 23 2.1.2 Sampling in Hai Phong 26 2.1.3 Sampling in Japan 27 2.2 Food sampling 30 2.2.1 Sampling in Hanoi 30 2.2.2 Sampling in Hai Phong 31 2.3 Food sample treatment 32 2.3.1 Collection of bacteria attached to food samples 32 2.3.2 Washing intervention 32 2.4 Water quality measurement 33 2.4.1 Electrical conductivity and temperature 33 2.4.2 Ammonium concentration 33 2.4.3 E coli and total coliform 34 2.4.4 Cultivation of colistin-resistant bacteria 35 ii 2.4.5 Total cell counts 36 2.5 Isolation of E coli possessing of mcr-1 36 2.5.1 Enrichment in liquid medium supplemented with antibiotic 36 2.5.2 Cultivation on Chromocult 37 2.5.3 Colony-direct PCR to check mcr-1 presence 37 2.6 Minimum inhibitory concentration (MIC) of E coli possessing mcr-1 37 2.7 Molecular biological analysis 38 2.7.1 DNA extraction 38 2.7.2 PCR and gel agarose electrophoresis 39 2.7.3 Quantitative PCR (qPCR) 41 2.7.4 SmartChip qPCR system analysis 43 2.7.5 Next-Generation Sequencing (NGS) analysis 44 2.8 Quantitative microbial risk assessment 44 CHAPTER PREVALENCE OF MCR-1 IN WASTEWATER AND WATER ENVIRONMENT 46 3.1 Profiles of ARGs in wastewater in Vietnam and Japan 46 3.2 Prevalence of mcr-1 in wastewater and water environment in Vietnam 48 3.2.1 Water quality 48 3.2.2 Detection of colistin-resistant bacteria 50 3.2.3 Prevalence of mcr-1 in wastewater and water environment 51 3.2.4 Correlation between mcr-1 and blaNDM-1 in wastewater and water environment 54 3.2.5 Correlation of mcr-1 with crAssphage in water samples 55 3.3 Behavior of mcr-1 in wastewater treatment plants in Japan 57 3.3.1 Water quality 57 3.3.2 Detection of colistin-resistant bacteria 60 3.3.3 Prevalence of mcr-1 in wastewater 61 3.3.4 Removal efficiency of mcr-1 in WWTPs 62 3.4 Conclusion 63 CHAPTER OCCURRENCE OF MCR-1 IN FOOD AT LOCAL MARKETS 64 4.1 E coli and total coliform in fresh food 64 4.2 mcr-1 contaminated food in local markets 65 4.3 Correlation of mcr-1 with blaNDM-1 and crAssphage in food samples 66 4.4 Conclusion 68 CHAPTER TRANSMISSION OF MCR-1 AMONG THE ENVIRONMENT AND HUMAN HEALTH RISK ASSESSMENT 69 5.1 Overview of mcr-1 circulation in water-food chain 69 iii 5.2 Polluted water and vegetables in vegetable production and distribution chain 70 5.2.1 E coli and total coliform at aquatic vegetable field 70 5.2.2 mcr-1 pollution at aquatic vegetable field 71 5.3 Isolation of 16 cultures possessing mcr-1 and transmission of mcr-1 in aquatic vegetable field 72 5.3.1 Enrichment 72 5.3.2 Selection of 16 cultures 74 5.3.3 Minimum inhibitory concentration (MIC) 75 5.3.4 DNA sequence analysis 76 5.4 Quantitative microbial risk assessment (QMRA) for E coli possessing mcr-1 in fresh vegetables 80 5.4.1 Exposure assessment 80 5.4.2 Measurements of pathogen 82 5.4.3 Dose-response model 82 5.4.4 QMRA analysis and risk characterization 84 5.5 Conclusion 85 CONCLUSION AND RECOMMENDATION 86 Conclusion 86 Recommendation 86 REFERENCES 88 iv LIST OF TABLES Table 1.1 Pathogens susceptible and resistance to colistin naturally (WHO (Global Antimicrobial Resistance Surveillance System), 2018) 13 Table 1.2 The detections of mcr family gene 15 Table 2.1 Water samples collected in Hanoi 25 Table 2.2 Water samples collected in Hai Phong 27 Table 2.3 Description of sampling sites in Japan 29 Table 2.4 Description of food samples in Hanoi 30 Table 2.5 Food samples in Hai Phong 32 Table 2.6 Classification of colonies cultivated on CHROMagar™ COL-APSE (CHROMagarTM The Chromogenic Media Pioneer, 2019) 36 Table 2.7 Enrichment conditions for E coli possessing mcr-1 isolation 36 Table 2.8 The PCR mixture components list 40 Table 2.9 Primer sequences of target genes 41 Table 2.10 The qPCR mixture components list 42 Table 3.1 Water quality of wastewater samples in Hanoi 49 Table 3.2 Water quality in WWTPs and river in Japan 58 Table 5.1 Efficiency of different enrichment conditions on selecting mcr-1 positive culture 73 Table 5.2 Descriptions of 16 cultures 75 Table 5.3 MIC test results of 16 cultures 76 Table 5.4 Genome information of 16 cultures (HSP: High-Scoring Segment Pair, a concept used in heuristic sequence alignment programs) 76 Table 5.5 Mapped plasmid sequences information 77 Table 5.6 Reference plasmid for genomic background of mcr-1 79 Table 5.7 Baseline and three scenarios for E coli O157:H7 possessing mcr-1 infection assessment 82 Table 5.8 Description of constant and variables in Equation 5.2 83 v LIST OF FIGURES Figure 1.1 Four main mechanisms of antimicrobial resistance Figure 1.2 Three main mechanisms of horizontal of genetic material transfer between bacteria Figure 1.3 One Health approach in context of AMR (modified from Korean National Institue of Health, 2019) Figure 1.4 The global spread of mcr-1 (colored parts indicate the countries where mcr1 was detected) (R Wang et al., 2018; Xiuna Wang et al., 2017) 17 Figure 1.5 Global presence of mcr-1 in One Health concept 20 Figure 2.1 Water sampling sites in Hanoi 24 Figure 2.2 Sampling at vegetables field in Hanoi (26 February 2020) 25 Figure 2.3 Water sampling sites in Hai Phong (December 2019) 27 Figure 2.4 Sampling sites in Japan (October – November 2019) 28 Figure 2.5 Sampling points in wastewater treatment plant in Japan 28 Figure 2.6 QMRA as a tool for synthesizing quantitative scientific data to improve water safety management (World Health Organization, 2016) 45 Figure 3.1 Number of detected genes and classification 47 Figure 3.2 Venn diagram of genes detected in TL and A1 47 Figure 3.3 Ratio of relative abundances of common target genes (target gene/16S rRNA genes) of TL and A1 48 Figure 3.4 E coli counts and Total coliform counts in water samples 50 Figure 3.5 Abundances of colistin-resistant E coli, coliforms, Pseudomonas, and Acinetobacter in wastewater in Hanoi 51 Figure 3.6 Absolute abundance of mcr-1 and ratio of mcr-1 to 16S rRNA genes in wastewater and water environment Open bar denotes levels below the limit of quantification 52 Figure 3.7 16S rRNA genes abundances in wastewater and water environment 52 Figure 3.8 Correlation of mcr-1 and 16S rRNA genes in water samples 53 Figure 3.9 blaNDM-1 absolute abundance in wastewater and water environment 55 Figure 3.10 Correlation of mcr-1 and blaNDM-1 in wastewater and water environment55 Figure 3.11 crAssphage abundance in wastewater and water environment 56 Figure 3.12 Correlation of mcr-1 with crAssphage in wastewater and water environment 57 Figure 3.13 Total cell counts, total coliform counts, and E coli counts in wastewater 59 vi Figure 3.14 Abundances of total and colistin-resistant Escherichia coli, coliforms, Pseudomonas, and Acinetobacter in the influent of plant A (A1), plant D (D1), and plant E (E1), effluent from the primary settlement basin of plant B (B2), and plant C (C2) The numbers indicate the percentages of resistant bacteria 61 Figure 3.15 Absolute abundances of mcr-1 in wastewater samples 62 Figure 3.16 Log reduction values of TCCs, total coliform, E coli and mcr-1 in wastewater treatment 63 Figure 4.1 E coli and total coliform counts in fresh food samples in local markets 65 Figure 4.2 A) mcr-1 abundance and ratio of mcr-1 to 16S rRNA genes in fresh food B) blaNDM-1 abundance in fresh food C) crAssphage abundance in fresh food in local markets Open bar denotes levels below the limit of quantification 66 Figure 4.3 Correlation between mcr-1 and blaNDM-1 in fresh food samples 67 Figure 5.1 Estimated circulation of mcr-1 in water-food chain in Vietnam 70 Figure 5.2 E coli and total coliform of samples in aquatic field areas 71 Figure 5.3 Absolute abundance of mcr-1 in water samples and vegetables sample at aquatic field 72 Figure 5.4 Electrophoresis of mcr-1 74 Figure 5.5 Gene map of plasmid T2 from E coli isolated from To Lich (33,320 bp) 78 Figure 5.6 Genomic background of mcr-1 79 Figure 5.7 Exposure assessment for infection of E coli possessing mcr-1 in fresh vegetables 81 Figure 5.8 Illness risk of diarrhea caused by E coli O157:H7 possessing mcr-1 84 Figure 5.9 Log reduction value (LRV) of risk 85 vii LIST OF ABBREVIATIONS AMR ARB ARGs BLAST bp COL-R ESBL HGT HSP LOQ LPS LRV MGE MIC N/A PB QMRA SDG TCCs WWTP Antimicrobial resistance Antibiotic resistant bacteria Antimicrobial resistance genes Basic Local Alignment Search Tool base pair Colistin-resistant Extended-spectrum β-lactamases Horizontal gene transfer High-Scoring Segment Pair Limit of quantification Lipopolysaccharide Log reduction value Mobile gene elements Minimum inhibitory concentration Not available Phosphate buffer Quantitative microbial risk assessment Sustainable development goals Total cell counts Wastewater treatment plant viii case study (chapter 3) Monte Carlo simulation was carried out for 10,000 times to estimate illness risk Table 5.7 Baseline and three scenarios for E coli O157:H7 possessing mcr-1 infection assessment No treatment from WWTP Treatment from WWTP No intervention Baseline scenario 2nd scenario Intervention 1st scenario (Fact) 3rd scenario 5.4.2 Measurements of pathogen To apply the DRM for pathogens, the abundance of mcr-1 gene copy numbers was converted to E coli possessing mcr-1 cell numbers The pure cultures of E coli possessing mcr-1 were analyzed to estimate the ratio of mcr-1 and 16S rRNA gene Since the average copy number of 16S rRNA genes is per cell, the conversion was performed by applying the Equation 5.3: 𝑚𝑐𝑟 − 1/𝐸 𝑐𝑜𝑙𝑖 𝑐𝑒𝑙𝑙 𝑐𝑜𝑝𝑖𝑒𝑠 ) × 7(16𝑆 𝑟𝑅𝑁𝐴 𝑔𝑒𝑛𝑒𝑠 𝑐𝑜𝑝𝑖𝑒𝑠/𝑐𝑒𝑙𝑙) µ𝐿 16𝑆 𝑟𝑅𝑁𝐴 𝑔𝑒𝑛𝑒 𝑐𝑜𝑝𝑦 𝑛𝑢𝑚𝑏𝑒𝑟𝑠 (𝑐𝑜𝑝𝑖𝑒s/µ𝐿) 𝑚𝑐𝑟 − 𝑔𝑒𝑛𝑒 𝑐𝑜𝑝𝑦 𝑛𝑢𝑚𝑏𝑒𝑟𝑠 ( = In Equation 5.3 (E.q 5.3): 7: average number of 16S rRNA gene per E coli genome The ratio of pathogenic E coli O157:H7 to E coli is 7.6 × 10-4 – × 10-2 5.4.3 Dose-response model Applying the Beta-Poisson dose response model, the illness probability of E coli O157:H7 carrying mcr-1 was calculated following Equation 5.4 (E.q 5.4) with the description of constant and variables shown in Table 5.8 82 𝑑 = 𝐶/𝑎 × 𝑏 × 10−(𝐿𝑅𝑉.𝑉+𝐿𝑅𝑉.𝑊) × 𝐹 × 𝑐 (E.q 5.4) 𝑃 = {1 − [1 + (𝑑/𝛽)]−∝ } × 𝑖 Table 5.8 Description of constant and variables in Equation 5.3 Description Unit Distribution and References value(s) C: mcr-1 abundance in fresh copies/g vegetable Triangular This study (min: 664, median: 8533, max: 85595) a: Ratio mcr-1 to E coli cell copies/cell Uniform (0.3-1.9) This study b: Ratio of pathogenic E coli CFU/CFU Uniform (Fuhrimann et O157:H7 to overall E coli (7.6 × 10-4–1 × 10-2) al., 2017) LRV.V: Log reduction value of Uniform E coli abundance in fresh (0.55-5.32) This study vegetable after intervention LRV.W: Log reduction value of Triangular E coli abundance (min: 3.4, max: 4.0, This study median: 3.9) F: Total vegetable consumptions g/day/adult 282.59 per person per day c: Fraction of raw vegetables (Global Nutrion Report, 2020) g/g Uniform (0.01-0.1) This study β in Beta-Poisson model 229.2928 (Pang α in Beta-Poisson model 0.267 83 2017) et al., i: Probability of illness from 0.35 infection of E coli O157:H7 (Fuhrimann et al., 2017) 5.4.4 QMRA analysis and risk characterization By applying Monte Carlo simulation on Beta-Poisson model for 10000 times, the risk of illness caused by E coli O157:H7 possessing mcr-1 was summarized as shown in Figure 5.8 The median value indicates that in a baseline scenario, no WWTP or intervention is applied, 15,110 people among 100,000 eating raw vegetables suffer from diarrhea caused by E coli O157:H7 carrying mcr-1 That high risk alerts us to consider more to the safety of fresh vegetables In the 1st scenario (the fact scenario), QMRA result indicated that in 80 people among 100,000 people eating raw vegetable develop diarrhea even they apply washing intervention Figure 5.8 Illness risk of diarrhea caused by E coli O157:H7 possessing mcr-1 Figure 5.9 describes the log reduction value of illness probability for the application of each mitigation If we apply washing intervention or WWTP to treat wastewater individually, the risk will reduce 2.3 or 3.4 log10, respectively The efficiency of WWTP in reducing LRV is higher than washing intervention, showing the importance of the 84 wastewater treatment for irrigation water From this scenario, WWTP can not only control the water quality but also reduce the risk of infection and disease If washing intervention and WWTP are applied together, LRV increases to 6.3 log10, suggesting the best scenario needs participation from all stakeholders, as described in One Health concept (World Health Organization, 2017b) In Hanoi, current situation is the 1st scenario applying only washing intervention If someone does not wash fresh vegetables before eating, the risk of diarrhea will be 212 times higher than the baseline Even in the second scenario, if WWTP takes place, fresh vegetables still can be contaminated during transportation or storage Therefore, intervention plays a critical role in diarrhea prevention Human behavior is the most important in health protection Figure 5.9 Log reduction value (LRV) of risk 5.5 Conclusion  Genomic evidence proved the transmission of mcr-1 among different factors in aquatic field  QMRA results evaluated the illness risk probability of diarrhea caused by E coli O157:H7 carrying mcr-1 in the current situation in Hanoi is 8.06 × 10-4  Applying intervention and WWTP together is the best scenario to reduce the infection risk 85 CONCLUSION AND RECOMMENDATION Conclusion The comparable levels of mcr-1 in urban sewage in Vietnam and Japan showed the prevalence of the transmissible colistin resistance gene Domestic wastewater is thus considered an important monitoring target for mcr-1 in urban cities The presence of mcr-1 is significantly correlated with 16S rRNA genes, blaNDM-1 and crAssphage, suggesting the co-occurrence of these genes in polluted water samples Whereas wastewater treatment could reduce mcr-1 copy numbers, final effluents after chlorination still contained mcr-1 In addition to conventional indices of total coliform and E coli, it is necessary to control mcr-1 released into aquatic environments from wastewater treatment plants Vegetables and meats in local markets in Vietnam were polluted by mcr-1 accounting for major exposure of mcr-1 Polluted irrigated water could be a source of mcr-1 in vegetables The high correlation of mcr-1 and blaNDM-1 in pork samples poses a threat of “superbug” in the food source Genomic evidence suggested the transmission of mcr-1 among different media including wastewater, field water and vegetables in the aquatic field Thus, irrigated water polluted by untreated wastewater could be the main source of mcr-1 in vegetables QMRA results estimated the illness risk probability of diarrhea caused by E coli O157:H7 carrying mcr-1 in the current situation of Hanoi is 8.06 × 10-4 The combination of intervention and WWTP could reduce the risk by 6.3 log10, which is the significant impact to prevent this potential infection Recommendation Wastewater is evaluated as the source of all infectious bacteria and ARGs in this study Therefore, to reduce the pollution and prevent the dissemination of mcr-1 as well as other ARGs, treatment of wastewater plays a pivotal role Further study is necessary to evaluate the removal performance of ARGs 86 To protect residents’ health from infection risk, under the condition without WWTP, wastewater has to be isolated from food production and distribution chains Irrigation water should be safe for urban and agricultural activity Meat processing should be controlled in terms 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MY HANH BEHAVIOR OF PLASMID- MEDIATED COLISTIN RESISTANCE GENE IN URBAN WATER ENVIRONMENT AND FOOD CHAIN MAJOR: ENVIRONMENTAL ENGINEERING CODE: 8520320.01 RESEARCH SUPERVISOR: Associate Prof Dr... mcr-1 in different water environment and food supply chain (food chain) in urban cities in northern Vietnam Specific objectives of our study are: 1) To investigate the behavior of mcr-1 in water environment. .. discovery of mcr-1 was reported in many environments, including food, human, wastewater, water environment and drinking water, which are indispensable parts of the One Health concept Colistin was