Behavior of plasmid mediated colistin resistance gene in urban water environment

1 uu 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 MA

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uu

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

MASTER'S THESIS

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VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY

VU THI MY HANH

BEHAVIOR OF PLASMID-MEDIATED COLISTIN RESISTANCE GENE IN URBAN

WATER ENVIRONMENT AND

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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

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TABLE OF CONTENTS

ACKNOWLEDGMENT i

TABLE OF CONTENTS ii

LIST OF TABLES v

LIST OF FIGURES vi

LIST OF ABBREVIATIONS viii

INTRODUCTION 1

CHAPTER 1 LITERATURE REVIEW 3

1.1 Antimicrobial resistance (AMR) 3

1.1.1 AMR definition and mechanism 3

1.1.2 One Health approach: Human-Animal-Environment interfaces in AMR 7

1.1.3 Antibiotic uses and resistance situation 9

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 2 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

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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 3 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 bla NDM-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 4 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 bla NDM-1 and crAssphage in food samples 66

4.4 Conclusion 68

CHAPTER 5 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

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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

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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

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LIST OF FIGURES

Figure 1.1 Four main mechanisms of antimicrobial resistance 4

Figure 1.2 Three main mechanisms of horizontal of genetic material transfer between bacteria 6

Figure 1.3 One Health approach in context of AMR (modified from Korean National Institue of Health, 2019) 8

Figure 1.4 The global spread of 1 (colored parts indicate the countries where mcr-1 was detected) (R Wang et al., 20mcr-18; Xiuna Wang et al., 20mcr-17) mcr-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 bla NDM-1 absolute abundance in wastewater and water environment 55

Figure 3.10 Correlation of mcr-1 and bla NDM-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

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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) bla NDM-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 bla NDM-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

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LIST OF ABBREVIATIONS

AMR Antimicrobial resistance

ARB Antibiotic resistant bacteria

ARGs Antimicrobial resistance genes

BLAST Basic Local Alignment Search Tool

COL-R Colistin-resistant

ESBL Extended-spectrum β-lactamases HGT Horizontal gene transfer

MIC Minimum inhibitory concentration

QMRA Quantitative microbial risk assessment SDG Sustainable development goals

WWTP Wastewater treatment plant

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INTRODUCTION

The discovery of penicillin and the invention of many antibiotics has made a new era of medical development in the fight with bacterial infections However, scientist Alexander Fleming, who discovered the world's first antibiotic and received the Nobel Prize in Physiology or Medicine in 1945 for this study, warned in his Nobel Lecture about the antibiotic resistance ability of bacteria if antibiotics were not used properly (Fleming,

n.d.) Shortly thereafter, a report on a penicillin-resistant Staphylococus strain, was first

observed in 1947 (Friedmann, 1948) Since then, more than 100 types of antibiotics have been identified However, the overuse and misuse of antibiotics help to create drug-resistant bacteria Besides its important role in human health, antibiotics are also used for treating and preventing diseases in agriculture, especially in livestock Animals continuously exposed to antibiotics are hot spots of antibiotic resistant bacteria (ARB) Moreover, water environment which receives ARB released from human and animals is

of great concern in terms of the fate of ARB Thus, occurrence of ARB should be monitored in the framework of “One Health” which emphasizes the linkage of human-animal-environment

Nowadays, antibiotic resistance is not a new problem, but it has become an urgent issue

in the whole world ARB cause at least 2.8 million infections and more than 35,000 deaths each year in USA, according to the U.S Centers for Disease Control and Prevention (CDC) (U.S Centers for Disease Control and Prevention (CDC), 2019) Drug-resistant forms of tuberculosis, gonorrhea, and staph infections are critical Huge efforts are required to prevent from returning to time when no antibiotics were available The World Health Organization (WHO) claims that we are living in an era of antibiotics dependency and global demand is responsible for protecting precious antibiotic resources for the next generation

Among critically important antibiotics, colistin is regarded as the last resort of antibiotics With the dangers of spreading colistin resistance, plasmid-mediated colistin resistance

gene (mcr) is receiving a special attention Currently, mcr has been reported to appear in

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many countries around the world (Lekunberri, Balcázar, & Borrego, 2017; Tuo et al., 2018; Xiuna Wang, Zhang, Sun, Liu, & Feng, 2017) In Vietnam, the level of antibiotic resistance is rated as one of the highest in the world There have been several studies on the occurrence of antibiotic resistance genes (ARGs) in wastewater as well as domestic water in Vietnam (Binh, Dang, Anh, Ky, & Thai, 2018; Nguyen, Kasuga, Liu, &

Katayama, 2019) However, at present, little is known about mcr in the environment in

Vietnam It is necessary to evaluate the reservoir of ARGs in water environment, as well

as the epidemiologic estimation of ARB in citizen health

In this study, we aimed at evaluating the abundance and dissemination of colistin

resistance gene 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 in urban cities in northern

Vietnam

 The concentration of mcr-1 in different aquatic environments

 Domestic wastewater reflecting loading of mcr-1 from urban cities

2) To compare situation of mcr-1 in wastewater treatment systems between Vietnam

and Japan

 The removal efficiency of mcr-1 during wastewater treatment

3) To reveal the prevalence of mcr-1 in foodstuffs in urban cities in northern Vietnam 4) To assess human health risk related to mcr-1 exposure

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1.1 Antimicrobial resistance (AMR)

1.1.1 AMR definition and mechanism

1.1.1.1 AMR definition

According to the definition of WHO (2018), “Antimicrobial resistance happens when

microorganisms (such as bacteria, fungi, viruses, and parasites) change when they are exposed to antimicrobial drugs (such as antibiotics, antifungals, antivirals, antimalarials, and anthelmintics)” (World Health Organization, 2018b) Antibiotic

resistance (ABR) usually only refers to the case of bacteria (World Health Organization, 2018a) Microbes that enhance the ability to resist multiple antimicrobials are described

“superbugs”(World Health Organization, 2018b) Pathogenic superbugs pose a serious human health risk (World Health Organization, 2018b)

1.1.1.2 AMR types and mechanisms

Antibiotics are one of the most efficient treatments in medicine for bacterial infections Facing the danger of AMR issue, a comprehensive understanding of its mechanisms will become the foundation to find out solutions for this threat In the context of AMR history,

four bacterial defense mechanisms are known (Figure 1.1)

(1) Reduced permeability of outer membrane to inhibit the penetration of antimicrobial agents into the cell;

(2) Active efflux to pump the antimicrobial agents to the outside of the cell;

(3) Modification of antimicrobial agents by producing some enzymes to destroy or change the structure of antimicrobial agents;

(4) Modification of target sites (both surface-expose and intracellular targets) which make antimicrobial agents ineffective (Boerlin & White, 2013)

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Figure 1.1 Four main mechanisms of antimicrobial resistance

Some bacteria can inherently save themselves by intrinsic characteristics while others can adapt to antimicrobial conditions by acquiring the resistance (Food and Agriculture Organization, 2016)

Intrinsic resistance

As well as innate immune systems in the human body, some specific genus or species of bacteria own the natural resistance ability to antibiotics even though they have not been exposed to those medicines This resistance phenotype is often based on their unique individual structural or biochemical characteristics (Boerlin & White, 2013) Lack of penicillin-binding proteins is a natural trait of Gram-positive bacteria including some

Enterococci Their characteristic inhibits antibiotic to bind and be active on bacteria,

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thus they can deactivate β-lactams agents (Rice, 2012) Other examples of intrinsic resistance related to the permeability of outer membrane The innate outer membrane structure of some Gram-negative bacteria is impermeable to large glycopeptide such as

vancomycin (Nelson, 1999) On the other hand, ampicillin-resistant Klebsiella bacteria

can destroy penicillin by producing β-lactamase (Fu et al., 2007) Intrinsic resistance is transferred directly to next generation via DNA multiplication

Acquired resistance

Susceptible bacteria can develop the acquired resistance The acquisition of antimicrobial resistance can result from vertical transmission and horizontal transmission (Boerlin & White, 2013)

Vertical transmission includes spontaneous and induced gene mutation events Mutations can either affect target or regulatory genes While target mutations affect the structural genes that encode the specific targets of antimicrobial action, regulatory mutations often change gene expression mechanisms (Courvalin, 2008) Although these mutation events happen rarely in bacteria, bacterial mutation frequencies can increase under stress condition such as the selective pressure of antibiotics (Couce & Blázquez, 2009) The development and prevalence of antimicrobial resistance is attributed to the acquisition of extrachromosomal resistance genes via horizontal transmission

Horizontal transmission occurs in three main cases of horizontal gene transfer (HGT)

among bacteria populations (Figure 1.2):

(1) Transformation: uptake of naked DNA present in the environment by naturally competent bacteria and may recombine with homologous sequences in the recipient’s genome

(2) Transduction: transfer of injected DNA from one bacterium to another by bacteriophages, and in the occurrence of a lysogenic phase, this DNA is integrated into the chromosome of the recipient cell

(3) Conjugation: transfer of plasmids between bacteria through a mating-like process (transfer is coupled with replication and a copy of the plasmid remains in the

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donor) to recipient cell in which it can replicate Plasmids may have acquire a

transposon carrying antimicrobial resistance genes (Boerlin & White, 2013) HGT can occur between many similar species of bacteria, such as between Escherichia

coli (E coli) causing food poisoning and E coli causing urinary tract infections (Javadi

et al., 2017); or between different species of bacteria, such as vancomycin resistance via

plasmid carrying vanA gene from enterococci to methicillin-resistant Staphylococcus

aureus (MRSA) (Kohler et al., 2018) HGT can also occur between natural and

pathogenic bacteria in the human gut Therefore, our gut microbiota can serve as a source

of antibiotic resistance genes This explains why people should take antibiotics only when they really need them Otherwise, bacteria can immediately turn into many complex resistance mechanisms and quickly resist to multiple antibiotics

Figure 1.2 Three main mechanisms of horizontal of genetic material transfer

between bacteria

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1.1.2 One Health approach: Human-Animal-Environment interfaces in AMR

"One Health" is the concept which emphasizes the interfaces among the human being, animals and environment for AMR control This concept produces a comprehensive and beneficial policy, research, program, and regulation on reducing the risk of AMR (World Health Organization, 2017) In the context of AMR, finding the exposure route of ARB plays a pivotal role in preventing and solving this issue Plenty of bacteria and microorganisms infect humans and animals as they exist everywhere AMR crosses the interfaces between human and animal or among country boundaries Therefore, a “One Health” perspective is necessary to solve the problem All stakeholders such as environmental health, human health, and animal health should coordinate to prevent, control and treat the AMR issue comprehensively and effectively (World Health Organization, 2017) Three global organizations WHO, FAO and OIE, which are respectively responsible for human health, food and agriculture, and animal health, have collaborated and issued a Global Action Plan on Antimicrobial Resistance to prevent exacerbation of AMR issues all over the world (World Health Organization, 2017)

Figure 1.3 shows the concept of One Health in the context of AMR The misuse and

abuse of antibiotics are accelerating AMR Antibiotics, which are structurally similar and even identical to those for humans, are also used for veterinary health care The utilization of antibiotics in animal health has been extended from treatment to precaution and growth promoters without expert oversight (World Bank, 2017) Antibiotic use in food-producing animals can influence human health by the residues of medicines in foods and especially by the selection of ABR pathogens in animals through direct contact

or consumption of contaminated food (World Health Organization, 2001) Wastewater can disseminate AMR rapidly and AMR may circulate through food supply chains due

to the contamination and inappropriate processing of food Therefore, resistance agents can arise from animal or human, and spread to other environments where the risk of horizontal gene transfer is concerned

To comply with the international recommendations of multi-sectoral surveillance policies in line with the One Health concept, the Vietnamese government has enacted a strategy for AMR by inter-ministerial surveillance with an integrated monitoring system (Bordier et al., 2018) Following the instruction from the WHO Advisory Group (World

1.1.3 Antibiotic uses and resistance situation

1.1.3.1 Situation in the world

Antibiotic use

According to WHO Report on Surveillance of Antibiotic Consumption in early implementation period (2016-2018), overall consumption of antibiotics including community and hospital aspect in 65 countries from different regions in the world varied from 1 to 2225 tons per year (total absolute weighs), equivalent to 4.4 to 64.4 DDD per

1000 inhabitants per day (World Health Organization, 2018c) DDD stands for Defined

Daily Dose, as defined by WHO, is “the assumed average maintenance dose of an

antimicrobial substance(s) used for its main indication in adults per day” (World Health

Organization, 2018c) The usage of antibiotics is extremely high in some regions worldwide, indicating possible overuse, whereas it presents low use in other parts, suggesting inadequate access to these drugs (World Health Organization, 2018c) A worldwide analysis report from WHO in 2015 revealed that the residents are able to buy antibiotic drugs without a prescription (17-64% of the countries) (WHO, 2015) The WHO multi-nation awareness analysis report also revealed that 36% of respondents believe that they can stop using drugs if they feel better (World Health Organization, 2015a) It is also possible that antibiotic resistant bacteria (ARB) can survive if the patients stop to take antibiotic as prescribed Patients sometimes do not take antibiotics well or stop the use of antibiotics, resulting in the occurrence of ARB under a lower

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concentration of antibiotics in the body Another situation leading to the occurrence of ARB is 64% of patients use antibiotics to treat colds and flu (World Health Organization, 2015a) The general misunderstanding and lack of awareness in the use of antibiotics indicate that AMR perhaps spread further

Antimicrobial resistance (AMR)

Although antimicrobial resistance occurs as a natural evolutionary direction of microorganisms, the abuse and improper use of antibiotics in humans and animals is accelerating this bad process Globally, due to resistance in diseases such as bacterial infections, malaria, HIV / AIDS or tuberculosis, at least 700,000 people die each year

In Europe and the United States, about 50,000 lives are lost each year due to resistant infections (Intergency Coordination Group on Antimicrobial Resistance, 2019)

antibiotic-In Japan, about 8000 deaths were attributed to methicillin-resistant Staphylococus

aureus (MRSA) and fluoroquinolone-resistant E coli (FQREC) in 2017 (Tsuzuki et al.,

2020) As the most alarming scenario predicted by the World Bank, unless sustained responses are taken, by 2050, the number of worldwide deaths related to AMR will be projected to reach 10 million per year which is equivalent to the rate that about 1 person will die every 3 seconds (World Bank, 2017) In April 2019, the Intergency Coordination Group on Antimicrobial Resistance issued a warning about the AMR situation and called for urgent action worldwide (Intergency Coordination Group on Antimicrobial

Resistance, 2019) For instance, antibiotics that could treat Shigella dysentery effectively

(such as ampicillin, nalidixic acid, tetracycline and chloramphenicol) are not effective, thus ciprofloxacin is currently recommended by WHO (Lichnevski, 1996) However, a dramatic increase in ciprofloxacin resistance rate decreases both the effectiveness of the

treatment Moreover, new AMR such as colistin resistance mcr-1 and

metallo-β-lactamase NDM-1 have emerged among gram-negative bacteria This may invalidate the effect of high-level antibiotics which are the last options for multidrug-resistant bacteria The spread of untreatable drug-resistant diseases poses a serious threat to the process of achieving sustainable development goals (SDGs) even though it is not directly

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mentioned in the list of 17 goals (WHO, 2019a) Antibiotic resistance is considered a barrier to the achievement of SDG 3 for human health and against food security (SDG 2), clean water and sanitation SDG (SDG 6) Since this problem is directly related to the production, consumption, distribution, and use of antibiotics, SDG 12 about responsible consumption and production is an additional relevant SDG Socio-economic consequences of AMR also indirectly threaten efforts to achieve the goal for poverty reduction and inequality (SDG 16) (WHO, 2019a)

1.1.3.2 High level of AMR in Vietnam

Antibiotic use

In recent years, Vietnam is among the nations that have observed a growing of AMR threats, induced by the inappropriate usage of antibiotics at all health care system levels and in livestock and aquaculture production (World Health Organization, n.d.) Survey

of antibiotics sold in drugstores in northern Vietnam which conducted by Global Antibiotic Resistance Partnership (GARP) showed that 88% (urban) – 91% (rural) of antibiotics were sold without prescriptions despite the law’s prohibition (Global Antibiotic Resistance Partnership - Vietnam National Working Group, 2010) According

to the WHO survey in Western Pacific regions (12 countries) about antibiotic awareness, Vietnamese people's responses indicate a higher misunderstanding in terms of proper antibiotic use than the average in the area (World Health Organization, 2015a) In hospitals, 274.7 DDD/100 day-bed This frequency is significantly higher compared to the data of 139 hospitals from 30 European countries (49.6 DDD/100 day-bed in 2001) (Vietnam Ministry of Health, 2013)

Not only in social health, but antibiotics are also commonly used in animal husbandry in Vietnam In the livestock industry, there is so much abuse of synthetic antibiotics 27%

of pig farms for meat, 24% of farms of small pigs and 10% of chicken farms use antibiotics 3-6 active elements in livestock (Vietnam Ministry of Health, 2013) The abuse of antibiotics as growth promotion drugs often leads to an increase in doses and treatment regimens Farmers use antibiotics mostly based on symptoms of animals

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(44%), instruction by veterinary staff (33%), recommendations from the manufacturer (17%) Only 6% of the farm owners use antibiotics following the antimicrobial susceptibility testing results (Vietnam Ministry of Health, 2013) This makes AMR problem more serious in Vietnam

Antimicrobial resistance (AMR)

According to data submitted in the period 2008–2009 from 15 hospitals under the Ministry, Provincial General Hospitals in Hanoi, Hai Phong, Hue, Da Nang, Ho Chi Minh city, 30–70% of gram-negative bacteria were resistant to cephalosporins of 3rd and

4th generation and nearly 40–60% of gram-negative bacteria were resistant to aminoglycosides and fluoroquinolones in 2009 (Vietnam Ministry of Health, 2009)

Nearly 40% of Acinetobacter were resistant to imipenem (Vietnam Ministry of Health,

2009) In 2009-2010, most antibiotics are reported with high resistance rates above 50%, while resistance rates for carbapenem, vancomycin and colistin were below 50% in the intensive care units at some health facilities (Le & Ha, 2011) The resistance rates to 3rdand 4th generation cephalosporins are particularly high (66–83%), followed by those to aminoglycosides and quinolones (60%) (Le & Ha, 2011) At the time of this report (2015), colistin has not been registered for human use in Vietnam hospital, this drug is not yet available in hospital pharmacies and can only be purchased from the free market

at relatively high prices (Vietnam Ministry of Health, 2009) Some patients trying to get colistin by themselves without professional instruction leads to the insufficient and improper use of this drug (Vietnam Ministry of Health, 2009) This situation poses an alarming risk that the bacteria will become resistant to colistin and bacterial diseases will become incurable (Vietnam Ministry of Health, 2009) The correlation between antibiotic use and antibiotic resistance is evident when the rate of gram-negative bacteria resistant to 4th generation cephalosporins is high in line with greater consumption of this antibiotics (Vietnam Ministry of Health, 2009) Nowadays, many developed countries using 1st generation antibiotics still treat diseases effectively, whereas Vietnam has to use 3rd and 4th generation antibiotics As the evaluation of MOH, AMR is a growing threat to the people and economy of Vietnam (Vietnam Ministry of Health, 2013) The

A, B, and C (or Polymyxin E1 and E2)

Colistin, which is a polycationic peptide, acts on pathogen bacteria as detergents on the cell membrane It attaches to the outer membrane of gram-negative bacteria cells and then binds to lipopolysaccharide (LPS) to displace calcium and magnesium cations, resulting in the osmotic instability and disintegration of membrane structures

1.2.2 Colistin resistance mechanism

1.2.2.1 Colistin intrinsic resistance

Since colistin acts on outer membrane of Gram-negative bacteria, it is not effective for all Gram-positive bacteria (WHO (Global Antimicrobial Resistance Surveillance System), 2018) The activity of colistin on selected bacteria is presented as colistin-

susceptible bacteria in Table 1.1

Table 1.1 Pathogens susceptible and resistance to colistin naturally (WHO (Global

Antimicrobial Resistance Surveillance System), 2018)

Colistin-susceptible bacteria Colistin-intrinsically resistant bacteria

1.2.2.2 Acquisition of colistin resistance

Mutational colistin resistance

Although the resistance mechanism is uncertain, there are various molecular mechanisms that have been distinguished in several bacterial species It has been reported that acquired colistin resistance in naturally susceptible species is mostly related to the modification of LPS structure (Bialvaei & Samadi Kafil, 2015) In

some Klebsiella pneumoniae isolates, they found shedding of capsular

polysaccharides, which trap or bind polymyxins, is the mechanism of colistin

resistance (Olaitan et al., 2014) In Acinetobacter baumannii, loss of LPS, and the

system modification affecting lipid modification and reducing bacterial membrane permeability are two primary chromosomally mediated colistin resistance mechanisms have been described (WHO (Global Antimicrobial Resistance Surveillance System), 2018) Resistance ability originating from chromosomal mutations does not transfer horizontally (WHO (Global Antimicrobial Resistance Surveillance System), 2018)

Transferrable colistin resistance – plasmid-mediated colistin resistance gene (mcr)

the import and export of food and the development of the tourism industry, mcr-1 is

spreading rapidly with amazing speed

Table 1.2 illustrates the continuous detections of different mcr genes including mcr-2,

mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, mcr-8, mcr-9 (mcr-1 variants) in only a short time

These publications showed rapid changes and development in the mcr gene family

Table 1.2 The detections of mcr family gene

mcr Country Publication year Sample origins Ref

human patients

(Y Y Liu et al., 2016)

mcr-4

Belgium, Italy

(Carattoli et al., 2017)

2018)

porcine

(Hammerl et al., 2018)

animals and food

(Borowiak et al., 2020)

1.3 Plasmid-mediated colistin resistance gene (mcr-1)

1.3.1 Global dissemination of mcr-1

In China in 2016, a transmissible colistin resistance gene (mcr-1) was reported to present

in commensal Escherichia coli isolated during surveillance in pig farms and the

possibility of the transfer of colistin-resistance to other bacterial species was shown (Y

Y Liu et al., 2016) Thereafter, the mcr-1 has been increasingly reported worldwide

among different bacterial pathogens, thereby reducing the potential of this last-resort

antibiotic Figure 1.4 illustrates the distribution and spread of mcr-1 in the international

scope (R Wang et al., 2018; Xiuna Wang et al., 2017) mcr-1 has appeared in many

regions and even in the most developed countries, alarming a serious threat to the health

of global citizens

17

Figure 1.4 The global spread of mcr-1 (colored parts indicate the countries where

mcr-1 was detected) (R Wang et al., 2018; Xiuna Wang et al., 2017)

The 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 simply used as medicine for humans from 1970 to

1994 (Lim et al., 2010) Then, farmers have used colistin for livestock production for

more than 5 decades This fact suggests that mcr-1 may root from microorganisms in

animals or human bodies then transfer to other carriers through the food chain or water

route Through daily activities, mcr-1 is discharged from humans and animals to

wastewater, then present in the water environment and even drinking water supply systems if the treatment process can not remove this gene effectively With replication

ability and through horizontal gene transfer events, mcr-1 has been circulating in our

living environment

18

Figure 1.5 shows the distribution of mcr-1 in food, human, sewage, drinking water, and

environment in the world

<Food>

Since 2016, mcr-1 has been reported in food-producing animals in many countries such

as mcr-1 – positive E coli strains isolated from swine in Japan (Kusumoto et al., 2016)

and Taiwan (J Y Liu et al., 2018), from breeding farms in China (Zhang et al., 2019), poultry farms in Vietnam (Campbell et al., 2017), food animals in Germany (Irrgang et al., 2016) and the USA (Meinersmann et al., 2017), and also fresh vegetables from China

(B T Liu & Song, 2019) The mcr-1 pollution in food suggests the contamination of

food is the major exposure route to human

flora (Hu et al., 2017) These researches state that mcr-1 can be intaken by anyone, in

any situation of health and different level of ages

<Wastewater>

Every day, a huge amount of municipal wastewater is discharged from livestock,

cultivation area and community, which makes sewage a potential of mcr-1 hot spot The abundance of mcr-1 in wastewater usually reported at high levels in raw sewage and even after the wastewater treatment process In Europe, mcr-1 was detected in effluents

in the range of 104–105 copies/L from 16 wastewater treatment plants (WWTP) in urban

areas in 10 countries detected (Cacace et al., 2019) The level of mcr-1 abundance in raw

sewage reported in China was 3.3 × 109 copies/L (R N Wang et al., 2019) and in Vietnam and Japan was in the order of 106–107 copies/L (Vu & Kasuga, 2020)

<Drinking water>

19

Recent study pointed out that advanced technology in drinking water treatment plans can

not completely remove mcr-1 (Khan et al., 2020) Although the reports about the occurrence of mcr-1 in drinking water are limited, it will be a great concern since it is a direct way for mcr-1 to penetrate human bodies

<Environment>

Due to the impact of mcr-1 in WWTP effluents, seawater, rivers, lakes and ponds are also polluted with mcr-1 The detection of mcr-1 in seawater in Algeria and public beaches in Spain indicates the spread of mcr-1 in many parts of the environment

Freshwater is an important source for drinking water supply and irrigation water, directly affects food safety and human health However, these water sources were also polluted

by mcr-1 in China (Tuo et al., 2018; R N Wang et al., 2019; D Yang et al., 2017),

Switzerland (Zurfuh et al., 2016), Italy (Caltagirone et al., 2017) and Lebanon (Hmede

et al., 2019)

20

Country References

F O O

D

China (Zhang et al., 2019),

(B T Liu & Song, 2019)

Taiwan (J Y Liu et al., 2018)

Vietnam (Campbell et al., 2017) Lebanon (Sulaiman & Kassem, 2019)

USA (Meinersmann et al., 2017)

U M A N

WATER ENVIRONMENT

China (Hu et al., 2017)

Vietnam (Yamamoto et al., 2018)

Denmark (Torpdahl et al., 2017)

China

(D Yang et al., 2017)

Country References

S E W A G

E

(Tuo et al., 2018)

Japan (Hayashi et al., 2019) Switzerland (Zurfuh et al., 2016)

Germany (Hembach et al., 2017) Italy (Caltagirone et al., 2017) Spain (Lekunberri et al., 2017) Lebanon (Hmede et al., 2019) Italy (Caltagirone et al., 2017) Algeria (Drali et al., 2018)

Europe (Pärnänen et al., 2019),

(Cacace et al., 2019) Brazil (Fernandes et al., 2017)

Figure 1.5 Global presence of mcr-1 in One Health concept

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1.3.2 Correlation of mcr-1 with other genes

1.3.2.1 Bacteria co-harboring mcr-1 genes and bla NDM genes

Besides mcr-1, the plasmid-mediated gene bla NDM which produces New Delhi β-lactamase enzyme is also of great concern since carbapenems currently represent the treatment of choice for severe infections caused by multidrug-resistant strains producing extended-spectrum β-lactamases (ESBLs) (Cornaglia et al., 2007) Since then, several

metallo-reports have identified bla NDM genes worldwide that have typically been associated with multidrug-resistant strains (Chen et al., 2011; Poire et al., 2011; Poirel et al., 2010) The

coexistence of mcr-1 with a carbapenemase is especially worrisome, as therapeutic options in such cases are very limited The researches on co-harboring mcr-1 gene and

bla NDM genes have been conducted in many regions in the world with different types of

samples For instance, a carbapenem-resistant and colistin-resistant E coli producing

MCR-1 and NDM-9 was collected from a chicken sample (Yao et al., 2016) Two strains

of Escherichia coli in fresh vegetables co-harboring mcr-1 and bla NDM-5/9 were reported

in 2019 in China Another E coli strain isolated from a fecal sample of a 43-year-old man in Venezuela harbors both mcr-1 and bla NDM-1 (Delgado-Blas et al., 2016) This resistance combination in a human pathogen is worrisome, as it impedes the use of the

last-resort antibiotics In Vietnam, bla NDM-1 is the frequently reported type of bla NDM in the environment, Vietnamese patients, healthy volunteers and a surgical hospital (Dao et al., 2014; Hoang et al., 2013; Isozumi et al., 2012; Tran et al., 2015) To our knowledge,

there is very little research about pathogenic bacteria co-harboring blaNDM and mcr in

Vietnam(Le-Vo et al., 2019; Yamaguchi et al., 2018)

1.3.2.2 Co-existence of mcr-1 and crAssphage

crAssphage is a bacteriophage which infects Bacteroides intestinalis (Karkman,

Pärnänen, & Larsson, 2019) It is identified in the human intestine with high abundance and rarely found in animals feces (Karkman et al., 2019) For real-time PCR, crAssphage shows better performance than other known fecal markers (Karkman et al., 2019) Therefore, it is used for tracking levels of human fecal pollution in the environment since

22

its significantly higher abundance in human fecal metagenomic than other phages (Karkman et al., 2019) crAssphage has been observed to correlate with other ARGs (Karkman et al., 2019) and thus, it could be a candidate for estimating the effect of fecal

contamination in mcr-1 surveilance

1.4 Research gaps

In a rural area in Vietnam, mcr-1 was detected inside healthy residents (Yamamoto et al., 2018) However, there has been no study that reveals the source of mcr-1 and how it can penetrate into human body Although transmission of mcr-1 in small-scale poultry farms was described in Vietnam (Campbell et al., 2017), an entire exposure route of mcr-1 in the environment has not been investigated In addition, the abundance of mcr-1 in water,

as well as the food chain, has not been measured in Vietnam

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2.1 Water sampling

2.1.1 Sampling in Hanoi

The list of all water samples collected in Hanoi is shown in Table 2.1

Drinking water sample

Tap water samples were collected from a domestic faucet in Nam Tu Liem District, Hanoi on 1 March 2020 This area is supplied with clean water by the system of Song

Da Water Investment Joint Stock Company 2 L of tap water was directly taken as original flushing water sample (coded TW) In addition, 2 L of tap water was stored in glass bottles outside without lid in 24 hours to get rid of chlorine from water During this stagnation process, water sample was placed in a clean space to avoid bacteria contamination (code TW24)

Wastewater samples collected from urban drainages

Water sampling were performed at To Lich River (TL), Nhue River (NH) on 13 September 2019 and 23 December 2019 On 26 February 2020, water samples were collected at To Lich River (TL) and Kim Nguu River (KN) in Hanoi, Vietnam as shown

in Figure 2.1 These urban drainages receive domestic wastewater directly from urban

area in Hanoi For each site, 500 mL water collected in sterilized bottles were kept on ice and delivered to the laboratory

24

Figure 2.1 Water sampling sites in Hanoi

Sampling at aquatic vegetable fields at Hoang Mai District

In vegetable fields in Hoang Liet Ward, Hoang Mai District, water from To Lich River

was directly used for irrigation (Figure 2.2) The sampling at aquatic vegetable fields

was conducted 1 time in dry season (on 26 February 2020) and two times in rainy season (on 19 May and 8 June 2020) For each time, 500 mL water in the aquatic fields and To Lich River (TH) were collected in sterilized glass bottles All samples were immediately delivered to the laboratory

25

Figure 2.2 Sampling at vegetables field in Hanoi (26 February 2020)

Table 2.1 Water samples collected in Hanoi

Urban

drainage

To Lich river (Dong Da)

TH02

N20.963179, E105.818064

26 February 2020

Nhue river (Nam Tu Liem)

NH12

N21.030920, E105.758604

26

Irrigation

water

Water from aquatic field (Hoang Mai)

water

Tap water TW

N21.031499, E105.762148 1 March 2020 Stagnated

tap water TW24

2.1.2 Sampling in Hai Phong

Water sampling were conducted at Re River (RE), Lach Tray River (LT), An Kim Hai chanel (KH), An Bien Lake (AB) and Cau Tre chanel (CT) in urban districts in Hai Phong, Vietnam, on 9 December 2019 All sampling sites in Hai Phong were marked on

map in Figure 2.3 Re River is the drinking water source of An Duong Drinking water

plant supplying clean water for urban residents The rest urban drainages receive domestic wastewater directly from urban districts Water samples collected in sterilized bottles were kept on ice and transported to the laboratory The list of all water samples

collected in Hai Phong is shown in Table 2.2

27

Figure 2.3 Water sampling sites in Hai Phong (December 2019)

Table 2.2 Water samples collected in Hai Phong

date

Urban

drainage

An Kim Hai channel AK N20.839241, E106.735992

Lach Tray river LT N20.842047, E106.650838

An Bien lake AB N20.849996, E106.693242 Drinking

water source Re river RE N20.861734, E106.634452

2.1.3 Sampling in Japan

In Japan, water sampling was conducted at five different domestic wastewater treatment plants in the Kanto Region on 3 October (plant B and C), 9 October (plant A) and 6 November 2019 (plant D and E) In addition, one sample was collected at Kanda Rive (KD) which is the river receiving effluent water from plant E on 28 October 2019 All

sampling sites in Japan were marked on map in Figure 2.4 For each WWTP, three or

28

five samples were collected following the treatment process as showing in Figure 2.5 and Table 2.3 For each sample, 500 mL water collected in sterilized bottles were kept

on ice and delivered to the laboratory

Figure 2.4 Sampling sites in Japan (October – November 2019)

Figure 2.5 Sampling points in wastewater treatment plant in Japan

E WWTP (Tokyo) 293,700 m3/day

Influent (raw sewage) E1 Effluent from primary settlement basin E2 Final effluent (after chlorination

Kanda River Receiving effluent from E WWTP KD

30

2.2 Food sampling

2.2.1 Sampling in Hanoi

Sampling at local markets

Food sampling was conducted in local markets in Hanoi Various types of daily food

were collected including fresh vegetables and meats (see in Table 2.4) All samples were

immediately transferred to the laboratory and subjected to treatment

Sampling at aquatic vegetable fields at Hoang Mai District

Seasonal vegetables were directly harvested in the fields at Hoang Mai District In addition, food samples were collected from a nearby local market that sells agricultural

products there Information on these foodstuff samples is included in Table 2.4

Table 2.4 Description of food samples in Hanoi Category Code Name of food Sampling date Sampling place

Vegetable

V1 Shiso

2019/12/04 Dong Da District V2 Lemongrass

V3 Lettuce V4 Coriander V9 Lettuce

2019/12/23 Dong Da District V10 Shiso

V11 Coriander V12 Field mint V13 Water spinach*

2020/02/26 Hoang Mai District V14 Water cress*

V15 Water cress V16 Water celery V17 Water mimosa

2020/05/19 Hoang Mai District V18 Water mimosa