Occurrence of antibiotic resistance genes in urban water environment in hanoi

PREVALENCE OF ANTIBIOTIC RESISTANCE GENES ARGS IN DIFFERENT WATER ENVIRONMENTS IN JAPAN.... PREVALENCE OF ANTIBIOTIC RESISTANCE GENES ARGs IN DIFFERENT WATER ENVIRONMENTS IN HANOI.... Pr

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

my quality of master thesis Without their guidance, it is impossible for me to finisheffectively the thesis.

Many thanks go to the other professors in Master's program of EnvironmentalEngineering 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-NAGASAKIFriendship Laboratory that supported us having opportunity to do the mostimportant experiment via their facilities (Real-time PCR system)

Last but not least, I would like to thank The Japan International CooperationAgency (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 Ihope to continue cooperation with all of you in the future

Sincerely thank

1 INTRODUCTION 1

2 LITERATURE REVIEW 4

2.1 Antibiotic resistance mechanism and spreading pathway 4

2.1.1 Gene mutations 4

2.1.2 Horizontal gene transfer 5

2.2 Wastewater treatment plant as the hot-spot of ARB and ARGs 7

2.2.1 Wastewater treatment plant - meeting place for selection of antibiotic resistance 7

2.2.2 Removal efficiency of antibiotic, ARB and ARGs in WWTP 8

2.3 Antibiotic resistance in water environment 14

2.3.1 Two approaches antibiotic resistance in waster environments including ARB and ARGs 14

2.3.2 General review for ARB and ARGs in water environments 15

2.3.3 Antibiotic, ARB, ARGs in different water environments in Vietnam 17 2.3.4 Emergence of colistin resistance genes in water environments 20

3 METHODS 21

3.1 Sampling 21

3.1.1 Sampling sites in Japan 21

3.1.2 Sampling sites in Hanoi 24

3.2 Persistence of ARGs in lab-scale experiment 28

3.3 Water quality measurement 29

3.3.1 Temperature and Electrical conductivity 29

3.3.2 Ammonium 29

3.3.3 E coli and total coliform 30

3.3.4 Flow cytometry 31

3.4 Molecular biological analysis 31

3.4.1 DNA extraction 31

3.4.2 PCR and gel agarose electrophoresis for presence of colistin resistance gene (mcr-1) 33

3.4.3 Real time PCR (qPCR) 34

4. 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

5. 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 6. 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 5

Figure 2.2 Transfer of ARGs in microorganism community 6

Figure 2.3 Integron is involved in HGT (Gillings et al 2015) 7

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 aeration (a); INF-No aeration (b); EFF-aeration (c); EFF-INF-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

Clinical & Laboratory Standards Institute

Escherichia coli

Electric conductivityEffluent

Extended spectrum beta-lactamaseHorizontal gene transfer

InfluentMobile gene elementsMinimum inhibitory concentrationsMacrolide-lincosamide-streptogramin BNext generation sequencing

Polymerase Chain ReactionQuantitative polymerase chain reactionWastewater treatment plant

of great concern For example, among E coli isolates, 64 percent of them were

resistant to third- generation cephalosporins, and 50 percent were resistant tofluoroquinolones (Gelband et al 2015) In 2000–2001, Vietnam had the highest

occurrence of Streptococcus pneumoniae resistance to penicillin (71%) and

erythromycin (92%) of all countries participating in the Asian Network forSurveillance of Resistant Pathogens (Gelband et al 2015)

According to State of the world’s antibiotics 2015 (Gelband et al 2015), the antibioticconsumption of Vietnamese is much higher than some other countries (China,Indonesia, Philippines) Although most of the countries in East Asia and Pacific areadecreased amount of antibiotic consumption during 2000 – 2010 period, Vietnam is one

of three countries which still increased antibiotic utilization year by year Antibioticconsumption was also indicated as a high rate in Vietnamese hospitals (5,104 of 7,571patients were receiving antibiotic therapy (67.4%)), and also a high occurrence ofinappropriate indications for antibiotic prescriptions (Thu et al 2012)

Furthermore, almost antibiotics were sold without prescription respectively 88% inurban and 91% in rural pharmacies (Thi et al 2014) Hence, people in urban area canpurchase antibiotics at drug store, pharmacies without any caution It can be suspected

that improper use of antibiotics for human can promote spread of antibioticresistance in Vietnam condition.

In 2011, antibiotic resistance is believed as one of the three biggest priorities of theTripartite Alliance (FAO, OIE, WHO) in terms of preventing health risks in thecirculation of human–animal–ecosystems Human and animals play vital role as asource of antibiotic and antibiotic resistant genes (ARGs) due to the antibioticconsumption (Rodriguez-Mozaz et al 2015) As a consequence, almost all of theused antibiotic and ARGs are eventually discharged into sewage system, and theninto wastewater treatment plant (WWTP) Therefore, WWTP can perform function

of removal of antibiotic resistance or be a reservoir for antibiotic resistance wherehorizontal gene transfer (HGT) is expected (Karkman et al 2018) HGT is animportant microbial activity and the spreading of genes (ARGs) by HGT to othermicroorganisms is regarded as augmentation strategies (Smets and Barkay 2005).Due to the rising concern about antibiotic and ARGs pollution in aquaticenvironment, some studies have been implemented to assess the prevalence of thisemerging pollution in WWTP effluent and receiving river (Rodriguez-Mozaz et al

2015, Urase and Sato 2016), whereas other studies have concentrated on fate ofARGs in livestock breeding wastewater (Jia et al 2017) In Vietnam, there areseveral studies about antibiotics, antibiotic resistant bacteria (ARB) and ARGsincluding occurrence of ARGs in foodborne (Van et al 2007), ARGs from hospitalwastewater (Lien et al 2017), ARB and ARGs in aquatic environment (Phuong Hoa

et al 2008, Hoa et al 2011, Takasu et al 2011, Nakayama et al 2017) However,only Nakayama et al (2017) mentioned quantities of ARGs in water environment inMekong Delta, Vietnam Therefore, little information on level of ARGs in waterenvironment was available, and the fate of ARGs in northern area remainsunknown Our study will fill in the gap and contribute to clarify importance ofantibiotic resistance in Vietnam with two main objectives below:

1) Reveal the prevalence of ARGs in water environment in Hanoi

• To investigate presence of ARGs in urban water environments

• To evaluate the role of urban water environment in terms of spreading ARGs

as well as the efficiency of removal ARGs of sewage treatment plant

2) Comparison ARGs situation between Vietnam and Japan aquatic

environment

• To compare ARGs pollution in aquatic environment situation betweenVietnam (Hanoi) and Japan (Tokyo, Saitama, Ibaraki) and the significance of ARGs inVietnam condition could be asserted

2 LITERATURE REVIEW

Following WHO definition, antimicrobial resistance (AMR) or antibiotic resistance

is the situation where microorganism such as bacteria, viruses or parasites canresistant to antimicrobial agents Consequently, common antibiotic becomeineffective and antibiotic resistance spread to others

Bacteria contains specific genes allowing them to adapt with environmental threatincluding the environment with presence of antibiotic It is one of the most uniqueability of bacteria during their evolution process In particular, there are two mainreasons to make bacteria become resistant to antibiotics: (i) Gene mutations and (ii)Accumulation foreign DNA coding resistance gene by horizontal gene transfer(Smith 2017)

(iii) Discharge antibiotic by efflux pump

Three antibiotic resistance mechanisms were described in Figure 2.1.

Figure 2.1 Antibiotic resistance mechanism - Gene mutations

2.1.2 Horizontal gene transfer

HGT is the phenomena which allow bacteria to exchange chromosomal and plasmidDNA among bacteria community via one of three mechanisms includingconjugation, transformation and transduction (Hurst 2007)

Conjugation: Bacteria directly transfer conjugative plasmid DNA from cell to other

cell via protein pilus (see in Figure 2.2.)

Transformation: Bacteria uptakes free DNA from surrounding medium which can

come from cellular elements of died cells (both chromosomal and plasmid DNA)

(see in Figure 2.2.)

Transduction: Bacteria receive the genetic material via transducing bacteriophage

(phage) When a virus (phage) infects to the recipient cell, genetic elements iscombined with genome of recipient Finally, recipient bacteria developes and

metabolizes with foreign DNA (see in Figure 2.2.)

Figure 2.2 Transfer of ARGs in microorganism community

Integron and HGT:

Integron is a mobile gene element located in transposons, plasmid or chromosome

which can capture and express the genes from other sources IntI1 was discovered

as the first class of Integron (Gillings et al 2015) Figure 2.3 describes the function

of IntI1 gene on transmission of ARGs among microorganism community IntI1

contains a gene encoding an enzyme (integrase) that catalyzes the site-specific

recombination (attI) An integron-encoded promoter (Pc) inserts the new integrated gene cassette (blaTEM is an example) into the array of genes so that several cassettes

of ARGs could be located in the same array Especially, sul1 (sulphonamides

resistance gene) and blaTEM gene (extended spectrum β-lactamase (ESBL)) were

associative in Figure 2.3.

Figure 2.3 Integron is involved in HGT (Gillings et al 2015)

2.2.1 Wastewater treatment plant - meeting place for selection of antibiotic

resistance

It is obvious that a large amount of antibiotics is used in hospitals for healthtreatment And, hospitals contribute significantly to the generation and spread ofARB and ARGs However, hospital effluents only contribute less than 1% of totalvolume of municipal wastewater so that hospital wastewater is diluted largely in theWWTPs (Kümmerer 2004) Nevertheless, wastewater treatment plants (WWTPs) isone of the main sources for antibiotics released into various environment (Michael

et al 2013)

In particular, a huge number of antibiotics is given to humans and utilized directly

in household scale and finally discharged to the sewage Exposure to antibioticsaccelerates the selection pressure for antibiotic resistance in wastewater (Rodriguez-Mozaz et al 2015) Then, WWTPs receive wastewater from different sourcesincluding domestic sewage, industry or hospital treated effluent Therefore, WWTPs

are the appropriate meeting place for various bacteria from different environments,making opportunity for interaction of bacteria Especially, WWTPs have beenreported as hot-spots for HGT and spread of dissemination of ARGs (Berendonk et

al 2015, Karkman et al 2018, Rodriguez-Mozaz et al 2015)

2.2.2 Removal efficiency of antibiotic, ARB and ARGs in WWTP

Basically, the conventional WWTP consists of a primary stage, secondary stage,and a tertiary stage can be applied Each stage is identified with different biologicaland physicochemical treatment processes

• Primary stage is aimed to decrease the solid elements in wastewater (sand, oils/fats and settleable solids)

• The secondary stage typically intends to remove organic matter and nutrientsvia a biological process (aerobic or anaerobic systems) such as conventional activatedsludge (CAS)

• The tertiary stage such as activated carbon adsorption, membrane filtrationand disinfection are for removal of the components which cannot be eliminated byprimary and secondary stage

In practice, WWTP are not optimized to remove pharmaceutical compounds.Advanced treatment shows a significant removal of antibiotics However, theycannot remove antibiotics in wastewater completely (Michael et al 2013)

Efficient removal rate for antibiotics was observed in Taiwanese WWTP Forexample, tetracyclines were eliminated by 66 – 91% in all studied WWTP Otherplant where antibiotic groups of sulfonamides, cephalosporins, lincosamides, andquinolones were removed up to 100% or inadequately removed (Lin, Yu, and Lateef2009) A WWTP in Girona (Spain) also presented a good removal rate with 80% ofantibiotics including ofloxacin, sulfamethoxazole, clarithromycin (Rodriguez-Mozaz et al 2015)

2.2.2.2 Removal of ARGs in WWTP

Several papers demonstrated that WWTP are very effective to eliminate ARB andARGs, but others gave the proof about the proliferation of ARGs during WWTP

Effective removal of ARGs in WWTP

In Girona WWTP in Spain, it receives approxiamately 1500 m3/d of the untreatedhospital wastewater along with nearly 55,000 m3/d of domestic wastewater fromGirona city (Rodriguez-Mozaz et al 2015) Five targeted ARGs were evaluated

including blaTEM (extended spectrum β-lactamase (ESBL)), qnrS (resistance to fluoroquinolones), sul1 (resistance to sulphonamide), ermB (resistance to macrolide- lincosamide-streptogramin B (MLSB)), tetW (resistance to tetracyclines) The concentration of ermB, qnrS in hospital effluent was revealed at nonsignificant

difference with WWTP influent Influent contained 107 (copies/L) of blaTEM, 108

(copies/L) of sul1, 109 (copies/L) of ermB On the other hand, a critical reduction of

ARG was observed in wastewater effluents that decreased more than hundredfold in

some cases such as ermB or blaTEM The copies number of qnrS was also detected at similar level to blaTEM (108 copies/L), however, this WWTP showed lower removal

rate for qnrS in comparison to blaTEM suggesting the removal performance of WWTPwas various depending on types of each ARGs (Rodriguez-Mozaz et al 2015)

For the better understanding about how WWTP works for reducing ARGs, Xu et al.(2017) showed the presence of tetracycline and AmpC β-lactamase resistance genes

in 4 non-urban WWTPs in China In this study, 11 types of tetracycline resistance

genes (tetA, tetB,tetC, tetE, teG, tetI, tetM, tetO, tetQ, tetS, tetX) and four types of AmpC β-lactamase genes (EBC, MOX, FOX, CIT) were analyzed by quantitative

polymerase chain reaction (qPCR) assays Among tetracycline resistance genes,50% of them were remained in both effluents and excess sludge samples Inaddition, AmpC β-lactamase genes were existed in excess sludge but WWTPseffluents (Y Bin Xu et al 2017) This suggests the WWTPs performance to reduceARGs was different depending on type and mechanism of ARGs

Xu et al (2017) also revealed the removal rates of ARGs in 4 WWTPs (A, B, C and

D) were 76.92%, 54.55%, 58.33%, 61.54%, respectively (see in Table 2.1.).

Besides, the water quality of influent samples, treatment process played an important role

on ARGs elimination Plant A with cyclic activated sludge system (CASS) showed thehighest removal rate of ARGs The removal rates were different in four WWTPs, whichcould be due to the different microbial community structure in treatment processes.Sedimentation stage and accumulation of excess sludge were the key stages for thereduction of ARGs in any plants By sedimentation and enrichment through biologicalreaction, the microorganisms containing ARGs accumulated in sludge during treatmentprocess ( Xu et al 2017)

Table 2.1 Removal rates of ARGs in WWTPs in China

A (cyclic Biological reaction 12

sludge system)

Excess sludge 8

B (cyclic Biological reaction 9

11

Proliferation of ARGs in WWTP

In contrast, Mao et al (2015) investigated the enrichment of ARGs through

different treatment processes in two WWTPs in northern China including the

following samples: raw influent (RI), primary clarifier tank (PCT), anaerobic tank(AaT), anoxic tank (AT), aerated tank (AeT), secondary clarifier (SCT), final

effluent (FE), recycled active sludge (AS) and dewatered sludge In total 10 ARGs

were targeted in this study including tetracyclin resistance genes (tetB, tetG, tetH,

tetS, tetT, tetX) sulphonamides resistance genes (sul1, sul2), quinolones resistance genes (qnrB) and MLSB resistance genes (ermC) 16S rRNA genes was also

included as a representative for total bacteria

The enrichment of various ARGs through WWTP was analyzed by normalizing the

total genes released by the corresponding inflow, whereas total effluent was

calculated considering final effluent (FE) plus dewatered sludge (DS) genes

Log (Enrichment ratio) = log(

If the enrichment ratio > 0 indicating an increase in ARGs flow through WWTP

If the enrichment ratio < 0 indicating a decrease in ARGs flow through WWTP

ARGs concentration showed the enrichment in cases of tetA, tetB, tetE, tetG, tetH,

tetS, tetT, tetX, sul1, sul2, qnrB, ermC within WWTP.

Among above enrichment genes, ten of ARGs (tetB, tetG, tetH, tetS, tetT, tetX, sul1,

sul2, qnrB, ermC) were enriched significantly in comparison to 16S rRNA genes.

The enrichment of different ARGs in the two WWTP was also identified by

normalizing concentration of ARGs to 16S rRNA value (Total bacteria), named

“Relative abundance of ARGs” (Mao et al 2015)

The relative abundance of total 10 ARGs in FE (final effluent), AS (activate sludge)

and DS (dewatered sludge) were much higher than RI (raw influent) The ranges of

enrichment ratio of ARGs were 8 ± 1 and 268 ± 248 for tetG and tetT, respectively,

which emphasized the emergence for improved understanding of WWTPperformance to control and monitor ARGs (Mao et al 2015) Rysz et al (2013) alsopointed out the reduction of food/microorganism ratio or extension of contact time

in anaerobic digesters could prevent the ability of ARBs from harvesting energywhich was profitable to loss of antibiotic resistance

From part of 2.2.2.2, Water quality of influents, plant performance, nutrientscompound, biological treatment processes were suspected as the factors affectingARGs removal efficiency within WWTP In addition, other factors of selectionpressure could be antibiotics concentration or some stress factors such as heavymetals, pesticides, toxic elements etc (Xu et al 2017, Mao et al 2015, Pal et al

2015, Berendonk et al 2015)

The strong correlation between the relative abundance of ARGs with heavy metals

in WWTPs were demonstrated with tetracycline resistance genes and AmpC lactamase resistance genes ( Xu et al 2017) The relative abundance of tetracyclineresistance gene was correlated to the presence of Cu (r=0.714, p<0.05) and Zn (r=0.881, p < 0.01) Similarly, the concentration of Cu also corresponded to the relativeratio of AmpC β-lactamase (r=0.847, p< 0.01)

β-The genes encoding antibiotic resistance and heavy metals resistance wereidentified in the same location of chromosomes or mobile genetic elements (MGE)that might promote microorganism to capture multiple resistances (Chihomvu,Stegmann, and Pillay 2015)

2.3 Antibiotic resistance in water environment

2.3.1 Two approaches antibiotic resistance in waster environments including

ARB and ARGs

• Culture-dependent approaches are to investigate the status of ARB in waterenvironment via comparison of resistance percentage, that is calculated by the differencebetween amount of colonies formed on culture media with and without supplementedantibiotic doses (Rizzo et al 2013)

• Culture-independent approaches are comprehensively based on informationabout ARGs which have also been perfectly developed For example, quantitative PCRsystem provide an approximation of estimation of pollution level of known ARGs inenvironmental samples (Berendonk et al 2015)

The comparable table between two approaches is described as below:

Table 2.2 Culture-dependent method and culture-independent method

Culture-dependent method Culture-independent methodAdvantages Direct evidence of Direct evidence of living

quantification of DNA target in microorganism

Diagnose without concern of reliable

No standardized protocols missing

Boolchandani, D’Souza, and Dantas (2019) summarized a review of method anddata for antibiotic resistance studies and presented a comprehensive sequencing-based resistance approach (Culture-independent method) Therefore, culture-independent method is potential source for antimicrobial resistance research.

2.3.2 General review for ARB and ARGs in water environments

2.3.2.1 Monitoring ARB in water system

Escherichia coli (E coli) is bacteria that is mainly located in the lower intestine of warm-blooded organisms Additionally, E coli is the most common bacteria for antibiotic resistance susceptibility test Urase and Sato (2016) investigated the E coli resistant to 14 antimicrobial agents by the Kirby-Bauer disk-diffusion method

with Mueller-Hinton agar in Tama river, Tokyo, Japan

After incubation, blue colonies were selected as E coli and again cultured with

minimum inhibitory concentrations (MICs) of antimicrobial agents which wasproposed in CLSI 2015 (Clinical and Laboratory Standards Institute 2015) The

resistant ratios of E coli in different water samples in Tama river were evaluated 12.3% of E coli were resistant to ABPC (penicillins), 2.1% were resistant to CTX

(third-generation cephalosporin), 2.1% were resistant to GM (aminoglycosides),2.1% were resistant to LVX (fluoroquinolones), 7.0% were resistant to ST/TMP(sulfonamide) and 7.9% were resistant to TC (tetracycline) No strains with MPM(carbapenems) were observed in this study In mountainous area, smaller ratios ofresistances were observed in water samples However, ratios of resistance from themiddle river to downstream were higher due to the impacts of WWTPs Specially,the water samples from WWTP demonstrated smaller or equal ratios of resistancescompared to Tame River (Urase et al 2016) The ratio of ESBL resistance (with

CTX resistant bacteria) among isolates of E coli strains from water samples in

Yeongsan River, South Korea was 2.5% (Jang et al 2013) which was equivalent tothe data of Urase and Sato (2016)

Similarly, resistant E coli strains were also investigated from water samples in Yodo River basin, Osaka, Japan In this study, 74% isolates of E coli from WWTP

effluent samples were resistant to at least one antibiotic agent and 46% of thosewere multiple antimicrobial resistance suggesting that WWTPs effluent increaselevel of contamination with multiple antimicrobial resistance in water environments.(Yamashita, Katakawa, and Tanaka 2017)

2.3.2.2 Prevalence of ARGs in water system

In 2019, five ARGs including sul1 (encoding sulphonamides resistance), blaTEM,

investigated in Lake Tai which is one of three largest freshwater lakes in China Intotal, 82 surface water samples were collected in northern region of Lake Tai Afterextraction DNA, Quantitative PCR (qPCR) assays were applied to quantify theseARGs Absolute concentrations of each target genes were described in the following

figure The results showed that sul1 gene was the most abundance in this study since

it was detected in 100% water samples ranging between 1.9 × 105 and 7.9 × 108

(copies/L) and, 67.1% of water samples was detected for blaTEM gene The other

third-generation cephalosporin resistance genes including blaNDM-1, blaCTX-M-32 were

analyzed with lower concentration Specially, mcr-1 or colistin resistance gene was

not detected in any samples in Lake Tai (Stange et al 2019)

An overview about presence of ARGs in global lakes was summarized in 2018 inorder to identify possible potential risks affecting to human health and ecosystem

No significant variations were observed in the copy numbers of sulfonamide

resistance genes including sul1 and sul2 in lake water and river water all over the world This is also similar to previous findings, sul1 is the most frequent abundant

ARGs in water environment (Stange et al 2019, Phuong Hoa et al 2008)

Furthermore, the WWTP affected concentrations of ARGs such as ermB, qnrS, sulI

in downstream river (Rodriguez-Mozaz et al 2015) Therefore, the prevalence ofARGs in water system was affected by WWTP

16

2.3.3 Antibiotic, ARB, ARGs in different water environments in Vietnam

2.3.3.1 Antibiotic consumption and antibiotic residues in water system

There is no official report of antibiotic consumption in Vietnam due to complicatedabuse of antibiotic Most of antibiotics are sold without requirement of doctor'sprescription up to 88% in urban and 91% in rural pharmacies (Thi et al 2014) Inhospitals, 67.4% patients were receiving antibiotic therapy with the most commonlyantimicrobial agents regarding to cephalosporins, penicillins, aminoglycosides at70.2%, 21.6%, 18.9%, respectively (Thu et al 2012)

Figure 2.4 describes the antibiotic consumption of countries in East Asia and Pacific Developed countries such as Japan, Hong Kong, South Korea etc showed

the reduction of antibiotic consumption, while those in Vietnam, China andThailand still increased (Gelband et al 2015)

Figure 2.4 Antibiotic consumption in Vietnam reference (Gelband et al 2015)

In addition, the residues of antibiotics were reported with significant concentration inwater environments such as lake, river, and canal Antibiotics concentrations weredetected at higher values in canals rather than those in the lake and river ranging fromng/L to some mg/L Sulfonamide antibiotics were commonly detected in canal andriver water followed by macrolide and quinolones Especially, sulfamethoxazole anderythromycin are the most prevalent in the two groups of sulfonamide and macrolide.Quinolones concentration was lower in comparison with the others (Binh et al 2018)

2.3.3.2 Monitoring ARB in water system

In 2011, Hoa et al (2011) investigated the correlation between antibiotic and ARB

in water environments in northern Vietnam

Water samples were collected at 10 locations including 3 Hanoi canals (HNC-1–3),

3 pig farms/fish ponds in Hanoi (HNP-1–3) and 4 coastal shrimp ponds in Haiphong(HNAQ-1–4) Then, water samples were incubated in Nutrient Broth at 30°Cimplemented with 60 (μg/mL) of sulfamethoxazole (SMX) and 60 (µg/mL)g/mL) of sulfamethoxazole (SMX) and 60 (µg/mL)erythromycin (ERY) (Hoa et al 2011) Formed colonies were enumerated after 7days for calculation of resistance rate by the following equation:

( )

Resistance rate % = ×100

0( )

Whereas,

N: number of colonies with antibiotic (CFU/mL)

N0: number of colonies without antibiotic (CFU/mL)

According to (Hoa et al 2011) the occurrence of SMX resistant bacteria were basicallygreater than those of ERY resistant bacteria at every location within 2 sampling times.SMX resistant bacteria was shown as the main types of bacteria in water sample when

it occupied up to 94.44% of total viable count in canal sample in January In addition,the resistance rate was observed higher in January rather in July

18

which may be caused by high rainfall level in rainy season of July (Hoa et al 2011).Sulfamethoxazole resistant bacteria were diverse in water system (canal, fish pond,

coastal shrimp pond) Though many types of bacteria possessed sul genes, Acinetobacter and Aeromonas were recorded as the major sulfamethoxazole resistant bacteria in aquatic environment in northern of Vietnam (Hoa et al 2011)

Takasu et al (2011) also checked fluoroquinolone resistant bacteria in variousaquatic environments in both Vietnam and Thailand The water samples were takenfrom canal, VAC system (Vegetable-Aquaculture-Cage) and aquaculture However,the tested antibiotic concentration was 16 mg/L of ofloxacin (OFL), norfloxacin(NOR) and ciprofloxacin (CIP) The results revealed the prevalence offluoroquinolone resistant bacteria which were detected at almost all samplingpoints; the resistant rate was between 0.1 to 15% for all samples Additionally,fluoroquinolone resistant bacteria were identified They were affiliated with

microorganisms including enteric bacteria (E coli), Acinetobacter, Brevundimonus, Actinobacteria etc (Takasu et al 2011).

Furthermore, 83% of E coli isolates from wastewater in hospital in Hanoi, Vietnam

resisted to at least one type of antibiotic, and 32% of those were multidrug

resistance For E coli isolates which resists to third generation cephalosporins, high

occurrence of ESBL resistance gene were observed (Lien et al 2017)

2.3.3.3 Prevalence of ARGs in water system

The distribution of sulphonamides resistance genes (sul) and plasmids in

sulfonamide resistant bacterial isolates were evaluated for different aquatic

environments in northern Vietnam Sul genes were frequently detected in all water

environments including swine farm, shrimp pond and city canal However, the

quantitation of sul genes was not investigated due to limitation of molecular

materials (Phuong Hoa et al 2008)

An advanced study was conducted in Mekong Delta, Vietnam In this study, freshwater samples were collected in backyard aquacultures, Can Tho, Vietnam The

qPCR results showed that almost freshwater water samples presented a positive result

with third generation cephalosporin resistance genes (blaCTX-M-1, blaCTX-M-9, blaTEM,

blaSHV), tetracycline (tetC), sulphonamides resistance genes (sul1, sul2) (Nakayama et

al 2017) Since backyard-based aquaculture is managed by the local farmers so theprevalence of ARGs in water system can be affected by farmer's management It wasnoted that some farmers loaded wastewater from human and livestock into

aquacultures so bla was prevalent in all samples And, sul1 was detected at

higher concentration rather than blaCTX-M-1 gene that was consistent with previousstudies (Nakayama et al 2017)

So far, little is known about 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 the last resort of antibiotic (polymyxins) Antibiotic resistance gene

(mcr-1) was the antibiotic resistance gene (ARGs) related to first resistance mechanism of

colistin which was first reported in China in 2015 (Liu et al 2016) Particularly, mcr-1 gene has been identified by plasmid mediated resistance consisting of other antibiotic

resistance genes, for example, ESBL group "extended-spectrum βs-lactamases" (Haenni

et al 2016) Recently, mcr-1 gene has been determined in 31 countries in five continents

from 2008 to 2017 Among those countries, Vietnam was identified as the second

country with the greatest number of mcr-1 positive isolates (58), just behind China (212)

(Wang et al 2018)

Recently, Campbell et al (2017) investigated the prevalence of mcr-1 genes in

chickens and human fecal samples in backyard chicken farms, Vietnam It is also

reported that Escherichia coli isolated from fecal microbiota of healthy residents in a Vietnam rural community contains “mcr” gene or colistin resistance genes (Yamamoto

et al 2019) At the time of writing, the presence of mcr-1 in water environment in

Vietnam have not revealed Therefore, monitoring ARGs in water environment plays a

crucial role to control the expand of mcr-1 in water environment.

CTX-M-1

3 METHODS

3.1 Sampling

3.1.1 Sampling sites in Japan

Sampling in the watershed of Tama River in Tokyo was conducted on 24 October

2018 The coverage of sewer system in the watershed reaches almost 99% Alongthe river, 9 WWTPs directly discharge effluent to main flow of Tama River As aresult, river flow in the middle and downstream is mainly composed of wastewatereffluent Sampling sites were selected to evaluate the impacts of wastewater

treatment plant effluent as shown in Figure 3.1 As a reference sample, additional

sample was collected from Kanda River in Bunkyo Ward in Tokyo on 19 October

2018 Almost all flow in Kanda River is derived from effluent from a WWTP

Figure 3.1 Sampling sites in Tama River, Tokyo

Another sampling was performed in the watershed of Lake Kasumigaura on 7November 2018 Surface water samples were collected at five points in the lake as

shown in Figure 3.2 In addition, influent and effluent of a WWTP were collected

on the same occasion Moreover, river water sample was collected from SakuraRiver which was the major inflow to Lake Kasumigaura

In both watersheds, 2 liters of water was collected and transferred with ice to thelaboratory Additionally, pH and temperature and electrical conductivity weremeasured with a HI98129 (Hanna) on site The detailed of sampling sites weredescribed in below figures

Figure 3.2 Sampling sites in Lake Kasumigaura, Ibaraki prefecture

Total collected samples in Japan are included in Table 3.1 as below.

Table 3.1 Description of sampling points in Japan Location

Category (Name, Prefecture) Code Note

Tama River, Tokyo TM1 Upstream of river

Tama River, Tokyo TM2 Receive 3 WWTPs discharges

TM3 discharge

River Tama River, Tokyo TM4 Without WWTPs discharge

TM5 dischargeTama River, Tokyo TM6 Without WWTPs discharge

Kanda River, Tokyo KD Almost 100% wastewater

Ara River, Tokyo AR For drinking water source

Lake Kasumigaura, Nearest point to the WWTP discharge

Influent WWTP (Saitama) SINF Domestic WWTP

SINF KasumigauraEffluent WWTP (Saitama) SEFF Domestic WWTP

Domestic WWTP next to Lake

KSEFF Kasumigaura

23

3.1.2 Sampling sites in Hanoi

Seasonal samples:

Water samples were collected from September, 2018 to April, 2019 including KN1,KN2, KN3, KN4 (Kim Nguu river) and YS1, YS2, YS3, DP (Yen So lake) in Hanoi

urban water environment (Figure 3.3 and Figure 3.4) Kim Nguu river was selected

as the target site because it receives all types of wastewater such as domestic,hospital, industry and bring the sewage to Yen So wastewater treatment plant

Gamuda Land Vietnam Ltd., Company invested and operated Yen So WWTP from

2010 with capacity of 200.000 m3/day It is the largest wastewater treatment plant inHanoi until 2019 with advanced treatment system including sand basin, oilseparating tank; sequencing batch reactor (SBR), specially, disinfection process byultraviolet array applied for treatment of wastewater from Kim Nguu river and Setriver However, due to a large treated volume per day, a part of effluent was nottreated by UV disinfection The treated effluent (KN4 w/o disinfection and YS1with disinfection) has discharged to this river and nearby lake (Yen So lake) whichcan be utilized for other purposes (irrigation and water resource for livestock farm –fish and duck)

Figure 3.3 Kim Nguu river and Yen So WWTP

The additional information for seasonal sampling points was included in Table 3.2.

Table 3.2 Seasonal sampling points in Hanoi Location

Kim Nguu River, Upstream of river

urban canals, 4 rivers, 7 lakes) and 3 groundwaters (see in Figure 3.4.) Six

wastewater treatment plants were operated in sampling area, to compare differentwater system with/without impacts of WWTPs Temperature and electricalconductivity were measured on site All water samples were taken and transferred

on ice to laboratory

25

Hanoi, Vietnam

(Source: Wikipedia Vietnam)

Figure 3.4 Extended sampling sites in Hanoi

Table 3.3 Extended sampling points in Hanoi Category Location (Name, District) Code Note

Linh Dam Lake, Hoang No WWTP discharge

Van Quan Lake, Ha Dong VQ-1802 No WWTP dischargeTruc Bach Lake, Ba Dinh TB-1802 Receive WWTP dischargeWest Lake, Tay Ho W-1802 No WWTP dischargeLake

Hoan Kiem Lake, Hoan No WWTP discharge

Set River, Hoang Mai S-1902 100% of raw wastewater

Lu River, Hoang Mai L-1902 100% of raw wastewater

Canal

To Lich River, Hoang Mai

TL2- 100% of raw wastewater1902

Kim Nguu River, Hai Ba KN3- 100% of raw wastewater

Duong River, Long Bien D-1802 For drinking source

River Day River, Chuong My DA-0303 For agriculture purpose

Nhue River, Tu Liem N1-2002 Close to Red riverNhue River, Ha Dong N2-2002 20 kilometers from N1

1004Groundwater Hoan Kiem HCGW- Shallow well, downtown