INTRODUCTION
Huge amounts of antibiotic elements have been used for several decades not only for human health but also veterinary and agricultural purposes (Rodriguez-Mozaz et al
2015) Even though antibiotic resistances are derived naturally, the abuse of antibiotics in humans and animals mainly contributes to accelerate ability of microorganisms that can stop antibiotics from working against (WHO) A system of Resistance Map created by Center for Disease Dynamics, Economics & Policy (CDDEP) warned the spread of antibiotic resistance all over the world It has been alerted that antibiotic resistance will kill 10 million people per year which is more than deaths caused by cancer in 2050 (O’Neill 2014) Thus, antibiotic resistance is critical health issue in this century In Vietnam, a high rate of antibiotic resistance is of great concern For example, among E coli isolates, 64 percent of them were resistant to third- generation cephalosporins, and 50 percent were resistant to fluoroquinolones (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 for Surveillance of Resistant Pathogens (Gelband et al 2015)
According to State of the world’s antibiotics 2015 (Gelband et al 2015), the antibiotic consumption of Vietnamese is much higher than some other countries (China, Indonesia, Philippines) Although most of the countries in East Asia and Pacific area decreased amount of antibiotic consumption during 2000 – 2010 period, Vietnam is one of three countries which still increased antibiotic utilization year by year
Antibiotic consumption was also indicated as a high rate in Vietnamese hospitals (5,104 of 7,571 patients were receiving antibiotic therapy (67.4%)), and also a high occurrence of inappropriate indications for antibiotic prescriptions (Thu et al 2012)
Furthermore, almost antibiotics were sold without prescription respectively 88% in urban and 91% in rural pharmacies (Thi et al 2014) Hence, people in urban area can purchase antibiotics at drug store, pharmacies without any caution It can be suspected that improper use of antibiotics for human can promote spread of antibiotic resistance in Vietnam condition
In 2011, antibiotic resistance is believed as one of the three biggest priorities of the Tripartite Alliance (FAO, OIE, WHO) in terms of preventing health risks in the circulation of human–animal–ecosystems Human and animals play vital role as a source of antibiotic and antibiotic resistant genes (ARGs) due to the antibiotic consumption (Rodriguez-Mozaz et al 2015) As a consequence, almost all of the used antibiotic and ARGs are eventually discharged into sewage system, and then into wastewater treatment plant (WWTP) Therefore, WWTP can perform function of removal of antibiotic resistance or be a reservoir for antibiotic resistance where horizontal gene transfer (HGT) is expected (Karkman et al 2018) HGT is an important microbial activity and the spreading of genes (ARGs) by HGT to other microorganisms is regarded as augmentation strategies (Smets and Barkay 2005)
Due to the rising concern about antibiotic and ARGs pollution in aquatic environment, some studies have been implemented to assess the prevalence of this emerging pollution in WWTP effluent and receiving river (Rodriguez-Mozaz et al
2015, Urase and Sato 2016), whereas other studies have concentrated on fate of ARGs in livestock breeding wastewater (Jia et al 2017) In Vietnam, there are several studies about antibiotics, antibiotic resistant bacteria (ARB) and ARGs including occurrence of ARGs in foodborne (Van et al 2007), ARGs from hospital wastewater (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 in Mekong Delta, Vietnam Therefore, little information on level of ARGs in water environment was available, and the fate of ARGs in northern area remains unknown Our study will fill in the gap and contribute to clarify importance of antibiotic 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 between Vietnam (Hanoi) and Japan (Tokyo, Saitama, Ibaraki) and the significance of ARGs in Vietnam condition could be asserted.
LITERATURE REVIEW
Antibiotic resistance mechanism and spreading pathway
Following WHO definition, antimicrobial resistance (AMR) or antibiotic resistance is the situation where microorganism such as bacteria, viruses or parasites can resistant to antimicrobial agents Consequently, common antibiotic become ineffective and antibiotic resistance spread to others
Bacteria contains specific genes allowing them to adapt with environmental threat including the environment with presence of antibiotic It is one of the most unique ability of bacteria during their evolution process In particular, there are two main reasons to make bacteria become resistant to antibiotics: (i) Gene mutations and (ii) Accumulation foreign DNA coding resistance gene by horizontal gene transfer (Smith 2017)
In the scenario of Gene mutations, antibiotic resistance occurs through one of listed mechanisms (Smith 2017)
(i) Modifications of target of antibiotics by producing enzymes to inactivate the antibiotic
(ii) Alteration of target site of antibiotics by preventing antibiotics from approach target binding locations
(iii) Discharge antibiotic by efflux pump Three antibiotic resistance mechanisms were described in Figure 2.1
Figure 2.1 Antibiotic resistance mechanism - Gene mutations
HGT is the phenomena which allow bacteria to exchange chromosomal and plasmid DNA among bacteria community via one of three mechanisms including conjugation, 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 is combined 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 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 (bla TEM 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 bla TEM gene (extended spectrum β-lactamase (ESBL)) were associative in Figure 2.3
Figure 2.3 Integron is involved in HGT (Gillings et al 2015)
Wastewater treatment plant as the hot-spot of ARB and ARGs
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 health treatment
And, hospitals contribute significantly to the generation and spread of ARB and ARGs However, hospital effluents only contribute less than 1% of total volume of municipal wastewater so that hospital wastewater is diluted largely in the WWTPs (Kümmerer 2004) Nevertheless, wastewater treatment plants (WWTPs) is one of the main sources for antibiotics released into various environment (Michael et al
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 antibiotics accelerates the selection pressure for antibiotic resistance in wastewater (Rodriguez-Mozaz et al 2015) Then, WWTPs receive wastewater from different sources including 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 been reported 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 biological and 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 nutrients via a biological process (aerobic or anaerobic systems) such as conventional activated sludge (CAS)
• The tertiary stage such as activated carbon adsorption, membrane filtration and disinfection are for removal of the components which cannot be eliminated by primary and secondary stage
In practice, WWTP are not optimized to remove pharmaceutical compounds
Advanced treatment shows a significant removal of antibiotics However, they cannot remove antibiotics in wastewater completely (Michael et al 2013)
Efficient removal rate for antibiotics was observed in Taiwanese WWTP For example, tetracyclines were eliminated by 66 – 91% in all studied WWTP Other plant where antibiotic groups of sulfonamides, cephalosporins, lincosamides, and quinolones were removed up to 100% or inadequately removed (Lin, Yu, and Lateef
2009) A WWTP in Girona (Spain) also presented a good removal rate with 80% of antibiotics 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 and ARGs, 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 m 3 /d of the untreated hospital wastewater along with nearly 55,000 m 3 /d of domestic wastewater from Girona city (Rodriguez-Mozaz et al 2015) Five targeted ARGs were evaluated including bla TEM (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 10 7 (copies/L) of bla TEM, 10 8 (copies/L) of sul1, 10 9 (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 bla TEM The copies number of qnrS was also detected at similar level to bla TEM (10 8 copies/L), however, this WWTP showed lower removal rate for qnrS in comparison to bla TEM suggesting the removal performance of WWTP was 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 In addition, AmpC β-lactamase genes were existed in excess sludge but WWTPs effluents (Y
Bin Xu et al 2017) This suggests the WWTPs performance to reduce ARGs 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 the highest removal rate of ARGs The removal rates were different in four WWTPs, which could be due to the different microbial community structure in treatment processes Sedimentation stage and accumulation of excess sludge were the key stages for the reduction of ARGs in any plants By sedimentation and enrichment through biological reaction, the microorganisms containing ARGs accumulated in sludge during treatment process ( Xu et al 2017)
Table 2.1 Removal rates of ARGs in WWTPs in China WWTPs Water sample Positive samples ARG Removal rates
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
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 WWTP performance to control and monitor ARGs (Mao et al 2015) Rysz et al (2013) also pointed out the reduction of food/microorganism ratio or extension of contact time in anaerobic digesters could prevent the ability of ARBs from harvesting energy which was profitable to loss of antibiotic resistance
2.2.2.3 Special factors affect to ARGs removal efficiency in WWTP
From part of 2.2.2.2, Water quality of influents, plant performance, nutrients compound, biological treatment processes were suspected as the factors affecting ARGs removal efficiency within WWTP In addition, other factors of selection pressure could be antibiotics concentration or some stress factors such as heavy metals, 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 β- lactamase resistance genes ( Xu et al 2017) The relative abundance of tetracycline resistance gene was correlated to the presence of Cu (r=0.714, p