Characterization of extended spectrum β lactamase producing escherichia coli in urban water environment in northern vietnam

VIETNAM NATIONAL UNIVERSITY, HANOI
VIETNAM JAPAN UNIVERSITY

NGUYEN BACH DUONG

CHARACTERIZATION OF
EXTENDED-SPECTRUM -LACTAMASE
PRODUCING ESCHERICHIA COLI IN
URBAN WATER ENVIRONMENT IN
NORTHERN VIETNAM

MASTER’S THESIS


VIETNAM NATIONAL UNIVERSITY, HANOI
VIETNAM JAPAN UNIVERSITY

NGUYEN BACH DUONG

CHARACTERIZATION OF
EXTENDED-SPECTRUM -LACTAMASE
PRODUCING ESCHERICHIA COLI IN
URBAN WATER ENVIRONMENT IN
NORTHERN VIETNAM

MAJOR: ENVIRONMENTAL ENGINEERING
CODE: 8520320.01

RESEARCH SUPERVISOR:
Associate Prof. Dr. IKURO KASUGA
Dr. TAKEMURA TAICHIRO

Hanoi, 2021


ACKNOWLEDGEMENT
Doing science research is a long journey that I am so grateful that I have received a
great deal of support and assistance over the last 12 months.
First and foremost, I would like to express my deepest thank to my supervisor –
Associate Professor Kasuga Ikuro for his patient guidance, valuable advice, continuous
support and encouragement. His immense knowledge and varied experience have
inspired me a lot during the research life at the graduate school.
I would also like to extend my deepest gratitude to my co-supervisor – Dr. Takemura
Taichiro for providing me the chance to carry out molecular biology experiments at
NIHE-Nagasaki Friendship Laboratory. His insightful suggestions have contributed
greatly to this master’s thesis.
I must also thank all the staff at NIHE-Nagasaki Friendship Laboratory, especially My
Hanh san, for their guidance and useful advice during the biological experiment.
The experiment related to this master thesis would not have been done without the
financial support from the Japan Agency for Medical Research and Development
(AMED) via the project “Development of Integrated Surveillance for Antimicrobial
Resistance”.
I would like to acknowledge lecturers at the Master’s Program in Environmental
Engineering (Vietnam Japan University) for giving constructive criticism to improve
the quality of my research.
Thanks also go to my classmates, my lab mates, as well as staffs at Vietnam Japan
University, with whom I have the pleasure to work while doing the thesis.
Last but not least, my sincere thanks are given to my family, my friends for their
profound belief in me. I would not have been able to complete this master thesis
without them.
Sincerely thank.

TABLE OF CONTENTS
CHAPTER 1. INTRODUCTION ...................................................................... 1
CHAPTER 2. LITERATURE REVIEW .......................................................... 4
2.1. Antimicrobial resistance ....................................................................................... 4
2.1.1. Antimicrobials and antimicrobial resistance ..................................................4
2.1.2. Molecular genetics of antimicrobial resistance ..............................................5
2.1.3. Mechanisms of antimicrobial resistance.........................................................7
2.1.4. Strategies to control antimicrobial resistance ............................................... 10
2.2. Antimicrobial resistance in the water environment ............................................10
2.3. Wastewater treatment plants – hot spots of AMR .............................................. 11
2.4. Occurrence of extended-spectrum -lactamase-producing Escherichia coli
(ESBL E. coli) ............................................................................................................11
2.4.1. Extended-spectrum -lactamase-producing Escherichia coli ......................11
2.4.2. -lactam antibiotics.......................................................................................12
2.4.3. Extended-spectrum -lactamases ................................................................. 16
2.4.4. ESBL E. coli in One Health ..........................................................................17

CHAPTER 3. METHODS ................................................................................ 25
3.1. Sampling .............................................................................................................25
3.2. Quantification of E. coli and ESBL E. coli (cefotaxime-resistant E. coli) .........28
3.3. Antimicrobial susceptibility testing .................................................................... 33
3.4. Persistence of ESBL E. coli in oligotrophic water environment ........................ 37
3.5. Genotyping of ESBL-encoding genes.................................................................39
3.6. Statistical analysis ............................................................................................... 44

CHAPTER 4. RESULT AND DISCUSSION ................................................. 45
4.1. Occurrence of ESBL E. coli ................................................................................45
4.1.1. Validation of culture method ........................................................................45

4.1.2. Occurrence of ESBL E. coli in urban drainage ............................................46
4.1.3. Occurrence of ESBL E. coli in river water ...................................................48
4.1.4. Resistance ratios of ESBL E. coli in water environments ............................49
4.2. Antimicrobial susceptibility of ESBL E. coli .....................................................51
4.3. Genotyping of ESBL-encoding genes in ESBL E. coli ...................................... 54
4.4. Persistence of ESBL E. coli in oligotrophic water environment ........................57
4.5. Removal of ESBL E. coli by wastewater treatment plant .................................. 58

CONCLUSION .................................................................................................. 60
REFERENCE .................................................................................................... 61
APPENDIX ........................................................................................................ 66


LIST OF TABLES
Table 2.1. Major mechanisms of resistance to antibiotic classes (Opal and Pop-Vicas,
2014) ................................................................................................................................9
Table 2.2. Classification of cephalosporins (Nguyễn, 2011)........................................ 14
Table 2.3. Studies on the occurrence of ESBL-producing E. coli in water environment
.......................................................................................................................................21
Table 3.1. Sampling points in Hanoi and Bac Ninh ..................................................... 25
Table 3.2. MALDI-TOF MS result interpretation ........................................................33
Table 3.3. Antibiotic disks used for susceptibility testing ............................................ 34
Table 3.4. Criteria of susceptibility of E. coli ...............................................................36
Table 3.5. Primer set for multiplex PCR CTX-M group 1, 2, 9 (Dallenne et al., 2010)
.......................................................................................................................................40
Table 3.6. Primer mixture CTX-M group 1, 2, 9 .......................................................... 40
Table 3.7. PCR mixture for multiplex PCR .................................................................. 41
Table 3.8. Primer set for multiplex PCR CTX-M group 8/25 (Dallenne et al., 2010) . 42
Table 3.9. PCR mixture for monoplex PCR .................................................................42
Table 4.1. Numbers of ESBL E. coli isolates tested and percentages of isolates

resistant to at least 4 antibiotics ..................................................................................... 53
Table 4.2. Genotyping of ESBL E. coli isolated from urban drainage.........................55
Table A1. Water quality in Hanoi samples ...................................................................66
Table A2. Water quality in Bac Ninh samples ............................................................. 70
Table A3. Water quality in extended sampling sites (surface water) ........................... 72
Table A4. Water quality in extended sampling site (WWTP) ...................................... 75

i


LIST OF FIGURES
Figure 1.1. Transmission of AMR in One Health approach...........................................2
Figure 2.1. Antibiotics and its target sites on bacterial cells .......................................... 4
Figure 2.2. Horizontal gene transfer in bacteria ............................................................. 6
Figure 2.3. lactam ring (i) and its subclasses: (ii) Penicillins, (iii) Cephalosporins,
(iv) Carbapenems, and (v) Monocyclic -lactams ........................................................ 13
Figure 2.4. Prevalence of healthy people carrying intestinal ESBL E. coli in six WHO
regions (Bezabih et al., 2021) ........................................................................................18
Figure 2.5. Global trend on the presence of ESBL E. coli in the intestine of healthy
people (Bezabih et al., 2021) ......................................................................................... 19
Figure 3.1. Sampling sites in Hanoi and Bac Ninh ...................................................... 26
Figure 3.2. E. coli appears as blue-green colony on TBX agar plate ...........................31
Figure 3.3. Bruker MALDI Biotyper (Microflex LT/SH) ............................................32
Figure 3.4. Result of species identification by MALDI-TOF MS ............................... 33
Figure 3.5. Growth of bacteria on the surface of agar plate after overnight incubation
(16 hours) ....................................................................................................................... 36
Figure 4.1. Composition of isolates identified by MALDI-TOF MS .......................... 45
Figure 4.2. Abundance of ESBL E. coli and total E. coli and resistance ratios in
different water samples in Hanoi and Bac Ninh (from Sep 2020 to May 2021)........... 47
Figure 4.3. Resistance ratios in different water samples in Hanoi and Bac Ninh ........49

Figure 4.4. Resistance ratios in upstream and downstream water in Hanoi, Bac Ninh
and other Northern provinces ........................................................................................50
Figure 4.5. Antibiotic resistance profile of ESBL E. coli isolated from (i) Hanoi urban
drainage; (ii) Bac Ninh urban drainge; (iii) WWTP effluent ........................................52
Figure 4.6. Image of gel electrophoresis of blaCTX-M group 1, group 2, and group 9 ... 54
Figure 4.7. Result of genotyping blaCTX-M-type ESBL-encoding gene in ESBL E. coli
.......................................................................................................................................55
Figure 4.8. Relationship of ESBL-encoding genes and number of antibiotics resistance
.......................................................................................................................................56
Figure 4.9. Log reduction of ESBL E. coli and non-ESBL E. coli in oligotrophic water
with time ........................................................................................................................57
Figure 4.10. Concentration of E. coli and ESBL E. coli in influent and effluent of
WWTP ...........................................................................................................................58
Figure 4.11. Correlation of log reduction value of E. coli and ESBL E. coli without
disinfection and with disinfection .................................................................................59

ii


LIST OF ABBREVIATIONS

ABP:

Ampicillin (antibiotic)

ARB:

Antimicrobial resistant bacteria

ARGs:

Antimicrobial resistance genes

AMR:

Antimicrobial resistance

bla:

gene encoding -lactamase

bp:

base pair

CAZ:

Ceftazidime (antibiotic)

CFN:

Cefdinir (antibiotic)

CFU:

Colony-forming unit

CTX:

Cefotaxime (antibiotic)

CTX-M:

Cefotaximase-Munich (-lactamase)

CP:

Chloramphenicol (antibiotic)

CPR:

Cefpirome (antibiotic)

CLSI:

Clinical and Laboratory Standards Institute

E. coli:

Escherichia coli

ESBL:

Extended-spectrum -lactamase

HGT:

Horizontal gene transfer

GES:

Guiana extended-spectrum (-lactamase)

GM:

Gentamycin (antibiotic)

iii


IPM:

Imipenem (antibiotic)

IZD:

Inhibition zone diameter

KM:

Kanamycin (antibiotic)

LVX:

Levofloxacin (antibiotic)

LRV:

Log reduction value

MDR:

Multidrug resistance

MPM:

Meropenem (antibiotic)

OXA:

Oxacillin-hydrolyzing (-lactamase)

PCR:

Polymerase chain reaction

SHV:

Sulfhydryl-variable (-lactamase)

ST:

Sulfamethoxazole +Trimethoprim (antibiotic)

TEM:

Temoneira (-lactamase)

TC:

Tetracycline

VEB:

Vietnamese extended-spectrum -lactamase

WHO:

World Health Organization

WWTP:

Wastewater treatment plant

iv


CHAPTER 1. INTRODUCTION
Antimicrobial resistance (AMR) – the ability of bacteria to fight against the
antimicrobial medicines – is listed as one of ten global health issues that urgently
needs tracking in 2021 (WHO, 2020). The global emergence and spread of AMR
drives human to face the lack of available effective treatment for the infection caused
by AMR bacteria. AMR is so serious that it is predicted to bring about 10 million
deaths in 2050 (O’Neill, 2014).
AMR will continue remaining as a key challenge to human health in the years ahead.
To deal with AMR challenge, the United Nations (UN) has encouraged the application
of the holistic approach – One Health. This approach involved the collaborative work
among specialized agencies working with the heath of human, animal, and
environment. Environment, especially the water environment, plays an important role
in the emergence and transmission of AMR since it is not only a reservoir of AMR

discharge from human and animal, but also a supply of water for agricultural irrigation,
and recreational activities (see in Figure 1.1). Figure 1.1 also indicates that
wastewater treatment is a factor in discharging of AMR into the environment.
According to the Ministry of Natural Resources and Environment, only 13% of
wastewater in Vietnam is treated while the remaining 87% is disposed directly into the
environment (Bộ Tài nguyên và Môi trường, 2018).
Since AMR is a One Health problem, a multisectoral surveillance system, which is a
powerful tool to provide the whole picture of AMR, is needed. However, such
surveillance is still lacking. To tackle this problem, the World Health Organization
(WHO) has developed the Tricycle protocol for surveillance of a single bacteria that
possesses a specific resistant mechanism, which is extended-spectrum -lactamaseproducing Escherichia coli (ESBL E. coli). The name “Tricyle” implies the idea that
the data of ESBL E. coli will be collected in three sectors: human, food chain (animal),
and the environment. Clearly, ESBL E. coli does not represent the overall situation of
AMR in the world since there still exists several infectious microorganisms and other
1


resistant traits. However, ESBL E. coli were selected as the target of the surveillance
protocol based on the following reasons (World Health Organization, 2021):
(i)

Existence of great variation in the rate of ESBL E. coli colonization in
humans and among countries, as well as the prevalence over time

(ii)

Existence of ESBL E. coli among farm animals

(iii)

Existence of proof that some of human deaths that are linked to ESBL E.
coli caused by either antibiotic use in food production or by ESBL E. coli in
the environment

(iv)

Interventions that aim to decrease antibiotics use or exposure in human and
animals have been accompanied with the decline in in ESBL E. coli
occurrence rates

(v)

ESBL production is an important resistant mechanism that makes critically
important antimicrobials ineffective.

Figure 1.1. Transmission of AMR in One Health approach
The research on ESBL E. coli in Vietnam to date has tended to focus on the
occurrence in human and animal rather than in the water environment. Only 2 papers
reported the occurrence of ESBL E. coli in the pig farm and slaughterhouse
2


wastewater in Vietnam (Hinenoya et al., 2018) (Nguyen et al., 2021). However, these
papers have been limited to the small number of ESBL E. coli isolates. Thus, the
occurrence of ESBL in the water environment in Vietnam remains unclear.
This thesis research in Environmental Engineering aims to unravel the characteristics
of extended-spectrum -lactamase-producing Escherichia coli (ESBL E. coli) in
urban water environment in Northern Vietnam in line with WHO Tricycle Project
which will help to address the research gap. Hereafter, cefotaxime-resistant E. coli
were regarded as ESBL E. coli in this study. Specifically, the research is expected to:

(i)

Determine the characteristics of ESBL E. coli in different urban water
environment in Northern Vietnam,

(ii)

Evaluate the role of wastewater treatment plant to reduce ESBL E. coli
discharge into the water environment.

3


CHAPTER 2. LITERATURE REVIEW
2.1. Antimicrobial resistance
2.1.1. Antimicrobials and antimicrobial resistance
Antimicrobials, which are commonly called as antibiotics, are the effective therapy for
treatment of bacterial infections by killing or slowing down the growth of the bacteria.
Antibiotics are classified based on their action on the site of bacterial cell. The main
sites of target of these agents are the synthesis or the activity of one of the following
components of bacterial cell: cell wall, cell membrane, ribosome and nucleic acid
(Sauberan and Bradley, 2018). The target of each antibiotic is shown in Figure 2.1.

Figure 2.1. Antibiotics and its target sites on bacterial cells
The discovery of antibiotics in early 20th century was a milestone in the history of
human. Since then, antibiotics have saved countless lives from several bacterial
infections. The magic of this invention, however, did not last long, as the bacteria
4

rapidly developed the resistance to these drugs, which is called as “antimicrobial
resistance” (AMR). AMR is the ability of bacteria to survive under the use of
antibiotics. While susceptible strains are killed by the antibiotics, resistant bacteria can
grow without any competition.
Although development of resistance in bacteria is a natural selection process, it is
accelerated by misuse and overuse of antibiotics in human and food animal production.
In fact, the rate of emergence of AMR is faster than the rate of new antibiotics is
developed. Since the 1980s the rate of discovery new antibiotics has fallen
dramatically (O’Neill, 2016). In other words, humans are facing the lack of available
treatment due to the prevalence of drug-resistant bacterial infections. Doctors now
have to prescribe antibiotics that is used to be avoided due to its bad side effects
(O’Neill, 2016). For example, last-line antibiotics like colistin, which can cause kidney
failure, now are given to patients to treat drug-resistant Gram-negative bacterial
infections. However, resistance to colistin has already emerged (O’Neill, 2016). In
former times, resistant infections were mainly associated with hospital settings,
however, in the past decade, they were frequently observed in community (O’Neill,
2016). An underestimate showed that every year, 700,000 deaths associated with
infections by drug-resistant bacteria (O’Neill, 2014). Moreover, AMR is costly as
infection by AMR bacteria results in longer hospital stay and higher treatment cost.
Every year, two million AMR infections cost the US health care system 20 billion US
dollars excessively (O’Neill, 2016).
2.1.2. Molecular genetics of antimicrobial resistance
Antimicrobial resistance genes (ARG) can be located either in chromosomal DNA or
plasmid DNA. The occurrence of antimicrobial resistance genes arises in three level:
(i) microevolutionary change, (ii) macroevolutionary change, and (iii) acquisition of
large segments of foreign DNA.
Microevolutionary changes include point mutation, that may result in change the target
site or the enzyme-substrate specificity of the antibiotics. For example, point mutation
in “classic” -lactamase genes (blaTEM-1, blaSHV-1, etc.) resulted in the formation of
extended-spectrum -lactamases (ESBLs) (Opal and Pop-Vicas, 2014).

5


Macroevolutionary change is the result of re-arrangement of large segment of DNA in
a single event. These re-arrangements can be: inversions, duplications, insertions, or
transpositions. Transpositions are generated by some specific genetic elements such as
transposons.
The last level of the occurrence of antimicrobial resistance are acquisition of large
segments of foreign DNA. Foreign DNA are carried by plasmids, bacteriophages, free
sequences of DNA, or by mobile genetics elements. The event of release and uptake
foreign DNA is called horizontal gene transfer (HGT) (Opal and Pop-Vicas, 2014).
HGT includes:
(i)

Transformation: Bacteria uptakes of DNA from the surroundings, these free
DNA are released from dead bacterial cell (see in Figure 2.2) (Opal and
Pop-Vicas, 2014)

(ii)

Transduction: Bacteria receives foreign DNA carried by transducing
bacteriophages. Bacteriophages carry and transfer genetic material from
donor cell to recipient cell (see in Figure 2.2) (Opal and Pop-Vicas, 2014)

(iii)

Conjugation: Bacteria transfers plasmid DNA directly via a mating bridge
between two cells (see in Figure 2.2) (Opal and Pop-Vicas, 2014)

Figure 2.2. Horizontal gene transfer in bacteria

6


HGT is largely responsible for the wide spread of AMR. Once antimicrobial resistance
genes (ARGs) are emerged, they can be spread among bacteria through HGT.
Plasmid
Plasmids are double-stranded circular DNA molecule that are widely common in
bacteria. Multiple copies of a plasmid, or different plasmids or both can exist within a
single bacteria cell. Plasmids can replicate independently from chromosome
duplication. Some small plasmids can be transferred to other bacteria via conjugation
(Opal and Pop-Vicas, 2014). Due to this characteristic, plasmids are responsible for
the wide spread of large amount of plasmid-borne antibiotic resistance gene.
Transposons
Transposon is a kind of transposable genetic element. They are small pieces of DNA
that encode functional genes, for example, antibiotic resistance. It can translocate (or
“jump”) between the chromosome and plasmid and vice versa (Opal and Pop-Vicas,
2014). Transposons cannot replicate themselves, thus, they must exist on a replicon
such as chromosome or plasmid. Transposition of transposons is an ongoing process in
the revolution of bacteria, they play a vital role in maintenance of ARG.
Integron
Integrons, which are also known as gene cassettes, are genetic elements that are able to
integrate, exchange, and express specific DNA sequences (Domingues et al., 2012).
Integrons can incorporate one or several gene-cassettes (Domingues et al., 2012), thus,
they can carry several antimicrobial resistance genes within one integron (Domingues
et al., 2012). Integrons are not mobile genetic elements because they are lack of genes
for self-mobility. The mobilization of integrons are due to their location on other
plasmids or transposons (Domingues et al., 2012). Integrons might also locate on
chromosome and acquired via transformation.
2.1.3. Mechanisms of antimicrobial resistance
Until now, eight mechanisms of antibiotic resistance have been discovered in bacteria

(Opal and Pop-Vicas, 2014), which are:
(i)

enzyme inactivation: bacteria produce enzyme that can inactivate the drug,
7


(ii)

decrease membrane permeability: bacteria change the permeability of its cell
membrane (either outer membrane or inner membrane or both) so that the
drug cannot enter the cell,

(iii)

promotion of efflux pump: bacteria cell forms membrane transport system
that operate active efflux drug pump. The drugs are pumped out right after
they enter the cell. These pumps can be universal (pumping several classes
of antibiotics) or drug specific. Efflux pumps are one of the mechanisms of
multi-drug resistant.

(iv)

alteration of target site: the bacteria modify the target of the drug (i.e.,
ribosomal target site, cell wall binding site, target enzyme) so that the drug
cannot bind to react with,

(v)

protection of target site: the bacteria cell produces an enzyme that prevent

the drug from binding to the target site,

(vi)

overproduction of target: bacteria cell produces an excess amount of the
target so that the drug cannot bind to all these targets,

(vii)

bypass of inhibited process: bacteria change from producing a specific
growth factor to taking it from the environment. Therefore, even if the drug
is inhibiting the synthesis process, the bacteria can still survive.

(viii) bind up antibiotic: bacteria produce enzymes that can modify the drug, thus,
make the drug unable to bind with the target site.
There are several resistance mechanisms toward a particular drug, which are shown in
Table 2.1.
More than one mechanism of antimicrobial resistance can exist simultaneously within
one bacterial cell, resulting in multidrug resistance (MDR) or pan-resistance (Opal and
Pop-Vicas, 2014). In this case, the bacteria are called as multidrug-resistant bacteria
or superbug. Superbugs’ infections leave the doctor limited or even no options of
antibiotics to use, thus, result in increasing treatment cost, and longer hospital stays.

8


9

+++

+++

+ (gram- negative)

+

inhibited

-

-

-

-

-

-

-

-

+

-

++

+

-

-

-

-

+++

++

+++: most common mechanism; ++: common; +: less common.

Bind up antibiotic

Bypass of
process

Protection of target
site
Overproduction
of
target

Alteration of target stie ++

Efflux Pump

-

+

++

-

++

-

-

Trimethoprim

+

Quinolone

-

-

-

-

++

-

+

++

-

+(Helicobact +++
er pylori)

+++

-

-

-

+

+++

+

+
(gram- +
(gram- +(gramnegative)
negative)

negative)

-

Amino- Chloram- Macrolide Sulfonamide Tetracycline
glycoside phenicol

Decrease Permeability + (gram- + (gram- + (gram- ++ (gram- negative) negative) negative) negative)

Enzymatic Inactivation +++

Betalactam

Table 2.1. Major mechanisms of resistance to antibiotic classes (Opal and Pop-Vicas, 2014)


2.1.4. Strategies to control antimicrobial resistance
In order to provide a useful reference for managing AMR, WHO Advisory Group on
Integrated Surveillance of Antimicrobial Resistance (AGISAR) has made a ranking of
medically important antibiotics for risk management of antimicrobial resistance. In
this document, 35 medically important antimicrobials are categorized as: critically
important antimicrobials, highly important antimicrobials, and important
antimicrobials. Critically important antimicrobials (CIA) are further classified into
highest priority CIA and high priority CIA (WHO Advisory Group on Integrated
Surveillance of Antimicrobial Resistance (AGISAR), 2019). Highest priority CIAs
includes antimicrobial classes that need prudent use and should only be used where
other antibiotics have not worked due to AMR. Highest priority CIAs are: (i)
Quinolones (ii) 3rd and higher generation Cephalosporins, (iii) Macrolides and
Ketolides, (iv) Glycopeptides, (v) Polymyxins.
Besides the list of important antimicrobials, WHO also published a global list of

antibiotic resistant bacteria to provide guideline for the prioritization of research and
funding to combat antibiotic-resistant bacteria (World Health Organization, 2017).
WHO’s experts have stratified the pathogens into three priority tiers: (i) critical, (ii)
high, and (iii) medium (World Health Organization, 2017). Enterobacteriaceae,
which is resistant to carbapenem and 3rd generation cephalosporins, is listed as a
pathogen at critical priority.
2.2. Antimicrobial resistance in the water environment
As discussed before, the water environment is an important reservoir and transmission
routes of AMR. Waters receives antibiotics residuals, ARG, antimicrobial resistant
bacteria (ARB) mainly from human and animal sources. Use of antibiotics and
residuals of antibiotics in food pose a selection pressure on the intestinal bacteria,
leading to the development of AMR (Amarasiri et al., 2020). In addition, consumption
of food contaminated with ARG or ARB might lead to HGT with the normal organism
living in the gastrointestinal tract (Amarasiri et al., 2020). These AMR elements are
discharged in feces, and eventually end up in the wastewater.

10


2.3. Wastewater treatment plants – hot spots of AMR
Wastewater treatment plants (WWTPs) are suggested to be hotspot for emergence of
new AMR mechanisms (Amarasiri et al., 2020). WWTPs receive wastewater from
different sources (i.e. domestic wastewater, hospital discharge, agricultural wastewater,
etc.). Under the selection pressure posed by several contaminants (microbes, antibiotic
residuals, metals), horizontal gene transfer can occur (Amarasiri et al., 2020). Research
has proved that WWTPs cannot completely remove ARB, ARG and antibiotic
residuals, thus, a portion of these AMR elements remaining in the effluent and further
released into the surface water, and further lead to development of antibiotic resistance
among natural microorganisms (Amarasiri et al., 2020).
To date, WWTP mainly focus on control of bacteria, thus, ARG is not included.

Controlling techniques can be broadly divided into: (i) removal processes, and, (ii)
inactivation processes (LeChevallier and Kwok-Keung, 2004). The processes
contributing to the removal of microbes include pretreatment, filtration, coagulationflocculation, while inactivation process involves application of either strong oxidizing
compounds (chlorine, chlorine dioxide or ozone) or UV light. Activation, or
disinfection, is a major contributor to the overall reduction of microbes in WWTPs.
Removal efficiency of different techniques are varied and depends on several factors
(for example, chemical doses, contact time, and so on). Selection of treatment process,
therefore, is crucial to reduce AMR loads discharged into natural environment. It is so
important as reclaimed water is used for several purposes, such as agricultural
irrigation, aquaculture, or used as recreational waters. Thus, humans can be exposed to
these AMR elements through aquatic sport or exposure during irrigation or
consumption of food that was previously irrigated with reclaimed water.
2.4. Occurrence of extended-spectrum -lactamase-producing Escherichia coli
(ESBL E. coli)
2.4.1. Extended-spectrum -lactamase-producing Escherichia coli
Escherichia coli (E. coli) belongs to the family Enterobacteriaceae. E. coli strains can
be broadly classified into 3 groups: (i) harmless commensal strains that are a part of
the normal microbiota of the gastrointestinal tract, (ii) strains that cause diarrheal
11


intestinal disease, and (iii) strains that cause extraintestinal infections (Poolman, 2016).
E. coli are the main agent causing diarrheal diseases, which accounts for around 9%
children death worldwide (Poolman, 2016). It is also estimated that approximately
80% of bacterial-related diarrheal disease in developing countries are caused by the
diarrheagenic E. coli (Poolman, 2016). Infections caused by E. coli are caused by
several ways: via contact between person affected or via transmission from animals,
food chains or unsanitary water.
The genes determining AMR in E. coli are likely to be found in pathogenicity island
(which commensal E. coli are lack of) and mobile genetic elements (MGEs) (Poolman,

2016). These AMR-encoding elements have been found in other pathogenic species,
which suggest the history of genetic transfer and/or exchange (Poolman, 2016).
ESBL E. coli is critical priority antibiotic-resistant bacteria that poses the resistant trait
to the highest priority critically important antibiotics. The emergence of ESBL E. coli
is attributed to the huge consumption of extended-spectrum-lactam antibiotics
(Chong et al., 2018). ESBL is a group of enzymes that can inactivate (or hydrolyze) lactam antibiotics. Further details of -lactam antibiotics and ESBL enzymes are
discussed in part 2.4.3.
2.4.2. -lactam antibiotics
-lactams are cell-wall active agents that inhibit the formation of bacterial cell wall.
Bacterial cell wall cannot be formed in the presence of -lactam ring, which is the core
of all -lactam antibiotics (see in Figure 2.3). This ring interrupts the cell-wall
formation by binding to the penicillin-binding proteins (PBPs). PBPs are the enzymes
that are responsible for synthesis of the cell wall. The cell wall maintains the cell
structures, gives the protection to the cellular organelles, and expands during cell
division. Thus, when a -lactam bind to PBP, the cell wall cannot be
assembled/maintained, resulting in the death of the cell.
Similar to other antibiotics, -lactams have undergone several modifications to
enhance their spectrum of activity, pharmacokinetic or to deal with the emergence of
antimicrobial resistance. -lactam antibiotics are classified based on the functional
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groups attaching the -lactam ring. At present, there are 4 subclasses of -lactam
antibiotics that are used in human. These subclasses include penicillins, cephalosporins,
carbapenems and monocyclic -lactams. The chemical structures of these antibiotics
are shown in Figure 2.3.

Figure 2.3. lactam ring (i) and its subclasses: (ii) Penicillins, (iii) Cephalosporins,
(iv) Carbapenems, and (v) Monocyclic -lactams
(i) Penicillins

Penicillins are the first -lactams to be discovered. They are non-toxic and are
considered to be one of the safest antimicrobial agents, besides cephalosporins. The lactam rings of penicillins are easily being hydrolyzed by a wide range of -lactamases,
which are enzymes that can break down the -lactam ring. Spectrum of this -lactam
class is narrow, it poses higher effect on Gram-positive bacteria than on Gramnegative ones.
(ii) Cephalosporins
Cephalosporins are classified into generations based on their spectrum, and their
stability with -lactamases. Later generations lose their potency against Gram-positive
but gain their bactericidal activity against Gram-negative bacteria. Cephalosporins 2nd

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and higher generations are relatively stable to -lactamases. Detail of the spectrums of
each generation are expressed in Table 2.2.
Table 2.2. Classification of cephalosporins (Nguyễn, 2011)
Spectrum

Antibiotic agents

1st generation
- More effective against Gram-positive bacteria, somewhat
effective against some Gram-negative bacteria

Cefazolin
Cephalexin

- Unstable, easily being hydrolyzed by -lactamases
2nd generation
- Effective against both Gram-negative and Gram-positive
bacteria

Cefaclor
Cefuroxime

st

- Less effect against Gram-positive bacteria compared to 1
generation

Cefoperazone

- Relatively stable to -lactamases
3rd generation
- Broad-spectrum, less active than 1st generations against
Gram-positive bacteria
- More active to MDR bacteria

Cefotaxime
Ceftazidime
Cefdinir

- More stable to -lactamases than 2nd generation
4th generation
- Similar effect on Gram-negative as 3rd generation, more
effectively against Gram-positive in comparison with 3rd
generation

Cefepim
Cefpirome

- Stable to a wide range of -lactamases

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(iii) Carbapenems
Carbapenems can bind to up to 4 kinds of PBPs, thus adding supplementary killing
effect and lessening the risk of resistance (Bush and Bradford, 2016). These antibiotics
are also known as the “last-resort” antibiotics for treatment of infections caused by
multidrug-resistant bacteria. Carbapenems stand out for its stability to almost all lactamases, except for carbapenemases (Bush and Bradford, 2016).
(iv) Monocyclic -lactams
Monocyclic -lactams are also known as monobactam. Until now, only Aztreonam in
this group has been approved for therapeutic use (Nguyễn, 2011). It is effective
against aerobic enteric bacteria. It is stable against all the common -lactamase except
for ESBLs and carbapenemase (Bush and Bradford, 2016).
-lactamase inhibitors
In the middle of the 1960s, scientists started to pay attention to develop compounds
that could inhibit -lactams to deal with the increasing occurrence of -lactamaseproducing pathogens (Bush and Bradford, 2016). Now, four common -lactamase
inhibitors are used in human medicine. They are clavulanic acid, sulbactam,
tazobactam, and avibactam. These -lactams inhibitors have similar structures as
penicillin but have relatively weak effect on the formation of the bacteria cell wall.
However, when they are used in combination with other -lactams, they can bind
irreversibly with -lactamases, thus, protect the -lactam from hydrolysis by lactamase. However, the activity levels and extent of substrates of each inhibitors
differ among these inhibitors (Toussaint and Gallagher, 2015).
Consumption of -lactams
A report on antibiotics consumption in 55 countries in different regions in the world
showed that -lactams are the most used class of antibiotics (World Health
Organization, 2018). In the United States, it accounts for 65% prescribed injectable
antibiotics, and almost half of the prescription is cephalosporins (Bush and Bradford,
2016). In Vietnam, -lactams are also the most prescribed antibiotics in the hospital.

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These -lactams include 2nd and 3rd generation cephalosporins, and carbapenem (Binh
et al., 2018). In Vietnam, using drugs without prescription is common, 71% of patients
before going to the hospitals have used antibiotics, and 76% of these antibiotics are lactams (Binh et al., 2018).
2.4.3. Extended-spectrum -lactamases
Resistance to -lactam antimicrobials is mainly production of -lactamase (see in
Table 2.1). -lactamases are enzyme that can break the bond of  -lactam ring of the
drug (see in Figure 2.3), thus, make the drug ineffective. The rapid widespread of lactamases has been attributed to its location on mobile genetic elements, such as
plasmids or transposons.
-lactamases have been presented in natural environment long before -lactam
antibiotic were introduced to human. However, the “old” -lactamases occurred at
very low frequency until -lactams became widely used in human and animals (Wilke
et al., 2005). Extended spectrum -lactamases (ESBL) are considered to be the “new”
-lactamases (Jacoby and Munoz-Price, 2005). It can be explained that the emergence
of extended spectrum -lactamases (ESBL) is attributed to the common use of
carbapenems, cephalosporins (3rd or higher generations), and monobactam (Wilke et
al., 2005). The term “extended-spectrum” describes the fact that these -lactamases
pose hydrolyzing effect to a wider spectrum of -lactam antibiotics in comparison with
the “classic” -lactamases (Livermore, 2008).
TEM-type, SHV-type, CTX-M-type, OXA-type are four main types of ESBLs (Jacoby
and Munoz-Price, 2005).
TEM-type ESBLs and SHV-type ESBLs
TEM-type ESBLs and SHV-type ESBLs are the very first ESBLs to be discovered
(Bush and Bradford, 2020). They are variants of the “classic” TEM-type -lactamases
and SHV-types -lactamases, respectively (Opal and Pop-Vicas, 2014). These
enzymes used to be the dominant ESBLs until the late 20th century (Bush and Bradford,
2020).
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CTX-M-type ESBLs
CTX-M-type ESBLs was discovered in 1980s (Bush and Bradford, 2020). Since the
beginning of this millennium, CTX-M has been the most prevalent ESBLs in the world
(Jacoby and Munoz-Price, 2005). Genes encoding CTX-M ESBLs originated from the
chromosomal DNA of Kluyvera spp., an environmental species of Enterobacteriaceae .
These genes were transferred to other species via horizontal gene transfer (Bush and
Bradford, 2020). CTX-M-type -lactamases are commonly detected in E. coli and
Klebsiella pneumoniae and other species of Enterobacteriaceae (Bush and Bradford,
2020). The name of this ESBL type emphasizes their great affinity to Cefotaxime (3rd
generation cephalosporins). Based on the similarity in the amino acid sequences
(>94%), CTX-M are categorized into 5 groups: Group1, Group 2, Group 8, Group 9,
Group 25 (Bush and Bradford, 2020). CTX-M-type enzyme can be inhibited by all
available -lactamases inhibitors (Bush and Bradford, 2020).
OXA-type ESBLs
OXA-type ESBLs differ from the other three ESBL types by the ability to confer the
resistant to the inhibition effect of clavulanic acid (Jacoby and Munoz-Price, 2005).
Other ESBLs
Other types of ESBLs have been reported (Jacoby and Munoz-Price, 2005). However,
they are uncommon and occurred at low frequency within specific geographic regions
(Jacoby and Munoz-Price, 2005). For example, VEB-type ESBL were detected in
Southeast Asian countries, while GES-type ESBLs were found in isolates from South
Africa, France and Greece (Jacoby and Munoz-Price, 2005).
2.4.4. ESBL E. coli in One Health
In Human
In the world
A meta-analysis of 62 articles covering the studies on the prevalence of healthy
residents (n=29,872) carrying fecal ESBL E. coli over the period 2003-2018 shows
that the global ratio of healthy persons carrying ESBL E. coli in their intestine was

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