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

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

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

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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

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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

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LIST OF ABBREVIATIONS

ABP: Ampicillin (antibiotic)

ARB: Antimicrobial resistant bacteria

ARGs: Antimicrobial resistance genes

AMR: Antimicrobial resistance

bla: gene encoding -lactamase

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)

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IPM: Imipenem (antibiotic)

IZD: Inhibition zone diameter

OXA: Oxacillin-hydrolyzing (-lactamase)

PCR: Polymerase chain reaction

SHV: Sulfhydryl-variable (-lactamase)

ST: Sulfamethoxazole +Trimethoprim (antibiotic)

TEM: Temoneira (-lactamase)

VEB: Vietnamese extended-spectrum -lactamase

WHO: World Health Organization

WWTP: Wastewater treatment plant

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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 -lactamase-producing 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

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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

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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

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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

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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)

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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

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,

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(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

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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

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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, coagulation-flocculation, 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

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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

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 Gram-negative 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)

1st generation

- More effective against Gram-positive bacteria, somewhat

effective against some Gram-negative bacteria

- Unstable, easily being hydrolyzed by -lactamases

Cefazolin Cephalexin

3rd generation

- Broad-spectrum, less active than 1st generations against

Gram-positive bacteria

- More active to MDR bacteria

- More stable to -lactamases than 2nd generation

Cefotaxime Ceftazidime Cefdinir

4th generation

- Similar effect on Gram-negative as 3rd generation, more

effectively against Gram-positive in comparison with 3rd

generation

- Stable to a wide range of -lactamases

Cefepim Cefpirome

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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 -lactamase-producing 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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16.5% (Bezabih et al., 2021) Over the period, among 6 WHO regions, highest ratios were observed in Southeast Asia, followed by Western Pacific region (27% and 25%, respectively), the lowest prevalence ration was reported in Europe (6%) (Bezabih et al., 2021) Tanzania, Vietnam, Laos, China, Thailand, Egypt, and Lebanon (in ascending order) were reported as countries with the highest community prevalence of ESBL E coli carriage (Bezabih et al., 2021) (Figure 2.4)

Figure 2.4 Prevalence of healthy people carrying intestinal ESBL E coli in six WHO

regions (Bezabih et al., 2021) The global prevalence of ESBL E coli in human intestine followed an upward trend (see Figure 2.5), from 2.6% in 2003-05 to 21.1% in 2015-18 (Bezabih et al., 2021) Healthy carriers is an ideal spread vehicle for the spread of ESBL E coli as people now can travel across countries boundaries (Nakayama et al., 2018) Those ESBL E coli might bring about serious infection not only in the carrier themselves but in others who contact to them ESBL E coli infection can only be cured by the last resort drugs

- carbapenems, which is now becoming less effective due to the emergence of carbapenemase-producing bacteria The higher the occurrence rate is the higher risk of

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transmission of ESBL E coli among healthy humans as well as between the healthy and the sick one increase

Figure 2.5 Global trend on the presence of ESBL E coli in the intestine of healthy

people (Bezabih et al., 2021) Another comprehensive review on the occurrence of ESBL E coli among healthy humans in Lebanon, Japan, China, Laos, Sweden, Portugal, Spain, and the Netherlands indicates the prevalence of CTX-M-type ESBL (Kawamura et al., 2017) In addition, a study in 3 countries in the Southeast Asia showed that the ratios of healthy residents from Laos, Thailand, and Vietnam carrying ESBL E coli are 72%, 69% and 51% respectively Most of these ESBL E coli harbored blaCTX-M group 1 and group 9 (Nakayama et al., 2015)

In Vietnam

CTX-M-type ESBL E coli were detected in workers in large-scale farm and healthy asymptomatic individuals in Ba Vi (Hanoi) at the rates of detection of 83% and 55% respectively (Bui et al., 2018) CTX-M-1 group accounted for 70 and 79% of ESBL E coli isolates in farm workers and community individuals respectively (Bui et al., 2018)

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While those data for CTX-M-9 group are 30% and 25% (Bui et al., 2018) Those isolates showed high rate of resistance (>50%) to ampicillin, sulfamethoxazole/trimethoprim and tetracycline (Bui et al., 2018) The ratios of resistance to each antibiotics in farm-workers were generally higher than in community residents (Bui et al., 2018)

Similarly, another study in 199 healthy residents in Ba Vi indicates that 50% of healthy residents in carried ESBL E coli (Bui et al., 2015) 87.9% of those ESBL E coli harbored the blaCTX-M genes (Bui et al., 2015) However, CTX-M-9 group was the dominant ESBL (Bui et al., 2015) Resistance to ampicillin, streptomycin, tetracycline, trimethoprim-sulfamethoxazole were relatively high in those ESBL E coli (>50%) (Bui et al., 2015)

A study in 2 tertiary hospitals in Northern Vietnam indicates the rate of occurrence of ESBL producer among E coli causing bloodstream infections to be 39.29% (Hung et al., 2019), blaTEM gene, blaCTX-M, blaSHV were detected in 100%, 81.82%, 18.18% of these isolates, respectively These isolates were highly resistant to ampicillin, sulfamethoxazole/ trimethoprim (Hung et al., 2019)

In Animal (Food)

In the world

ESBL-producing E coli were detected in food samples, including: chicken meat (Japan, China, Turkey, Sweden, the Netherlands, Germany, the UK, Italy, Brazil), raw pork (China, Vietnam), cooked pork products (China), beef (Vietnam, Turkey), and fish (Vietnam) (Kawamura et al., 2017) In those findings, CTX-M is the most common ESBL (Kawamura et al., 2017) In the Philippines, 67% of E coli isolates collected from farmed chicken was ESBL producer and 87% these ESBL producers were CTX-M positive (Gundran et al., 2019) ESBL E coli were highly resistant to ampicillin, ceftazidime, sulfamethoxazole/trimethoprim (Gundran et al., 2019)

In Vietnam

CTX-M-type ESBL E coli were detected in randomly selected chicken in large-scale farm and small-scale farm at the rates of detection of 71% and 14% respectively (Bui

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et al., 2018) CTX-M-9 group was dominant in those ESBL E coli (Bui et al., 2018) Those ESBL E coli exerted high ratios of resistance (>50%) to ampicillin, sulfamethoxazole/trimethoprim, chloramphenicol, kanamycin (Bui et al., 2018) ESBL

E coli in farmed chicken showed higher resistance ratio than community chicken (Bui

et al., 2018)

industrial farms in Me Kong Delta (Southern Vietnam) While most of these blaCTX-M

in chicken farm belong to group 1 while most of those from pig samples belong to group 9 (Hinenoya et al., 2018) Most of them were resistant to cefotaxime, ampicillin, chloramphenicol, tetracycline, sulfamethoxazole/ trimethoprim (Hinenoya et al., 2018)

In the Environment

In the world

Compared to the number of research working on ESBL E coli isolates from water environment are far less than those in human and food (animal) Table 2.3 summarizes

a number of research of this organism in water environment in the world

Table 2.3 Studies on the occurrence of ESBL-producing E coli in water environment

Area of

study

Contamination source

Occurrence rate (%)

ESBL genes

Resistance to antibiotics References Yodo river

basin, Japan

WWTP effluent

cefotaxime, atrezonam, kanamycin, gentamycin, levofloxacin

2.9% − 5.3%

group(15%)

cefotaxime (100%), ceftazidime (50%),

Tsutsui and Urase,

2019

22

group(15%), blaTEM

(5%)

cefpirome (90%), levofloxacin (60%), ciprofloxacin (65%)

WWTP in

Tokyo,

Japan

WWTP effluent (before disinfection)

1.9%

(WWTP-A) 1.2% − 2.0%

(WWTP-B)

group(45%)

group(60%) blaTEM

(5%)

cefotaxime (100%), ceftazidime (15%), cefpirome (95%), levofloxacin (50%), ciprofloxacin (50%)

Tsutsui and Urase,

2019

Colorado,

the USA

Domestic wastewater

<1%

(Sewer water, WWTP influent, WWTP effluent, surface water)

(except for cefotaxime and

ceftazidime)

Schmiege

et al.,

2021

(healthcare institution WW), 6.83%

(WWTP influents),

<1%

(WWTP effluent)

WWTP influents:

Blaak et al., 2015

Recreational

water, the

Netherlands

WWTP effluent

(>50%) resistant to ampicillin, cefotaxime, ceftazidime, tetracycline, trimethopro

me

Blaak et al., 2014

1.1%-6.4% blaCTX-M-9

group,

group, blaTEM-

blaSHV

tetracycline (97.3%), ticarcillin-clavulanic acid

(90.8%),

Liu et al.,

2018

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cephalothin (89.5%), nalidixic acid (81.6%), cefotaxime (77.6%), ciprofloxacin (69.7%), etc

In Vietnam

A literature review on the occurrence and characterization of ESBL E coli in the water environment in Vietnam was conducted in PubMed by using the key word “ESBL E coli water Vietnam” Only 2 articles related to this topic were found The first paper reported the presence of ESBL E coli isolates from wastewater in pig farm in Can Tho and Hau Giang (Southern Vietnam), (Hinenoya et al., 2018) Those isolates possessed

cefotaxime (n=6), ceftazidime (n=3), ampicillin (n=6), fosfomycin (n=6), streptomycin (n=4), kanamycin (n=2), gentamycin (n=1), chloramphenicol (n=6), tetracycline (n=6), sulfamethoxazole/ trimethoprim (n=6) (Hinenoya et al., 2018) The second paper reported the occurrence of ESBL E coli in slaughterhouse environment (surface swab, water) (Nguyen et al., 2021) The ratio of ESBL-producer among those samples was 19% (n=9) and 78% harbored CTX-M encoding genes (group 9 and group 1) (Nguyen

et al., 2021)

No previous study has addressed the occurrence of ESBL E coli in natural waters as well as in domestic wastewater in Vietnam The study presented in this thesis fills the gap in the literature by providing a fresh insight in the occurrence and characteristics

of ESBL E coli in urban water environment in the Northern cities of Vietnam It also enhances our understanding the role of wastewater treatment plant in controlling ESBL E coli discharge into the natural environment

Three types of water were collected:

(i) River water in upstream: this sample represents the pre-city impact

(ii) River water in downstream: this sample represents the impact of the city (iii) Human communal wastewater: this sample represents the intestinal microbiome of the city’s population

In addition, extended samples of the effluent of wastewater treatment plant were collected in each sampling events The effluent samples in Bac Ninh were collected in stabilization pond before discharging into the agricultural canal, in this WWTP, no disinfection was applied In Hanoi, those samples were collected right after discharging into the surface water Detail of sampling sites are provided in Table 3.1

Table 3.1 Sampling points in Hanoi and Bac Ninh

Kim Nguu river (before WWTP) Upstream surface water Nhue river upstream

Downstream surface water Nhue river in urban residence Effluent WWTP Kim Nguu river (after WWTP)

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Upstream surface water Ngu Huyen Khe river (upstream) Downstream surface water Ngu Huyen Khe river (downstream)

Location of the sampling sites are shown in Figure 3.1

Figure 3.1 Sampling sites in Hanoi and Bac Ninh For comparison of occurrence of ESBL E coli in Northern Vietnam, besides the regular sampling events in Hanoi and Bac Ninh, surface water samples were collected

in other Northern cities (Ninh Binh, Nam Dinh, Thai Binh, Hung Yen) from September to October 2020 Further information of the samples can be found in Table A3

In order to study how much ESBL E coli can be removed from wastewater, besides the samples described in Table 3.1, extended sampling events were caried out from

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October to December 2020 to collect in Yen So WWTP (Hanoi), Bay Mau WWTP (Hanoi), Vinh Niem (Hai Phong) Location of the sampling sites are described in Table A4 Influent and effluent WWTP were collected Yen So WWTP applied UV light disinfection; while in Vinh Niem WWTP and Bay Mau WWTP, chlorine disinfection was used

Sampling time

The sampling time stratified over the year, from September 2020 to May 2021 Samples in Hanoi were collected every month, while samples in Bac Ninh were collected every two months Due to the emergence of Covid-19 within the community

of Bac Ninh in May 2021, the sampling event was canceled

Material

Autoclaved Duran® original GL45 bottles (500 mL or 250 mL) or SPL® Wide-mouth

PP bottles (500 mL or 250 mL), tissue paper, insulated bag, cooling gel ice, label, pH strips, waterproof conductivity meter AS650, clean bucket tied with rope, clean water, gloves

Procedure

o Step 1: Use clean bucket to collect sample During sampling time, wearing gloves is compulsory to minimize the risk of infection Sample is taken at around 20 cm below the surface

o Step 2: Sample is poured into autoclaved bottle, close the cap tightly to prevent leakage Rinse the outside of the bottle with clean water and wipe with tissue paper

o Step 3: Record the temperature, conductivity, and pH of the sample by measuring the remaining in the bucket Write down all the information in the label, put the label on the bottle

o Step 4: The sample is kept in insulated bag filled with gel ice bags and transferred as soon as possible to the laboratory (temperature below 4°C) Sample should be analyzed within 24 hours

The concentration of ESBL E coli and E coli in the water samples were determined

by the membrane filtration method

Quantification of E coli and ESBL E coli is carried out within 24 hours after the samples have been collected First, the sample is diluted using 10-fold serial dilution Then membrane filtration is applied to trap the bacteria on the surface of the membrane filter; subsequently, the filter is placed on appropriate medium and incubate

to enumerate in the next day

In this experiment, two types of agars are used for each sample:

(i) For quantification of E coli: Tryptone Bile X-glucuronide (TBX) agar

(Merck Millipore)

(ii) For quantification of ESBL E coli: TBX supplemented with cefotaxime

(CTX) (FUJIFILM Wako Pure Chemical Corporation) at 4 g/mL The concentration of CTX added into TBX medium is the minimum inhibitory concentration (MIC) breakpoint for verification of ESBL E coli by Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2021)

o Sterilized pipette tips (1000 L), micropipette (1000 L)

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Procedure

o Step 1: Take the sample out of insulated cooling bag or refrigerator, mix well

to resuspend the sediments

o Step 2: Pipette 1 mL of the sample into 9 mL of PBS, mix the sample dilution

by hand

o Step 3: Discard the pipette tip and use a new one to take 1 mL of this dilution

to make the next dilution in the same manner in Step 2

o Step 4: Repeat step 3 until all the expected dilutions are made

Membrane filtration

Materials

o Autoclaved membrane filtration apparatus, vacuum pump, sterile membrane filter (0.45 µm pore size and 47 mm diameter, gridded, cellulose mixed ester type, Advantec), sterile forceps, sterile flat-blade pipette tips (1000 L), pipette, sterile graduated cylinders (volume 15 mL and 50 mL), petri disks (Nuova Aptaca, Italy)

o Serial dilutions of sample: for each agar of each sample, two to four of fold serial dilution are tested

tenth-o Ceftenth-otaxime sttenth-ock stenth-olutitenth-on (4 g/L): Disstenth-olving 160 mg ceftenth-otaxime stenth-odium salt (FUJIFILM Wako Pure Chemical Corporation) in 40 mL double distilled water

in a sterile tube, mix well and filter the solution using 0.2 m membrane filter into another sterile tube Cover the tube with aluminum foil cover, keep it in the refrigerator A new stock solution is prepared every month

o Agar plates: Preparing the agar plates by dissolving Chromocult TBX agar powder (Merck KGaA, Germany) with the ratio 31.6 g per 1 litter double distilled water, heat the solution until boiling, agitate the solution frequently until completely dissolved Autoclave the medium at 120°C for 15 minutes After the medium has cooled down (around 50°C), pour into plates (12-15 mL/plate) For TBX medium with supplemented cefotaxime, cefotaxime stock solution should be added (adjust to final concentration in the agar is 4 g/mL)

o Step 2: Starting with the greatest sample dilution, pipette the amount of sample needed onto the filter (avoid touching the pipette tip onto the filter surface) Apply vacuum until all the amount of sample added is filtered Vacuum should not be applied once no liquid is left on the membrane Remove the funnel aseptically, place the membrane filter (grided side up) onto appropriate agar plate Each dilution should be filter in triplicate Use sterile PBS solution to rinse the interior walls of the filter funnel and the filter base

o Step 3: Filter the next most diluted sample using the same method in step 1 and step 2

o Note: Use new sterile filtration system for filtration of new sample

o Step 4: Place the agar plates inverted in the incubator and incubate them overnight (16-18 hours) at 37°C

o Step 5: Enumeration: After overnight incubation, E coli appears as blue or blue-green colonies on TBX agar; ESBL E coli also appears as blue or blue-green colonies on TBX agar supplemented with cefotaxime The number of blue/ blue-green colonies in each plate are recorded The colony forming unit (CFU) concentration was calculated as CFU/ 100 mL from the plates with countable colonies (>10)

Isolation of ESBL E coli

Blue-green colonies growing on TBX agar is purified for further experiment using streak plate method Each colonies is purified at least twice

Confirmation of E coli

The blue-green colonies growing on TBX agar is taken for species identification to confirm whether they are ESBL E coli The method used for species identification is MALDI-TOF MS

MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry is a revolutionary technology for rapid and reliable identification of bacteria species The MALDI-TOF MS system measures the unique peaks of ribosomal protein of the bacteria and identify its species by matching those peaks with

an intensive reference library

Material

o Experimental equipment:

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 Bruker MALDI Biotyper (Model: Microflex LT/SH)

 MALDI Biotyper Compass Software

o Experimental consumables:

 Bruker HCCA matrix portioned

 Pure formic acid (99-100%)

 Clean reusable MALDI target plates

 10 L pipette and 10 L pipette tips

 Sterile wooden applicator sticks (toothpicks)

Figure 3.3 Bruker MALDI Biotyper (Microflex LT/SH) Procedure

o Step 1: Prepare fresh overnight culture of tested isolates;

o Step 2: Use a sterile toothpick to apply a small amount of the tested colony to a spot on target plate;

o Step 3: Add 1L formic acid 70% on the spot;

o Step 4: Put the plate on heater to let the acid quickly evaporate;

o Step 5: Add 1L matrix on the spot and wait until the spot completely dry;

o Step 6: Put the target plate into MALDI-TOF MS machine and run the analysis

to identify the colony;

o Step 7: Result interpretation