Khóa luận tốt nghiệp Công nghệ sinh học: Determination of antibiotic resistance genotypes of non-typhoidal Salmonella isolated from human

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITY PASTEUR INSTITUTE HO CHI MINH CITY GRADUATION THESIS DETERMINATION OF ANTIBIOTIC RESISTANCE GENOTYPES OF NON-TYPHOI

MINISTRY OF EDUCATION AND TRAINING

NONG LAM UNIVERSITY-HO CHI MINH CITY

PASTEUR INSTITUTE HO CHI MINH CITY

GRADUATION THESIS

DETERMINATION OF ANTIBIOTIC RESISTANCE

GENOTYPES OF NON-TYPHOIDAL Salmonella

ISOLATED FROM HUMAN

Majors : BIOTECHNOLOGY

Student : NGUYEN TIEN DAT

Student code : 19126245

Academic year : 2019 - 2023

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYPASTEUR INSTITUE HO CHI MINH CITY

GRADUATION THESIS

DETERMINATION OF ANTIBIOTIC RESISTANCE

GENOTYPES OF NON-TYPHOIDAL Salmonella

ISOLATED FROM HUMAN.

First and foremost, I would like to express fully gratitude toward the Board of Nong

Lam University for facilitating me to the new environment in learning and studying

Continuously, I am so thankful to my teachers and the Faculty of Biological Sciences

for supporting and creating all favourable conditions for my thesis

In addition, I would like to sincerely thank the Institute Pasteur Ho Chi Minh City and

the staffs of the Department of Immunology for providing the most favourable

conditions for me to work and study there Especially, I would like to express my

deepest gratitude to my adviser, MSc Nguyen Hoang Vu, for his enthusiastic support

and immense knowledge of my study He has kindly guided me during the period of

my thesis

And last but not least, I would like to thank my family and my classmates for

supporting me to overcome the problems and challenges during my study that helped

me to complete my graduation essay

Sincerely, thank you!

CONFIRMATION AND COMMITMENT

My name is Nguyen Tien Dat Student ID: 19126245 Class: DH19SHC (email:19126245@st.hcmuaf.edu.vn), Faculty of Biological Sciences, Nong Lam University

Ho Chi Minh City I guarantee that this graduate thesis was completely conducted bymyself as well as that all the data and results are utterly accurate, truthful and objective

I will take all the responsibility before the Council for this commitment

Thu Due City, March 2023Student’s signature

il

DETERMINATION OF ANTIBIOTIC RESISTANCE GENOTYPES OF

NON-TYPHOIDAL Salmonella [ISOLATED FROM HUMAN

The research was conducted at the Pasteur Institute in Ho Chi Minh City,performed 20 samples classified as Salmonella enterica subsp enterica (I) The main

objective of the study is to classify and determine antibiotic resistance genes in serovars

of Salmonella enterica subsp enterica (I) Serovar identification was performed by theslide agglutination technique for somatic antigen (O) and the flagellar antigens wasidentified by mPCR: 8 primer pairs for H1 antigen fljC and 7 primer pairs for H2 antigen

fljB combining with slide agglutination Firstly, the results revealed that the serovar S.Enteritidis was the most prevalent with 25% (5/20), followed by S Agona 10% (2/20),

S Anatum 10% (2/20), S Infantis 5% (1/20), S Stanley 5% (1/20), S Saintpaul 5%(1/20) and 6 serogroups that could not be determined fully antigens including S$ GroupO9 15% (3/20), S Group O8 (HI:r) 5% (1/20), S Group O4 (HI:e,h) 5% (1/20), S

Group O4 (HI:r) 5% (1/20), S Group O13 5% (1/20), S Group O16 5% (1/20).Secondly, the results of antimicrobial susceptibility testing include group quinolones(nalidixic acid: 40%), fluoroquinolones - the new generation of quinolone (pefloxacin:70%), B-lactams (ampicillin: 40%, cefotaxime: 20%, amoxicillin + clavulanic acid:

15%, meropenem: 0%), aminoglycosides (gentamicin: 15%), sulfonamides

(trimethoprim + sulfamethoxazole: 10%), and macrolides (azithromycin: 10%) wasconducted by disk diffusion following the CLSI standards Finally, this study determinedresistance genes including: bla tem 25% (5/20), bla oxa 0%, qnr A 0%, qnr S 15%(3/20), mcr - 1 0% using single PCR techniques The analysis results revealed that 75%(15/20) of the strains exhibited at least one antibiotic resistance phenotypes Particularly,33% (5/15) of them are MDR strains, with serogroups accounting for 40% (2/5) of the

MDR strains Noticeably, 100% (2/2) S Agona harboured multiple antibiotic resistance

genes.

Key words: serovars Salmonella, qnr Š, qnr A, bla tem, bla oxa, mcr - 1

1H

TOM TAT

XAC DINH KIEU GENE KHANG KHANG SINH CUA SALMONELLA

KHÔNG THUONG HAN DUOC PHAN LAP TỪ NGƯỜI

Nghiên cứu được thực hiện tại Viện Pasteur thành phố Hồ Chí Minh, sử dụng

được 20 mau đã được phân loài là Salmonella enterica subsp enterica (1) Mục tiêuchính của nghiên cứu là phân loài serovars và xác định các genes khang kháng sinh trong

serovars Salmonella enterica subsp enterica (1) Định danh serovars được sử dụng kỹ

thuật ngưng kết huyết thanh cho kháng nguyên soma (O) và kháng nguyên tiêm mao (H) được xác định bằng mPCR gồm: 8 cặp mồi cho kháng nguyên HI và 7 cặp mồi cho kháng nguyên H2 kết hợp cùng với ngưng kết huyết thanh Kết quả cho thấy serovars S.

Enteritidis chiếm ti lệ cao nhất 25% và còn 5 serovars khác bao gồm S Agona 10%

(2/20), S Anatum 10% (2/20), S Infantis 5% (1/20), S Stanley 5% (1/20), S Saintpaul

5% (1/20) Tiếp theo yy như nhóm quinolones (nalidixic acid: 40%), fluoroquinolones

-thế hệ mới của quinolone (pefloxacin: 70%), B - lactams (ampicillin: 40%, cefotaxime:

20%, amoxicillin + clavulanic acid: 15%, meropenem: 0%), aminoglycosides

(gentamicin: 15%), sulfonamides (trimethoprim + sulfamethoxazole: 10%), and

macrolides (azithromycin: 10%) bằng phương pháp kháng sinh đồ theo tiêu chuẩn của

CLSI Cùng với đó trong nghiên cứu này xác định được các gen kháng thuốc với tỉ lệ

bla tem 25% (5/20), bla oxa 0%, qnr A 0%, gnr S 15% (3/20), mcr — 1 0% bang kỹ thuật PCR Kết quả phân tích đã cho thay 75% (15/20) chủng có kiều hình kháng ít nhất một

loại kháng sinh, trong đó có 60% (9/15) là serovar và 40% (6/15) còn lại là của

serogroup Đặc biệt trong số đó có 33% (5/15) là các chủng đa kháng thuốc mà

serogroup đã chiếm 40% (2/5) trong số các chủng đa kháng thuốc Ngoài ra, 100% (2/2) chủng S Agona sở hữu đa kiểu gene kháng sinh.

Từ khóa: serovars Salmonella, qnr S, qnr A, bla tem, bla oxa, mcr - 1

iv

TABLE OF CONTENTS

Page

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Centers for Disease Control and Prevention

Clinical and Laboratory Standards Institute

Cefotaxime

Hektoen enteric agar

Multiplex polymerase chain reaction

SXT Trimethoprim - sulfamethoxazole

LIST OF TABLES

Page

Table 2.1 Old and new designation of O antigens (Grimont and Weill, 2007) 4

Table 3.1 List positive controls for H1 antigen of Salmonella - - - 18

Table 3.2 List positive controls for H2 antigen of Salmonella - - 18

Table 3.3 List primer of antibiotic resistance øenes - -+c+-<<+<c+sc<+s 23 Table 4.1 Antigen serovars Salmonella enterica subsp.enterica identified 25

Table 4.2 Antigen serogroup Salmonella enterica subsp.enterica identified 25

Table 4.3 The quantity of antibiotic resistance strains with antibiotic groups 26

Table 4.4 List typical phenotype of antibiotic resistance stra1ns - - 37

Table 4.4 (continue) List phenotype of typical antibiotic resistance strains 28

Table 4.5 Comparing the typical antibiotic resistant phenotype and genotype of SOLO VALS vere mer G06g918 A8350 610/8943084001348054459600i:Q000150u1118306E:38014:L883000046,G010011g30p2.9ogbpcaggigu230000u 000.086 29 Table 4.5 (continue) Comparing the typical antibiotic resistant phenotype and F00 40500.i0 11212777 29

Table 4.6 Comparing the typical antibiotic resistant phenotype and genotype of

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LIST OF FIGURES

PageFigure 2.1 Antimicrobial susceptibility testing with positive confrol 13

Figure 3.1 Research Works - 51k TT TH TH TH nh ng 17Figure 4.1 The percentage of serovars and serogroups of Salmonella 24

Figure 4.2: The rate of MDR phenotype» s:ccssccseecereecsceeeseesceseeserarmennaennnaverreeess 27

Figure 4.3 Quantity antibiotic resistance ؀nes - 5c cess ceeeeeeeeeeeeeeeees 29

CHAPTER 1 INTRODUCTION

1.1 Problem statement

Salmonella infection is one of the prevalent bacterial infections affecting both

humans and animals, primarily impacting the gastrointestinal tract and often beingeliminated through feces as it resides in the digestive system This disease is categorizedinto two main types: typhoidal (typhoid fever) and non-typhoidal, with non-typhoidal

Salmonella causing significant economic losses and mortality worldwide A commonmanifestation of this infection is diarrhea According to the World Health Organization(WHO) statistics in 2017, nearly 1.7 billion children, predominantly those under 5 yearsold, suffer from diarrheal diseases annually, resulting in approximately 525,000 deaths

each year, making it the second-leading cause of death in children under 5 years old(https://www.who.int) In 2010, an epidemiological study in Ho Chi Minh City recorded

77 out of 1419 cases of diarrhoea (5.4%) in children under 5 years old as Salmonella

non-typhoidal positive (Thompson et al., 2013) The prevalence increased to a minimum

of 271 cases per 1000 children in 2015 (Anders et al., 2015) Although Salmonellainfections accounted for only 18% of the total cases, the reinfection rate by Salmonellawas the highest (Anders ef al., 2015), emphasizing its highly infectious nature,

particularly among children living with carriers of the pathogen, as reported by theOUCRU (https://www.oucru.org) Comparing Salmonella infection rates with other

continents, non-typhoidal Salmonella (NTS) in Asia is under the most significantimpact Therefore, Vietnam, situated in this region, is heavily affected by this disease,leading to increased diversity in NTS serovars (Lan Huong Phu et al., 2016) However,antibiotic treatments are becoming less effective as resistance rates continue to rise Astudy in 2019 on 15 strains by Nguyen Ly Hoang Ngan et al reported a resistance rate

of 66.7% to Trimethoprim-Sulfamethoxazole, with other antibiotics showing similarlyhigh resistance levels (Ngan et al., 2019) However, for Salmonella infections, antibiotic

usage remains the most effective intervention Over time, researchers have madeadjustments in antibiotic usage to tailor them to specific Salmonella serovars, including

combining enzyme-inhibiting agents targeting antibiotic resistance genes Despite these

efforts, bacteria continue to encode genes to adapt to various antibiotics Antibiotic

resistance has become a global concern, especially in human populations, impacting anation's development as commonly used antibiotics gradually lose their effectiveness

Therefore, identifying antibiotic resistance genes in Salmonella serovars is crucial, aseach serovar may harbour distinct antibiotic resistance genes The objective of this study

is to identify common antibiotic resistance genes in non-typhoidal Salmonella fromindividuals in the Southern region (https://www.cdc.gov)

1.2 Objectives

1 Successful identify of Salmonella serovars to consolidate information on bacterial

diversity in the Southern provinces

2 Determine antimicrobial susceptibility testing and the presence of antibioticresistance genes in 20 randomly selected samples based on current antibiotic resistancegene data

1.3 Contents

1 Determine serovars Salmonella

2 Perform antimicrobial susceptibility testing including these antibiotic group: lactamase; fluoroquinolones; Macrolide; Trimethoprim - sulfamethoxazoles and

ÿ-determine resistance genes bla tem, bla oxa gnr A, gnr S, and mcr - | in serovars

Salmonella

CHAPTER 2 LITERATURE REVIEW

2.1 Salmonella and serovars of Salmonella

Salmonella is a genus in the family Enterobacteriaceae which are Gram-negative,

oxidase negative, catalase positive, non-spore forming rods They are also facultativeanaerobes The bacterial size is approximately 2-5 microns in length and 0.5-1.5 microns

in width (Andino and Hanning, 2015) This genus is divided into 2 species: S enterica

and S bongori, each of which is further subdivided into numerous serotypes S enterica

is categorized into six common subspecies (I, II, IHa, IIb, IV, VI) whilst S bongori isgrouped into subgroup V These species and subspecies can be distinguished based on

differential characteristic Together, they include over 2500 serovars which are

subdivided based on the White-Kauffman-Le Minor (WKL) scheme These serovars areclassified according to three determinant factors of the main antigens: O antigen

(somatic), H antigen (flagella), and capsular K antigens related to virulence (Vi) The

presence of antigens (O) or (Vi) determines the Salmonella group, while the H antigenidentifies specific serovars (Agasan eft al., 2002) Currently, serotyping is now

extrapolated through the characteristics of surface antigens O and H, with Videtermination invoked only in cases where there is suspicion regarding the presence of

a specific serovar This standardized approach was issued by “The Salmonella of the

nomenclature committee of the international society for microbiology” in 1934

The O antigen, also known as the somatic antigen, is a stable polysaccharidelocated in the outer membrane of bacteria and represents major component of the surfacelipopolysaccharide (LPS) of Gram-negative bacteria and is highly variable in structure

It is formed by repetitive oligosaccharide units (O units), typically containing 2 to 8

sugar residues In the historical classification of O groups, the initial designations werebased on letters Due to the limited availability of letters, the system extended to include

numbers from 51 to 67 However, in a more contemporary and logical approach, each

O group is now identified using the specific O factor characteristics This revised method

provides a more systematic and descriptive means of categorizing O groups (Grimontand Weill, 2007)

Table 2.1 Old and new designation of O antigens (Grimont and Weill, 2007)

Old New Old New Old New

diversity observed among O antigen groups (Reeves and Wang, 2002; Doyle et al.,

2020)

The H antigen, also known as the flagellar antigen, is a long filamentous protein

responsible for the bacterial motility Each Salmonella serovar expresses a specific type

of H antigen, either monophasic (single-phase) or diphasic (two-phase), depending onthe presence of one or two distinct flagellin genes (fliC and fljB) (Versalovic, 2011)

Nonetheless, both phases could not be detected in a culture as a whole cause individualcells of diphasic isolates express only one H antigen phase at any given time Thisindicates that the expression of the fljB gene is apparent only when the fliC gene is

exhibited (Ryan et al., 2017; Bonifield and Hughes, 2003)

The Vi antigen, also referred to as the capsular antigen, constitutes the outermost

layer of the cell, also a polysaccharide-based capsid This external structure is pivotal as

both a significant virulence factor for bacteria and a crucial element for protective

mechanisms Notably, the Vi antigen is prevalent in Sưửmonella strains associated with

typhoid fever, emphasizing its importance in bacterial pathogenicity and immune

evasion (Jennings, 1990)

The Kauffmann-White scheme, introduced in 1934, initially validated 44

Salmonella serovars (Kauffmann, 1934), forming a crucial basis for the systematicclassification of these bacterial strains After F Kauffmann's retirement in 1964, thescheme consisted of 958 serovars Subsequently, L Le Minor introduced an annual

supplement in the Annales de l'Institut Pasteur, later known as Research in

Microbiology At the time of L Le Minor's retirement, the number of serovars had

expanded to 2,267 Following M.Y Popoffs departure, the count reached 2,555serovars Since L Le Minor had a significant role in describing most of the serovarsknown at that time, the list of antigenic formulae, formerly recognized as the

"Kauffmann-White scheme," was renamed the White-Kauffmann-Le Minor scheme

(Grimont and Weill, 2007)

Originally, the nomenclature of serovars were initially determined by factors such

as the diseases linked to infection, the geographical region of isolation, or the

characteristic habitats of the bacteria Nevertheless, nowadays in the contemporaryparadigm, the nomenclature for serovars is exclusively determined by geographic place

names associated with the specific areas of their initial isolation (Ryan et al., 2017)

These methods have contributed an evolution in naming conventions, facilitating more

convenient identification and tracking of Salmonella serovars in research For the firstmention in a publication, the full name “Salmonella enterica” is utilized This is thenfollowed by the word “serovar” or the abbreviated version “ser.” along with the name

of the serovar The serovar name is presented in non - italicized Roman alphabet letterswith the initial letter capitalized Consequently, the complete name would be Salmonella

enterica subsp./ssp enterica ser Enteritidis Subsequent mentions of the name can be

followed by only the serovar name, such as Salmonella Enteritidis or S Enteritidis A

full serovar name is exclusively assigned to isolates of Salmonella enterica subsp.enterica that meet the complete antigenic criteria for a serovar For instance, Salmonellaenterica subsp./ssp enterica 1,9,12:g,m:- represents a commonly encountered

5

monophasic variant of Salmonella enterica subsp./ssp enterica ser Enteritidis which

cause these strains do not express the fljB - encoded phase-2 flagellar antigen

(Guibourdenche ef al., 2010)

In the present context, a comprehensive classification method has identified atotal of 2700 Salmonella serovars Nevertheless, some variants of S enterica aredifferentiated from the typical by lacking flagellar structures (non - flagella), resulting

in these bacteria being non-motile This characteristic distinguishes them from other

Salmonella types such as Salmonella Gallinarum or Salmonella Pullorum (Yang et al.,2020) Salmonella enterica spp Enterica (1) is a subspecies group of bacteria commonly

linked with causing gastroenteritis in both humans and warm-blooded animals Incontrast, other subspecies within the Salmonella enterica genus often contribute todiseases in cold-blooded animals The primary mode of human exposure to this

subspecies is through contact with warm-blooded animals such as dogs and cats, as well

as the consumption of food derived from these animal groups, including poultry, cattle,and pigs (Jones, 2011) As a result, subspecies group (I) has become the most prevalentdue to the highest number of serovars within all subspecies, approximately 1500 out of

2700 serovars (Achtman et al., 2012) Given this prevalence, this study is going toconcentrate on determining the antibiotic resistance profiles of serovars within theSalmonella enterica subsp enterica (1) currently to consolidate information fortreatment strategies

2.1.2 Morphological and cultural characteristic

Currently, there are various culture media for isolating Salmonella spp However,the commonly used media for isolation are XLD and HEK agar Nevertheless, these two

media are gradually being replaced by more specific isolation media (Park et al.,

2012;Rambach, 1990)

2.2 Pathogenesis

The most common route of Salmonella transmission is from animals to humans,

particularly through the consumption of raw food originating from animals Animals,

acting as carriers, can harbor bacterial strains, particularly Salmonella, even in the

absence of obvious symptoms, thereby maintaining good health Nevertheless, in

instances of severe infection, these animals may still exhibit signs such as diarrhea

Non-typhoidal Salmonella, a bacterial pathogen causing gastroenteritis, predominantlyresides in the stomach and intestines The survival of this threat when excreted in feces

is notable, showcasing a remarkable ability to persist in soil for an extended duration,

estimated to be between 203 and 231 days (Islam ef al., 2004) This emphasizes capacity

of Salmonella about endurance and existence without a specific host, maintaining

viability in the surrounding environment Moreover, environments characterized bywater or low humidity levels can create favorable conditions for the growth and

prolonged persistence of Salmonella (Waldner et al., 2012; Islam et al., 2004) This

robust environmental adaptability implies a continuous potential for the bacterium to

exist and pose a threat, emphasizing the need for comprehensive preventive strategiesand awareness in both agricultural and public health domains

2.2.1 Clinical Manifestation Disease

The signs indicating the risk of Salmonella infection include abdominal painaccompanied by frequent diarrhea, which typically occurs within 12 - 36 hours after

infection, depending on an individual's health and can last for 2 - 7 days(https://www.who.int) Additionally, there are other symptoms that can help to identify

this bacterial infection more clearly, such as fever, abdominal pain, nausea, and

sometimes vomiting, along with persistent diarrhea and occasionally blood in the stool

The reason diarrhea can cause the death is often attributed to dehydration The loss ofwater is exacerbated during vomiting or fever episodes When the body loses water, aportion of water and electrolytes (sodium, chloride, potassium, and bicarbonate) are

depleted and cannot be naturally replenished unless supplemented(https:/Awww.who int)

In impoverished countries, the lack of access with clean water has become a

significant challenge, making the recovery process more difficult, especially forvulnerable populations such as children, the elderly, and those with compromised

immune systems like people with HIV Adults with higher immune system are morelikely to overcome the disease without significant complications (https://www.cdc.gov)

In the global healthcare context, monitoring and controlling Salmonella are becomingincreasingly crucial to ensure food safety and community health Developing rapid and

effective diagnostic methods, researching antibiotic resistance, and enhancingcommunity awareness of preventive measures are crucial steps in the fight against

Salmonella With increased knowledge and management, we can prevent the dangerousspread of Salmonella, safeguarding our health and animals, preventing transmission

from animals to humans and vice versa

2.3 Prevention

Following the research of CDC, emphasizing the crucial role of hygiene and

safety measures when interacting with animals to prevent the spread of bacteria such asSalmonella These measures include thorough handwashing with soap and water afterproximity with animals and their living environments Besides, avoiding to the high-risk

animal groups are essential Cleaning and disinfecting the living environment of pets

and regularly taking them to the veterinarian are also recommended to keep both pets

and their surroundings safe These practices not only protect the health of individualsbut also contribute the research on management and controlling bacteria in the

relationship between humans and animals (https://www.cdc.gov)

2.3.1 Treatment

Currently, scientists have researched vaccines against infections caused by

Non-Typhoidal Salmonella (NTS) However, existing vaccines have not completelyprevented and controlled diseases caused by this bacterial species Therefore, WHO and

researchers worldwide are still in the process of developing a vaccine with broadprotective capabilities (https://www.who.int) As the treatment and vaccine

development are still in progress, the healthcare facilities provide antibiotic treatmentfor patients infected with Salmonella For adults with Salmonella infection, mild

symptoms often resolve on their own after a few days without specific treatment,

requiring only fluid supplementation On the other hand, seeking medical assistance iscrucial for severe infections Medical facilities may prescribe antibiotics, and in more

serious cases, supplementary antibiotic support may be provided However, timely

treatment at healthcare facilities is essential for children under 5 and individuals with

weakened immune systems to prevent infections from becoming worse

2.4 Mechanism resistance to antibiotic of Salmonella

2.4.1 Efflux pump

Efflux pumps constitute a crucial primary defense mechanism againstantimicrobials, playing a pivotal role in expelling antibiotics across the bacterial cell(Du et al., 2018) The exposure to antibiotics triggers diverse gene expression profiles,resulting in the efflux of antibiotics through these intricate pumps Bacterial species

exhibit the capacity to upregulate specific efflux pump genes as a response to challenge

environments where toxic molecules are prevalent (Piddock, 2006)

The cell envelope of Salmonella is characterized by two distinct membranes - theouter membrane and the inner membrane - separated by a thin periplasm (Du ef al.,

2018) Tripartite efflux pumps, spanning this cellular envelope, play a pivotal role in

antibiotic export and involve a complex interplay of multiple transporter proteins In thiscomplex process, antibiotics are initially transported from the inner membrane to theperiplasmic space and subsequently expelled from the bacterial cell through the outer

membrane (Du et al., 2018; Tal and Schuldiner, 2009) This efflux pump-mediated

resistance mechanism operates by diminishing the concentration of specific antibioticcompounds, facilitating their exports across the cellular membranes into the external

environment For instance, common antibiotic groups are reduced by this mechanism

including tetracyclin, macrolide, clindamycin, hoặc fluoroquinolones (Alenazy, 2022;

V., 2006)

2.4.2 Reduces antibiotic permeability

Specially, Salmonella-gram negative bacteria, exhibit lower intrinsic

permeability to antibiotics compared to gram-positive bacteria This characteristiccontributes to their increased antibiotic resistance The selective permeability of both

the outer and inner membranes play a crucial role in regulating the entry of substances

such as antibiotics, bile salts, and nutrients, (Nikaido, 2003) This bacteriumpossesses the ability to make structural modifications to their cell membranes or cell

walls, creating obstacles that impede the effective penetration of antibiotics These

modifications often arise from alterations in porins or membrane proteins, key

components responsible for facilitating the entry of antibiotics into the bacterial cell Byundergoing changes in these crucial elements, bacteria develop mechanisms to hinderthe efficacy of antibiotics, thereby reducing the ability of antibiotic to reach theirintended targets within the bacterial cell (Delcour, 2009) This adaptive responseemphasizes the remarkable resilience and adaptability of bacteria in the face of antibiotic

exposure, posing challenges for effective treatment strategies and highlighting theongoing need for innovative approaches in combating antibiotic resistance

through the modification of the chemical properties of antibiotics, are exemplified bythe presence of aminoglycoside-modifying enzymes (AME) These enzymes play acrucial role in altering the conjugate charge of hydroxyl or amino groups within the

molecular structure of aminoglycosides This adaptive mechanism underscores the

remarkable biochemical strategies employed by bacteria

2.4.4 Mutation in Antibiotic Target

To resist antibiotics, bacteria undergo mutations in the genes responsible for

encoding the target of antibiotic This adaptive strategy prevents the antibiotic fromeffectively binding to the bacteria's cells or entering the intracellular For instance,

penicillin typically targets PBPs in bacteria, disrupting their function However,

mutations occurring in this region make penicillin unable to bind, thus impeding its

ability to eliminate the bacteria Mutations in genes that encode ribosomal proteins canalso contribute to antibiotic resistance, particularly against antibiotics targeting bacterialprotein synthesis, such as macrolides or tetracyclines (Wong ef al., 2014)

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2.5 Antibiotic resistance groups mechanism

2.5.1 B — Lactam

Beta-lactam antibiotics inhibit the final step in the synthesis of peptidoglycan byacylation transpeptidases, thereby preventing peptide bonds formation to createpeptidoglycan membrane These antibiotics bind to the PBP regions and disrupt the

bacterial transpeptidation process, leading to the inability to form a cell wall and

resulting in death (Pandey and Cascella, 2023)

2.5.2 Quinolone

This antibiotic group inhibits the DNA synthesis by the effect on bacterial type

II topoisomerases, DNA gyrase, and topoisomerase IV It breaks permanently double —

strand in bacterial chromosome and when the DNA strand breaks overwhelm the cell’s

ability to repair the DNA, it leads to cell death The first generation is nalidixic acid,

later generations have fluoride added to the ring so it is called fluoroquinolone (Yan andBryant, 2023)

2.5.3 Macrolides

In the progress of protein synthesis, these antibiotics disrupt the function of the

bacterial 30S ribosomal subunit by impending the binding of tRNA to mRNA This

action effectively brings the bacterial protein synthesis progress to a halt (Patel andHashmi, 2023)

2.5.4 Sulfonamide

This group of antibiotics is capable of inhibiting the synthesis of folate, a crucial

component in bacterial DNA synthesis, by specifically blocking the conversion of aminobenzoic acid to dihydropteroate Additionally, to disrupt sequential steps in thefolate metabolic pathway, for instance Sulfamethoxazole - antibiotic in this group iscombined with Trimethoprim Trimethoprim acts by preventing the transformation ofdihydrofolate into tetrahydrofolate This combination leads to maximal antibacterialactivity by targeting multiple steps in the bacterial folate synthesis pathway

p-lãi

2.6 Resistance genes mechanism of Salmonella

2.6.1 Bla rem and Bla oxa

The bla tem genes 1s a specific type of beta-lactamase gene commonly found in

bacteria Its codes for the production of the TEM beta-lactamase enzyme, which confersresistance to beta - lactam antibiotics by hydrolysing the beta-lactam ring, rendering theantibiotic ineffective This gene is frequently associated with resistance to penicillinsand early - generation cephalosporins It is one of the most prevalent beta-lactamasegenes found in clinical isolates of Gram-negative bacteria (Matagne ef aí., 1998)

2.6.2 Qnr

The gur genes belong to a group of plasmid-mediated quinolone resistance

determinants The proteins encoded by gur genes protect the bacterial DNA gyrase andtopoisomerase IV which are the targets of quinolone antibiotics, from inhibition bythese drugs This protection occurs through directing the binding of the gnr proteins tothe target enzymes, reducing the binding affinity of quinolones to DNA gyrase and

topoisomerase IV As a result, the bacteria become less susceptible to the bactericidal

effects of quinolone Remarkably, their presence on mobile genetic elements like

plasmids facilitates their rapid spread among bacterial populations through horizontalgene transfering (Jacoby ef al., 2015)

2.6.3 Mcr

The mcr genes are plasmid-borne genes that contribute to colistin resistance The

mechanism underlying the resistance conferred by the mcr gene involves a lipid Aphosphoethanolamine transferase This enzyme facilitates the transfer of a

phosphoethanolamine residue to the lipid A component located in the cell membrane of

Gram-negative bacteria (Rebelo et al., 2018)

2.7 Methods overview

2.7.1 Multiplex PCR

Multiplex PCR is a type of polymerase chain reaction that utilizes multiple

primer pairs to amplify different target sequences in the same reaction mixture Whendesigning Multiplex PCR reactions, it is essential to select primer pairs with equivalent

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annealing temperatures and ensure that they do not interact or form cross-reactive pairs.The advantage of this method is quick and simple because of requiring only a minimal

amount of DNA sample and incurring low costs However, it demands high-quality

DNA samples to ensure optimal reaction performance Moreover, effective primer

design is crucial to amplify the specific target gene segments It is important that the

size of the amplified gene segments should not be exceeded 3 kb

2.7.2 Antimicrobial susceptibility testing

The disk diffusion susceptibility method is a simple and well-standardized

approach The procedure involves applying a bacterial inoculum, approximately 1 2x108 CFU/mL, onto the surface of a large Mueller-Hinton agar plate with a diameter

-of 150 mm Subsequently, up to 12 commercially - prepared antibiotic disks, each

featuring a fixed concentration, are positioned on the inoculated agar surface Following

an incubation period of 16 - 24 hours at 35°C, the zones of growth inhibition aroundeach antibiotic disk are meticulously measured to the nearest millimeter The diameter

of the growth inhibition zone correlates with the susceptibility of the isolate and theantibiotic of diffusion rate through the agar medium Interpretation of the zone diametersfor each antibiotic is conducted according to the criteria outlined by the Clinical and

Laboratory Standards Institute (CLSI, formerly known as the National Committee for

Clinical Laboratory Standards or NCCLS) The outcomes of the disk diffusion test are

qualitative, providing a susceptibility category (susceptible, intermediate, or resistant)rather than a Minimum Inhibitory Concentration (MIC)

Figure 2.1 Positive control E.coli ATCC 25922 by disk diffusion

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The disk test was standardized and tested by strains Escherichia coli ATCC

25922 and Staphylococcus aureus ATCC 25923 The results will be recorded by thelatest tables published by the CLSI The advantages of the disk method are the test

simplicity that does not require any special equipment, the provision of categorical

results easily interpreted by all clinicians, and flexibility in selection of disks for testing

The disadvantages of the disk test are lack of mechanization or automation of the test

2.7.3 Serotyping

The traditional serotyping method, known as the White—Kauffmann—Le minor

scheme, has been employed for nearly 90 years since its introduction in 1934 (“TheGenus Salmonella Lignières, 1900.,” 1934) Serotyping is a definitive typing method

used for epidemiological characterization of bacteria Serotyping of Salmonella strains

is carried out by identification of surface antigens (LPS, O-antigens) and flagella

antigens (proteins, H-antigens) Most commonly, strains of Salmonella express twophases of H- antigens but aphasic, monophasic and triphasic variants are known Thedefinition of the serotypes is based on the antigen combination present and is given inthe “Kauffmann-White scheme”, Popoff and Le Minor, WHO Centre for Reference and

Research on Salmonella, Institut Pasteur, France, 1997

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CHAPTER 3 MATERIALS AND METHODS

3.1 Time and location of the research

This research was performed from August 2023 to January 2024 at Bacteriology

1 -Department of Microbiology and Immunology, Pasteur Institute, Ho Chi Minh city

3.2 Material

3.2.1 The subject of research

A total of 20 Salmonella strains were isolated from feces of human from 2013 to

2021 in southern provinces Vietnam The strains were identified as subspeciesSalmonella enterica subsp enterica (1) by biochemical methods from previous study

and it were storage at -70°C at Pasteur Institute in Ho Chi Minh City

3.2.2 Positive and negative control strains

Quality control strains for antimicrobial susceptibility testing were Escherichiacoli ATCC 25922 and Staphylococcus aureus ATCC 25923 Positive controls for

resistance genes are provided by ATCC

3.2.3 Chemical

For verification strain: Hektoen Enteric Agar (Oxoid) medium

Medium growth: Blood agar (Oxoid)

DNA extraction: Tris-EDTA buffer solution (Fluka)

PCR for determining H antigen serotypes and resistance genes: GoTaq® Master Mix 2X

(Promega), Nuclease-Free Water (Promega), specific primers

Electrophoresis: Agarose, TBE 0.5X buffer solution (Promega), Ethidium Bromide(Promega) staining Solution

Serotyping using antisera: Salmonella Antisera Kit (Denka Seiken)

Determination phenotype antibiotic susceptibility testing: Mueller Hinton Agar(Liofilchem)

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3.2.4 Devices and equipment

Microwave (Panasonic) Gel documentation system (Syngene) Water bath (Grant)

Incubator (Incucell) Electronic scale (Sartorius)

3.2.5 Antibiotics in antimicrobial susceptibility testing

The isolated serovars Salmonella strains were tested for their antimicrobic

susceptibility by the diffusion method on Mueller Hinton Agar using the followingantibiotic disks from Liofilchem company: ampicillin (10 pg), azithromycin (15 ng),trimethoprim + sulfamethoxazole (25 wg), cefotaxime (30 ug), nalidixic acid (30 ng),meropenem (10 ug), gentamicin (10 ug), pefloxacin (5 ug), and amoxicillin + acidclavulanic (30 wg) Evaluation of the results was done according to the criteria of M100

CLSI 31* edition E coli 25922 and S aureus ATCC 25923 were used as qualitycontrols

3.3 Methods

3.3.1 Research works

Determine the ratio of antibiotic resistance phenotypes and genotypes of 20

Salmonella enterica subsp enterica (1) isolated from human from 2013 to 2021 insouthern provinces of Vietnam The experiment performed by 3 methods: combine slide

agglutination method and mPCR methods to determine the serovars, antimicrobial

susceptibility testing for determine phenotype, and single PCR methods to determine

antibiotic resistance genotype

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BA medium

Determination of resistance genes:

Bla rey, bla oxa, nể S, gnr A, mer - |

Figure 3.1 Research works3.3.2 Recover Salmonella on HEK medium

The 20 strains of Salmonella enterica subsp enterica (1) were isolated on HEK

medium This is a selective medium for confirming Salmonella The cultured plate wasincubated at 37°C for 24 hours The colonies that were round, transparent, with or

without a black center are Salmonella

3.3.3 Sub-culture Salmonella on BA medium

After recover Salmonella on HEK medium, the colonies were sub - cultured

directly on BA medium This is a nutritional environment that helps Salmonellaenterica subsp enterica (1) growth The inoculated plate was incubated at 37°C for 24

hours Colonies on BA medium is round, opaque white, and have a circular center

3.3.4 Determination of serovars Salmonella

3.3.4.1 Multiplex PCR method for detecting H1 and H2 antigens

Before performing slide agglutination testing, this study performed detecting byPCR to save time Utilizing 8 primers (1; lv; b; d; r; e,h; ø,m; z10) for detecting phase

HI and 7 primers (1,2; 1,5; 1,6; 1,7; e,n,x; e,n,z1s; l,w) for detecting phase H2 withmultiplex PCR method The positive controls are collected from ATCC

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Table 3.1 List positive controls for HI antigen of Salmonella

STT Antigens Target antigens Positive controls

1 H:i fliC - 1 S Typhimurium (4,[5],12:1:1,2)

2 H:d fliC - d S Schwarzergrund (4,2:d:1,7)

3 H: e,h fliC - e,h S Meleagridis (3,10:e,h:1,w)

4 H: gym fliC - gm S Enteritidis (1,9,12:g,m:-)

5 H:r fliC -r S Infantis (6,7,14:r:1,5)

6 H: Zio fliC - Z10 S Mbandaka (6,7,14:z1o:e,n,Z1s)

7 H: b fliC - b S Minnesota (21:b:e,n,x)

8 H: Lv fliC - Iv S London (3,10,15:1,v:1,6)

Table 3.2 List positive controls for H2 antigen of Salmonella

STT Antigens Target antigens Positive controls

6 H: e,n,x fljB - e,n,x S Minnesota (21:b:e,n,x)

T7 H: e,n,Z15 fljB - e,n,z15 S Mbandaka (6,7,14:z10:e,n,Z15)

Primers using in this study from (Herrera-Leon et al., 2004; Cardona-Castro et

al., 2009; Santos et al., 2020) were listed in Appendix 1 The thermal cycle of theseprimers was adjusted by previous study at Pasteur Institute HCM city The mPCR was

performed in a 25 ul reaction volume containing 12.5 wl of GoTaq® Master Mix 2X(Promega) (amplified products were then analyzed by electrophoresis in a 1% (w/v)

agarose, TBE 0.5X buffer solution (Promega), Ethidium Bromide (Promega) staining

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Solution The standard DNA (Invitrogen) was included in each DNA gel electrophoresis

to indicate the PCR product size

3.3.4.2 DNA extraction

DNA of 20 strains was extracted by the boiling method Use TE buffer as a

solvent Process: Suspend 2 full loops of colonies into tube 1.7 mL containing 300 uL

of TE buffer Vortex to suspend bacteria Boil with a heater at 100°C for 10 minutes.Centrifuge at 10000 rpm for 5 minutes Collect DNA from the supernatant and removedebris The DNA was tested in Nanodrop for testing purify

3.3.4.3 Slides agglutination method for detecting O antigens and H antigens

Due to the abundance of O antigens in Salmonella so performing slides

agglutination would save on specimen and time compared to conducting multiplex PCR.Procedure based on WHO guidelines “Serotyping of Salmonella enterica O and H

antigen” 6th Ed January 2004 Using colonies cultured in BA medium to perform serumagglutination on glass slides 20 strains of Salmonella enterica subsp enterica (1) wereagglutinated with antisera O, including OMA (O2; O4; O7; 08; O9; O9, 46; 03,10;O1,3,19) or OMB (O11; O13; 06,14; O16; O18; O21; O35) Record the O agglutinationresults to find and select antisera H for the next experiment

After O agglutination, continue to agglutinate with antisera H, including H-phase

1 (g; m; s; t; f; p; e; h; n; x; a; b; c; ds r; l; k; 1; v3 W; 213; Z2s; Y; Z¡ Z10;) and antisera phase 2 (1; 2; 5; 6; 7; e; n; x; z15; 1; w)

H-a) O-typing: Place two loops full of saline on a slide next to each other Remove

a loop full of growth from the inoculated nutrient agar plate and mix it into both salinedrops on the slide Ensuring a smooth, opaque suspension in both drops In the first drop,add a drop of poly - O antisera with or without Vi antiserum Mix antisera and culture

(antigen) for up to 1 minute Hold the slide still If drop 2 has lumps in it, the culture is

auto agglutinating, and no further typing is possible Drop an O - serum on a slide andmix a loop full of culture from the nutrient agar Rock the slide gently for a maximum

of 2 minutes Lumping is a positive reaction A homogenous suspension is a negativereaction The O - sera pools are used to test the strains initially The strains are next

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examined in each of the positive unique O - sera of O - pool Both positive and negative

reactions are noted

b) H-typing: Due to the method of determining HI and H2 antigens by PCR is

limited in primers so using slide agglutination method is a supplement Sub-culture from

nutrient agar to Sven Gard agar Inoculate in one spot at the centre of a Sven Gard agar

Incubation overnight at 37°C Place two drops of saline with a loop close to one another

on a slide On Sven Gard agar, take a loop full of growth from the edge of the motilityzone and mix it with the first saline drop The second drop should be repeated (negativecontrol test) In both drops, make sure the suspension is opaque Add a drop of Poly H

antisera to the first suspension and mix for up to one minute Avoid tipping the slide.Mix a loop full of culture from the edge of the motility zone with a drop of an H - serum

on a slide Rock the slide gently for a maximum of 2 minutes The negative reaction is

a homogeneous suspension Lumping is a positive reaction The H - antisera pools are

used to evaluate the strains initially The strains are then tested against each of the

positive H - pool's individual H-antisera H -antigens detected (Phase 1) are noted Bothpositive and negative reactions are noted Approximately 5 ml of Sven Gard agar (55 -

60 °C) and 10 pl of antisera against the identified H-antigen (Phase 1) are added to asmall-sized petri dish

Continue with phase 2, these H - antigens are detected by the same methods as

described for the phase 1 H - antigens (phase 1) Both positive and negative reactionsare noted by the Microsoft Excel Identification of the serotype using the "Kauffmann-

White scheme and 2007", combine the O and H responses to determine the serovar based

on the list of “Antigenic formulae of the Salmonella serovars 2007 9" edition”.

3.3.5 Antibiotic Susceptibility Testing

The procedure is standardized by CLSI M02 - A11 page 12 volume 32

a) Inoculation of test plates: Following the adjustment of the inoculumsuspension turbidity, it is optimal to dip a sterile cotton swab into the suspension within

15 minutes Rotate the swab several times and firmly press it against the inside wall ofthe tube above the fluid level to remove excess fluid Inoculate the dried surface of an

20

MHA plate by streaking the swab across the entire sterile agar surface Repeat this

process two more times, rotating the plate approximately 60° each time for an even

distribution of inoculum As a final step, swab the agar rim Leave the lid slightly ajarfor three to five minutes (but no more than 15 minutes) to allow excess surface moisture

to be absorbed before applying on the antibiotic - impregnated disks NOTE: Avoid

extremes in inoculum density Never use undiluted overnight broth cultures or otherunstandardized inoculate for streaking plates

b) Application of disks to inoculated agar plates: In the procedure for

antimicrobial susceptibility testing, it is crucial to dispense a predetermined battery of

antimicrobial disks onto the surface of the inoculated agar plate To ensure optimalcontact with the agar surface, each disk should be pressed down, whether placed

individually or with a dispensing apparatus Even distribution of the disks is essential,with a minimum distance of 24 mm from center to center Ideally, no more than 12 disksshould be positioned on a 150 mm plate, or more than five disks on a 100 mm plate It

is advisable to arrange disks with predictably small zones next to those yielding largerzones to prevent overlap Attention should be paid to the proximity of disks to the edge

of plate, as this can affect the roundness of zones Once a disk has contacted the agarsurface, relocation is not recommended Instead, a new disk should be placed in anotherlocation on the agar

Following disk placement, the plates should be inverted and promptly placed in

an incubator set to 35 + 2°C Testing at temperatures above 35°C may fail to detect

methicillin-resistant staphylococci (MRS) Incubation in an increased CO2 atmosphere

is discouraged, except for specific bacterial species such as Haemophilus spp., N

gonorrhoeae, N meningitidis, and streptococci This caution is due to interpretivestandards being developed under ambient air incubation conditions, and CO2 exposure

can significantly alter the inhibitory zone sizes of certain antimicrobial agents

c) Reading plates and interpreting results: Following 16 - 18 hours of incubation,the examination of each plate is crucial Until the plate acceptably streaked with accurate

inoculum, the resulting zones of inhibition will be uniformly circular and there will be

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