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 TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY PASTEUR INSTITUE HO CHI MINH CITY GRADUATION THESIS DETERMINATION OF ANTIBIOTIC RESISTANCE GENOTYPES OF NON-TYPHOIDAL Salmonella ISOLATED FROM HUMAN. Advisor Student Msc. NGUYEN HOANG VU NGUYEN TIEN DAT Vee Thu Duc City, 04/2024 ACKNOWLEDGEMENTS 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. Class: DH19SHC (email: 19126245@st.vn), Faculty of Biological Sciences, Nong Lam University Ho Chi Minh City. I guarantee that this graduate thesis was completely conducted by myself 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 2023 Student’s signature il ABSTRACT 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. The main objective of the study is to classify and determine antibiotic resistance genes in serovars of Salmonella enterica subsp. Serovar identification was performed by the slide agglutination technique for somatic antigen (O) and the flagellar antigens was identified 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. Saintpaul 5% (1/20) and 6 serogroups that could not be determined fully antigens including S$. 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%) was conducted by disk diffusion following the CLSI standards.
Finally, this study determined resistance 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. 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. Mục tiêu chính của nghiên cứu là phân loài serovars và xác định các genes khang kháng sinh trong serovars Salmonella enterica subsp. 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. 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 ACKNOWLEDGEMENTS .:cceccesceeseeseesseeseeecerseescesecseessessceaeeseeeseeseeeseeseeeseeaeeeseess i CONFIRMATION AND COMMITMENT .:cc:ceceeceeseeseeeeseeseeseeseeseeseeseeseeseeseeneees ii ABSTRACT wermesenmcernien eerie enemy ee eS iii a as iv TABLE OF GCONTEN Teen n0 0 là hg11110 01534/05SGS005300800353G085LE80G/EEE4EE4030464013.1000040 Vv LIST OF ABBREVIATIONS S015 —. viii LIST OB TA BIGES ios: 55 2c i a et cố a ee CC. ee ee ix LIST OF FIGURES sen essitntueesgsgsgSegSigy2viilqosotlbkptareostsaptftesitlieixotktùxSiissglgbrstasgpasaprsesaosi X Wl5U21 21MEI-dM AAEMRsó 01930 901 89" .ốốốốố ốc 1 Leds .PRODLEM STALIN svenveocsanseemavenenesmennenemesmnerecmeaer NSE OEE 1 1.
2, DW TVETSr er ae a 63Eb20063Eul0nE0B1Sg62BGEGIRlBEBNIEHSGGUSUEEGSNEHLGISHIUiSS2N6S010-3660860ug8S8EE. LITERATURE RE VIEW các is60211256540655160436501183150031380838338590094 3 2. Salmonella and serovars Of SalmorielÏA. Morphological and cultural charaCf€T1SfIC.
Disease secsssexce61001606603515653860151463363095646933880466383358ãgẸ0300688 7 2555 TEVWGHHDEaeeseeeseseesesssseniisoasssstisakirigtigrssdilctitrregloirjsrirhiridbotigtualcbbxguougdirrùd ens artnsb ea 8 DES ae AMS ASIN cna ce tn sera neers i im aoe eran ea Semana 8 2. Mechanism resistance to antibiotic of SalmuorielÏA.-----5-<<<+<<+sc<+ecexeesees 9 22/5, ete ƯA OO ssa catego easel erence aera eee teem ee al 9 2:1;2; Reduces antibiotic permeability s:asossssesssasessosaosetossdaseogtglsodi4EIEESESS4642984813608601/00806 9 23/153, ITTAGHVAHHOTI'EH2WTHWLsseseeeseenadsskkiiitisiiotorikotovinrdoirosgasissucixEotoLAIEEAEKEEUDEIIBI0E0001382. Antibiotic resistance groups mechañ1SI.-- 5 <5 + *++*£++vEe+eeeeeeezeeeree I DS lL. D=—,LJMGEETLusossctesstgt5segttistseboettgssgfegyt932SRGSNEGiNEEDNGIEIBSt3SÿSIERqfiEỹ-SgitiSiGSiilsiigoililiGisjite 11 2.2, QUINOLONE si, s25 5626266262 sã6 56362 sve s a te SS Be nano ASN Seo OA A OND sa 80035 36050ã.4, Sultonainid Cxrcsenceeuscsscess seem asses eRe TS EERE 11 2.
Resistance genes mechanism Of SalMONel1A. 12 DiS Dis OU sưng nghĩ nhi ga GGGR0Gi40080359655380100185380.1, Methods OV er view ss 2gnánhh 20304003 46730030958/2E423/R08)50012045:485-49E35d050NGEEVSS4EG502031483300483330.2091)0008, 12 2 Tle Multiplex PCR wesssssesecssenewnnssvcsveresveseevestvenesnasconvavonissseuavcesewsnsvvesnesverremeveeorneneeess 12 2. Antimicrobial susceptibility tesfing.-- - cece cece 25 S21 2222 22 re.5 SQtOty PING ssiscstistrniassisEEL11515313665103160313356953 0595843853343 38843/354558095. MATERIALS AND METHODS.
Time and location ‘of the TGSGATCH.::ssssscs:xzssssicese 9565566636025 9Ẹag800/0980g58g68. The subject 0Êresearei. Positive and negative control SfTA11AS.3 GUO Cal Ebesissreseoiss0090240009809 GAGEE1634E88E0A083306040B93E6-g83585g5Sugi830SE86)4000i8603-0008/g3g88 15 3.4, Devices: and €dUÏDHi€Tis-eseexssssssssss15565192116180 151 L15814161461561400355581545381341S55E033558 550 16 3. Antibiotics in antimicrobial susceptibility testing.321 RESCATCH WOLKS wiesseesseaxsoxexcamsnsanersvasneensenauareeranameemnasereinessreanenenesearens 16 3.
Recover Salmonella on HEK medium. Sub-culture Salmonella on BA medium. Determination of serovars Salmonella .------ +5 <+<<++<+++<£+sc+sk+seseesseesee 17 3. Antibiotic Susceptibility Testing.
Determination resistance genes by PCR. RESULTS AND DISCUSSION. Determination serovars ,S2Ïwoi€ÏÏ4. Antimicrobial susceptibility t©sfing.-- --- --- 5 + + + sen re re re 26 4.
Determination resistance Qenes «0. 28 vi AA, DISCUSSION scccsacsns ccamincncusserevanmaciencexs iS a RR iO Seale oa waa a a alto acta aaa cae 30 CHAPTER 5. CONCLUSION AND SUGGESSTION.1, Conclisi On zsnccszemrsemuncss sees aE RRA ae eR SR aS EERE 33 52s SUSROSUION seesiseseesssebiESETEE08255510136966368951335536401310E21853-163505805311990155891585198070895/007408 33 REFERENCE .ceccescesceeseesseeseeeseseeecesecsceesecsecaecaecsaecsecaeeseeaecseeesecaeeeseeseeeaeeasesees 33 APRENIDIDS csssszessssmassessesescaawssiesasiencinscis sss aieu sea nei sos a So de SRO REN SRR 37 Vil LIST OF ABBREVIATIONS AMP Ampicillin AMC Amoxicillin-clavulanic acid AZM Azithromycin BA Blood agar CN Gentamicin CDC Centers for Disease Control and Prevention CLSI Clinical and Laboratory Standards Institute CTX Cefotaxime HEK Hektoen enteric agar mPCR Multiplex polymerase chain reaction MEM Meropenem MDR Multidrug resistance NA Nalidixic acid NC Negative control OUCRU Oxford University Clinical Research Unit PBSs Penicillin-binding proteins PBP Penicillin — binding proteins PEF Pefloxacin PC Positive control PCR Polymerase chain reaction Vill SXT Trimethoprim - sulfamethoxazole LIST OF TABLES Page Table 2. Old and new designation of O antigens (Grimont and Weill, 2007).
List positive controls for H1 antigen of Salmonella. List positive controls for H2 antigen of Salmonella. List primer of antibiotic resistance øenes. Antigen serovars Salmonella enterica subsp.
Antigen serogroup Salmonella enterica subsp. The quantity of antibiotic resistance strains with antibiotic groups. List typical phenotype of antibiotic resistance stra1ns. List phenotype of typical antibiotic resistance strains.
Comparing the typical antibiotic resistant phenotype and genotype of SOLO VALS vere mer G06g918 A8350 610/8943084001348054459600i:Q000150u1118306E:38014:L883000046,G010011g30p2. Comparing the typical antibiotic resistant phenotype and F00 40500. Comparing the typical antibiotic resistant phenotype and genotype of SOLO STOUP wes ceesenescosvenesenapewavemeesenseyaeeasursiwsense vance edna sexo say ervesseneesnd estes wee neeanuereyeeuneneneress 30 1X LIST OF FIGURES Page Figure 2. Antimicrobial susceptibility testing with positive confrol.- -- -- 51k TT TH TH TH nh ng 17 Figure 4.
The percentage of serovars and serogroups of Salmonella.2: The rate of MDR. phenotype» s:ccssccseecereecsceeeseesceseeserarmennaennnaverreeess 27 Figure 4. Quantity antibiotic resistance Ø€nes.-- ------ 5c cess ceeeeeeeeeeeeeeeees 29 CHAPTER 1. Problem statement Salmonella infection is one of the prevalent bacterial infections affecting both humans and animals, primarily impacting the gastrointestinal tract and often being eliminated through feces as it resides in the digestive system.
This disease is categorized into two main types: typhoidal (typhoid fever) and non-typhoidal, with non-typhoidal Salmonella causing significant economic losses and mortality worldwide. A common manifestation of this infection is diarrhea. According to the World Health Organization (WHO) statistics in 2017, nearly 1.7 billion children, predominantly those under 5 years old, 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. 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.
The prevalence increased to a minimum of 271 cases per 1000 children in 2015 (Anders et al. Although Salmonella infections accounted for only 18% of the total cases, the reinfection rate by Salmonella was the highest (Anders ef al., 2015), emphasizing its highly infectious nature, particularly among children living with carriers of the pathogen, as reported by the OUCRU (https://www. Comparing Salmonella infection rates with other continents, non-typhoidal Salmonella (NTS) in Asia is under the most significant impact. Therefore, Vietnam, situated in this region, is heavily affected by this disease, leading to increased diversity in NTS serovars (Lan Huong Phu et al.
However, antibiotic treatments are becoming less effective as resistance rates continue to rise. A study 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 similarly high resistance levels (Ngan et al. However, for Salmonella infections, antibiotic usage remains the most effective intervention.
Over time, researchers have made adjustments 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 a nation's development as commonly used antibiotics gradually lose their effectiveness. Therefore, identifying antibiotic resistance genes in Salmonella serovars is crucial, as each serovar may harbour distinct antibiotic resistance genes.
The objective of this study is to identify common antibiotic resistance genes in non-typhoidal Salmonella from individuals in the Southern region (https://www. Successful identify of Salmonella serovars to consolidate information on bacterial diversity in the Southern provinces.