MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY - HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES GRADUATION THESIS SEQUENCING AND ANALYZING GENETIC CHARACTERISTICS OF THE stn GENE OF Salmonella enterica subsp. enterica ISOLATED FROM SWIFTLET HOUSE ENVIRONMENTS Major: BIOTECHNOLOGY Student implementation: CHAU NGOC HUYNH NHU Student code: 19126125 Academic year: 2019-2023 Thu Duc City, 02/2024 ACKNOWLEDGEMENTS I would like to express my sincere gratitude towards the Board of Nong Lam University Ho Chi Minh City and the Board of Faculty of Biological Sciences for creating the most favorable conditions for me to finish this study. And especially, I would like to express my deep gratitude to Dinh Xuan Phat. PhD for wholeheartedly teaching, guiding, supporting, and giving me inspiration throughout the process of the thesis.
It would be impossible for the study to be completed if it hadn’t been the help of all people who has accompanied with me during this hard times. And for that, I want to send thanks of Ms. Nguyen Thi Mi Mi and other members of Gene Technology Laboratory BIO313 for helping me to reach my goals. Finally, I want to thank my family for always believing in me and encouraging me to continue on the route I've chosen.
Thank you for motivating me to keep trying. Thank you to my buddies for always supporting me, studying with me, and helping me achieve my goals. AlthoughI still have many unanswered thanks, I hope those who have supported me greatly remain well and successful in their careers. CONEIRMATION COMMITMENT My name is Chau Ngoc Huynh Nhu, Student ID: 19126125, Class: DH19SHB, Faculty of Biological Sciences, Nong Lam University Ho Chi Minh City.
I guarantee that this research was conducted by myself and that the results presented are utterly Thu Due City, February 2024 Student’s signature ABSTRACT This study was conducted to sequencing and analyze genetic characteristics of the sin gene of Salmonella enterica subsp. enterica isolated from swiftlet houses in Southern Vietnam. Ninety samples including 67 fecal samples and 23 nest surface swab samples were collected from 30 swiftlet houses in Binh Duong, Dong Nai provinces and Ho Chi Minh City. The samples were isolated and identified Salmonella enterica subsp.
enterica using PCR method. Samples subsequently tested positive for Salmonella enterica subsp. enterica were examined for the presence of the s7 gene, after then amplified full-length gene, sequenced and genetic analyzed.67%) were positive with Salmonella spp. and 6/6 (100%) were positive with Salmonella enterica subsp.
enterica by PCR. For the detection of the virulence stn gene of 6 isolated Salmonella samples, the results showed that this gene appeared with a detection rate of 100%. In this study, we had successfully designed primer for amplification and sequencing of stn gene with length of 750 bp. Then, we also constructed phylogenetic tree of this gene and analyzed of more 34 sequences from GeneBank with identity nucleotides of 98.4 - 100% and amino acids of 94.
It was noted that there were 18 positions with amino acid change. So, the study had successfully amplified and sequenced the full- length stn gene and analyzed the genetic characteristics of the stn gene in Salmonella enterica subsp. Key words: Salmonella, sequencing, stn gene, swiftlet house. TÓM TẮT Nghiên cứu này được thực hiện nhằm phân tích trình tự và đặc điểm di truyền của gen stn trên vi khuẩn Salmonella enterica subsp.
enterica trong nhà yên ở miền Nam Việt Nam. Chín mươi mẫu bao gồm 67 mẫu phân và 23 mẫu phết bề mặt tô được thu thập từ 30 nhà yến ở các tỉnh Bình Dương, Đồng Nai và Thành phố Hồ Chí Minh. Sau đó, các mau được phân lập và phát hiện Salmonella enterica subsp. enterica bằng phương pháp PCR.
Các mẫu sau đó được xét nghiệm dương tính với vi khuẩn Salmonella enterica subsp. enterica đã được kiểm tra sự hiện diện của gen sin, sau đó khuếch đại gen có chiều dài đầy đủ, giải trình tự và phân tích di truyền. Kết quả phân lập ghi nhận 29/90 (32,22%) mẫu nghi ngờ dương tính với Salmonella. 6/90 (6,67%) mẫu được phát hiện dương tinh với Salmonella spp.
và 6/90 (6,67%) dương tính với Salmonella enterica subsp. enterica bằng PCR. Kết quả phát hiện gen độc luc sin của 6 mau Salmonella đương tính trước đó cho thay gen này xuất hiện với ty lệ phát hiện là 100%. Trong nghiên cứu này, chúng tôi đã thiết kế thành công primer dé khuếch đại và giải trình tự gen sin có chiều là 750 bp.
Sau đó, nghiên cứu này còn xây dựng cây phát sinh loài của gen này và phân tích trình tự tương đồng của 34 trình tự có độ tương đồng nucleotide 98,4 - 100% va độ tương đồng các axit amin 94,8 - 100%. Ghi nhận có 18 vi trí có sự thay đôi về axit amin. Như vậy, nghiên cứu này đã thành công trong việc khuếch đại và giải trình tự toàn bộ gen sén, và phân tích các đặc điểm di truyền của gen sin ở Salmonella enterica subsp. Từ khoá: Salmonella, giải trình tự, stn gen, nhà yến.
TABLE OF CONTENTS Page ACKNOWLEDGEMENT S. sts:eausesecansansanasennancumanann an eneninecranecemnnenimers 1 CONFIRMATION COMMITMENT. iv TABLE OF CON TENTS wsssscsseazscussseszanvnar G162 13 0E to mieten d088548S7S8325840TgS48Gi8.q2:8qusabsl V LIST OF ABBREVIATIONS cán na nh 2020060103 61115818 c6 556814386425130613 8558348500488 Vill LIS-E OF TABLES sscsssccnvstiot mie emir eee arab ari enea eeu ees 1X ISIE OP BIGUR ES se. sessuseosennesscieigaugstgegudinbkioaotigbidlnsud05000ui002000004/Ó1400g-nÄuiipătagdgtggioibi20gaui S0g2A00g,gp x CHAPTER 1.
BACK GROUND sisesssssssunsiuveperenusagueansensveuaslsevedle se aneediadsuepuasueeacaataanaeemus 1 L1; IrIfOdG HO saneneneseedsneksiSiintAtEk BaySEE1SRStiEEDODEIAGSEEED. 1 [ee © DiCCU Vestas oe. 2 l5, COT TCHS luazesouliodetreshgtrsatiadjit2sii:SodaieduBitBiddusb dutgiisouisddoiiExorgalulBoaruSgioBuatigsaniagidSa2.cm223SganEĂL3i4csaisb. Introduction to Salmonella and Salmonella enterica subsp.
Taxonomy of Salmonella enterica subsp. Basic biological characteristics of SalmOnell A. Basic genomic characteristics Of S2Ï7owelÏ4. Salmonella enterotoxin (StH) 0 nnửutd4.
? 252:la: limicall STØTŠLszsxstssxtontiseig tia SDISG 95618500 S8-0L4S8084802339113391g31 uSSHS:.2: GLOSS LESIONS se si14E65150011365586806386808135345015848S535853S. Diagnosis, prevention, and treatment the dIsease.- --- --+--++++x++scc+cezeeezxrs 10 DigcdlHATO STS once nanos anintivonnaa wainenraedeneiseesaiioninion ia eomtvnniwaninontiwsi geben nlinnini dinner a 10 2. ¡šoy (on DonđÝắắđá“35Ö5. 11 23:04 LEERLTETIEIGTỢINGWRỨiiaeneiecuoabisbdbitbidptlotlA48exgbi38sggbastnsistasBxiEpoigtolSitBn9gigtisllatflaBs8ssa 11 2.
Laboratory diagnostic methods .1; Polymerase cham reaction = POR o. os cass, can cesses sesusets se EEháeg000<G6s38cl<gátEc choi aLSe 12 PIN N§xy 2)0 00591. 12 PSR ace hoi Cl ————————————— 14 2:32].SAfISGT SSC WTCGoss eens us servacesesnossssramenaruneannennemnmunuareuanemanenases 14 2. Step of Sanger se€qU€TCIHE.
16 2112| HELOITLBÌTGSGETG HBúcsszscsesi50o25810ni06056610100165208279002801:Ea50A0668/287.LHIESFHRNGIIII TGSGBTDĂ sau beisinDidinDGG1114110014834603804554893894358 285836 GHEH. MATERIALS AND METHODS.-- 572 Sc+cs+zsersrrrrrrrrrerree 18 3. Time and location of the researCH. Materials and methods ::0sccsossnesprsseesmenaezenaneasnnnnrnmmwenram meee 18 3.
Positive controls, negative controls, and field samples.-- --- --+---+<-+ 18 B22 Cle MIC lS) pse-zstg50150936155/2610E31002512058:515.SEHLEB-HESSESĐLEMBEAGIHSGBS:91BSS0S.EBIBES-SÌIGHDE-2TMD082 35g85 18 3. Equipment and instrument T0. Research worktlow sssusceccussmmsm em meensneu meme mee eee NEE 19 3. DNA extraction using WizPrepTM Viral DNA/RNA Mini Kit V2.
Designing primers used for amplifying full-length gene. Detection of the presence of Salmonella enterica subsp. enterica in the swiftlet house CNVITONMENE N06. Amplifying and sequencing Sf QeMe .-- --- + - + 5+ *S+*S2<£Es ren rrrkt 22 3.
RESULTS AND DISCUSSION ‘seesecsescussrusrnueeneneueneareraemernes: 24 6 Ale B.ESU TT ssisaesensieaiis0E11108310185183633608900680138380584. Designing primers used for amplifying full-length sin gene. Detection of the presence of Salmonella enterica subsp. enterica in swiftlet HO SỔ ie cscsiena:sacesneseaassemareena enema ceananeame ee seen EERE aenE ERE EEE RAEI 24 4.
Amplifying and sequencing full-length øene. 26 41:2: IOISGUSSÍOHii16455151205111 4301516110135 L251E3B3353543BSESEIBEESSESSGEISUEIGLBDRG.2SBSNNEXEBSEHEG40880U84 ATS 28 CHAPTER 5. CONLUSION AND RECOMMENDATION.3Ö oye Sores Olt heen ee ee ee ee ee ee eee 30 5.2 IRE COT SAS OD 05. csnasinanssinansind oicWasns nn dianelnomauianinahionnnsbtiednnwaeeinndnadbltuonhaehanaxaebstanuee DU REEEREN GE esscssence steers coarse xenenenieas mseesdurens tel as cauiskeaes sas iat east tatauseeases eeeraeewaneemevaeut 31 uujsI00802 5 —.Ô 39 LIST OF ABBREVIATIONS cAMP : Cyclic adenosine monophosphate CT : Cholera toxin DNA : Deoxyribonucleic acid ddNTPPs : Dideoxynucleotides dNTPs : Deoxynucleotide triphosphates LPS : Lipopolysaccharide LT-1 : Heat-labile enterotoxin PCR : Polymerase Chain Reaction sin : Salmonella enterotoxin Spp.
: subspecies CTSM : Chain-Termination Sequencing Method XLD : Xylose Lysine Deoxycholate WKL : The White-Kauffmann-Le Minor CDC : Centers for Disease Control BSA : Bismuth sulfate agar SSA : Salmonella Shigella agar tRNA : Transfer RNA LIST OF TABLES Page Table 3. Primers used for detection of Salmonella enterica subsp. Thermal cycles used for detection of Salmonella enterica subsp. The components used for PCR amplifying full-length sim gene.
Thermal cycles PCR amplifying full-length sim gene. Primers used for amplifying full-length stn øene. The results of Sa/monella isolation from swiftlet house samples. Percent similarity nucleotide and amino ac1d.- ---- +5 55s ++ss++s£+ss<+s 27 LIST OF FIGURES Page Figure 2.
Scanning electron microscopy of a Š. Circular map of the S. Typhimurium P-stx-12 chromosome. Gross pathology of an American cowbird that died of salmonellos1s.
Salmonella infection in wild passerine species. Gross lesions of Sa/monella infection in wild birds. The Sanger sequencing method in 7 steps.--- --+- 5555 +++ss++s+zeszsss Figure 4. Electrophoresis results of investigation of st øene.-- ¿+ --+cscc++xs++ Figure 4.
Electrophoresis results of amplifying of st øene. Phylogenetic trees based on the nucleotide sequence of stn øenes. Distribution of amino acid variability along stn sequenec. Introduction In Vietnam, bird's nest farming for commercial purposes has appeared since 2004 in some Southern provinces.
However, in the past 10 years, this profession has developed rapidly with many different types and scales. According to the Ministry of Agriculture and Rural Development, there are 42 out of 63 provinces nationwide currently involved in swiftlet farming with over 22,000 bird nest houses. The estimated production of bird nest products was estimated to be 150 tons per year, equivalent to about 600 million USD. The Mekong Delta had the highest number of swiftlet houses at 10,572, accounting for 44.67% of the region's total swiftlet houses.
The South-Central Coast has 5.965 houses, accounting for 25. (Vietnam Agriculture Newspaper: https://vietnamagriculture. The presence of bacteria in the bird's nest environment may produce low quality of edible bird’s nests, in addition, these bacteria can also cause many foodborne diseases posing risks to the public health (RAZAK, 2016; Worku ef a/, 2022)). Salmonellosis, infection caused by a wide range of Salmonella enterica strains, is a prevalent food- borne disease.
Salmonella infections are classified as typhoidal salmonellosis (TS, enteric fever) or non-typhoidal Salmonella (NTS) infections (Ngogo ef ai, 2020; Akinyemi et al. The major pathogenic serovars of Salmonella enterica that infected humans through the consumption of certain food products are Salmonella Enteritidis and Salmonella Typhimurium (Thung ef a/. The stn gene was found in Salmonella spp., regardless of their serotype (Dinjus et ai, 1997; Makino et al. Salmonella enterotoxin (sin) is a virulence factor that contributes to diarrhea in its host (Chopra ef a/.
Biological activities of stn are critical for Salmonella virulence, particularly during the enteric phase of the disease (Chopra ef al. Therefore, the thesis “Sequencing and analyzing genetic characteristics of the stn gene of Salmonella enterica subsp. enterica isolated from swiftlet house environments” was conducted. Objectives Successfully sequenced and analyzed genetic characteristics of the stn gene of isolated Salmonella enterica subsp.
enterica from swiftlet house environments. Contents Content 1: Amplifying and sequencing full-length gene. Content 2: Phylogenetic analysis. Introduction to Salmonella and Salmonella enterica subsp.
enterica Salmonella is named after American bacteriologist D. Salmon, who first isolated the bacteria from the intestines of pigs in 1884 (Su and Chiu, 2007). It is a Gram-negative, motile, hydrogen sulfide-producing, acid-labile and facultative intracellular organism that commonly causes gastroenteritis worldwide and contributes to cross-infection between humans and animals (AJmera and Shabbir, 2023). Taxonomy of Salmonella enterica subsp.
enterica The genus Salmonella belongs to the Enterobacteriaceae family, order Enterobacteriales, class Gamma-Proteobacteria, phylum Proteobacteria, the Kingdom Monera or Eubacteria (Tindall ef a/., 2005; Adeolu ef al. According to the Centers for Disease Control (CDC), the genus Sa/monella contained two species, Salmonella enterica and Salmonella bongori. Salmonella enterica is divided into six subspecies: enterica (1), salamae (Il), arizonae (Hla), diarizonae (IIIb), houtenae (IV), and indica (VD, whereas S. bongori has no subspecies.