งานนำเสนอกำลังจะดาวน์โหลด โปรดรอ

งานนำเสนอกำลังจะดาวน์โหลด โปรดรอ

Molecular epidemiology and antimicrobial susceptibility of Vibrio cholerae O1 and non-O1 isolated from patients and environment in Khon Kaen Bureau of.

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งานนำเสนอเรื่อง: "Molecular epidemiology and antimicrobial susceptibility of Vibrio cholerae O1 and non-O1 isolated from patients and environment in Khon Kaen Bureau of."— ใบสำเนางานนำเสนอ:

1 Molecular epidemiology and antimicrobial susceptibility of Vibrio cholerae O1 and non-O1 isolated from patients and environment in Khon Kaen Bureau of General Communicable Diseases* Department of Microbiology, Faculty of Medicine, Khon Kaen University** Waraluk Tangkanakul* Chariya Chomvarin** et al.

2 Today topics Details on Vibrio cholerae Background and research questions Objectives and scope of study Study design Methodology, results and conclusion of multiplex PCR, RAPD and disk diffusion method Overall conclusions Recommendations for future project

3 Details :1. History of Vibrio cholerae Since then advances in cholera research have significantly contributed to the understanding of the organism and the disease caused by it “Cholera” first mentioned by Hippocrates more than 2000 years, a broad spectrum of intestinal diseases Kommabazillen 1882, John Snow and Robert Koch first isolated comma-shaped organisms -V. cholerae-

4 Details : 2. General characteristics of V. cholerae 2.1 Morphology gram-negative curve rod, polar flagella family Vibrionaceae more than 200 serogroups O1,O139 severe disease and cholera pandemics non-O1/non-O139 gastroenteritis, septicemia and/or extraintestinal infection

5 2.2 Vibrio cholerae genome The smaller (chromosome II) 1.07 million bps. The larger (chromosome I) 2.96 million bps. Details : 2. General characteristics of V. cholerae

6 2.2 Vibrio cholerae genome Details : 2. General characteristics of V. cholerae Chr. I Contains the importance genes to essential cell functions; associated with virulence  DNA replication  Cell division  Gene transcription  Protein translation  Cell-wall biosynthesis Chr. I Contains the importance genes to essential cell functions; associated with virulence  DNA replication  Cell division  Gene transcription  Protein translation  Cell-wall biosynthesis Chr. II Plasmid; A circular piece of DNA  replicates autonomously  Drug resistance  Several DNA metabolism enzyme Chr. II Plasmid; A circular piece of DNA  replicates autonomously  Drug resistance  Several DNA metabolism enzyme

7 พ.ศ.พ.ศ. จำนวนผู้ป่วย อหิวาตกโรค จังหวัดขอนแก่น อัตรา ป่วย * เชื้อที่เป็น สาเหตุ VC.Inaba VC.Inaba VC.Inaba 25301, VC.Inaba VC.Inaba VC.Inaba VC.Inaba, VC. Ogawa VC. Ogawa VC. Ogawa VC. Ogawa (459), VC (2) 25371, VC (1442), VC. Ogawa (258)

8 VC. Ogawa VC. Ogawa VC. Ogawa VC. Ogawa VC.Inaba (9), VC. Ogawa (156) VC.Inaba (32), VC. Ogawa (6), VC.0139 (1) VC.Inaba (263), VC. Ogawa (1) VC.Inaba (11), VC. Ogawa (17), VC.0139 (1) VC.Inaba (127), VC. Ogawa (23) VC.Inaba (81), VC. Ogawa (2) VC.Inaba (2) VC. Ogawa (2)

9 สถานการณ์อหิวาตกโรค จ. ขอนแก่น ปี

10 จำนวนผู้ป่วยอหิวาตกโรค จังหวัด ขอนแก่น จำแนกตามเดือน เทียบกับค่าเฉลี่ยของ 5 ปีย้อนหลัง

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12 ตามรอยหอย ( แครง ) เจ๊วัฒนา ตลาด อ. จิระ กระสอบ ทุกวัน เจ๊แป้ง ตลาดรถไฟ 30 กระสอบ ทุกวัน ป้าเพ็ญ โรงหนังราชา 8-20 กระสอบ ทุกวัน ขอนแก่น กาฬสินธุ์ บ้าน หนองแก เหล่า นาดี ภูเวียง ชุมแพ กระนวน อุบลรัตน์ดอนโมง หนองบัว - ลำภู มหา - สารคาม ตลาด บางลำภู มหา - สารคาม บอระบือโกสุม ชัยภูมิ บ้านไผ่ DC วังน้อย Big C Fresh food Fast food Macro Lotus Fast food Fresh food สุราษฎร์ฯ ภุมเรียง อ. ไชยา ปากน้ำ อ. เมือง อ. ท่าฉาง อ. กาญ - จนดิษฐ์ สมุทรสาคร ตลาดมหาชัย กรุงเทพฯ วังหอย ท่าข้าม บางขุนเทียน อื่นๆ การขนส่ง - สุราษฎร์ฯ - สมุทรสาคร ใช้รถกะบะ - สมุทรสาคร - ขอนแก่น ใช้รถ 6 ล้อ ( คันเดียวกันทั้ง 3 ร้าน ) - ขอนแก่น - อำเภอ / จังหวัดต่างๆ ใช้รถประจำทาง และรถส่วนบุคคล ไม่ได้แช่เย็น ความเสี่ยง 4 และ 10 ต.ค.50 พบ V.parahaemolyticus ในเปลือกหอยด้านนอก และน้ำในวังเลี้ยงหอย ไม่พบในเนื้อหอย กาฬสินธุ์ (หมู่บ้านใกล้เคียง)

13 Epidemiology in Khon Kaen Thailand

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15 สถานการณ์การดื้อยาต้านจุลชีพของเชื้อ อหิวาตกโรคของ จ. ขอนแก่น 1. การดื้อยาของเชื้อ V. cholerae Ogawa ( แหล่งข้อมูล : ศูนย์วิทย์ฯ ขอนแก่น ) - ปี 2541 : เชื้อดื้อต่อยา Te ร้อยละ 92, Sxt ร้อยละ 97 - ปี 2542 : เชื้อดื้อต่อยา Te ร้อยละ 98, Sxt ร้อยละ การดื้อยาของเชื้อ V. cholerae Inaba - ปี 2543 : เชื้อดื้อต่อยา Te ร้อยละ 10, Sxt ร้อยละ 5 - ปี 2544 : เชื้อดื้อต่อยา Te ร้อยละ 0, Sxt ร้อยละ 0 - ปี 2545 : เชื้อดื้อต่อยา Te ร้อยละ 0, Sxt ร้อยละ 0

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17 การทดสอบความไวต่อยาต้านจุลชีพ ของเชื้อ V. cholerae Inaba โดยศูนย์วิทย์ฯ ขอนแก่น ปี

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20 ความไวของเชื้อ V. cholerae Inaba ต่อยาต้านจุล ชีพ ปี 2550 โดยศูนย์วิทย์ ขอนแก่น ( N = 3 )

21 ความไวของเชื้อ V. cholerae Hikogima ต่อยาต้านจุลชีพ ปี 2550 โดยศูนย์วิทย์ ขอนแก่น และรพ. ศรีนครินทร์ ( N = 1 )

22 Research questions ทำไมพบผู้ป่วยอหิวาตกโรคระบาดใน บางปี ทำไมมีผู้ติดเชื้อ Vibrio cholerae non o1, non o139 เข้ารับการรักษาใน โรงพยาบาลมากขึ้น มีการเปลี่ยนแปลงของเชื้อทางโมเลกุล อย่างไร การดื้อยาที่พบเกี่ยวข้องกับการ เปลี่ยนแปลงของเชื้ออย่างไร

23 Details :2. General characteristics of V. cholerae Details : 2. General characteristics of V. cholerae 2.4 Molecular epidemiology  Sign et al; 2001 (India); clinical and environmental V. cholerae O1 and non O1 V. cholerae O1 V. cholerae non O1 - ctxA - 96% - 0% - zot - 96%- 0% - ace - 96%- 0% - tcpA - 100%- 0% - toxR - 100%- 96% - stn/sto - 0% - 0% - hlyA - 100% -100% - ompU - 100% - 4%

24 Details :2. General characteristics of V. cholerae Details : 2. General characteristics of V. cholerae 2.5 Molecular epidemiology (cont.)  Rivera et al; 2001(South America) ; environmental V. cholerae O1 and non-O1 V. cholerae O1 V. cholerae non O1 - ctxA - 100% - 5.8% - zot - 100%- 0% - tcpA - 100%- 7.0% - toxR - 100%- 100% - stn/sto % % - hlyA - 100% % - ompU - 100%- 76.9%

25 Way out ทำไมพบผู้ป่วยอหิวาตกโรคลดลงในบางปี ทำไมมีผู้ติดเชื้อ Vibrio cholerae non o1, non o139 เข้ารับการรักษาในโรงพยาบาล มากขึ้น มีการเปลี่ยนแปลงของเชื้อทางโมเลกุล อย่างไร ใช้ การหา virulence gene, pattern และ similarity ของเชื้อทางโมเลกุล โดยวิธี Random Amplification Polymorphic DNA การดื้อยาที่พบเกี่ยวข้องกับการ เปลี่ยนแปลงของเชื้ออย่างไร หา เปอร์เซนต์การดื้อยา และแบบแผนการ ดื้อยา

26 1.To detect virulence genes including ctxA, tcpA, zot, ace, stn/sto, hlyA, ompU, and regulatory gene, toxR in clinical and environmental V. cholerae O1 and non–O1 performed by multiplex PCR. Objec tive 2. To compare the patterns of genomic diversity among clinical and environmental V. cholerae O1 and non-O1 by RAPD (Random Amplification Polymorphic DNA)

27 Objec tive 3. To compare the antimicrobial susceptibility patterns of the clinical and environmental V. cholerae O1 and non-O1 strains isolated in Khon Kaen.

28 1.Clinical and environmental V. cholerae O1 and non-O1 isolates were collected from : Srinagarind hospital : Khon Kaen hospital : Aquatic environment : water from patient’s house Scope and limitation of research

29 Scope and limitation of research (cont.) 3. Antimicrobial susceptibility test ; interpreted  disk diffusion method susceptible intermediate resistance

30 Table 1 V. cholerae O1 and non-O1 included in this study StrainSourceYear of isolate No. of isolate Total of isolate V. cholerae O1HumanDiarrheal patient Carrier EnvironmentWater from patient’s house Aquatic environment20031 V. cholerae non-O1 HumanDiarrheal patient EnvironmentWater from patient’s house Aquatic environment

31 Environmental samples - Aquatic environment - Water from patient’s house Rectal swab samples - Patients - Carriers Detection of Vibrio cholerae from specimens by culture method Detection of Vibrio cholerae serogroups by latex agglutination V. cholerae non-O1 V. cholerae O1 (N=6 2) (N=174) Study design

32 Molecular typing Antimicrobial susceptibility Detection of virulence associated genes including ctxA, tcpA, zot, ace, ompU, stn/sto, hlyA and toxR RAPDDisk diffusion Multiplex PCR V. cholerae O1, V. cholerae non-O1 Study design

33 Methodology : Objective 1 (1) Multiplex PCR for detection of virulence gene and their patterns Multiplex PCR for detection of virulence gene and their patterns gen es encodesfunctions ctxA cholera toxin- the major toxin - increase cAMP - the toxin-damaged cells become pumps for water and electrolytes ace accessory cholera toxin - increases transcellular ion transport - to contribute to diarrhea in cholera zot zonular occluden toxin opening of tight junctions of the small intestine tcpA TcpA, the major subunit of a toxin coregulated pilus (TCP) - the major colonization factor - the receptor for CTX ø

34 Methodology : Objective 1 (2) Multiplex PCR for detection of virulence gene and their patterns Multiplex PCR for detection of virulence gene and their patterns ge nes encodesfunctions tox R transmembran e protein the master regulator om pU outer membrane protein resistance to bile, anionic detergents  sodium dodecyl sulfate, cationic peptide  polymyxinB hly A El Tor haemolysin - a pore-forming toxin -lyses erythrocytes and other mammalian cells stn /st o heat stable enterotoxin increase cGMP that adversely effects electrolyte flux

35  VPI (Vibrio pathogenicity islands/TCP islands)  CTX phage (CTX  )  RTX gene clusters  Consist of at least three major pathogenicity islands Multiplex PCR for detection of virulence gene and their patterns Methodology : Objective 1 (3)

36 Methodology : Objective 1 (4) Multiplex PCR for detection of virulence gene and their patterns Multiplex PCR for detection of virulence gene and their patterns VPI (Vibrio pathogenicity islands/TCP islands) tcp gene clusters tcpP activates trancription of the toxT gene, essential activor for tcp gene cluster transcription - the major colonization factor - the receptor for CTX  tcpA encoding ToxT, activate ctx and tcp gene clusters toxT

37 Methodology : Objective 1 (5) Multiplex PCR for detection of virulence gene and their patterns Multiplex PCR for detection of virulence gene and their patterns CTX phage (CTX  ) ctxA, Bencoding the cholera toxin (CT)  cholera disease symptoms aceencoding accessory cholera toxin zotencoding zonular occluden toxin toxR encoding transmembrane protein ToxR ; The master regulatory which is itself regulated by environment

38 Methodology : Objective 1 (6) Multiplex PCR for detection of virulence gene and their patterns Multiplex PCR for detection of virulence gene and their patterns RTX (repeat in toxin) gene clusters rtxA encoding the presumptive cytotoxin rtxC encoding an acyltransferase rtxB encoding an associated ATP-binding cassette rtxD transporter system

39 Obj. 1 (7): Oligonucleotide primers, amplicon sizes, and PCR conditions used for detecting Gene and size of amplicon (bp) Primer sequencePCR condition Referen ce ctxA (302) F-5 / CTCAGACGGGATTTGTTAGGGACG 3 / R-5 / TCTATGTCTGTAGCCATT 3 / Multiplex PCR: 95  C, 30 sec; 60  C, 1 min; 72  C, 1 min (25 cycles); (16);(33) tcpA (472) F-5 / GAAGAAGTTTGTAAAAGAAGAACAC 3 / R-5 / GAAAGCACCTTCTTTCAGGTTG 3 / zot (947) F-5 / TCGCTTAACGATGGCGCGTTTT 3 / R-5 / AACCCCGTTTCACTTCTACCCA 3 / ace (600) F-5 / AGAGCGCTGCATTTATCCTTATTG 3 / R-5 / AACTCGGTCTCGGCCTCTCGTATC 3 / Multiplex PCR: 95  C, 30 sec; 60  C, 1 min; 72  C, 1 min (25 cycles); (21) (41) toxR (779) F-5 / CCTTCGATCCCCTAAGCAATAC 3 / R-5 / AGGGTTAGCAACGATGCGTAAG 3 / ompU (869) F-5 / ACGCTGACGGAATCAACCAAAG 3 / R-5 / GCGGAAGTTTGGCTTGAAGTAG 3 / stn/sto (140) F-5 / AAAACAGTGCAGCAACCACAAC 3 / R-5 / GCTGGATTGCAACATATTTCGC 3 / Duplex PCR: 95  C, 30 sec; 55  C, 1 min; 72  C, 1 min (25 cycles) (33);(41) hlyA (540) F-5 / -CTTAGCTGAGCTGCGCGATTTG-3 / R-5 / -GAGTTGATCATTCAGA-3 / of V. cholerae ctxA, tcpA, zot, ace, ompU, toxR, stn/sto and hlyA

40 zot 947 bp tcpA 472 bp ctxA 302 bp Figure 1 Agarose gel electrophoresis of ctxA, tcpA and zot gene amplicons performed by using multiplex PCR. Lane M, 100 bp molecular weight marker ; lane 1, postive control; lane 2 and 3, V. cholerae O1 isolated from clinical specimens; lane 4, negative control. Results : Multiplex PCR (1) M bp 1000

41 Results :Multiplex PCR (2) toxR 779 bp ompU 869 bp ace 600 bp bp M Figure 2 Agarose gel electrophoresis of ace, ompU and toxR gene amplicons performed by using multiplex PCR. Lane M, 100 bp molecular weight marker; lane 1, Postive control; lane 2 and 3, V. cholerae O1 isolated from clinical specimens; lane 4, negative control.

42 Results: Multiplex PCR (3) Table 1 The specificity of primers to other Vibrio spp. and other enteric pathogens Bacterial strains Genes ctxAzotacetcpAompUtoxRhlyAstn V. alginolyticus (NIH) V. vulnificus (NIH) V. mimicus (NIH) V. fluvialis (NIH) V. vulnificus V. mimicus V. parahaemolyticus Plesiomonas shigelloides Aeromonas hydrophila

43 Table 2 Distribution of virulence genes and regulatory gene in 236 V. cholerae isolates Strainssource Genotype presence(+) or absence(-) of genes Total of isolates (%) ctxAtcpAzotacetoxRompUstn/stohlyA Clinical V. cholerae O1 patients (80.0) carriers (20.0) Total isolates 110 Environmental V. cholerae O1 Water from patient’s house (50.0) Aquatic environment (50.0) Total isolates2 Clinical V. cholerae non-O1 patients (73.4) (7.8) (3.1) (4.7) (1.6) (3.1) (1.6) carriers (4.7) Total isolates64 Environmental V. cholerae non-O1 Water from V. cholerae O1 patient’s house (10.0) Aquatic environment (38.3) (48’3) (3.3) Total isolates60

44 Results: Multiplex PCR (4) Distribution of virulence genes and regulatory gene of Vibrio cholerae Figure 3 Distribution of virulence genes and regulatory gene in 115 clinical and environmental V. cholerae O1 and non-O1 isolates.

45 CONCLUSION AND DISCUSSION : OBJ.1 1.Detection of clinical and environmental V. cholerae O1 and non-O1 virulence genes by PCR. 1.1 Both clinical and environmental V. cholerae O1 strains have more several virulent genes than V. cholerae non-O Most V. cholerae non-O1 (both clinical and environmental strains) carried toxR and hlyA, indicating that El tor haemolysin may play role in diarrheal disease. 1.4 Some clinical V. cholerae non-O1 carried zot, ace and stn whereas some environmental strains carried stn gene. 1.2 All clinical and environmental V. cholerae O1 carried ctx and tcpA, indicating that those strains from both sources can cause severe diarrhea

46 RAPD Molecular typing for second objective - usefulness for epidemiology study - pathogenicity study - several molecular methods Characteristic of molecular typing method MethodPrincipleCharacteristic RAPD Random Amplification Polymorphic DNA - Amplification of genomic DNA - Various primers - high discriminatory power - easy in performance - short time - economic advantages over other DNA- based techniques

47 Methodology of Obj 2 (1) : Methodology of RAPD-typing of genomic diversity among clinical and environmental * DNA extraction V. cholerae O1 and non-O1 isolates from patient and environments blood agar incubated at 37  C for 24 h 1 loopful of V. cholerae cells 300  l of cell lysis solution

48 The lysate Incubate for 5 min at 80 o C Incubate for 1 hrs. at 37 o C 100  l of protein precipitation solution centrifugation 1,3000xg for 5 min. mixed and inverted Added 1.5 µl RNaseA cool at room tempt. mixed, incubated on ice 5 min.

49 at 94  C, 5 min. (1 cycle); 94  C, 30 sec.; 47  C, 30 sec; 72  C, 1 min. (38 cycles); 72  C, 5 min (1 cycle) amplified product Analyze by electrophoresis, 1% Nusieve agarose gel in ethidium bromide-staining amplification condition :

50 Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates RAPD types RAPD patternsNumber of isolates (year) Total number of isolates (%) Primer 1Primer 2 V. cholerae O1 V. cholerae non-O1 Patient (n=110) Environment (n=2) Patient (n=64) Environment (n=60) 1 aA 104(03, 04,07)2(03) 106(44.9) 2aC 1(04) 1 (0.4) 3aD 2(03), 1(04) 8 (03, 04, 05)5(04)16 (6.8) 4 aE1(03)1 (0.4) 5aK1 (04)1 (0.4) 6aL1(04)1 (0.4) 7aO1(05) 2 (0.8) 8aQ2(04)2 (0.8) 9aX1(05)1 (0.4) 10aZ1(05)1 (0.4) 11a2B1(05), 1(07)2 (0.8) 12a2D2(07)2 (0.8) 13a2H2(07)2 (0.8) 14a2N2(07)2 (0.8) 15a2O1(07)1 (0.4) 16a2Q1(07)1 (0.4) 17a7 (07)7 (3.0) 18a3E1 (07)1 (0.4) 19bC 1(03) 1 (0.4) 20bM1(05)1 (0.4)

51 Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates (cont.) RAPD types RAPD patternsNumber of isolates (year) Total number of isolates (%) Primer 1Primer 2 V. cholerae O1 V. cholerae non-O1 Patient (n=110) Environment (n=2) Patient (n=64) Environment (n=60) 21cH1(04)1 (0.4) 22cO1(04)1 (0.4) 23cD1(05)1 (0.4) 24cN1(05)1 (0.4) 25dD2(05)2 (0.8) 26dT1(04)1 (0.4) 27eD2(05)2 (0.8) 28eX1(03)1 (0.4) 29e2A1(05)1 (0.4) 30eJ1(05)1 (0.4) 31eS1(05)1 (0.4) 32fD1(05)1 (0.4) 33gY1(05)1 (0.4) 34gO1(05)1 (0.4) 35jC1(03)1 (0.4) 36iF1(03)1 (0.4) 37jE1(03)1 (0.4) 38kC1(03)1 (0.4) 39lU1 (04)1 (0.4) 40mX1 (04)1 (0.4)

52 Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates (cont.) RAPD types RAPD patternsNumber of isolates (year) Total number of isolates (%) Primer 1Primer 2 V. cholerae O1 V. cholerae non-O1 Patient (n=110) Environment (n=2) Patient (n=64) Environment (n=60) 41mD3(05)3 (1.3) 42nP1(04)1 (0.4) 43nU1(04)1 (0.4) 44nV1(04)1 (0.4) 45oM1(04)1 (0.4) 46oO1(04)1 (0.4) 47pD1(04)1 (0.4) 48qN1(04)1 (0.4) 49rR1(04)1 (0.4) 50sD1(04)1 (0.4) 51tO1(04)1 (0.4) 52uR1(04)1 (0.4) 53vW1(04)1 (0.4) 54wB1(03)1 (0.4) 55wD5(05)1(04)6 (2.5) 56wG1(04)1 (0.4) 57wI1(04)1 (0.4) 58wR1(04)1 (0.4) 59xI1(07)1 (0.4) 60yE1(07)1 (0.4) 61y2K2(07)2 (0.8) 62z1(07)1 (0.4) 63z2N1(07)1 (0.4) 64z2P1(07)1 (0.4) 65z2S1(06)1 (0.4)

53 Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates (cont.) RAPD types RAPD patternsNumber of isolates (year) Total number of isolates (%) Primer 1Primer 2 V. cholerae O1 V. cholerae non-O1 Patient (n=110) Environment (n=2) Patient (n=64) Environment (n=60) 662a2E2(07)2 (0.8) 672b2(07)2 (0.8) 682c2G1(07)1 (0.4) 692d2I1(07)1 (0.4) 702e2J1(07)1 (0.4) 712L1(07)1 (0.4) 722g2K1(07)1 (0.4) 732h2M1(07)1 (0.4) 742iE1(07)1 (0.4) 752j2R2(07)2 (0.8) 762j2Z1(07)1 (0.4) 772j3D1(07)1 (0.4) 782j1(07)1 (0.4) 792kC1(07)1 (0.4) 802k2T1(06)1 (0.4) 812l2U1(06)1 (0.4) 822m2V1(06)1 (0.4) 832n2W1(06)1 (0.4) 842o2P1(06)1 (0.4) 852p2X1(06)1 (0.4) 862q2Y1(06)1 (0.4) 872r2Z3(06)3 (1.3) 882s3A1(06)1 (0.4) 892t3B1(06)1 (0.4)

54 Figure 4 (A) RAPD patterns of the clinical and environmental V. cholerae O1 and non-O1 isolates generated by primer 1. Patterns are designed as in Table 3 and indicated on top of each lane. Molecular weight bands are indicated on the left. bp ~12, Results : RAPD (1) A.

55 B. Resul ts: RAPD (2) Figure 5 (B) Dendrogram representing the relatedness of V. cholerae O1 and non-O1 patterns. Groups with similarity were established using the UPGMA. The right of dendrogram consisted of total number of isolates and the strains were found. Each strain was assigned by use of a four-letter code that refers to the number of isolate, patient (P) or environment (E), and year (03 to 07)

56 bp bp Figure 6 RAPD patterns of the clinical and environmental V. cholerae O1 and non-O1 isolates generated by primer 2. Patterns are designed as in Table 8 and indicated on top of each lane. Molecular weight bands are indicated on the left and year (03 to 07) Results: RAPD (3)

57 Figure 7 Dendrogram representing the relatedness of V. cholerae O1 and non-O1 patterns generated by primer 2. Groups with similarity were established using the UPGMA. The right of dendrogram consisted of total number of isolates and the strains were found. Each strain was assigned by use of a four-letter code that refers to the number of isolate, patient (P) or environment (E), and year (03 to 07) Results : RAPD (7)

58 CONCLUSION AND DISCUSSION : OBJ.2 2. RAPD-typing of genomic diversity among clinical and environmental V. cholerae O1 and non-O V. cholerae non-O1 strains are very genetically heterogeneous indicating derived from different clone. 2.3 **Almost V. cholerae O1 strains isolated in the same and difference year are the same RAPD pattern indicating derived from the same clone. 2.1 The 89 different RAPD types were observed by using Primer 1 (45) and Primer 2 (57).

59 Obj 3. Methodology: Antimicrobial susceptibility by disk diffusion method (1) * Preparation of bacterial suspensions V. cholerae isolates from patients and environments blood agar incubated for 24 h at 37 o C turbidity to a 0.5 McFarland 1 colony was suspended in 4 ml of BHI incubated at 37  C for 3 h

60 Mueller Hinton agar incubated for 24 h, at 37 o C measured the inhibition zone antibiotic disk McFarland No. 0.5 (~ 1x10 8 CFU/ml) (AMP, C, CIP, E, SXT, NOR, TE, PB disk) Obj 3. Methodology: Antimicrobial susceptibility by disk diffusion method (cont.)

61 Obj 3. Methodology: The criteria for interpretation of antimicrobial susceptibility by disk diffusion methods (cont.) Antimicrobial agents Inhibition zone diameters (mm) Reference Resistant (R) Intermediate (I) Sensitive (S) Ampicillin   17 (BBL, 2004) Ciprofloxacin   21 (BBL, 2004) Co- trimoxazole   16 (BBL, 2004) Erythromycin   23 (BBL, 2004) Norfloxacin   21 (BBL, 2004) Tetracycline   19 (BBL, 2004) Polymyxin B   12 (BBL, 2004)

62 Table 4 Antimicrobial susceptibility (AS) types of 236 clinical and environmental V. cholerae O1 and non-O1 isolates. AS types Antimicrobial susceptibility patterns Number of isolates Total number of isolates (%) AMP C CIP E SXT NOR TE V. cholerae O1V. cholerae non-O1 Patients (n=110) Environments (n=2) Patients (n=64) Environments (n=60) I.SSSSSSS (4.2) II. RSSSSSS (0.4) III. SSSRSSS (0.8) IV. RSSRSSS (6.4) V. ISSRSSS (0.8) VI. SSSISSS (33.9) VII. RSSISSS (14.0) VIII. ISSISSS (5.1) IX. SSSIRSS (0.4) X. RRSRSSS (0.4)

63 Table 4 Antimicrobial susceptibility (AS) types of 236 clinical and environmental V. cholerae O1 and non-O1 isolates. (cont.) AS types Antimicrobial susceptibility patterns Number of isolates Total number of isolates (%) AMP C CIP E SXT NOR TE V. cholerae O1V. cholerae non-O1 Patients (n=110) Environments (n=2) Patients (n=64) Environments (n=60) XI. SISIRSS (0.4) XII. IISIRSS (0.8) XIII. SISSSSS (0.4) XIV. ISSSSSI (0.4) XV. SSSIRSI (0.8) XVI. SSSISSR (0.4) XVII. SSSIRSR61* 06*067 (28.4) XVIII SSIIRSR1* (0.4) XIX RSSIRSR3* 0003 (1.3) XX SRSRSSR1* 0001 (0.4) Total (100) R = resistant, I = intermediate, S = susceptible

64 Table 5 Antimicrobial susceptibility of 236 clinical and environmental V. cholerae O1 and non-O1 isolates to seven antimicrobial agents by disk diffusion method. Antimicrobial agents Number of isolates (%) V. cholerae O1 (n=112)V. cholerae non-O1 (n=124) RISRIS Ampicillin(AMP) 33 (29.5)9 (8.6)70 (62.5)19 (15.3)8 (6.4)97 (78.2) Chloramphenicol (C) 2 (1.8)1 (0.9)109 (97.3)0 (0)3 (2.4)121 (97.6) Co-trimoxazole (SXT) 65 * (58.0)0 (0)47 (42.0)12 (9.6)0 (0)112 (90.3) Ciprofoxacin (CIP) 0 (0)1 (0.9)111 (99.1)0 (0) 124 (100.0) Erythromycin (E) 18 (16.1)92 (82.1)2 (1.8)3 (2.4)110 (88.7)11 (8.9) Norfloxacin (NOR) 0 (0) 112 (100)0 (0) 124 (100.0) Tetracycline (TE) 66*(58.9)0 (0)46 (41.1)6 (4.8)3 (2.4)115 (92.7) R, resistant, I, intermediate, S, susceptible ( *, 2007)

65 No. of combinations of antimicrobial resistance Number of V. cholerae resistant isolates (%) V. cholerae O1V. cholerae non-O1 Patient (n=110 ) Environment (n= 2 ) Total isolates (n=112) Patient (n= 64) Environment (n= 60) Total isolates (n=124) 010 (9.1)2 (100) 12 (10.7) 50 (78.1)42 (70.0) 92 (74.2) 120 (18.1)020 (17.8)7 (10.9)17 (28.3)24 (19.3) 275 (68.2) 075 (67.0)7 (10.9)1 (1.7)8 (6.4) 35 (4.5) Total No. of V. cholerae resistant isolates (%) 100 (90.9)0 (0)100 (90.9)14 (21.9)18 (30.0) 32 (25.8) Table 6 Antimicrobial susceptibility of V. cholerae O1 and non-O1 isolates to seven antimicrobial agents by disk diffusion method clarified to the……………….

66 CONCLUSION AND DISCUSSION : OBJ.3 3. Antimicrobial susceptibility 3.2 No resistance to ciprofloxacin and norfloxacin were found. 3.3 V. cholerae O1 were more resistant to antimicrobial agents than V. cholerae non-O different antibiogram were found. 3.4 Resistance to tetracycline and cotrimoxazole of V. cholerae occurred in The environmental V. cholerae non-O1were more resistance to antimicrobial agents than clinical isolates. This result imply a role of natural environments to serve as a reservoir of multidrug resistance in V. cholerae This result imply a role of natural environments to serve as a reservoir of multidrug resistance in V. cholerae

67 In conclusion, we demonstrated that V. cholerae O1 carried the virulence-associated genes more than V. cholerae non O1. However clinical and environmental V. cholerae non-O1 carried other genes besides ctx gene that can cause diarrhea. Therefore, V. cholerae O1 and non O1 in the aquatic environment are potentially pathogenic and may affect people’s health. OVERALL CONCLUSION

68 Surveillance of V. cholerae O1 and non-O1 in the clinical and environmental sources, combined with genotype monitoring using virulence-associated genes, RAPD typing, and antibiogram should be useful to confirm the human health risk. RECOMMENDATION

69 THANK YOU FOR YOUR ATTENTION


ดาวน์โหลด ppt Molecular epidemiology and antimicrobial susceptibility of Vibrio cholerae O1 and non-O1 isolated from patients and environment in Khon Kaen Bureau of.

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