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

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

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

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

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

2.2 Vibrio cholerae genome Details : 2. General characteristics of V. cholerae 2.2 Vibrio cholerae genome 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

จำนวนผู้ป่วย อหิวาตกโรค พ.ศ. จำนวนผู้ป่วย อหิวาตกโรค จังหวัดขอนแก่น อัตราป่วย* เชื้อที่เป็นสาเหตุ 2527 1 0.1 VC.Inaba 2528 111 7.3 2529 23 1.5 2530 1,259 80.6 2531 71 4.3 2532 53 3.2 2533 122 VC.Inaba , VC. Ogawa 2534 102 6.0 VC. Ogawa 2535 47 2.8 2536 461 27.7 VC. Ogawa (459), VC. 0139 (2) 2537 1,700 103.8 VC. 0139 (1442), VC. Ogawa (258)

2538 141 8.3 VC. Ogawa 2539 40 2.3 2540 7 0.4 2541 66 3.8 2542 165 9.5 VC.Inaba (9), VC. Ogawa (156) 2543 39 2.2 VC.Inaba (32), VC. Ogawa (6) , VC.0139 (1) 2544 264 15.1 VC.Inaba (263), VC. Ogawa (1) 2545 29 1.7 VC.Inaba (11), VC. Ogawa (17) , VC.0139 (1) 2546 150 8.5 VC.Inaba (127) , VC. Ogawa (23) 2547 83 4.7 VC.Inaba (81), VC. Ogawa (2) 2548 2 0.11 VC.Inaba (2) 2549 VC. Ogawa (2)

สถานการณ์อหิวาตกโรค จ.ขอนแก่น ปี 2544 - 2550

จำนวนผู้ป่วยอหิวาตกโรค จังหวัดขอนแก่น จำแนกตามเดือน เทียบกับค่าเฉลี่ยของ 5 ปีย้อนหลัง 10

0-35.75 35.75-71.50 71.50-107.25 107.25-143.00

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

Epidemiology in Khon Kaen Thailand

สถานการณ์การดื้อยาต้านจุลชีพของเชื้ออหิวาตกโรคของ 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 98 2. 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

การทดสอบความไวต่อยาต้านจุลชีพของเชื้อ V การทดสอบความไวต่อยาต้านจุลชีพของเชื้อ V. cholerae Inaba โดยศูนย์วิทย์ฯ ขอนแก่น ปี 2541 - 2549

ความไวของเชื้อ V. cholerae Inaba ต่อยาต้านจุลชีพ ปี 2550 โดยศูนย์วิทย์ ขอนแก่น ( N = 3 )

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

ทำไมพบผู้ป่วยอหิวาตกโรคระบาดในบางปี Research questions ทำไมพบผู้ป่วยอหิวาตกโรคระบาดในบางปี ทำไมมีผู้ติดเชื้อ Vibrio cholerae non o1, non o139 เข้ารับการรักษาในโรงพยาบาลมากขึ้น มีการเปลี่ยนแปลงของเชื้อทางโมเลกุลอย่างไร การดื้อยาที่พบเกี่ยวข้องกับการเปลี่ยนแปลงของเชื้ออย่างไร

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%

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 - 10.5% - 28.2% - hlyA - 100% - 97.5% - ompU - 100% - 76.9%

ทำไมพบผู้ป่วยอหิวาตกโรคลดลงในบางปี Way out ทำไมพบผู้ป่วยอหิวาตกโรคลดลงในบางปี ทำไมมีผู้ติดเชื้อ Vibrio cholerae non o1, non o139 เข้ารับการรักษาในโรงพยาบาลมากขึ้น มีการเปลี่ยนแปลงของเชื้อทางโมเลกุลอย่างไร ใช้ การหา virulence gene, pattern และ similarity ของเชื้อทางโมเลกุล โดยวิธี Random Amplification Polymorphic DNA การดื้อยาที่พบเกี่ยวข้องกับการเปลี่ยนแปลงของเชื้ออย่างไร หา เปอร์เซนต์การดื้อยา และแบบแผนการดื้อยา

Objective 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. 2. To compare the patterns of genomic diversity among clinical and environmental V. cholerae O1 and non-O1 by RAPD (Random Amplification Polymorphic DNA)

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

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

Table 1 V. cholerae O1 and non-O1 included in this study Strain Source Year of isolate No. of isolate Total of isolate V. cholerae O1 Human Diarrheal patient 2003 19 88 2004 13 2007 56 Carrier 7 22 3 12 Environment Water from patient’s house 1 2 Aquatic environment V. cholerae non-O1 64 10 2005 26 2006 8 18 6 5 4 54 28

Study design (N=62) (N=174) V. cholerae O1 V. cholerae non-O1 Environmental samples - Aquatic environment - Water from patient’s house Rectal swab samples - Patients - Carriers (N=62) (N=174) Detection of Vibrio cholerae from specimens by culture method Detection of Vibrio cholerae serogroups by latex agglutination V. cholerae O1 V. cholerae non-O1

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

Methodology : Objective 1 (1) Multiplex PCR for detection of virulence gene and their patterns genes encodes functions 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 CTXø

Methodology : Objective 1 (2) genes encodes functions toxR transmembrane protein the master regulator ompU outer membrane protein resistance to bile, anionic detergents  sodium dodecyl sulfate, cationic peptide  polymyxinB hlyA El Tor haemolysin - a pore-forming toxin lyses erythrocytes and other mammalian cells stn/sto heat stable enterotoxin increase cGMP that adversely effects electrolyte flux Methodology : Objective 1 (2) Multiplex PCR for detection of virulence gene and their patterns

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

Methodology : Objective 1 (4) 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

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

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

of V. cholerae ctxA, tcpA, zot, ace, ompU, toxR, stn/sto and hlyA Obj. 1 (7): Oligonucleotide primers, amplicon sizes, and PCR conditions used for detecting of V. cholerae ctxA, tcpA, zot, ace, ompU, toxR, stn/sto and hlyA Gene and size of amplicon (bp) Primer sequence PCR condition Reference 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/ 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/

Results : Multiplex PCR (1)   M 1 2 3 4 bp 1200 1000 zot 947 bp 500 tcpA 472 bp ctxA 302 bp 100 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 (2) bp 1200 ompU 869 bp 1000 toxR 779 bp 500 ace 600 bp 100 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.

Results: Multiplex PCR (3) Table 1 The specificity of primers to other Vibrio spp. and other enteric pathogens Bacterial strains Genes ctxA zot ace tcpA ompU toxR hlyA stn V. alginolyticus (NIH) - V. vulnificus (NIH) V. mimicus (NIH) + V. fluvialis (NIH) V. vulnificus V. mimicus V. parahaemolyticus Plesiomonas shigelloides Aeromonas hydrophila

Genotype presence(+) or absence(-) of genes Table 2 Distribution of virulence genes and regulatory gene in 236 V. cholerae isolates Strains source Genotype presence(+) or absence(-) of genes Total of isolates (%) ctxA tcpA zot ace toxR ompU stn/sto hlyA Clinical V. cholerae O1 patients + - 88 (80.0) carriers 22 (20.0) Total isolates 110 Environmental Water from patient’s house 1 (50.0) Aquatic environment 2 V. cholerae non-O1 47 (73.4) 5 (7.8) 2 (3.1) 3 (4.7) 1 (1.6) 64 Water from V. cholerae O1 patient’s house 6 (10.0) Aquatic environment 23 (38.3) 29 (48’3) 2 (3.3) 60

Distribution of virulence genes and regulatory gene of Vibrio cholerae 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.

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-O1. 1.2 All clinical and environmental V. cholerae O1 carried ctx and tcpA, indicating that those strains from both sources can cause severe diarrhea 1.3 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.

RAPD Molecular typing for second objective usefulness for epidemiology study pathogenicity study several molecular methods Characteristic of molecular typing method Method Principle Characteristic 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

V. cholerae O1 and non-O1 isolates from patient and environments 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

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

94C, 30 sec.; 47C, 30 sec; 72C, 1 min. (38 cycles); amplification condition : 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

Number of isolates (year) Total number of isolates (%) Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates RAPD types RAPD patterns Number of isolates (year) Total number of isolates (%) Primer 1 Primer 2 V. cholerae O1 V. cholerae non-O1 Patient (n=110) Environment (n=2) (n=64) (n=60) 1 a A 104(03, 04,07) 2(03) 106(44.9) 2 C 1(04) 1 (0.4) 3 D 2(03), 1(04) 8 (03, 04, 05) 5(04) 16 (6.8) 4 E 1(03) 5 K 1 (04) 6 L 7 O 1(05) 2 (0.8) 8 Q 2(04) 9 X 10 Z 11 2B 1(05), 1(07) 12 2D 2(07) 13 2H 14 2N 15 2O 1(07) 16 2Q 17 7 (07) 7 (3.0) 18 3E 1 (07) 19 b 20 M

Number of isolates (year) Total number of isolates (%) Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates (cont.) RAPD types RAPD patterns Number of isolates (year) Total number of isolates (%) Primer 1 Primer 2 V. cholerae O1 V. cholerae non-O1 Patient (n=110) Environment (n=2) (n=64) (n=60) 21 c H 1(04) 1 (0.4) 22 O 23 D 1(05) 24 N 25 d 2(05) 2 (0.8) 26 T 27 e 28 X 1(03) 29 2A 30 J 31 S 32 f 33 g Y 34 35 j C 36 i F 37 E 38 k 39 l U 1 (04) 40 m

Number of isolates (year) Total number of isolates (%) Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates (cont.) RAPD types RAPD patterns Number of isolates (year) Total number of isolates (%) Primer 1 Primer 2 V. cholerae O1 V. cholerae non-O1 Patient (n=110) Environment (n=2) (n=64) (n=60) 41 m D 3(05) 3 (1.3) 42 n P 1(04) 1 (0.4) 43 U 44 V 45 o M 46 O 47 p 48 q N 49 r R 50 s 51 t 52 u 53 v W 54 w B 1(03) 55 5(05) 6 (2.5) 56 G 57 I 58 59 x 1(07) 60 y E 61 2K 2(07) 2 (0.8) 62 z 63 2N 64 2P 65 2S 1(06)

Number of isolates (year) Total number of isolates (%) Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates (cont.) RAPD types RAPD patterns Number of isolates (year) Total number of isolates (%) Primer 1 Primer 2 V. cholerae O1 V. cholerae non-O1 Patient (n=110) Environment (n=2) (n=64) (n=60) 66 2a 2E 2(07) 2 (0.8) 67 2b 68 2c 2G 1(07) 1 (0.4) 69 2d 2I 70 2e 2J 71 2L 72 2g 2K 73 2h 2M 74 2i E 75 2j 2R 76 2Z 77 3D 78 79 2k C 80 2T 1(06) 81 2l 2U 82 2m 2V 83 2n 2W 84 2o 2P 85 2p 2X 86 2q 2Y 87 2r 3(06) 3 (1.3) 88 2s 3A 89 2t 3B

Results : RAPD (1) bp ~12,000 1600 1000 850 650 500 400 300 200 100 A. 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. 54

Results: RAPD (2) B. 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)

Results: RAPD (3) 500 300 400 850 650 100 200 1000 3000 4000 1650 2000 5000 bp 500 300 650 100 200 400 850 3000 1650 4000 1000 2000 5000 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 (7) 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)

CONCLUSION AND DISCUSSION : OBJ.2      2. RAPD-typing of genomic diversity among clinical and environmental V. cholerae O1 and non-O1. 2.1 The 89 different RAPD types were observed by using Primer 1 (45) and Primer 2 (57). 2.2 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.

1 colony was suspended in 4 ml of BHI 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 oC 1 colony was suspended in 4 ml of BHI incubated at 37C for 3 h turbidity to a 0.5 McFarland

Obj 3. Methodology: Antimicrobial susceptibility by disk diffusion method (cont.) Mueller Hinton agar McFarland No. 0.5 (~ 1x108 CFU/ml) antibiotic disk incubated for 24 h, at 37oC measured the inhibition zone (AMP, C, CIP, E, SXT, NOR, TE, PB disk)

Inhibition zone diameters (mm) 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 13 14-16 17 (BBL, 2004) Ciprofloxacin 15 16-20 21 Co-trimoxazole 10 11-15 16 Erythromycin 14-22 23 Norfloxacin 12 13-16  21 Tetracycline 14 15-18 19 Polymyxin B  8 9-11 12

Antimicrobial susceptibility patterns Total number of isolates 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 O1 V. cholerae non-O1 Patients (n=110) Environments (n=2) Patients (n=64) Environments (n=60) I. S 5 10 (4.2) II. R 1 1 (0.4) III. 2 (0.8) IV. 13 15 (6.4) V. I 2 VI. 3 45 31 80 (33.9) VII. 15 12 32 (14.0) VIII. 7 12 (5.1) IX. X.

Antimicrobial susceptibility patterns Total number of isolates 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 O1 V. cholerae non-O1 Patients (n=110) Environments (n=2) Patients (n=64) Environments (n=60) XI. S I R 1 1 (0.4) XII. 2 2 (0.8) XIII. XIV. XV. XVI. XVII. 61* 6* 67 (28.4) XVIII 1* XIX 3* 3 (1.3) XX Total 110 64 60 236 (100) R = resistant, I = intermediate, S = susceptible

Antimicrobial agents Number of isolates (%) 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) R I S 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) 47 (42.0) 12 (9.6) 112 (90.3) Ciprofoxacin (CIP) 111 (99.1) 124 (100.0) Erythromycin (E) 18 (16.1) 92 (82.1) 110 (88.7) 11 (8.9) Norfloxacin (NOR) 112 (100) Tetracycline (TE) 66*(58.9) 46 (41.1) 6 (4.8) 115 (92.7) R, resistant, I, intermediate, S, susceptible (*, 2007)

V. cholerae O1 V. cholerae non-O1 Table 6 Antimicrobial susceptibility of V. cholerae O1 and non-O1 isolates to seven antimicrobial agents by disk diffusion method clarified to the………………. No. of combinations of antimicrobial resistance Number of V. cholerae resistant isolates (%) V. cholerae O1 V. cholerae non-O1 Patient (n=110 ) Environment (n= 2 ) Total isolates (n=112) Patient (n= 64) Environment (n= 60) Total isolates (n=124) 10 (9.1) 2 (100) 12 (10.7) 50 (78.1) 42 (70.0) 92 (74.2) 1 20 (18.1) 20 (17.8) 7 (10.9) 17 (28.3) 24 (19.3) 2 75 (68.2) 75 (67.0) 1 (1.7) 8 (6.4) 3 5 (4.5) Total No. of V. cholerae resistant isolates (%) 100 (90.9) 0 (0) 14 (21.9) 18 (30.0) 32 (25.8)

CONCLUSION AND DISCUSSION : OBJ.3 3. Antimicrobial susceptibility 3.1 20 different antibiogram were found. 3.2 No resistance to ciprofloxacin and norfloxacin were found. This result imply a role of natural environments to serve as a reservoir of multidrug resistance in V. cholerae 3.3 V. cholerae O1 were more resistant to antimicrobial agents than V. cholerae non-O1. 3.4 Resistance to tetracycline and cotrimoxazole of V. cholerae occurred in 2007. 3.5 The environmental V. cholerae non-O1were more resistance to antimicrobial agents than clinical isolates. 66

OVERALL CONCLUSION 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.

RECOMMENDATION 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.

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