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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,

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slide1

Molecular epidemiology and antimicrobial susceptibility of Vibrio cholerae O1andnon-O1isolated from patients and environment inKhon Kaen

Waraluk Tangkanakul* Chariya Chomvarin** et al.

Bureau of General Communicable Diseases*

Department of Microbiology, Faculty of Medicine,

Khon Kaen University**

today topics
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

Recommendationsfor future project

slide3

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-

slide4

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
slide5

Details :2. General characteristics of V. cholerae

2.2Vibriocholeraegenome

  • The larger
  • (chromosome I) 2.96 million bps.
  • The smaller
  • (chromosome II)
  • 1.07 million bps.
slide6

Details :2. General characteristics of V. cholerae

2.2Vibriocholeraegenome

  • 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
2544 2550
สถานการณ์อหิวาตกโรค จ.ขอนแก่น ปี 2544 - 2550
slide10
จำนวนผู้ป่วยอหิวาตกโรค จังหวัดขอนแก่น จำแนกตามเดือน เทียบกับค่าเฉลี่ยของ 5 ปีย้อนหลัง
slide11

0

0-35.75

35.75-71.50

71.50-107.25

107.25-143.00

slide12
ตามรอยหอย (แครง)

Fresh

food

Lotus

Fast

food

ตลาด

บางลำภู

Fast

food

บ้านไผ่

Big C

Fresh

food

กาฬสินธุ์

DC

วังน้อย

มหา-

สารคาม

บ้าน

หนองแก

เหล่า

นาดี

ภูเวียง

ชัยภูมิ

บอระบือ

โกสุม

กาฬสินธุ์

(หมู่บ้านใกล้เคียง)

ปากน้ำ

อ.เมือง

อ.กาญ-

จนดิษฐ์

มหา-

สารคาม

หนองบัว-

ลำภู

อุบลรัตน์

ดอนโมง

ชุมแพ

อื่นๆ

อ.ท่าฉาง

สุราษฎร์ฯ

กระนวน

กรุงเทพฯ

วังหอย

ท่าข้าม

บางขุนเทียน

เจ๊วัฒนา

ตลาด อ.จิระ

10-20 กระสอบ

ทุกวัน

ภุมเรียง

อ.ไชยา

สมุทรสาคร

ตลาดมหาชัย

Macro

ขอนแก่น

ป้าเพ็ญ

โรงหนังราชา

8-20 กระสอบ

ทุกวัน

เจ๊แป้ง

ตลาดรถไฟ

30 กระสอบ

ทุกวัน

ความเสี่ยง

4 และ 10 ต.ค.50 พบV.parahaemolyticus

ในเปลือกหอยด้านนอก และน้ำในวังเลี้ยงหอย

ไม่พบในเนื้อหอย

  • การขนส่ง
  • สุราษฎร์ฯ-สมุทรสาคร ใช้รถกะบะ
  • สมุทรสาคร-ขอนแก่น ใช้รถ 6 ล้อ (คันเดียวกันทั้ง 3 ร้าน)
  • ขอนแก่น-อำเภอ/จังหวัดต่างๆ ใช้รถประจำทาง
  • และรถส่วนบุคคล ไม่ได้แช่เย็น
slide15

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

v cholerae inaba 2541 2549
การทดสอบความไวต่อยาต้านจุลชีพของเชื้อ V. cholerae Inaba โดยศูนย์วิทย์ฯ ขอนแก่น ปี 2541 - 2549
v cholerae inaba 2550 n 3
ความไวของเชื้อ V. cholerae Inaba ต่อยาต้านจุลชีพ ปี 2550 โดยศูนย์วิทย์ ขอนแก่น ( N = 3 )
v cholerae hikogima 2550 n 1
ความไวของเชื้อ V. cholerae Hikogima ต่อยาต้านจุลชีพ ปี 2550 โดยศูนย์วิทย์ ขอนแก่น และรพ. ศรีนครินทร์ ( N = 1 )
research questions
Research questions

ทำไมพบผู้ป่วยอหิวาตกโรคระบาดในบางปี

ทำไมมีผู้ติดเชื้อ Vibrio cholerae non o1, non o139 เข้ารับการรักษาในโรงพยาบาลมากขึ้น

มีการเปลี่ยนแปลงของเชื้อทางโมเลกุลอย่างไร

การดื้อยาที่พบเกี่ยวข้องกับการเปลี่ยนแปลงของเชื้ออย่างไร

slide23

Details :2. General characteristics of V. cholerae

2.4 Molecular epidemiology

  • Sign et al; 2001 (India); clinical and environmental

V. choleraeO1 and non O1

  • V. choleraeO1 V. choleraenon 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%
slide24

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 O1V. 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%
way out
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

slide26

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)

slide27

Objective

3. To compare the antimicrobial susceptibility patterns of the

clinical and environmental V. cholerae O1 and non-O1

strains isolated in Khon Kaen.

slide28

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
slide29

Scope and limitation of research (cont.)

  • 3. Antimicrobial susceptibility test ; interpreted
      • disk diffusion method
  • susceptible
  • intermediate
  • resistance
slide31

Study design

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 Vibriocholeraeserogroups bylatex agglutination

V. cholerae O1

V. cholerae non-O1

slide32

Study design

V. cholerae O1, V. cholerae non-O1

Detection of virulence associated genes including ctxA, tcpA, zot, ace, ompU, stn/sto, hlyA and toxR

Molecular typing

Antimicrobial susceptibility

RAPD

Disk diffusion

Multiplex PCR

slide33

Methodology : Objective 1 (1)

Multiplex PCR for detection of virulence gene and their patterns

slide34

Methodology : Objective 1 (2)

Multiplex PCR for detection of virulence gene and their patterns

slide35

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
slide36

Methodology : Objective 1 (4)

Multiplex PCR for detection of virulence gene and their patterns

VPI (Vibrio pathogenicity islands/TCP islands)

tcp gene clusters

activates trancription of the toxT gene,

essential activor for tcp gene cluster

transcription

  • tcpP
  • tcpA

- the major colonization factor

- the receptor for CTX

  • toxT

encoding ToxT, activate ctx and tcp gene

clusters

slide37

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

encoding transmembrane protein ToxR ; The master

regulatory which is itself regulated by environment

toxR

slide38

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

slide39

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

slide40

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.

slide41

Results :Multiplex PCR (2)

M 1 2 3 4

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.

slide42

Results: Multiplex PCR (3)

Table 1 The specificity of primers to other Vibrio spp. and other enteric pathogens

slide44

Results: Multiplex PCR (4)

Distribution of virulence genes and regulatory gene of Vibriocholerae

Figure 3 Distribution of virulence genes and regulatory gene in 115 clinical and

environmental V. cholerae O1 and non-O1 isolates.

slide45

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. choleraenon-O1.

1.2 All clinical and environmental V. cholerae O1carried ctx and tcpA, indicating that those strains from both sources can cause severe diarrhea

1.3 Most V. choleraenon-O1 (both clinical and environmental strains) carried toxRand hlyA, indicating that El tor haemolysin may play role in diarrheal disease.

1.4 Some clinical V. choleraenon-O1carried zot, ace and stn whereas some environmental strains carried stngene.

slide46

RAPD Molecular typing for second objective

  • usefulness for epidemiology study
  • pathogenicity study
  • several molecular methods

Characteristic of molecular typing method

slide47

Obj 2 (1) : Methodology ofRAPD-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

slide48

The lysate

mixed and inverted

Incubate for 5 min at 80 oC

Added 1.5 µlRNaseA

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.

slide49

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

slide51

Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates (cont.)

slide52

Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates (cont.)

slide53

Table 3 RAPD patterns of the 236 clinical and environmental V. cholerae O1 and non-O1 isolates (cont.)

slide54

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.

slide55

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)

slide56

Results: RAPD (3)

bp

5000

4000

3000

2000

1650

1000

850

650

500

400

300

200

100

5000

4000

bp

3000

2000

1650

1000

850

650

500

400

300

200

100

  • 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)
slide57

Results :

RAPD (7)

Figure 7Dendrogram 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
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.

slide59

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

slide60

antibiotic disk

Obj 3. Methodology:Antimicrobial susceptibility by disk diffusion method (cont.)

Mueller Hinton agar

McFarland No. 0.5

(~ 1x108 CFU/ml)

incubated for 24 h, at 37oC

measured the inhibition zone

(AMP, C, CIP, E, SXT, NOR, TE, PB disk)

slide61

Antimicrobial agents

Inhibition zone diameters (mm)

Reference

Intermediate (I)

Resistant (R)

Sensitive (S)

Ampicillin

13

14-16

17

(BBL, 2004)

Ciprofloxacin

15

16-20

21

(BBL, 2004)

Co-trimoxazole

10

11-15

16

(BBL, 2004)

Erythromycin

13

14-22

23

(BBL, 2004)

Norfloxacin

12

13-16

 21

(BBL, 2004)

Tetracycline

14

15-18

19

(BBL, 2004)

Polymyxin B

 8

9-11

12

(BBL, 2004)

Obj 3. Methodology: The criteria for interpretation of antimicrobial

susceptibility by disk diffusion methods (cont.)

slide62

Table 4 Antimicrobial susceptibility (AS) types of 236 clinical and environmental

V. cholerae O1 and non-O1 isolates.

slide63

Table 4 Antimicrobial susceptibility (AS) types of 236 clinical and environmental V. cholerae O1

and non-O1 isolates. (cont.)

R = resistant, I = intermediate, S = susceptible

slide64

Table 5 Antimicrobial susceptibility of 236 clinical and environmental V. cholerae O1

and non-O1 isolates to seven antimicrobial agents by disk diffusion method.

R, resistant, I, intermediate, S, susceptible(*, 2007)

slide65

Table 6 Antimicrobial susceptibility of V. cholerae O1 and non-O1 isolates to seven

antimicrobial agents by disk diffusion method clarified to the……………….

conclusion and discussion obj 3
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.

3.4Resistance 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.

overall conclusion
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.RECOMMENDATION
ad