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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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Bureau of general communicable diseases department of microbiology faculty of medicine

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


Bureau of general communicable diseases department of microbiology faculty of medicine

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-


Bureau of general communicable diseases department of microbiology faculty of medicine

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


Bureau of general communicable diseases department of microbiology faculty of medicine

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.


Bureau of general communicable diseases department of microbiology faculty of medicine

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

. 2544 - 2550


Bureau of general communicable diseases department of microbiology faculty of medicine

5


Bureau of general communicable diseases department of microbiology faculty of medicine

0

0-35.75

35.75-71.50

71.50-107.25

107.25-143.00


Bureau of general communicable diseases department of microbiology faculty of medicine

()

Fresh

food

Lotus

Fast

food

Fast

food

Big C

Fresh

food

DC

-

()

.

.-

-

-

.

.

10-20

.

Macro

8-20

30

4 10 ..50 V.parahaemolyticus

  • -

  • - 6 ( 3 )

  • -/


Bureau of general communicable diseases department of microbiology faculty of medicine

Epidemiology in Khon Kaen Thailand


Bureau of general communicable diseases department of microbiology faculty of medicine

.

1. V. cholerae Ogawa( : )

- 2541 : Te 92 ,Sxt 97

- 2542 : Te 98 ,Sxt 98

2. V. cholerae Inaba

- 2543 : Te 10 ,Sxt 5

- 2544 : Te 0 ,Sxt 0

- 2545 : Te 0 ,Sxt 0


V cholerae inaba 2541 2549

V. cholerae Inaba 2541 - 2549


V cholerae inaba 2550 n 3

V. cholerae Inaba 2550 ( N = 3 )


V cholerae hikogima 2550 n 1

V. cholerae Hikogima 2550 . ( N = 1 )


Research questions

Research questions

Vibrio cholerae non o1, non o139


Bureau of general communicable diseases department of microbiology faculty of medicine

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%


Bureau of general communicable diseases department of microbiology faculty of medicine

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

Vibrio cholerae non o1, non o139

virulence gene, pattern similarity Random Amplification Polymorphic DNA


Bureau of general communicable diseases department of microbiology faculty of medicine

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

  • nonO1 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)


Bureau of general communicable diseases department of microbiology faculty of medicine

Objective

3. To compare the antimicrobial susceptibility patterns of the

clinical and environmental V. cholerae O1 and non-O1

strains isolated in Khon Kaen.


Bureau of general communicable diseases department of microbiology faculty of medicine

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 patients house


Bureau of general communicable diseases department of microbiology faculty of medicine

Scope and limitation of research (cont.)

  • 3. Antimicrobial susceptibility test ; interpreted

    • disk diffusion method

  • susceptible

  • intermediate

  • resistance


  • Bureau of general communicable diseases department of microbiology faculty of medicine

    Table 1V. cholerae O1 and non-O1 included in this study


    Bureau of general communicable diseases department of microbiology faculty of medicine

    Study design

    Environmental samples

    - Aquatic environment

    - Water from patients 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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    Methodology : Objective 1 (1)

    Multiplex PCR for detection of virulence gene and their patterns


    Bureau of general communicable diseases department of microbiology faculty of medicine

    Methodology : Objective 1 (2)

    Multiplex PCR for detection of virulence gene and their patterns


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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.


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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.


    Bureau of general communicable diseases department of microbiology faculty of medicine

    Results: Multiplex PCR (3)

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


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    Table 2 Distribution of virulence genes and regulatory gene in 236 V. cholerae isolates


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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.


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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.


    Bureau of general communicable diseases department of microbiology faculty of medicine

    RAPD Molecular typing for second objective

    • usefulness for epidemiology study

    • pathogenicity study

    • several molecular methods

    Characteristic of molecular typing method


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    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 37C for 24 h

    1 loopful of V. cholerae cells

    300 l of cell lysis solution


    Bureau of general communicable diseases department of microbiology faculty of medicine

    The lysate

    mixed and inverted

    Incubate for 5 min at 80 oC

    Added 1.5 lRNaseA

    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.


    Bureau of general communicable diseases department of microbiology faculty of medicine

    amplification condition :

    at 94C, 5 min. (1 cycle);

    94C, 30 sec.; 47C, 30 sec; 72C, 1 min. (38 cycles);

    72C, 5 min (1 cycle)

    amplified product

    Analyze by electrophoresis, 1% Nusieve agarose gel in ethidium bromide-staining


    Bureau of general communicable diseases department of microbiology faculty of medicine

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


    Bureau of general communicable diseases department of microbiology faculty of medicine

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


    Bureau of general communicable diseases department of microbiology faculty of medicine

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


    Bureau of general communicable diseases department of microbiology faculty of medicine

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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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.


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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)


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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)


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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.


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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 37C for 3 h

    turbidity to a 0.5 McFarland


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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)


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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.)


    Bureau of general communicable diseases department of microbiology faculty of medicine

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

    V. cholerae O1 and non-O1 isolates.


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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)


    Bureau of general communicable diseases department of microbiology faculty of medicine

    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 peoples 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


    Bureau of general communicable diseases department of microbiology faculty of medicine

    THANK YOU FOR YOUR ATTENTION


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