1. Welcome To
My presentation

2. DR. PARITOSH KUMAR BISWAS
An Investigation on clonal diversity of Escherichia coli from cattle in smallholdings while using selective media for O157 strains
Md. Zohorul Islam
Roll. No.: 0111/03
Reg. No.: 088
Session:
Department of Microbiology
Research Supervisor
DR. PARITOSH KUMAR BISWAS
Department of Microbiology, CVASU
6th October, 2012

3. Introduction>>>
Major categories of pathogenic E. coli
Intestinal pathogenic
Extra-intestinal pathogenic
Enterohemorrhagic (EHEC)
Enterotoxigenic (ETEC)
Enteropathogenic (EPEC)
Enteroinvasive (EIEC)
Enteroaggregative (EAEC) and
Diffusely adherent (DAEC)
Uropathogenic (UPEC )
Meningitis associated (MNEC)

4. Most Important Serotype: EHEC O157:H7
Why it is called notorious microbe?
- It is an emerging zoonotic pathogen (WHO,1997)
Enter into human food chain
It doesn't produce any disease in cattle
Hemorrhagic colitis
Hemolytic Uremic Syndrome (HUS)
Kidney failure
Death

5. EHEC O157 virulence factor
Genomic marker (gene)
Virulence factor
Hly
Hemolysin
Eae
Intimin
Stx
Shiga toxin

6. Cattle and other ruminant are largest reservoir of EHEC O157
Sources of EHEC O157
Cattle and other ruminant are largest reservoir of EHEC O157
Food and water contaminated with cattle feces are the major sources for human infection
Armstrong et. al., 1996

7. Large outbreak in Germany
History >>>
Large outbreak in Germany
2011
1993
R, Anderson, 2011.
Large western states outbreak, 500 cases and 4 deaths
1985
CDC-MMWR
Association of STEC with HUS
1983
Karmali et al. 1985
Description of Shiga toxin
1982
O’Brien and LaVeck.1983
First clinical isolation, “Rare E. coli serotype”
Riley et al. 1983

8. Objectives >>>
Major objectives of this study was
To determine the clonal diversity of probable EHEC O157 isolates grown on CT-SMAC
To estimate the prevalence of probable EHEC O157 being harbored by the smallholders’ cattle
To phenotypically discriminate the antimicrobial-resistant diversity of the probable EHEC O157 strains

9. <<<Methodology
Sample collection
Study area
Selective isolation in CT-SMAC
PFGE was performed following the standardized CDC PulseNet protocol (CDC, 2009)
Plasmid profiling was conducted by alkaline-lysis according to the protocol described by Kado and Liu (1981) with minor modifications
Primary enrichment in BPW
PCR for stx1, stx2 and hly gene
Confirmation in EMB
Plasmid profiling
Detection of 7 house keeping gene
Antimicrobial resistance pattern
Diversity of non EHEC by PFGE

10. Distribution of EHEC O157 in smallholders’ cattle
Results >>>
Distribution of EHEC O157 in smallholders’ cattle
No. samples
tested
No. (%) colorless colony producing isolates in CT-SMAC
No. (%) probable EHEC O157
No. isolates having ≥1 virulent gene of Stx1, Stx2 and hly (%)
518 57
(11%) 39
(7.5%) 31
(6%)
Stx1= 0.97%
Stx2=5%
hly=3.1%

11. 518 rectal swab sample Sorbitol non fermenter=57
Sorbitol fermenter =461
Confirmed as E. coli in EMB =39

12. Isolate carrying one or more virulent gene = 31
Probable EHEC O157 = 39
Isolate carrying one or more virulent gene = 31
Stx1= 5
Stx2 = 26
Hly = 16

13. Prevalence of Stx1 genotype of probable EHEC O157 in cattle in smallholdings
Variable
(N)
Prevalence % (No. positive )
95% CI P
OR (95% CI)
Age
Calf (208)
0.5 (1)
0.01 – 2.7
0.6527
2.71 ( )
Adult (310)
1.3 (4)
0.4 – 3.3
Breed
Local (448)
0.9 (4)
0.1373
0.62 ( )
Cross (70)
1.4 (1)
Sex
Male (186)
1.1 (2)
0.1 – 3.8
0.6589
1.19 ( )
Female (332)
0.9 (3)
0.2 – 2.6
Health status
Diseased (90)
1.1 (1)
0.03 – 6.0
0.593
1.19 ( )
Healthy (428)
0.3 – 2.4
Antibiotic used
Yes (64)
1.6 (1)
0.04 – 8.4
0.4842
1.79 ( )
No (454)
0.2 – 2.2 
Total
518
1.0 (5)
0.3 – 2.2 -

14. Prevalence of Stx2 genotype of probable EHEC O157 in cattle in smallholdings
Variable
(N)
Prevalence % (No. positive )
95% CI P
OR (95% CI)
Age
Calf (208)
3.4 (7)
0.2177
1.87 ( )
Adult (310)
6.1 (19)
Breed
Local (448)
5.2 (22)
0.5578
0.85 ( )
Cross (70)
6.1 (4)
Sex
Male (186)
8.1 (15)
0.0213
2.56 ( )
Female (332)
3.3 (11)
Health status
Diseased (90)
6.7 (6)
0.2854
1.46 ( )
Healthy (428)
4.7 (20)
Antibiotic used
Yes (64)
4.8 (3) 1
0.92 ( )
No (454)
5.1 (23)
Total
518
5 (26) -

15. Prevalence of hly genotype of probable EHEC O157 in cattle in smallholdings
Variable
(N)
Prevalence % (No. positive )
95% CI P
OR (95% CI)
Age
Calf (208)
1.9 (4)
0.5 – 4.9
0.301
2.05 ( )
Adult (310)
3.9 (12)
2.0 – 6.7
Breed
Local (448)
2.9 (13)
1.6 – 4.9
0.4645
0.67 ( )
Cross (70)
4.3 (3)
0.9 – 12
Sex
Male (186)
3.8 (7)
1.5 – 7.6
0.5981
1.4 ( )
Female (332)
2.7 (9)
1.3 – 5.1
Health status
Diseased (90)
5.6 (5)
1.8 – 12.5
0.1716
2.23 ( )
Healthy (428)
2.6 (11)
1.3 – 4.6
Antibiotic uesed
Yes (64)
7.8 (5)
2.6 – 17.3
0.0364
3.41 ( )
No (454)
2.4 (11)
1.2 – 4.3
Total
518
3.1 (16)
1.8 – 5 -

16. Diversity among the isolates lacking any virulent gene of Stx1, Stx2 and hly
Seven isolates was tested for 7 housekeeping gene of E. coli
Seven such isolates was tested for clonal diversity by PFGE
These isolates was also tested for plasmid profile

17. Detection of 7 housekeeping gene
probable EHEC O157 isolates, that lacked any of the 3 virulent genes, possessed all the seven house keeping genes - adk, fumC, gyrB, icd, mdh, purA and recA
It reconfirms their fidelity as E. coli at the molecular level

18. Gel Image of housekeeping gene of E. coli
Gyrb-911 bp
Icd-878 bp
recA-780 bp
fumC- 806 bp

19. Plasmid profiling
All the seven isolates had a >54.2 kb size plasmid
Six isolates contained ≥ 1-<3 plasmids
Four of the isolates contained very large plasmid of 147 kb size

20. PFGE findings
Except for two, all other E. coli isolates are genetically diverse, displaying six different band patterns

21. Phenotypic diversity based on Antimicrobial resistance patterns
Frequencies of Stx1, Stx2 and hly genotypic isolates resistant to the antimicrobials
Antimicrobial
No. resistant isolates (%)
Stx1 (n=5)
Stx2 (n=26)
hly (n=16)
AML
1 (20%)
8 (31%)
5 (31%)
CRO
2 (7%)
2 (13%) NA
4 (15%)
CIP
1 (6%) TE
15 (58%)
3 (19%)
AMP
2 (40%)
9 (35%) P
5 (100%)
26 (100%)
16 (100%)
DOC
3 (12%)
SXT
3 (60%)
14 (54%)
7 (44%) CN
5 (19%) C
0 (0%)

22. Overall antimicrobial resistance pattern
88%, 84% and 82% isolates were sensitive to chloramphenicle, gentamicine and ciprofloxacin, respectively
100% isolates were resistant to penicillin, and 53% to trimethoprim-sulfomethoxazole.

23. Multi drug resistant pattern
About 60% isolates carrying all the three EHEC O157 virulent genes were resistant to ≥ 2 antimicrobials, among them one isolate was resistant to six antimicrobials
Isolates having no virulent gene, but harboring 54.2 kb sized plasmid were resistant to tetracycline, salfomethoxazole, ampicillin and amoxicillin

24. Conclusion
Based on the possession of three virulent genes – Stx1, Stx2 and hly the strains of EHEC O157 isolated from this study are not clonal, but diverse
31 strains had ≥1 of these three genes; among them 28 were shiga toxin producing because they harbor either Stx1 or Stx2 gene or the both.
By PFGE assay and plasmid profiling of the 7 isolates lacking any of the mentioned three virulent genes, 6 genotypes were identified, suggesting that these isolates were not clonally related either except for two.
The antimicrobial resistance profiles of the colorless colony producing isolates to the 11 antimicrobials tested also varied.

25. Conclusion
Based on colorless colonies on a CT-SMAC agar plate we couldn’t predict it as EHEC O157 unless other confirmatory tests are applied, such as identification of the presence of at least one virulent gene by a PCR assay.

26. My Research Supervisor Department of Microbiology, CVASU
Acknowledgements
My Research Supervisor
&
RFLDC
PRTC
Jens P. Christensen, Department of Veterinary Disease Biology, Copenhagen University, Denmark
Department of Microbiology, CVASU

27. THANK YOU ALL