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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: 2010-2011 Department of Microbiology Research Supervisor DR. PARITOSH KUMAR BISWAS Department of Microbiology, CVASU 6th October, 2012

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)

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

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

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

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

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

<<<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

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%

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

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

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 (0.3-24.4) Adult (310) 1.3 (4) 0.4 – 3.3 Breed Local (448) 0.9 (4) 0.2-2.3 0.1373 0.62 (0.1-5.6) Cross (70) 1.4 (1) 0.04-7.7 Sex Male (186) 1.1 (2) 0.1 – 3.8 0.6589 1.19 (0.2-7.2) Female (332) 0.9 (3) 0.2 – 2.6 Health status Diseased (90) 1.1 (1) 0.03 – 6.0 0.593 1.19 (0.1-10.8) Healthy (428) 0.3 – 2.4 Antibiotic used Yes (64) 1.6 (1) 0.04 – 8.4 0.4842 1.79 (0.2-16.2) No (454) 0.2 – 2.2  Total 518 1.0 (5) 0.3 – 2.2 -

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) 1.4 - 6.8 0.2177 1.87 (0.8-4.5) Adult (310) 6.1 (19) 3.7 - 9.4 Breed Local (448) 5.2 (22) 3.1 - 7.3 0.5578 0.85 (0.3-2.6) Cross (70) 6.1 (4) 1.6 - 14 Sex Male (186) 8.1 (15) 4.6 - 13 0.0213 2.56 (1.2-5.7) Female (332) 3.3 (11) 1.7 - 5.9 Health status Diseased (90) 6.7 (6) 2.5 - 14 0.2854 1.46 (0.6-3.7) Healthy (428) 4.7 (20) 2.9 - 7.1 Antibiotic used Yes (64) 4.8 (3) 1.0 - 13 1 0.92 (0.3-3.2) No (454) 5.1 (23) 3.2 - 7.5 Total 518 5 (26) 3.3 - 7.3 -

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 (0.7-6.5) Adult (310) 3.9 (12) 2.0 – 6.7 Breed Local (448) 2.9 (13) 1.6 – 4.9 0.4645 0.67 (0.2-2.4) Cross (70) 4.3 (3) 0.9 – 12 Sex Male (186) 3.8 (7) 1.5 – 7.6 0.5981 1.4 (0.5-3.8) Female (332) 2.7 (9) 1.3 – 5.1 Health status Diseased (90) 5.6 (5) 1.8 – 12.5 0.1716 2.23 (0.8-6.6) Healthy (428) 2.6 (11) 1.3 – 4.6 Antibiotic uesed Yes (64) 7.8 (5) 2.6 – 17.3 0.0364 3.41 (1.2-10.2) No (454) 2.4 (11) 1.2 – 4.3 Total 518 3.1 (16) 1.8 – 5 -

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

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

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

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

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

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%)

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.

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

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.

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.

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

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