Zoonotic Study of Escherichia coli O157 H7 from Animals to Human through Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) Methods.

(1)

ZOONOTIC STUDY OF

ESCHERICHIA COLI

O157:H7 FROM ANIMALS TO HUMAN THROUGH

ARBITRARILY PRIMED POLYMERASE CHAIN REACTION (AP-PCR) METHOD

A. Introduction

B.

Escherichia coli

O157:H7 is a zoonotic agent of the type of Shiga toxin producing

Escherichia coli

that can cause disease in human, and cattle is known as

the main reservoir of these bacteria (Karmali

et al.,

2010). The infection by these bacteria in animals usually asymtomatic, whereas these bacterial infection

in humans usually show clinical symptoms i.e. diarrhea, colitis hemorrhagic and hemolytic uremic syndrome (HUS) (Acheson, 2010; Wani

et al.,

2004) . This

study report the application of AP-PCR method in order to study the zoonotic potency of

E. coli

O157:H7 from animals as a main reservoir of these

bacteria to human.

Fragments and total bands of 14 isolates of E. coli

O157:H7 generated by AP-PCR using primers M13F and M13R.

AP-PCR profile of genomic DNA of E. coli O157:H7 by using primers M13F (A) and M13R (B) on 1.5%. agarose gel. Line 1: ATCC 43894 (positive control); Line 2: KL52(7); 3: KL87(7); 4: KL30(4); 5: KL45(1); 6: KL(48(2); 7: KL85(1); 8: KL83(5); 9: KL24(5); 10: KL68(1); 11: 106(3); 12: KL-55(6); 13: SM-25(1); 14: SM-7(1); M: Marker 100 bp DNA

Ladder.

Methods

Results

C. Conclusion

D.

A

B

Similarity coefficient among isolates of E. coli O157:H7, which showed closely similarity between human and animal isolates

Phenogram of E. coli O157:H7, which was constructed using UPGMA, which placed both human and animal isolates in one clade

Arbitrarily primed polymerase chain reaction (AP-PCR) method provides for simples and rapid for tracing of zoonotic agent E. coli O157:H7, which were supported by its highly sensitivity

References

1. Acheson, D.W.K., 2000. J. Food Protect. (6): 819-821.

2. Karmali MA, Gannon V, Sargeant JM. 2010. Vet. Microbiol. 140: 360-370.

3. Wani, S.A., Samanta, I., Munshi, Z.H., Bhat, M.A., and Nishikawa, Y., 2004. J. Appl. Microbiol. 100:108–113 Cultivation of 14 isolates of E. coli O57 i.e.

ATCC 43894 (positive control), KL52(7),

KL87(7), KL30(4), KL45(1), KL(48(2), KL85(1), KL83(5), KL24(5), KL68(1), 106(3),

KL-55(6), SM-25(1),SM-7(1)

Extraction of bacterial DNA using QIAamp DNA Mini Kits

AP-PCR using primers M13F and M13R with PCR program: I. 94O_{C, 5}

min; II. 39 cycles (94O_{C, 5 min, 35}O_C,

1 min, 72O_{C, 1 min), III. 72}O_{C, 5 min.}

Evolutionary distance of each isolate was measured with algorithm unweighted pair group method using arithmetic averages (UPGMA)

Acknowledgments

The research was funded by the Directorate of Research and Community Services, Directorate General of Higher Education, Republic of Indonesia through Udayana

Research Grants with contract No.21.34 / UN 14 / SBRC / 2012, January 19th_{, 2012.}