Isolation and genotyping of sorbitol fermenting of E.coli O157:H- strain isolated from children and calves in AL –Samawa Province

  • Ahmed Sami Jarad College of Medicine, University of AL-Muthanna. Department of Pathology
  • Eyhab R. M. Al-Samawy College of Medicine, University of AL-Muthanna ,Department of Anatomy
Keywords: Shiga toxin Escherichia coli, , CHROMagar O157, rfbO157, flicH7, stx1, stx2, eaeA, and EHEC hlyA.

Abstract

Infections with Shiga toxin Escherichia coli (STEC) are the important cause of several sever disease in human and animal. Non-sorbitol-fermenting O157:H7 is fundamental and most regular isolated. However, sorbitol-fermenting (SF) O157: H- strains are significantly increased and cannot detected by routine lab test. Current study designed to isolate and genotyping of SF-O157: H- in samples collected from 125 children manifested with diarrhea in Pediatric Hospital and 135 calves with or without diarrhea from local farm in Al –Samawa Province. Isolation and diagnostic procedure depends on selective culturing media to then all samples cultured on CHROMagar O157 to detect E. coli O157 from other intestinal flora. Then, the isolated (O157) was streaking onto Sorbitol MacConkey agar and store in incubator at 37 °C for 24h and examined for typical E. coli O157:H7, then the isolated SF- O157: H- were subjected to various biochemical tests include IMViC (Indole, Methyl red, VogesProskauer, and Citrate) to affirm proof of  E.coli. And latex agglutination checking to serotyping of E.coli O157: H7 was done. .All isolated SF-O157: H- were tested for the carrying of flowing genes rfbEO157, FlicH7, stx1, stx2, EHEC hlyA, eaeA by PCR assey. Results showed the ratio of SF- O157: H- were 8 of out 125 at a percentage (6.4 %) in sample collected from children and 16 / 135 at a percentage (11.8 %) in calves and Prevalence of virulent genes (rfbO157, flicH7, stx1, stx2, eaeA, and EHEC hlyA) among SF- O157 were, rfbO157 gene of SF- O157:H- gave 87.5% (7/8) and 100%(16/16) in children and calves respectively, flicH7 gave negative result in all isolated, and 37.5 % (3/8) expressed stx1 gene in children and 31.2 % (5/16) in calves, the stx2 gene appeared in 12.5 %(1/8) and 6.25 %(1/16) in children and calves respectively, eaeA gene gave a positive results were 50%(4/8) in children and 43.7 %(7/16) in calves and EHEC hlyA gene results were 25%(2/8) and 6.25 %(1/16) from both children and calves respectively. SF- O157: H- strains have variant number of virulent factor, some strain has one or two, three and may have all virulence or no virulent detected with them in some case. In conclusion, SF- O157: H- is important pathogen to human especially children, and cow are the fundamental store to this pathogen particularly calves and can infect it, SF- O157: H- carry important virulence factors (stx1, stx2, eaeA and EHEC HlyA) and cannot detected by routine lab test as SMAC, so the best detected method by using PCR assay for the main virulent factor.

Downloads

Download data is not yet available.

References

1- Gunzer F. , Böhm H. , Rüssmann H. , Bitzan M. , Aleksic S. and Karch H. (1992). Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome. Journal of Clinical Microbiology 30(7):1807–1810.
2- Eklund M. , Bielaszewska M. , Nakari U-M , Karch H. and Siitonen A. (2006). Molecular and phenotypic profiling of sorbitol-fermenting Escherichia coli O157: H- human isolates from Finland. Clin Microbiol Infect. July , 12(7): 634–641.
3- Riley L. W. , Remis R. S. , Helgerson S. D. ,McGee H.B. , Wells J.G. , Davis B.R. , Hebert R.J. , Olcott E.S. , Johnson L.M. , Hargrett N.T. , Blake P.A. and Cohen M.L. (1983). Hemorrhagic colitis associated with a rare Escherichia coli serotype. New Eng. J. of Med. 308: 681-685.
4- Karch H. and Bielaszewska M. (2001). Sorbitol-Fermenting Shiga Toxin-Producing Escherichia coli O157:H− Strains: Epidemiology Phenotypic and Molecular Characteristics and Microbiological Diagnosis. Journal of Clinical Microbiology 39(6): 2043–2049.
5- Mellmann A. , Lu S. , Karch H. , Xu J. , Harmsen D. , Schmidt M. A. and Bielaszewska M. (2008). Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM . Applied and Environmental Microbiology 74(1) 67–72.
6- Siegler R. and Oakes R. (2005): Hemolytic uremic syndrome , pathogenesis treatment and outcome. Curr Opin Pediatr 2005 , 17: 200–204.
7- Karch H. , Friedrich A.W. , Gerber A. et al. (2006). New aspects in the pathogenesis of enteropathic hemolytic uremic syndrome. Semin Thromb Hemost. 32: 105–112.
8- Goldwater P.N. (2007): Treatment and prevention of enterohemorrhagic Escherichia coli infection and hemolytic uremic syndrome. Expert Rev Anti Infect Ther. 5: 653–663.
9- Lee S.Y. , Lee M.S. , Cherla R.P. , et al. (2008). Shiga toxin 1 induces apoptosis through the endoplasmic reticulum stress response in human monocytic cells. Cell Microbio. 10: 770–780.
10- Rangel J.M. , Sparling P.H. , Crowe C. , Griffin P.M. and Swerdlow D.L. (2005). Epidemiology of Escherichia coli O157:H7 outbreaks United States 1982–2002. Emerg. Infect. Dis. 11 603–609.
11- Savage P.J. , Campellone K.G. and Leong J.M. (2007). Interaction of enterohemorrhagic Escherichia coli (EHEC) with mammalian cells: Cell adhesion type iii secretion and actin pedestal formation. Curr. Protoc. Microbiol. doi:10.1002/9780471729259.mc05a01s05.
12- Schmidt H. , Scheef J. , Huppertz H. I. , Frosch M. and Karch H.(1999). Escherichia coli O157:H7 and O157:H- strains that do not produce Shiga toxin: phenotypic and genetic characterization of isolates associated with diarrhea and hemolytic-uremic syndrome. J. Clin. Microbiol. 37:3491–3496.
13- Ammon A. , Peterson L. R. and Karch. H. (1999). A large outbreak of hemolytic uremic syndrome caused by an unusual sorbitol-fermenting strain E. coli O157:H-. J. Infect. Dis. 179:1274–1277.
14- Allison L. , Harding S. , Locking M. , Pollock K. , Evans J. , Knight H. , Foster G. , Cowden J. and Hanson M. (2006). Sorbitol-fermenting E. coli O157 in Scotland—laboratory investigation of an outbreak of haemolytic ureamic syndrome abstr.1:1- 4.
15- Bielaszewska M. , Schmidt H. , Liesegang A. , Prager R. , Rabsch W. Tscha¨pe H. , Cizek A. , Janda J. , Blahova K. and Karch H. (2000). Cattle can be a reservoir of sorbitol-fermenting Shiga toxin-producing Escherichia coli O157:H- strains and a source of human diseases. J. Clin. Microbiol. 38: 3470–3473.
16- Proulx F. and Sockett P. (2005). Prospective surveillance of Canadian children with the haemolytic uraemic syndrome. Pediatr Nephrol. 20: 786–790.
17- Garvey P. , McKeown P. , Carroll A. and McNamara E. (2006). Epidemiology of Verotoxigenic E. coli in Ireland 2005. EPI-Insight 7 2–3.
18- Orth D. , Grif K. , Dierich M.P. and Würzner R. (2006). Sorbitol-fermenting Shiga toxinproducing Escherichia coli O157: indications for an animal reservoir. Epidemiology and Infection 134 719–723.
19- Buvens G. , Pierard D. , Hachimi-Idrissi S. and Lauwers S. (2009). First sorbitol-fermenting Verocytotoxin-producing Escherichia coli O157: H- isolated in Belgium. Acta Clinica Belgica 64 59–64.
20- Haugum K. , Lindstedt B.A. , Løbersli I. , Kapperud G. and Brandal L.T. (2012). Identification of the anti-terminator qO111: H- gene in Norwegian sorbitol-fermenting Escherichia coli O157: NM. FEMS Microbiology Letters 329 102–110.
21- Bettelheim K.A. , Whipp M. , Djordjevic S.P. , Ramachandran V. (2002). First isolation outside Europe of sorbitol-fermenting verocytotoxigenic Escherichia coli (STEC) belonging to O group O157. Journal of Medical Microbiology 51 713–714.
22- Kim J. , Nietfeldt J. , Ju J. et al.(2001). Ancestral divergence genome diversification and phylogeographic variation in subpopulations of sorbitol-negative b-glucuronidase negative enterohemorrhagic Escherichia coli O157. J. Bacteriol. , 183: 6885–6897.
23- Bockemu¨hl J. , Karch H. and Tscha¨pe H. (1998). Zur Situation der Infektionen des Menschen durch enteroha¨morrhagische Escherichia coli (EHEC) in Deutschland 1997. Bundesgesundhblatt Suppl. (October):2–5.
24- Liesegang A. , Sachse U. , Prager R. , Claus H. , Steinru¨ck H. , Aleksic S. , Rabsch W. Voigt W. , Fruth A. , Karch H. , Bockemu¨hl J. and Tscha¨pe H. (2000). Clonal diversity of Shiga toxin-producing Escherichia coli O157:H7/H- in Germany—a ten-year study. Int. J. Med. Microbiol. 290:269–278.
25- Karch H. , Janetzki-Mittmann C. , Aleksic S. and Datz M. (1996). Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation DNA-based methods and direct culture. J. Clin. Microbiol. 34:516–519.
26- Bielaszewska M. , Tarr P.I. , Karch H. , Zhang W. and Mathys W. (2005). Phenotypic and molecular analysis of tellurite resistance among enterohemorrhagic Escherichia coli O157:H7 and sorbitol-fermenting O157:NM clinical isolates. J. Clin. Microbiol. 43: 452–454.
27- Smith J.L. , Fratamico P.M. and Gunther N. (2013). Shiga Toxin-Producing Escherichia coli. Adv. Appl. Microbiol. 86:145.
28- Garrido P. , Blanco M. , Moreno-Paz M. , Briones C. , Dahbi G. , Blanco J. and Parro V. (2006). STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of the LEE pathogenicity island of human and animal Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains. Clin. Chem. 52(2): 192–201.
29- Etcheverria A.I. and Padola N.L. (2013). Shiga toxin-producing Escherichia coli: factors involved in virulence and cattle colonization. Virulence. 4: 366-372.
30- Taneike I. , Zhang H. M. , Wakisaka-Saito N. and Yamamoto T. (2002). Enterohaemolysin operon of Shiga toxin-producing Escherichia coli: a virulence function of inflammatory cytokine produc- tion from human monocytes. FEBS Letters 524(1-3): 219–224.
31- Sonntag A. , Prager R. , Bielaszewska M. , Zhang W. , Fruth A. Tscha¨pe H. and Karch H. (2004). Phenotypic and genotypic analyses of enterohemorrhagic Escherichia coli O145 strains from patients in Germany. J. Clin. Microbiol. 42:954–962.
32- Zhang W. , Mellmann A. , Sonntag A. , Wieler L. , Bielaszewska M. , Tscha¨pe H. , Karch H. and Friedrich A. W. (2007). Structural and functional differences between disease-associated genes of enterohaemorrhagic Escherichia coli O111. Int. J. Med. Microbiol. 297:17–26.
33- Khalid I. S. , Mahmoud A. M. , Asmaa M.A. and Tomohiro T.( 2013). Prevalence genetic characterization and virulence genes of sorbitol-fermenting Escherichia coli O157:H- and E. coli O157:H7 isolated from retail beef. Int J Food Microbiol. August 1 , 165(3): 295–301.
34- Stromberg Z. R. (2015). Detection Methods and Intestinal Adherence of non-O157 Shiga Toxin-Producing Escherichia coli. Dissertations in Vet. and Bio.Sci. Lincoln Nebraska.
35- Hara-Kudo J. (2003). Fluorogenic and chromogenic media The rapid technique for isolation of V. cholera. J. Appl. & Eviron. Microbial. 67: 5819-5823.
36- Tarr P. I. Gordon C. A and Chandler W.L. (2005). Shiga-toxin-producingEscherichia coli and haemolytic uraemic syndrome. Lancet 365:1073-1086.
37- Ngwa G. A , Schop R , Weir S , León-Velarde C.G , Odumeru J.A.(2013). Detection and enumeration of E. coli O157:H7 in water samples by culture and molecular methods. J Microbiol Methods. 15 ,92(2):164-72.
38- De L. , Garcia S. , Orden J.A. , Ruiz-quiteria J.A. , Diez R. and Cid D. (2002). Prevalence and characteristics of attaching and effacing strains of Escherichia coli isolated from diarrheic and healthy sheep and goats. Am. J. Vet. Res. 63: 262–266.
39- Karmali M. A , Petric M. and Bielaszewska M. (1999). Evaluation of a microplate latex agglutination method (Verotox-F Assay) for detecting and characterizing verotoxins(Shiga Toxins)in Escherichia coli. J.Clin. Microbiol. 37:396–399.
40- Al –Dawmy F. A.A. and Yousif A.A.(2013). Prevalence of E.coli O157:H7 in intestinal and Urinary tract infection in children .International Journal of Advanced Research Vol.1( 8): 111-120.
41- Dean-Nystrom E. A. , Bosworth B. T. , Moon H. W. and O’Brien A. D. (1998). Escherichia coli O157:H7 requires intimin for enteropathogenicity in calves. Infect. Immun. 66:4560–4563.
42- Dean-Nystrom E. A. , Bosworth B. T. , Cray W. C. and Moon H. W. (1997). Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatal calves. Infect. Immun. 65:1842–1848.
43- Eisenhauer P. B. , Chaturvedi P. , Fine R. E. , Ritchie A. J. , Pober J. S. , Cleary T. G. and Newburg D. S. (2001). Tumor necrosis factor alpha increases human cerebral endothelial cell Gb3 and sensitivity to Shiga toxin. Infect. Immun. 69:1889–1894.
44- Bielaszewska M. Schmidt H. , Karmali M. A. , Khakhria R. , Janda J. , Blahova K. and Karch H. (1998). Isolation and characterization of sorbitolfermenting Shiga toxin-producing Escherichia coli O157:H- strains in the Czech Republic. J. Clin. Microbiol. 36:2135–2137.
45- Tesh V. L. , Burris J. A. , Owens J. W. , Gordon V. M. , Wadolkowski E. A. , O’Brien A. D. and Samuel J. E. (1993). Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice. Infect. Immun. 61:3392–3402.
46- Lahti E.(2003). Cattle and reindeer as possible sources of Escherichia coli O157 infection in humans. PhD thesis. Helsinki: Faculty of Veterinary Medicine University of Helsinki.
47- Pohlenz J. F. Winter K. R. and Dean-Nystrom E. A. (2005). Shiga-ToxigenicEscherichia coli-Inoculated Neonatal Piglets Develop Kidney Lesions That Are Comparable to Those in Humans with Hemolytic-Uremic Syndrome . Infection and Immunity 73(1) 612–616.
48- Brooks J.T. , Sowers E.G. , Wells J.G. , Greene K.D. , Griffin P.M. , Hoekstra R.M. and Strockbine N.A. (2005). Non-O157 Shiga toxin-producing Escherichia coli infections in the United States 1983–2002. J. Infect. Dis. 92: 1422-1429.
49- Lathrop S. , Edge K. and Bareta J. (2009). Shiga Toxin-producing Escherichia coli New Mexico USA 2004-2007. Emerg. Infect. Dis. 15: 1289‐1291.
50- Friedrich A. W. Zhang W. , Bielaszewska M. , Mellmann A. , Ko¨ck R. , Fruth A. , Tscha¨pe H. and Karch H. (2007). Prevalence virulence profiles and clinical significance of Shiga toxin-negative variants of enterohemorrhagic Escherichia coli O157 infection in humans. Clin. Infect. Dis. 45:39–45.
51- Teel L. D. , Schmitt C. K. , Melton-Celsa A. R. and O’Brien A. D. (2002). One of two copies of the gene for the activatable Shiga toxin type 2d in Escherichia coli O91:H21 strain B2F1 is associated with an inducible bacteriophage. Infect. Immun. 70:4282–4291.
52- Siegler R., L.T. G. Obrig, T. J. Pysher, V. L. Tesh, N. D. Denkers, and F. B. Taylor. (2003). Response to Shiga toxin 1 and 2 in a baboon model of hemolytic uremic syndrome. Pediatr. Nephrol. 18:92–96.
53- Paton A. W. and Paton J. C. (1998). Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1 stx2 eaeA enterohemorrhagic E. coli hlyA rfbO111 and rfbO157. J. Clin. Microbiol. 36: 598–602.
54- Gannon V. P. , King R. K. , Kim J. Y. and Thomas E. J. (1992). Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Appl. Environ. Microbiol. 58: 3809–3815.
55- Fratamico P.M. , Sackitey S.K. , Wiedmann M. and Deng M.Y. (1995). Detection of Escherichia coli O157:H7 by multiplex PCR. J. Clin. Microbiol. 33: 2188-2191.
56- Quinn P.J. , Carter M.E. , Markey B. and Carter G.R. (2004). Clinical Veterinary microbiology. 6th ed. Mosby an imp. Wolf London.
Published
2018-01-15
How to Cite
Jarad, A. S., & R. M. Al-Samawy, E. (2018). Isolation and genotyping of sorbitol fermenting of E.coli O157:H- strain isolated from children and calves in AL –Samawa Province. Al-Qadisiyah Journal of Pure Science, 22(2), 277-290. Retrieved from https://journalsc.qu.edu.iq/index.php/JOPS/article/view/571
Section
Articles