Detection of Virulence Factors Shiga-Toxin 1 and 2 and Intimin in Escherichia coli O104

Samantha Fanelli November 17th, 2011 65 samples of Escherichia coli O104 of various origins were analyzed for the presence of intimin (Eae) and Shiga-toxin 1 and 2 (Stx1 and Stx2) via polymerase chain reaction (PCR) and gel electrophoresis. Associations between the serotype and the presence of known virulence factors were investigated in light of the recent deadly outbreak in Germany. All samples were obtained from the E. Coli Reference Center, Penn State University, University Park, Pennsylvania. Of the 65 samples, 12 were positive for Stx1, 4 were positive for Stx2, 49 were negative for all three virulence factors, and none were positive for EAE or both Stx1 and Stx2. Though the data suggest no definitive association between the O104 serotype and presence of these virulence factors, conclusions can still be drawn based on that lack of consistency. The presence of these virulence factors in the O104 serotype appears to be atypical based on these results, and the lack of correlation between the samples that did test positive suggests a random dispersion of these genes. __________________________________________________________

Introduction Escherichia coli O104:H4, a typically non-virulent strain, was responsible for 3816 reported cases of infection, of which 52 were fatal, in contaminated sprouts in the late spring and early summer of 2011. Between May 8th and July 4th, over 800 cases, including 361 of those that were fatal, reported hemolytic uremic syndrome (HUS), an acute form of kidney failure that is indicative of Shiga toxin-producing E. coli (STEC) infections.2 Epidemiological profiles of the O104:H4 strain determined it possessed a variant of the Shiga-toxin 2 (Stx2) gene, but lacked intimin or Shiga-toxin 1 (Stx1).1 These three genes are general indicators of pathogenicity of the strain. Intimin is an adhesive factor responsible for tightly attaching STEC strains to the epithelial lining of the intestine and is encoded by the E. coli attaching and effacing (Eae) gene3. Stx1 and Stx2 genes encode for the production of Shiga toxins 1 and 2, which are capable of causing enterohaemorrhagic disease in humans.4 STEC O104 is considered very rare, and this particular strain was abnormally pathogenic.5 The curiously high pathogenicity of this particular strain stimulated an investigation into other O104 strains to determine the occurrence of these virulence factors in the O104 serotype. Materials and Methods 65 isolates of the O104 serotype were obtained from the E. Coli Reference Center, Penn State University, University Park, Pennsylvania from various species and geographical origins. They were grown on tryptic soy agar plates for 24 hours, and the DNA was isolated by heating a small sample of each bacterium mixed with distilled water and centrifuging to 100oC. Multiplex polymerase chain reaction (PCR) was then performed on each isolate along with gel electrophoresis targeting Eae, Stx1, and Stx2. A working mix of 1.457 l of enzyme diluent, 0.319 l of deoxyribonucleotide diphosphates (dNTPs), 0.128 l of Taq, 2.363 l of cresol red, and 3.722 l of sterile water was prepared and mixed with each of the Eae, Stx1, and Stx2 pre-prepared primers in designated
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proportions to create the PCR working mix. 8l of working mix and 3l of template DNA from each sample, along with positive and negative controls, were placed in a 96 well plate with film and run through a thermocycler. A 1% agarose gel was used for electrophoresis of each sample along with a DNA ladder, and the amplified samples were visualized and photographed with the use of ultraviolet light. Once strains positive for any of the genes of interest were identified, these strains were analyzed again to test for false positives. A 3mM master mix was prepared for a standard PCR analysis using 184l of enzyme diluent, 40l of deoxyribonucleotide diphosphates (dNTPs), 16l of Taq, 220l of cresol red, and 540l of sterile water. Working mixes were made for each individual gene and used only with the samples that tested positive for that individual gene. The rest of the analysis proceeded the same as the previous PCR and gel electrophoresis. Results
Table 1. O104 Strains Tested for Presence of EAE, Stx1, and Stx2 Genes Sample 0 0122 0 3194 0 3359 0 3362 1 1952 1 2185 1 2386 1 2633 1 2673 1 2806 1 2807 1 2824 1 2825 1 3285 2 0936 3 3790 EAE Stx1 + + + + + + Stx2 + Sample 4 0898 4 2039 4 2366 4 2458 5 1515 5 3526 6 0778 6 0779 6 0800 6 0829 6 0830 6 1342 6 1343 6 1344 6 1345 6 1349 EAE Stx1 + + + + + + Stx2 Sample 6 1353 6 1354 7 0441 7 1708 9 0124 10 0293 78 0180 82 0531 82 0833 83 0711 83 0712 83 0713 84 0801 85 0858 86 1602 92 1238 EAE Stx1 Stx2 Sample 95 3781 95 3782 95 3783 95 3784 97 0061 97 0562 97 0563 97 1271 97 1272 97 1273 97 1274 97 1275 97 1276 99 0230 99 1282 99 1324 99 1936 EAE Stx1 Stx2 + + + -

Figure 1. Gel electrophoresis results for Stx1 positive E. coli O104 isolates. Lane 1 DNA ladder, Lane 2 Stx1 positive control (43895), Lane 3 Stx1 negative control (K12), Lane 4 Sample 1 2673, Lane 5 Sample 1 2806, Lane 6 Sample 1 2807, Lane 7 Sample 1 2824, Lane 8 Sample 1 2825, Lane 9 Sample 1 3285, Lane 10 DNA ladder, Lane 11 Sample 5 3526, Lane 12 Sample 6 0778, Lane 13 Sample 6 0779, Lane 14 Sample 6 0800, Lane 15 Sample 6 0829, Lane 16 6 0830, Lane 17 99 1936

Figure 2. Gel electrophoresis results of Stx 2 positive E. coli O104 isolates. Lane 1 DNA ladder, Lane 2 Stx2 positive control (43895), Lane 3 Stx2 negative control (K12), Lane 4 Sample 0 0122, Lane 5 Sample 97 1274, Lane 6 Sample 97 1275, Lane 7 Sample 97 1276,

Table 2. Sample Collection Data for Stx1 or Stx2 Positive Samples Sample 0.0122 1.2673 1.2806 1.2807 1.2824 1.2825 1.3285 5.3526 6.0778 6.0779 6.0800 6.0829 6.0830 97.1274 97.1275 97.1276 Stx1/Stx2 Stx2 Stx1 Stx1 Stx1 Stx1 Stx1 Stx1 Stx1 Stx1 Stx1 Stx1 Stx1 Stx1 Stx2 Stx2 Stx2 Species Unknown Bovine Bovine Bovine Bovine Bovine Water Ovine Carcass Carcass Carcass Carcass Carcass Bovine (Calf) Bovine (Calf) Bovine (Calf) Investigator National Institute of Health, Korea USDA-ARS-RLHUSMARC USDA-ARS-RLHUSMARC USDA-ARS-RLHUSMARC USDA-ARS-RLHUSMARC USDA-ARS-RLHUSMARC Kensico Laboratory USDA-ARS-RLHUSMARC USDA-ARS-RLHUSMARC USDA-ARS-RLHUSMARC USDA-ARS-RLHUSMARC USDA-ARS-RLHUSMARC USDA-ARS-RLHUSMARC Michigan State University Michigan State University Michigan State University Location Seoul, South Korea Clay Center, NE Clay Center, NE Clay Center, NE Clay Center, NE Clay Center, NE Valhalla, NY Clay Center, NE Clay Center, NE Clay Center, NE Clay Center, NE Clay Center, NE Clay Center, NE East Lansing, MI East Lansing, MI East Lansing, MI Date Collected 2/4/2000 6/5/2001 6/5/2001 6/5/2001 6/5/2001 6/5/2001 8/7/2001 12/15/2005 3/17/2006 3/17/2006 3/17/2006 3/17/2006 3/17/2006 9/19/1997 9/19/1997 9/19/1997

Discussion While a majority of the samples lacked any of these three virulence factors, a fair amount contained either Stx1 or Stx2 (Table 1). No information was available regarding the disease causing status of these strains, so no conclusions can be drawn on whether these samples expressed pathogenicity. Not all STEC are EHEC, thus it is impossible to conclude with certainty whether these strains were pathogenic without consulting further records.4 It is likely they did not, because all samples lacked intimin for the bacteria to adhere to the intestine, but the O104:H4 strain that caused the outbreak in Germany also lacked intimin, thus no conclusions can be drawn. The data available consisting of what species the sample was isolated from as well as the investigative facilities, geographical location, and date received proved little consistency and demonstrated that the distribution of these genes is fairly random (Table 2). Stx2 genes appeared in the earlier dated samples, while the more recent samples after 2001 only possessed the Stx1 gene. This may indicate a shift towards a prevalence of Stx1 over time, and further research could validate such a claim. The samples were also collected from various parts of the country and even the world, with samples from as far as South Korea. This indicates a world-wide prevalence of these genes that is not solely restricted to the United States or any other geographical location. The species were also inconsistent, implying the genes are not any more or less likely to appear in a given species. Distribution among species also appears to be random. Samples collected subsequently from the same herd or even same animal (the data does not specify) possessed the same genes, which is to be expected. Samples 97.1274, 97.1275, and 97.1276 for instance, were all collected at the same time and all possessed only an Stx2 gene. If these samples were collected from the same animal, it would make sense that the E. coli would all be the same. If a single herd was used to take multiple samples, a herd would likely be exposed to similar pathogens, including E. coli, in feed or feces and would possess the same strain. The appearance of Stx1 and Stx2 in these samples, though not very frequent, still offers important insights into general E. coli maintenance, as well as future experiments. The presence of these virulence factors is important to note, because it implies the possibility of an outbreak. O104 is considered a fairly nonpathogenic serotype,5 but as the summer of 2011 and these results indicate, that is certainly not always the case. O104 is more than capable of acquiring and possessing these virulence factors, and frequent monitoring that Stx genes are not becoming increasingly prevalent could be extremely advantageous in preventing another outbreak. Further experimentation on determining what, if any, species or geographical locations are more prone to obtaining these STEC strains could be instrumental in determining at-risk areas, herds, or crop species for outbreaks. Though outbreaks are often tragic, they can provide valuable information in ways to prevent future recurrences and better both food and animal safety.

References (1) Frank, C.; Werber, D.; Cramer, J.P. Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 outbreak in Germany. NE J. Med. 2011, 1 10. (2) Center for Disease Control. Investigation update: outbreak of Shiga toxin-producing E. coli O104 (STEC O104:H4) infections associated with travel to Germany. http://www.cdc.gov/ecoli/2011/ecolio104/ (accessed November 1, 2011). (3) Boerlin, P.; McEwen, S.A.; Boerlin-Petzold, F.; Wilson, J. B.; Johnson, R. P.; Gyles, C. L. Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J. Clin. Microbiol. 1999, 37, 497 - 503. (4) Taylor, C.M. Enterohaemorrhagic Escherichia coli and Shigella dysenteriae type 1-induced haemolytic uraemic syndrome. Pediatr. Nephrol. 2008, 23, 1425 1431. (5) European Food Safety Authority. Shiga toxin-producing E. coli (STEC) O104:H4 2011 outbreaks in Europe: taking stock. EFSA Journal. 2011, 9, 2390.