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Chapter 14 : Detection of Shiga Toxin-Producing from Nonhuman Sources and Strain Typing

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Detection of Shiga Toxin-Producing from Nonhuman Sources and Strain Typing, Page 1 of 2

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Abstract:

Shiga toxin (verotoxin)-producing (STEC) strains were first described in 1977 by their ability to cause cytotoxic effects on Vero cells ( ). In the early days, production of Shiga toxin (Stx) by was thought to be associated with certain human enteropathogenic (EPEC) strains ( ). STEC was recognized as a zoonotic agent when the first known outbreaks occurred in 1982. STEC O157:H7, a rare serotype of , was isolated from patients developing hemorrhagic colitis (HC) after they ingested undercooked beef in restaurant chains ( ). STEC O157 was isolated from the incriminated beef, indicating a possible transmission from a bovine reservoir. In the following years cattle were identified as a worldwide natural reservoir for STEC O157 and non-O157 strains and as an important source of food contamination ( ). Repeated sampling of cattle revealed that the agent was occasionally present in the majority of cattle farms in Europe and America ( ). However, recent findings on the epidemiology of enteroaggregative Shiga toxin-producing (EAEC-STEC) O104:H4 indicate that not all STEC strains have a zoonotic origin ( ).

Citation: Beutin L, Fach P. 2015. Detection of Shiga Toxin-Producing from Nonhuman Sources and Strain Typing, p 299-319. In Sperandio V, Hovde C (ed), Enterohemorrhagic and Other Shiga Toxin-Producing . ASM Press, Washington, DC. doi: 10.1128/microbiolspec.EHEC-0001-2013

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Figure 1

Identification and isolation of STEC from mixed samples of bacteria with the Stx colony immunoblot. Mauve-stained Stx2-positive STEC colonies are detected from a sample containing STEC mixed with Stx-negative bacteria. doi:10.1128/microbiolspec.EHEC-0001-2013.f1

Citation: Beutin L, Fach P. 2015. Detection of Shiga Toxin-Producing from Nonhuman Sources and Strain Typing, p 299-319. In Sperandio V, Hovde C (ed), Enterohemorrhagic and Other Shiga Toxin-Producing . ASM Press, Washington, DC. doi: 10.1128/microbiolspec.EHEC-0001-2013
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Figure 2

Suitability of conventional PCRs for detection of genes in enriched samples from minced meat. The same panel of enrichment cultures (#84–92) was tested with two PCR systems using the MK1/MK2 primer system (A) and the Lin-up/Lin-down primer system (B) ( ). Primers MK1/MK2 generate 230-bp-long PCR products (arrow), and a ladder of nonspecific bands of different sizes is visible in almost all meat samples. (B) Lin-up/Lin-down generates 900-bp size PCR products (arrow), and only two STEC-positive meat samples (#88+92) give corresponding PCR products. M, molecular weight standard, K, positive STEC control; arrow, position of the specific PCR product. doi:10.1128/microbiolspec.EHEC-0001-2013.f2

Citation: Beutin L, Fach P. 2015. Detection of Shiga Toxin-Producing from Nonhuman Sources and Strain Typing, p 299-319. In Sperandio V, Hovde C (ed), Enterohemorrhagic and Other Shiga Toxin-Producing . ASM Press, Washington, DC. doi: 10.1128/microbiolspec.EHEC-0001-2013
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Figure 3

Importance of cultural enrichment for the detection of viable STEC in meat samples by conventional and real-time PCR. Agarose gel showing Stx2-specific PCR products in enrichment cultures. The PCR was performed with common stx2 PCR primers LP43/LP44 as described in reference . The corresponding CT values from real-time PCR are indicated below. (A) Lanes: m, molecular weight standard; –c, Stx-negative control strain; +c, Stx2-positive control strain. (B) Sample containing nonamplifiable genes. PCR and real-time PCR are performed after 6, 8, and 18 h of enrichment. gene-specific signals are decreasing following prolonged enrichment with both conventional and real-time PCR. (C) Sample containing amplifiable genes. PCR and real-time PCR are performed after 6, 8, and 18 h of enrichment. gene-specific signals are increasing following prolonged enrichment with both conventional and real-time PCR. doi:10.1128/microbiolspec.EHEC-0001-2013.f3

Citation: Beutin L, Fach P. 2015. Detection of Shiga Toxin-Producing from Nonhuman Sources and Strain Typing, p 299-319. In Sperandio V, Hovde C (ed), Enterohemorrhagic and Other Shiga Toxin-Producing . ASM Press, Washington, DC. doi: 10.1128/microbiolspec.EHEC-0001-2013
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Figure 4

Stool sample of a human patient with HUS infected with EHEC O157:H7 grown on enterohemolysin agar ( ). Morphological differences between the hemolysis zones on enterohemolysin agar facilitate the detection of enterohemolytic, -positive STEC strains. Plating of 10-fold dilutions from stool enrichment cultures grown in tryptic soy broth for detection of enterohemolytic, presumptive STEC colonies. Four morphologically different types of bacteria designated from A to D were detected: A, alpha-hemolysin producing, Stx-negative B, hemolysin-negative C, enterohemolytic ()-positive STEC O157:H7; D, . doi:10.1128/microbiolspec.EHEC-0001-2013.f4

Citation: Beutin L, Fach P. 2015. Detection of Shiga Toxin-Producing from Nonhuman Sources and Strain Typing, p 299-319. In Sperandio V, Hovde C (ed), Enterohemorrhagic and Other Shiga Toxin-Producing . ASM Press, Washington, DC. doi: 10.1128/microbiolspec.EHEC-0001-2013
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Figure 5

Enhancement of selectivity for isolation of STEC from bovine fecal samples on enterohemolysin agar supplemented with vancomycin. (A) Growth of enterohemolytic STEC colonies from a sample of bovine feces on enterohemolysin agar supplemented with 8 mg/liter of vancomycin. The gram-positive flora is suppressed. (B) Growth of enterohemolytic STEC colonies from the same sample of bovine feces on nonselective enterohemolysin agar. The abundant gram-positive flora overgrows STEC present in the sample. doi:10.1128/microbiolspec.EHEC-0001-2013.f5

Citation: Beutin L, Fach P. 2015. Detection of Shiga Toxin-Producing from Nonhuman Sources and Strain Typing, p 299-319. In Sperandio V, Hovde C (ed), Enterohemorrhagic and Other Shiga Toxin-Producing . ASM Press, Washington, DC. doi: 10.1128/microbiolspec.EHEC-0001-2013
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