Dataset Information

Development of a Sandwich ELISA for EHEC O157:H7 Intimin ?1.

ABSTRACT: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin ?1) is one of the most important outer membrane proteins required for EHEC's intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin ?1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples.

SUBMITTER: Zhang X

PROVIDER: S-EPMC5014315 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1.

Zhang Xuehan X Li Meng M Zhang Bicheng B Chen Kangming K He Kongwang K

PloS one 20160907 9

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC's intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O1 ...[more]

PMID: 27603508

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Project description:While significant advances have been made in EHEC pathogenesis, we still do not fully understand the impact of environmental stress on EHEC virulence. During the course of infection, EHEC must evade or overcome several biological barriers, the first of which is the gastric acidity encountered during passage through the stomach. EHEC is remarkable in its ability to tolerate this acidity. There are four different acid resistance systems that provide E. coli O157:H7 protection against exposure to low pH (2-2.5). Interestingly, EHEC uses these acid resistance systems differentially for survival in foods versus the bovine intestinal tract. The glutamate-dependent acid-resistance system is thought to offer the best protection below pH 3. Since the infectious dose of EHEC is so low (50-100 organisms), acid resistance becomes an important virulence trait. Studies of EHEC response to acid stress have focused primarily on levels of acid tolerance and the molecular basis of tolerance. However, the impact of acid stress on EHEC virulence is less well understood. In the related pathogen, EPEC, the plasmid-encoded regulator, Per, that regulates expression of many EPEC virulence factors, is regulated negatively at pH 5.5 and positively at pH 8.0, suggesting that virulence gene expression is repressed during mild acid stress and enhanced in alkaline pH typical of the small intestine. Expression of EPEC type III secreted factors involved in A/E lesion formation has been shown to be influenced by factors including culture media, iron and calcium levels. Protein secretion was inhibited at pH 6 and 8. In a third study, a gadE (encoding acid resistance regulator) mutation resulted in increased adhesion of E.coli O157:H7 to colonic epithelial cells, suggesting negative regulation of one or more adhesins. Other studies have reported that shiga toxin production is sensitive to culture conditions including pH. However, there are no studies of EHEC virulence changes after more severe acid stress nor studies linking stressed EHEC virulence phenotype with transcriptional changes. The goal of this study was to determine how acid stress affects EHEC virulence properties and through microarray analysis, define the genetic basis for these changes. Understanding how acid stress modulates the virulence potential of this pathogen is essential for delineating the pathogenesis of disease caused by EHEC infection and may offer novel approaches to prevent and treat EHEC infections.

Project description:Enterohemorrhagic Escherichia coli (EHEC) are important human pathogens, causing hemorrhagic colitis and hemolytic uraemic syndrome in humans. E. coli O157:H7 is the most common serotype associated with EHEC infections worldwide, although other non-O157 serotypes cause life-threatening infections. Cattle are a main reservoir of EHEC and intervention strategies aimed at limiting EHEC excretion from cattle are predicted to lower the risk of human infection. We have previously shown that immunization of calves with recombinant versions of the type III secretion system (T3SS)-associated proteins EspA, intimin and Tir from EHEC O157:H7 significantly reduced shedding of EHEC O157 from experimentally-colonized calves, and that protection could be augmented by the addition of H7 flagellin to the vaccine formulation. The main aim of the present study was to optimize our current EHEC O157 subunit vaccine formulations by identifying the key combinations of these antigens required for protection. A secondary aim was to determine if vaccine-induced antibody responses exhibited cross-reactive potential with antigens from other EHEC serotypes. Immunization with EspA, intimin and Tir resulted in a reduction in mean EHEC O157 shedding following challenge, but not the mean proportion of calves colonized. Removal of Tir resulted in more prolonged shedding compared with all other groups, whereas replacement of Tir with H7 flagellin resulted in the highest levels of protection, both in terms of reducing both mean EHEC O157 shedding and the proportion of colonized calves. Immunization of calves with recombinant EHEC O157 EspA, intimin and Tir resulted in the generation of antibodies capable of cross-reacting with antigens from non-O157 EHEC serotypes, suggesting that immunization with these antigens may provide a degree of cross-protection against other EHEC serotypes. Further studies are now required to test the efficacy of these vaccines in the field, and to formally test the cross-protective potential of the vaccines against other non-O157 EHEC.

Dataset Information

Development of a Sandwich ELISA for EHEC O157:H7 Intimin ?1.

Publications

Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1.

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