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The mutation Glu151Asp in the B-component of the Bacillus cereus non-hemolytic enterotoxin (Nhe) leads to a diverging reactivity in antibody-based detection systems.

Didier A, Jeßberger N, Krey V, Dietrich R, Scherer S, Märtlbauer E - Toxins (Basel) (2015)

Bottom Line: The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection.Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody.Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, Faculty of Veterinary Medicine, Ludwig-Maximilians Universität München, 85763 Oberschleißheim, Germany. a.didier@mh.vetmed.uni-muenchen.de.

ABSTRACT
The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.

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(A) Examples of the Duopath® Assay results for the mutant strains MHI 2970, MHI 1668, and MHI 1541. Undiluted supernatants were added to the lateral flow device. All strains showed a positive reaction for Nhe; (B) dilution series of a typical low-producer (MHI 3178) compared to one of the mutant strains (MHI 2970). A positive reaction is visible up to a dilution of 1:100 for the low-producer but only up to 1:10 for the mutant strain. The arrows point to the NheB band; the pink C on the device shows control band.
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toxins-07-04655-f002: (A) Examples of the Duopath® Assay results for the mutant strains MHI 2970, MHI 1668, and MHI 1541. Undiluted supernatants were added to the lateral flow device. All strains showed a positive reaction for Nhe; (B) dilution series of a typical low-producer (MHI 3178) compared to one of the mutant strains (MHI 2970). A positive reaction is visible up to a dilution of 1:100 for the low-producer but only up to 1:10 for the mutant strain. The arrows point to the NheB band; the pink C on the device shows control band.

Mentions: In order to assay the consequences of the point mutation in a further test system, the Duopath® assay was performed. Interestingly, all strains under study reacted positively in the Duopath® test (Table 1, examples depicted in Figure 2A). Only when a dilution series of the supernatants was applied, the signal of the mutant strains faded out earlier than that of the low-producer MHI 3178 (comparatively depicted for MHI 2970 in Figure 2B). These results are interesting since the mAbs included in the Duopath® test are the 1E11 and 2B11.


The mutation Glu151Asp in the B-component of the Bacillus cereus non-hemolytic enterotoxin (Nhe) leads to a diverging reactivity in antibody-based detection systems.

Didier A, Jeßberger N, Krey V, Dietrich R, Scherer S, Märtlbauer E - Toxins (Basel) (2015)

(A) Examples of the Duopath® Assay results for the mutant strains MHI 2970, MHI 1668, and MHI 1541. Undiluted supernatants were added to the lateral flow device. All strains showed a positive reaction for Nhe; (B) dilution series of a typical low-producer (MHI 3178) compared to one of the mutant strains (MHI 2970). A positive reaction is visible up to a dilution of 1:100 for the low-producer but only up to 1:10 for the mutant strain. The arrows point to the NheB band; the pink C on the device shows control band.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663526&req=5

toxins-07-04655-f002: (A) Examples of the Duopath® Assay results for the mutant strains MHI 2970, MHI 1668, and MHI 1541. Undiluted supernatants were added to the lateral flow device. All strains showed a positive reaction for Nhe; (B) dilution series of a typical low-producer (MHI 3178) compared to one of the mutant strains (MHI 2970). A positive reaction is visible up to a dilution of 1:100 for the low-producer but only up to 1:10 for the mutant strain. The arrows point to the NheB band; the pink C on the device shows control band.
Mentions: In order to assay the consequences of the point mutation in a further test system, the Duopath® assay was performed. Interestingly, all strains under study reacted positively in the Duopath® test (Table 1, examples depicted in Figure 2A). Only when a dilution series of the supernatants was applied, the signal of the mutant strains faded out earlier than that of the low-producer MHI 3178 (comparatively depicted for MHI 2970 in Figure 2B). These results are interesting since the mAbs included in the Duopath® test are the 1E11 and 2B11.

Bottom Line: The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection.Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody.Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, Faculty of Veterinary Medicine, Ludwig-Maximilians Universität München, 85763 Oberschleißheim, Germany. a.didier@mh.vetmed.uni-muenchen.de.

ABSTRACT
The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.

Show MeSH
Related in: MedlinePlus