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The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coli.

Snelling AM, Macfarlane-Smith LR, Fletcher JN, Okeke IN - BMC Microbiol. (2009)

Bottom Line: The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains.Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies.This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biomedical Sciences and Bradford Infection Group, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK. A.M.Snelling@Bradford.ac.uk

ABSTRACT

Background: The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed.

Results: We demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies.

Conclusions: A PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.

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Pair-wise alignment between the daaD and aafB gene regions used as a basis for a discriminatory PCR-RFLP. Identities are asterixed. Oligonucleotide binding sites for the PCR-RFLP protocol are underlined and AluI restriction sites are highlighted in boldface.
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Figure 3: Pair-wise alignment between the daaD and aafB gene regions used as a basis for a discriminatory PCR-RFLP. Identities are asterixed. Oligonucleotide binding sites for the PCR-RFLP protocol are underlined and AluI restriction sites are highlighted in boldface.

Mentions: The daaC, aafC and similar genes are predicted to encode ushers for adhesin export and are highly similar across the entire length of the genes, both to each other and to usher genes from other adhesin operons (Figure 2). Downstream of the usher genes is a smaller open reading frame. In the case of the EAEC aafC, the downstream gene, aafB, has not been experimentally defined and may encode a protein that represents the AAF/II tip adhesin [22]. The aafB predicted product shares 59% identity with the DAEC AfaD/DaaD, a non-structural adhesin encoded by a gene downstream of afaC/daaC [21]. At the DNA level, aafB and daaD/afaD genes also share some identity (63% over the most similar 444 bp region), but this is less than that of the usher genes (Figure 3).


The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coli.

Snelling AM, Macfarlane-Smith LR, Fletcher JN, Okeke IN - BMC Microbiol. (2009)

Pair-wise alignment between the daaD and aafB gene regions used as a basis for a discriminatory PCR-RFLP. Identities are asterixed. Oligonucleotide binding sites for the PCR-RFLP protocol are underlined and AluI restriction sites are highlighted in boldface.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803494&req=5

Figure 3: Pair-wise alignment between the daaD and aafB gene regions used as a basis for a discriminatory PCR-RFLP. Identities are asterixed. Oligonucleotide binding sites for the PCR-RFLP protocol are underlined and AluI restriction sites are highlighted in boldface.
Mentions: The daaC, aafC and similar genes are predicted to encode ushers for adhesin export and are highly similar across the entire length of the genes, both to each other and to usher genes from other adhesin operons (Figure 2). Downstream of the usher genes is a smaller open reading frame. In the case of the EAEC aafC, the downstream gene, aafB, has not been experimentally defined and may encode a protein that represents the AAF/II tip adhesin [22]. The aafB predicted product shares 59% identity with the DAEC AfaD/DaaD, a non-structural adhesin encoded by a gene downstream of afaC/daaC [21]. At the DNA level, aafB and daaD/afaD genes also share some identity (63% over the most similar 444 bp region), but this is less than that of the usher genes (Figure 3).

Bottom Line: The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains.Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies.This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biomedical Sciences and Bradford Infection Group, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK. A.M.Snelling@Bradford.ac.uk

ABSTRACT

Background: The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed.

Results: We demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies.

Conclusions: A PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.

Show MeSH
Related in: MedlinePlus