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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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BLAST alignment of a diffuse adherence dafa/daa operon (Accession number AF325672) and region 2 of the aaf/II operon from strain 042 (Accession number AF114828). Genbank Annotated orfs are shown for dafa (top) and aaf, region 2 (bottom). Connectors show regions of 80% or more identity at the DNA level. The figure was generated using the Artemis Comparison Tool (ACT)[45].
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Figure 2: BLAST alignment of a diffuse adherence dafa/daa operon (Accession number AF325672) and region 2 of the aaf/II operon from strain 042 (Accession number AF114828). Genbank Annotated orfs are shown for dafa (top) and aaf, region 2 (bottom). Connectors show regions of 80% or more identity at the DNA level. The figure was generated using the Artemis Comparison Tool (ACT)[45].

Mentions: A BLAST search of the recently completed genome of cross-hybridizing EAEC strain 042 at http://www.sanger.ac.uk/cgi-bin/blast/submitblast/escherichia_shigella, revealed that the most similar target for the daaC probe that can be identified in the 042 genome in silico is the aafC gene, part of the AAF/II-encoding operon, with 294 (84%) identical nucleotides and only five single nucleotide gaps over the length of the homologous 344 nucleotide daaC probe region, at the DNA level (Figure 2). The aafC gene is located on the large virulence plasmid of strain 042 and other AAF/II-positive EAEC [21]. The daaC gene, on the other hand, may be chromosomally or plasmid located [7]. Therefore, although genuine target strains often have only one copy of daaC, cross hybridizing strains could potentially have one or more copies of the aafC gene, a factor that could also contribute to the hybridization signals of aafC-positive EAEC. Elias et al. have previously noticed that enteroaggregative E. coli strains hybridize to the daaC probe and proposed that the cross-hybridizing region was within the AAF/II fimbrial biogenesis cluster [21]. In this study, all but one strain possessing the aafA gene from the AAF/II biogenesis cluster hybridized with the daaC probe. We hybridized the panel of 26 well-studied strains to a DNA fragment probe for the aggregative adherence fimbrial usher gene, aggC, which has been demonstrated by Bernier et al. to hybridize to both aggC and aafC [18]. All the aafA-positive, daaC-positive strains hybridized with this probe (Table 2). In summary, we report that daaC cross-hybridization arises from an 84% identity between the probe sequence and the EAEC aafC gene, and that this degree of similarity significantly compromises diagnostic use of the existing daaC probe for the detection of DAEC.


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)

BLAST alignment of a diffuse adherence dafa/daa operon (Accession number AF325672) and region 2 of the aaf/II operon from strain 042 (Accession number AF114828). Genbank Annotated orfs are shown for dafa (top) and aaf, region 2 (bottom). Connectors show regions of 80% or more identity at the DNA level. The figure was generated using the Artemis Comparison Tool (ACT)[45].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: BLAST alignment of a diffuse adherence dafa/daa operon (Accession number AF325672) and region 2 of the aaf/II operon from strain 042 (Accession number AF114828). Genbank Annotated orfs are shown for dafa (top) and aaf, region 2 (bottom). Connectors show regions of 80% or more identity at the DNA level. The figure was generated using the Artemis Comparison Tool (ACT)[45].
Mentions: A BLAST search of the recently completed genome of cross-hybridizing EAEC strain 042 at http://www.sanger.ac.uk/cgi-bin/blast/submitblast/escherichia_shigella, revealed that the most similar target for the daaC probe that can be identified in the 042 genome in silico is the aafC gene, part of the AAF/II-encoding operon, with 294 (84%) identical nucleotides and only five single nucleotide gaps over the length of the homologous 344 nucleotide daaC probe region, at the DNA level (Figure 2). The aafC gene is located on the large virulence plasmid of strain 042 and other AAF/II-positive EAEC [21]. The daaC gene, on the other hand, may be chromosomally or plasmid located [7]. Therefore, although genuine target strains often have only one copy of daaC, cross hybridizing strains could potentially have one or more copies of the aafC gene, a factor that could also contribute to the hybridization signals of aafC-positive EAEC. Elias et al. have previously noticed that enteroaggregative E. coli strains hybridize to the daaC probe and proposed that the cross-hybridizing region was within the AAF/II fimbrial biogenesis cluster [21]. In this study, all but one strain possessing the aafA gene from the AAF/II biogenesis cluster hybridized with the daaC probe. We hybridized the panel of 26 well-studied strains to a DNA fragment probe for the aggregative adherence fimbrial usher gene, aggC, which has been demonstrated by Bernier et al. to hybridize to both aggC and aafC [18]. All the aafA-positive, daaC-positive strains hybridized with this probe (Table 2). In summary, we report that daaC cross-hybridization arises from an 84% identity between the probe sequence and the EAEC aafC gene, and that this degree of similarity significantly compromises diagnostic use of the existing daaC probe for the detection of DAEC.

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