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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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Colony blot of representative reference strains hybridized to the daaC probe from pSLM862. The blot was hybridized with biotinylated probe under high stringency conditions. Strains spotted on the membrane were 1: E. coli K-12 DH1, 2: E. coli NCTC 10418, 3: enteropathogenic E. coli E2348/69, 4: enterohaemorrhagic E. coli C412, 5: enterotoxigenic E. coli H10407, 6: enteroaggregative E. coli 042, 7: diffusely adherent E. coli DAEC1, 8: enterotoxigenic E. coli P006413, 9: enterotoxigenic E. coli P006371, 10: enteroinvasive E. coli G24; 11: Cytotoxic necrotizing toxin-producing E. coli P006254, 12: diffusely-adherent E. coli DA57-1166.
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Figure 1: Colony blot of representative reference strains hybridized to the daaC probe from pSLM862. The blot was hybridized with biotinylated probe under high stringency conditions. Strains spotted on the membrane were 1: E. coli K-12 DH1, 2: E. coli NCTC 10418, 3: enteropathogenic E. coli E2348/69, 4: enterohaemorrhagic E. coli C412, 5: enterotoxigenic E. coli H10407, 6: enteroaggregative E. coli 042, 7: diffusely adherent E. coli DAEC1, 8: enterotoxigenic E. coli P006413, 9: enterotoxigenic E. coli P006371, 10: enteroinvasive E. coli G24; 11: Cytotoxic necrotizing toxin-producing E. coli P006254, 12: diffusely-adherent E. coli DA57-1166.

Mentions: In the course of an aetiologic study of diarrhoea focused on diarrhoeagenic E. coli, we observed that in addition to recognizing diffusely adherent E. coli strains, the daaC probe was hybridizing to colony blots of some test and control strains that showed aggregative adherence. We hybridized the daaC probe with colony blots of a well-studied panel of 26 EAEC strains and seven DAEC strains. We found that five of these EAEC strains hybridized with the daaC probe, including prototypical EAEC strain 042, even when conditions were of slightly greater stringency than those reported in the literature [11]. All five had previously been documented to carry the aafA gene, encoding the structural subunit of the AAF/II fimbriae [17]. As shown in Figure 1, hybridization was noticeably weaker than to the DAEC strains, but sufficiently strong to confound strain categorization. Twenty-one strains lacking aafA did not hybridize with the daaC probe, irrespective of whether they hybridized to the probe for aggA, the structural subunit gene for AAF/I fimbriae (Table 2).


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)

Colony blot of representative reference strains hybridized to the daaC probe from pSLM862. The blot was hybridized with biotinylated probe under high stringency conditions. Strains spotted on the membrane were 1: E. coli K-12 DH1, 2: E. coli NCTC 10418, 3: enteropathogenic E. coli E2348/69, 4: enterohaemorrhagic E. coli C412, 5: enterotoxigenic E. coli H10407, 6: enteroaggregative E. coli 042, 7: diffusely adherent E. coli DAEC1, 8: enterotoxigenic E. coli P006413, 9: enterotoxigenic E. coli P006371, 10: enteroinvasive E. coli G24; 11: Cytotoxic necrotizing toxin-producing E. coli P006254, 12: diffusely-adherent E. coli DA57-1166.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Colony blot of representative reference strains hybridized to the daaC probe from pSLM862. The blot was hybridized with biotinylated probe under high stringency conditions. Strains spotted on the membrane were 1: E. coli K-12 DH1, 2: E. coli NCTC 10418, 3: enteropathogenic E. coli E2348/69, 4: enterohaemorrhagic E. coli C412, 5: enterotoxigenic E. coli H10407, 6: enteroaggregative E. coli 042, 7: diffusely adherent E. coli DAEC1, 8: enterotoxigenic E. coli P006413, 9: enterotoxigenic E. coli P006371, 10: enteroinvasive E. coli G24; 11: Cytotoxic necrotizing toxin-producing E. coli P006254, 12: diffusely-adherent E. coli DA57-1166.
Mentions: In the course of an aetiologic study of diarrhoea focused on diarrhoeagenic E. coli, we observed that in addition to recognizing diffusely adherent E. coli strains, the daaC probe was hybridizing to colony blots of some test and control strains that showed aggregative adherence. We hybridized the daaC probe with colony blots of a well-studied panel of 26 EAEC strains and seven DAEC strains. We found that five of these EAEC strains hybridized with the daaC probe, including prototypical EAEC strain 042, even when conditions were of slightly greater stringency than those reported in the literature [11]. All five had previously been documented to carry the aafA gene, encoding the structural subunit of the AAF/II fimbriae [17]. As shown in Figure 1, hybridization was noticeably weaker than to the DAEC strains, but sufficiently strong to confound strain categorization. Twenty-one strains lacking aafA did not hybridize with the daaC probe, irrespective of whether they hybridized to the probe for aggA, the structural subunit gene for AAF/I fimbriae (Table 2).

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