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Methods

Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method

C. Almeida, J. M. Sousa, R. Rocha, L. Cerqueira, S. Fanning, N. F. Azevedo, M. J. Vieira
C. Almeida
IBB–Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, Braga, PortugalLEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, PortugalBioMode, Biomolecular Determination S.A., SpinPark–Centro de Incubação de Base Tecnológica, Avepark–Zona Industrial da Gandra, Caldas das Taipas Guimarães, Portugal
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J. M. Sousa
IBB–Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal
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R. Rocha
BioMode, Biomolecular Determination S.A., SpinPark–Centro de Incubação de Base Tecnológica, Avepark–Zona Industrial da Gandra, Caldas das Taipas Guimarães, Portugal
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L. Cerqueira
BioMode, Biomolecular Determination S.A., SpinPark–Centro de Incubação de Base Tecnológica, Avepark–Zona Industrial da Gandra, Caldas das Taipas Guimarães, Portugal
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S. Fanning
UCD Centre for Food Safety, School of Public Health, Physiotherapy & Population Science, UCD Centre for Molecular Innovation & Drug Discovery, University College Dublin, Belfield, Dublin, Ireland
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N. F. Azevedo
LEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, PortugalBioMode, Biomolecular Determination S.A., SpinPark–Centro de Incubação de Base Tecnológica, Avepark–Zona Industrial da Gandra, Caldas das Taipas Guimarães, Portugal
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M. J. Vieira
IBB–Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal
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DOI: 10.1128/AEM.01009-13
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    Fig 1

    PNA-FISH outcome for ground beef samples artificially inoculated with 10 CFU/25 g of E. coli O157:H7 CCC-05-12. Results were obtained using a direct hybridization protocol without any additional sample pretreatment (A) or using a pretreatment with 1% Triton X-100 (B). It is possible to observe a decrease in the autofluorescence intensity for panel B in both red (I) and green (II) channels.

Tables

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  • Table 1

    Results of the EcoPNA1169 probe specificity and sensitivity testc

    StrainaSerotypeIsolation originVerotoxin productionPNA FISH outcome
    E. coli CECT 4267O157:H7Human stool from outbreak of hemorrhagic colitisStx1, Stx2+
    E. coli CECT 4782O157:H7Human stool from outbreak of hemorrhagic colitisStx1, Stx2+
    E. coli CECT 4783O157:H7Raw hamburger meat implicated in hemorrhagic colitis outbreakStx1, Stx2+
    E. coli CECT 5947O157:H7stx2 gene has been replaced+
    E. coli NCTC 12900O157:H7NT+
    E. coli CCC-1-12O157:H7Fecal swabStx2+
    E. coli CCC-5-12O157:H7Fecal swabStx2+
    E. coli CCC-7-12O157:H7Fecal swabStx2+
    E. coli CCC-10-12O157:H7Fecal swabStx2+
    E. coli CCC-11-12O157:H7Milk filterStx2+
    E. coli CCC-12-12O157:H7Milk filterStx2+
    E. coli CCC-13-12O157:H7Milk filterStx2+
    E. coli CCC-14-12O157:H7Bovine milk filterStx2+
    E. coli CCC-15-12O157:H7Bovine milk filterStx2+
    E. coli CCC-16-12O157:H7Caprine milk filterStx2+
    E. coli CCC-18-12O157bBovine milk filterStx2+
    E. coli CCC-23-12O157:H7Milk filterNT+
    E. coli CCC-24-12O157:H7Milk filterNT+
    E. coli CCC-25-12O157:H7Milk filterNT+
    E. coli CCC-26-12O157bMilk filterNT+
    E. coli CECT 352O127a:K63(B8):H-EPEC−
    E. coli CECT 504O141:K85(B):H4Swine edemaND−
    E. coli CECT 515TO1:K1(L1):H7Human urine–cystitisND−
    E. coli CECT 533O103:K-:H-ND−
    E. coli CECT 727O111:K58(B4):H-Infantile gastroenteritisEPEC−
    E. coli CECT 730O55:K59(B5):H-ND−
    E. coli CECT 736O28a,28c:K73(B18):H-FecesND−
    E. coli CECT 740O125a,125b:K70(B15):H19GastroenteritisND−
    E. coli CECT 744O158:K-:h23Feces of infant with diarrheaND−
    E. coli CECT 832O111:K58(B4):H-Infantile gastroenteritisND−
    E. coli CECT 4537O10:K5(L5):H4Human peritonitisND−
    E. coli CECT 4555O97:K-:H-ND−
    E. coli CCC-2-12O103Fecal swabNT−
    E. coli CCC-3-12O26Fecal swabStx1, Stx2−
    E. coli CCC-4-12O26Fecal swabNT−
    E. coli CCC-8-12O26Fecal swabNT−
    E. coli CCC-9-12O26Fecal swabNT−
    E. coli CCC-19-12O26Caprine milk filterStx1−
    E. coli CCC-20-12O26Caprine milk filterStx1−
    E. coli CCC-21-12O26Bovine milk filterStx1−
    E. coli CCC-22-12O26Bovine milk filterStx1−
    E. coli CECT 434O6Clinical isolateND−
    E. coli N9NDPorcine fecesND−
    E. coli N5NDBovine fecesND−
    E. coli ATCC 29425 (K12)OR:H48:K-ND−
    Escherichia hermannii ATCC 33650NRHuman isolate−
    Escherichia vulneris ATCC 29943Human wound−
    Shigella boydii ATCC 9207ND−
    Salmonella enterica serovar Typhimurium NCTC 12416−
    Salmonella enterica serovar Typhi SGSC 3036−
    Salmonella enteritidis SGSC 2476−
    Cronobacter sakazakii CECT 858Child's throat−
    Cronobacter sakazakiiMilk−
    Klebsiella pneumoniae ATCC 11296−
    • ↵a SGSC, Salmonella Genetic Stock Centre; ATCC, American Type Culture Collection; NCTC, National Collection of Type Cultures; CECT, Spanish Type Culture Collection.

    • ↵b E. coli O157 strains that tested negative for the presence of H7 antigen.

    • ↵c NT, nontoxigenic E. coli; EPEC, enteropathogenic E. coli (epidemiologically implicated as a pathogen, but the virulence mechanism is not related to the excretion of enterotoxins); ND, not determined; NR, nonrelevant information for the present study.

  • Table 2

    PNA-FISH results obtained for the detection of E. coli O157:H7 on different food matrices inoculated with concentrations between 0.01 and 100 CFU per 25 g or mlc

    Concn (CFU/25 g or ml)PNA-FISH result (PP/TPa) for:
    Ground beefUnpasteurized milk
    37°C41.5°C
    mTSB+NbBPWmTBS+NBPW37°CmTBS+Nb41.5°CmTBS+N
    100+ (6/6)+ (6/6)+ (6/6)+ (6/6)+ (6/6)+ (6/6)
    10+ (6/6)+ (4/6)+ (6/6)+ (2/6)+(6/6)+ (6/6)
    1+ (5/6)− (0/6)+ (5/6)− (0/6)+ (6/6)+ (6/6)
    0.1− (0/0)− (0/0)− (0/0)− (0/0)− (0/0)− (0/0)
    0.01− (0/0)− (0/0)− (0/0)− (0/0)− (0/0)− (0/0)
    • ↵a PP/TP, number of samples that tested positive by PNA-FISH/total number of positive samples as determined by the culture method.

    • ↵b Results were considered for the determination of the method sensitivity, specificity, and accuracy.

    • ↵c Results for the three independent assays, performed for two E. coli O157:H7 strains, are provided.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 -

      Comparison between the culture (ISO 16654:2001) and PNA-FISH results regarding E. coli O157:H7 detection in 60 food samples artificially contaminated, after a pre-enrichment in mTSB and BPW at 37°C (Table S1); partial alignment of 23S rDNA sequences for probe selection (Fig. S1); PNA-FISH outcome for heat-inactivated and viable E. coli CECT 4267 cells (Fig. S2).

      PDF, 360K

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Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method
C. Almeida, J. M. Sousa, R. Rocha, L. Cerqueira, S. Fanning, N. F. Azevedo, M. J. Vieira
Applied and Environmental Microbiology Sep 2013, 79 (20) 6293-6300; DOI: 10.1128/AEM.01009-13

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Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method
C. Almeida, J. M. Sousa, R. Rocha, L. Cerqueira, S. Fanning, N. F. Azevedo, M. J. Vieira
Applied and Environmental Microbiology Sep 2013, 79 (20) 6293-6300; DOI: 10.1128/AEM.01009-13
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