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Bacteriology

Genetic Diversity of Intimin Genes of Attaching and Effacing Escherichia coli Strains

W. L. Zhang, B. Köhler, E. Oswald, L. Beutin, H. Karch, S. Morabito, A. Caprioli, S. Suerbaum, H. Schmidt
W. L. Zhang
1Institut für Hygiene der Westfälischen Wilhelms-Universität, 48149 Münster
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B. Köhler
2Institut für Hygiene und Mikrobiologie der Bayerischen Julius-Maximilians-Universität, 97080 Würzburg
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E. Oswald
3UMR960 INRA de Microbiologie Moleculaire, Ecole Nationale Veterinaire, 31000 Toulouse, France
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L. Beutin
4Division of Emerging Bacterial Pathogens, Robert Koch Institut, 13353 Berlin, Germany
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H. Karch
1Institut für Hygiene der Westfälischen Wilhelms-Universität, 48149 Münster
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S. Morabito
5Laboratorio di Medicina Veterinaria, Istituto Superiore di Sanita, 00161-Rome, Italy
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A. Caprioli
5Laboratorio di Medicina Veterinaria, Istituto Superiore di Sanita, 00161-Rome, Italy
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S. Suerbaum
2Institut für Hygiene und Mikrobiologie der Bayerischen Julius-Maximilians-Universität, 97080 Würzburg
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H. Schmidt
2Institut für Hygiene und Mikrobiologie der Bayerischen Julius-Maximilians-Universität, 97080 Würzburg
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  • For correspondence: Herbert.Schmidt@mailbox.tu-dresden.de
DOI: 10.1128/JCM.40.12.4486-4492.2002
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  • FIG. 1.
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    FIG. 1.

    Phylogenetic tree of intimin variant genes constructed by the UPGMA method of Mega 2.1. Numbers on the branches represent the branch length and denote the genetic distance (e.g., number of substitutions per unit time) between the two taxa they connect. Bold numbers in italics represent bootstrap values.

  • FIG. 2.
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    FIG. 2.

    Split decomposition analysis of intimin variant genes with Splitstree. By this method, evolutionary data are canonically decomposed into a sum of weakly compatible splits and represented by a splits graph. For ideal data, this is a tree; less ideal data will give rise to a tree-like network, which can be interpreted as evidence for different and conflicting phylogenies, such as recombination in the evolutional history. Splitstree demonstrates network-like structures in the 5′ region (positions 1 to 2112) (A), the entire gene (B), and the 3′ region (positions 2112 to end) (C) of intimin alleles.

  • FIG. 3.
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    FIG. 3.

    Illustration of polymorphic sites in the 5′ regions of intimin alleles by Happlot. Vertical lines indicate polymorphic sites.

Tables

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  • TABLE 1.

    Designations, sequence characteristics, and origin of the intimin alleles used in this study

    Designation of intimin alleleORF length (bp)G+C content (%)Reference strain (serotype)Accession no.
    α (alpha)2,82042.09E2348/69 (O127:H6)M58154
    β (beta)2,82042.98RDEC-1 (O15:H−)AF200363
    γ 1 (gamma 1)2,80542.67EDL933 (O157:H7)Z11541.1
    γ 2 (gamma 2)2,80842.9595NR1 (O111:H−)AF025311
    ε (epsilon)2,84742.78PMK5 (O103:H2)AF116899
    ζ (zeta)2,81742.60537/89 (O84:NM)AJ298279
    4795/95 (O84:H4)AJ271407
    η (eta)2,84742.50CF11201 (O125:H−)AJ308550
    θ (theta)2,80843.07CL37 (O111:H8)AF449418
    ι (iota)2,81441.707476/96 (O145:H4)AJ308551
    κ (kappa)2,82042.066044/95 (O118:H5)AJ308552
  • TABLE 2.

    PCR primers and conditions for amplification of intimin alleles

    PrimercSequence of primer (5′ → 3′)Target sequenceReference strain(s)PCR programSize of product (bp)Reference
    SK1CCCGAATTCGGCACAAGCATAAGCConserved re- gion of eae94°C, 30 s; 52°C, 60 s; 72°C, 60 sa,b863 32
    SK2CCCGGATCCGTCTCGCCAGTATTCGEDL933, E2348/69
    LP2CCCGAATTCTTATTTTACACAAGTGGC eae-αE2348/6994°C, 30 s; 55°C, 60 s; 72°C, 120 sa,b2,807 33
    LP3CCCGAATTCTTATTCTACACAAACCGC eae-γEDL93394°C, 30 s; 48°C, 60 s; 72°C, 90 sa, b2,792 33
    94°C, 30 s; 55°C, 60 s; 72°C, 90 sb, e
    LP4CCCGTGATACCAGTACCAATTACGGTC eae-βRDEC-194°C, 30 s; 55°C, 60 s; 72°C, 120 sa,b2,287 28
    LP5AGCTCACTCGTAGATGACGGCAAGCG eae-εPMK594°C, 30 s; 55°C, 60 s; 72°C, 120 sa,b2,608 28
    LP6BTAGTTGTACTCCCCTTATCC eae-ζ4795/9594°C, 30 s; 53°C, 60 s; 72°C, 150 sa,b2,430This study
    LP7TTTATCCTGCTCCGTTTGCT eae-ι7476/9794°C, 30 s; 52°C, 60 s; 72°C, 150 sa,b2,685This study
    LP8TAGATGACGGTAAGCGAC eae-ηCF1120194°C, 30 s; 52°C, 60 s; 72°C, 150 sa,b2,590This study
    LP10GGCATTGTTATCTGTTGTCT eae-κ6044/9594°C, 30 s; 52°C, 60 s; 72°C, 150 sa,b2,769This study
    LP11BGTTGATAACTCCTGATATTTTA eae-θCL-3794°C, 30 s; 50°C, 60 s; 72°C, 150 sa2,686This study
    94°C, 30 s; 50°C, 60 s; 72°C, 150 sb
    • ↵ a Before the first cycle the sample was denatured for 2 min at 94°C.

    • ↵ b After the last cycle, the sample was extended for 5 min at 72°C.

    • ↵ c Primer SK1 was used as forward primer in all PCR approaches in combination with SK2, LP2, LP3, LP4, LP5, LP6B, LP7, LP8, LP10, and LP11B.

    • d Three cycles

    • ↵ e Twenty-eight cycles.

  • TABLE 3.

    Sequence pair distances of intimin genes on the basis of a ClustalW alignment using sequence distance matrix from Bioedit

    GeneDistance with respect toa:
    αβγ1γ2εζηθικ
    α1.0000.8330.8570.8450.824 0.916 0.8470.8450.8700.879
    β1.0000.8300.8400.8630.8270.8180.8380.828 0.886
    γ11.000 0.913 0.8320.8460.841 0.909 0.8700.856
    γ21.0000.8400.8290.828 0.992 0.8590.843
    ε1.0000.816 0.921 0.8390.8300.819
    ζ1.0000.8350.8280.8500.858
    η1.0000.8280.8460.853
    θ1.0000.8580.841
    ι1.0000.868
    κ1.000
    • ↵ a Values represent ratios of identities to the length of the longer sequence of a given pair. Values in bold type are mentioned in the text.

  • TABLE 4.

    Number and distribution of singleton polymorphic sites and parsimony-informative polymorphic sites with two, three, and four different nucleotides in the 5′ and 3′ regions of 10 aligned intimin genes

    Sitea (no. of nucleotides)No. of sites in region:
    5′3′
    SPM (2)10845
    PIPM (2)200185
    SPM (3)69
    PIPM (3)27157
    SPM (4)00
    PIPM (4)757
    • ↵ a SPM, singleton polymorphic sites; PIPM, parsimony-informative polymorphic sites.

  • TABLE 5.

    Frequency of eae types in 111 stx-negative E. coli isolates from patients and carriers

    eae typeNo. of strainsSerotype (no. of strains)
    α3ONT:HNT, O63:H29; O132:H4
    β38O26:H− (12), O26:H11 (9), O14:H−, O81:H−, O90:H−, O108:H−, O103:H2, O5:H4, O121:H−, O20:H−, O33:H6, O33:H2, ONT:H6, Orough:H26, ONT:H32, Orough:HNT, Orough:H11, O145:HNT (2)
    γ35O157:H− (11), O145:H− (7), O145:HNT, O145:H19, O25:H28, O70:H11, O76:H8 (2); O76:H−, O2:H37, O8:H− (2), Orough:H19, ONT:H33, O111:H2, O118:HNT, O125:HNT, O156:H25, O156:H35
    ε14O103:H3, O103:H2 (3), O103:H− (3), O96:H19, ONT:HNT, O121:H−, O157:H−, O8:H−, Orough:H− (2)
    ζ5O156:H25, O104:H−, O85:H− (2), O105:H31
    η3O125:H−; O2:H49,a O12:H19a
    θ0
    ι6O145:H4 (3), ONT:H8, Orough:HNT, O98:H2
    κ2O118:H5; O157:H42a
    NDb5ONT:H−/HNT (2), O5:H−, O132:H4, O54:H−
    • ↵ a Isolates from the RKI, Berlin, Germany. The O2:H49 strain originates from a healthy carrier.

    • ↵ b ND, not determined.

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Genetic Diversity of Intimin Genes of Attaching and Effacing Escherichia coli Strains
W. L. Zhang, B. Köhler, E. Oswald, L. Beutin, H. Karch, S. Morabito, A. Caprioli, S. Suerbaum, H. Schmidt
Journal of Clinical Microbiology Dec 2002, 40 (12) 4486-4492; DOI: 10.1128/JCM.40.12.4486-4492.2002

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Genetic Diversity of Intimin Genes of Attaching and Effacing Escherichia coli Strains
W. L. Zhang, B. Köhler, E. Oswald, L. Beutin, H. Karch, S. Morabito, A. Caprioli, S. Suerbaum, H. Schmidt
Journal of Clinical Microbiology Dec 2002, 40 (12) 4486-4492; DOI: 10.1128/JCM.40.12.4486-4492.2002
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KEYWORDS

Adhesins, Bacterial
Carrier Proteins
Escherichia coli
Escherichia coli Proteins
Genetic Variation

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