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Food Microbiology

Viability and DNA Maintenance in Nonculturable Spiral Campylobacter jejuni Cells after Long-Term Exposure to Low Temperatures

Beatriz Lázaro, Jose Cárcamo, Ana Audícana, Ildefonso Perales, Aurora Fernández-Astorga
Beatriz Lázaro
Departamento de Inmunologı́a, Microbiologı́a y Parasitologı́a, Facultad de Farmacia, Universidad del Paı́s Vasco (UPV/EHU), 01080 Vitoria-Gasteiz, and
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Jose Cárcamo
Laboratorio Normativo de Salud Pública, Departamento de Sanidad, Gobierno Vasco (GV/EJ), 48010 Bilbao, Spain
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Ana Audícana
Laboratorio Normativo de Salud Pública, Departamento de Sanidad, Gobierno Vasco (GV/EJ), 48010 Bilbao, Spain
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Ildefonso Perales
Laboratorio Normativo de Salud Pública, Departamento de Sanidad, Gobierno Vasco (GV/EJ), 48010 Bilbao, Spain
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Aurora Fernández-Astorga
Departamento de Inmunologı́a, Microbiologı́a y Parasitologı́a, Facultad de Farmacia, Universidad del Paı́s Vasco (UPV/EHU), 01080 Vitoria-Gasteiz, and
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DOI: 10.1128/AEM.65.10.4677-4681.1999
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    Fig. 1.

    Survival curves of C. jejuni C-1 during incubation in PBS at 4°C (A) and 20°C (B) and of its derivative C-1RR at 4°C (C) and 20°C (D). Initial numbers of respiring cells in experiments at 4°C were 9.77 × 108 and 1.23 × 109 ml−1 and numbers of culturable cells were 3.14 × 108 and 2.3 × 109 CFU ml−1 for strains C-1 and C-1RR, respectively. At 20°C, initial numbers of respiring cells were 1.22 × 108 and 1.02 × 109 ml−1 and the numbers of culturable cells were 1.85 × 108 and 1.05 × 109 CFU ml−1 for strains C-1 and C-1RR, respectively. Points are mean values from triplicate determinations.

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    Fig. 2.

    Transmission electron micrographs showing the different morphologies of C. jejuni cells at several times during incubation at 4°C. Bacterial cells were viewed on 300-mesh grids by transmission electron microscopy (Philips CM10 microscope) following negative staining with 2% (wt/vol) potassium phosphotungstate. (A) Typical spiral cell; (B and C) coccoid and concomitant spiral and coccoid forms showing the presence of the flagella; (D and E) cells showing bleb formation (arrows).

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    Fig. 3.

    Two-dimensional, silver-stained protein profiles ofC. jejuni cells. Profiles are shown for culturable cells of strain C-1 (A) and strain C-1RR (B) from late log phase at day 0 (control) and for nonculturable cells of strain C-1 after 196 days of incubation in PBS at 4°C (C) and 20°C (D). Isoelectric focusing was performed with a gel containing 4.1% (vol/vol) acrylamide and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 12.5% (wt/vol) polyacrylamide gel (Mini Protean II two-dimensional system; Bio-Rad). A low-range sodium dodecyl sulfate-polyacrylamide gel electrophoresis standard (14.4 to 97.4 kDa; Bio-Rad) was run in all the gels. Individual proteins that were usually observed in both strains are labeled with arrows. Numbers 1, 2, 3, 4, 5, and 6 show major characteristic protein bands. Number 7 denotes an up-regulated protein after 196 days at 20°C.

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    Fig. 4.

    PFGE of DNA extracted from C. jejuni and digested with SalI (lanes a to e and k) and SmaI (lanes g to i and l). The restriction fragments were separated in a 1.2% agarose gel by electrophoresis for 24 h at 170 V and 14°C, with ramped pulse times from 10 to 35 s (contour-clamped homogeneous electric field DR II PFGE apparatus; Bio-Rad). After electrophoresis, the gels were stained with ethidium bromide. Lanes: a to c, strain C-1 at day 0 of starvation at 1 × 109, 5 × 109, and 1 × 1010 cells ml−1, respectively; d, strain C-1 at day 20 of starvation at 20°C; e, strain C-1 at day 116 of starvation at 4°C; f, λ DNA ladder ranging from 48.5 to 485 kb; g, strain C-1 at day 0 of starvation at 5 × 109 cells ml−1; h, strain C-1 at day 20 of starvation at 20°C, i, strain C-1 at day 116 of starvation at 4°C; j, strain C-1 at day 116 of starvation at 4°C and not digested; k and l, strain C-1 at day 61 of starvation at 20°C.

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Viability and DNA Maintenance in Nonculturable Spiral Campylobacter jejuni Cells after Long-Term Exposure to Low Temperatures
Beatriz Lázaro, Jose Cárcamo, Ana Audícana, Ildefonso Perales, Aurora Fernández-Astorga
Applied and Environmental Microbiology Oct 1999, 65 (10) 4677-4681; DOI: 10.1128/AEM.65.10.4677-4681.1999

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Viability and DNA Maintenance in Nonculturable Spiral Campylobacter jejuni Cells after Long-Term Exposure to Low Temperatures
Beatriz Lázaro, Jose Cárcamo, Ana Audícana, Ildefonso Perales, Aurora Fernández-Astorga
Applied and Environmental Microbiology Oct 1999, 65 (10) 4677-4681; DOI: 10.1128/AEM.65.10.4677-4681.1999
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KEYWORDS

Campylobacter jejuni
DNA, Bacterial

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