Viability and DNA maintenance in nonculturable spiral Campylobacter jejuni cells after long-term exposure to low temperatures

Abstract

Survival of Campylobacter jejuni at 4 and 20 degrees C was investigated by using cellular integrity, respiratory activity, two-dimensional (2D) protein profile, and intact DNA content as indicators of potential viability of nonculturable cells. Intact DNA content after 116 days, along with cellular integrity and respiring cells, was detected for up to 7 months at 4 degrees C by pulsed-field gel electrophoresis. Most changes in 2D protein profiles involved up- or down-regulation.

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-1_RR 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-1_RR, 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-1_RR, respectively. Points are mean values from triplicate determinations.

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).

FIG. 3

Two-dimensional, silver-stained protein profiles of C. jejuni cells. Profiles are shown for culturable cells of strain C-1 (A) and strain C-1_RR (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.

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.