Inducible gene expression by nonculturable bacteria in milk after pasteurization

Abstract

The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5 degrees C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.

FIG. 1.

Viability of bacteria in pasteurized milk. Viability was assayed by PI exclusion (DVC) (open bars) and CFU counts on PCA (shaded bars). Significant differences between the two methods by a two-tailed test are indicated by single asterisks (P < 0.05) and double asterisks (P < 0.01).

FIG. 2.

The gfp gene encoding GFP was introduced into P. putida under the control of a lac promoter and induced by IPTG. (A) Cells in LB medium before induction; (B) cells in LB medium after induction with IPTG; (C) P. putida inoculated into UHT milk; (D) P. putida inoculated into UHT milk and induced with IPTG; (E) P. putida inoculated into UHT milk, followed by heat treatment (63.5°C for 30 min); (F) P. putida inoculated into UHT milk, followed by heat treatment (63.5°C for 30 min) and induction with IPTG. MF, mean fluorescence (arbitrary scale); CV, coefficient of variation. In panel F, the dead-cell population with low green fluorescence equivalent to that seen in panel E has been gated out in order to show the high-fluorescence subpopulation distinctly.

FIG. 3.

Comparison of CFU and GFP-based total counts of E. coli in pasteurized and unpasteurized milk. The asterisk indicates a significant (P < 0.05) difference. Error bars, standard errors of the means of three replicates. UHT milk was inoculated with gfp-tagged E. coli (2.5 × 10⁶ ml⁻¹) and heat treated (63°C for 30 min) before analysis. GFP-based viable counts were measured by epifluorescence microscopy. Solid bars, numbers of GFP-expressing cells per milliliter; open bars, numbers of CFU per milliliter.

FIG. 4.

Comparison of CFU and GFP-based total counts of P. putida in pasteurized and unpasteurized milk. Asterisks indicate significant (P < 0.05) difference. Error bars, standard errors of the means of three replicates. UHT milk was inoculated with gfp-tagged P. putida (5.8 × 10⁵ ml⁻¹) and heat treated at 63.5°C for 30 min. GFP-based viable counts were measured by epifluorescence microscopy. Solid bars, numbers of GFP-expressing cells per milliliter; open bars, numbers of CFU per milliliter.