Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes

Abstract

Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes were investigated. For part 1 of this analysis, L. monocytogenes ATCC 19115 was grown to log, stationary, death, and long-term-survival phases at 35 degrees C in tryptic soy broth with yeast extract (TSBYE). Cells were diluted in whole milk that had been subjected to ultrahigh temperatures (UHT whole milk) and then high-pressure processed (HPP) at 400 MPa for 180 s or thermally processed at 62.8 degrees C for 30 s. As cells transitioned from the log to the long-term-survival phase, the D(400 MPa) and D(62.8 degrees C) values increased 10- and 19-fold, respectively. Cells decreased in size as they transitioned from the log to the long-term-survival phase. Rod-shaped cells transitioned to cocci as they entered the late-death and long-term-survival phases. L. monocytogenes strains F5069 and Scott A showed similar results. For part 2 of the analysis, cells in long-term-survival phase were centrifuged, suspended in fresh TSBYE, and incubated at 35 degrees C. As cells transitioned from the long-term-survival phase to log and the stationary phase, they increased in size and log reductions increased following HPP or heat treatment. In part 3 of this analysis, cells in long-term-survival phase were centrifuged, suspended in UHT whole milk, and incubated at 4 degrees C. After HPP or heat treatment, similar results were observed as for part 2. We hypothesize that cells of L. monocytogenes enter a dormant, long-term-survival phase and become more barotolerant and thermotolerant due to cytoplasmic condensation when they transition from rods to cocci. Further research is needed to test this hypothesis and to determine the practical significance of these findings.

FIG. 1.

Growth of L. monocytogenes ATCC 19115 in TSBYE at 35°C for different times to yield log-phase (A), stationary-phase (B), death-phase (C), or long-term-survival-phase (D) cells. The data points and error bars represent means and standard deviations based on three replications of the experiment.

FIG. 2.

Growth of L. monocytogenes at different phases of the life cycle and subsequent survival after HPP and heat treatments. Cells of L. monocytogenes ATCC 19115 were grown in TSBYE at 35°C for different times to yield log-phase (A), stationary-phase (B), death-phase (C), or long-term-survival-phase (D) cells. Milk inoculated with L. monocytogenes was pressure-treated at 400 MPa for 180 s or heat treated at 62.8°C for 30 s. The data points and error bars represent means and standard deviations based on three replications of the experiment.

FIG. 3.

Barotolerance (A) and thermotolerance (B) of L. monocytogenes at different growth phases. Cells of L. monocytogenes ATCC 19115 were grown in TSBYE at 35°C for 12 h, 16 h, 22 h, 41 h, or 30 days to yield late-log-phase, stationary-phase, early-death-phase, late-death-phase, or long-term-survival-phase cells. Milk inoculated with L. monocytogenes was pressure treated at 400 MPa for different times or heat treated at 62.8°C for different times. The data points and error bars represent means and standard deviations based on three replications of the experiment.

FIG. 4.

LM, SEM, and TEM images of L. monocytogenes at different growth phases. Cells of L. monocytogenes ATCC 19115 were grown in TSBYE at 35°C for various times to yield cells at different phases and then observed using LM (Gram stain), SEM, and TEM. Bars: 10 μm (LM), 1 μm (SEM), and 0.5 μm (TEM).

FIG. 5.

Growth of long-term-survival-phase cells of L. monocytogenes after inoculation into fresh TSBYE at 35°C and subsequent survival after HPP and heat treatments. Cells of L. monocytogenes ATCC 19115 were grown in TSBYE at 35°C for 30 days to yield long-term-survival-phase cells, and 10-ml portions of cultures were centrifuged. Pellets were suspended in 10 ml of fresh TSBYE and then incubated at 35°C for various times before being diluted 1:100 in UHT whole milk. Milk inoculated with L. monocytogenes was pressure treated at 400 MPa for 180 s or heat treated at 62.8°C for 30 s. The data points and error bars represent means and standard deviations based on three replications of the experiment.

FIG. 6.

Phase-contrast photomicrographs of long-term-survival-phase cells of L. monocytogenes after inoculation into fresh TSAYE. Photomicrographs were taken at 0 h (A), 1.5 h (B), 2.5 h (C), 3.5 h (D), 5 h (E), and 15 h (F) of incubation. Arrows indicate the transition from a coccoid cell to a rod-shaped cell that formed a microcolony. Bars, 10 μm.

FIG. 7.

Growth of long-term-survival-phase cells of L. monocytogenes after inoculation into UHT whole milk at 4°C and subsequent survival after HPP and heat treatments. Cells of L. monocytogenes ATCC 19115 were grown in TSBYE at 35°C for 30 days to yield long-term-survival-phase cells, and 10-ml portions of cultures were centrifuged. Pellets were suspended in 10 ml of UHT whole milk and then incubated at 4°C for various times before being diluted 1:100 in UHT whole milk. Milk inoculated with L. monocytogenes was pressure treated at 400 MPa for 180 s or heat treated at 62.8°C for 30 s. The data points and error bars represent means and standard deviations based on three replications of the experiment.