Selection and identification of a Listeria monocytogenes target strain for pulsed electric field process optimization

Abstract

Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm. When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23 degrees C for 144 micro s, inactivation ranged from 0.7 to 3.7 log(10) CFU/ml. Inactivation by 72- micro s PEF treatments at 37 degrees C ranged from 0.3 to 2.5 log(10) CFU/ml. L. monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains. The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2). Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A. Use of L. monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L. monocytogenes. The nine L. monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques. These strains were better differentiated with PFGE than with AP-PCR. The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.

FIG. 1.

Decrease in log₁₀ CFU per milliliter (PEF inactivation) of L. monocytogenes strains caused by PEF treatment at 25 kV/cm when cells were suspended in 0.1% NaCl. Cell suspensions were adjusted to ∼10⁸ CFU/ml before treatment. (A) Pretreatment temperature, 23°C; and treatment time, 144 μs; (B) pretreatment temperature, 37°C; and treatment time, 72 μs. Different letter designations indicate strains with statistically different (P < 0.05) PEF inactivation levels, based on one-way ANOVA and Tukey mean comparison, within each treatment temperature data set.

FIG. 2.

Survivor plot of L. monocytogenes Scott A and OSY-8578, treated with PEF at 25 kV/cm and 23°C, in 0.1% NaCl or 50% acid whey. Symbols: ○, Scott A; ♦, OSY-8578; dashed lines, 0.1% NaCl; and continuous line, 50% acid whey.

FIG. 3.

Growth (in log₁₀ CFU/milliliter) and PEF inactivation (decrease in log₁₀ CFU/milliliter) of L. monocytogenes Scott A and OSY-8578 after different incubation periods. Treatment with PEF was done with 0.1% NaCl suspension medium (15°C) with an electric field of 25 kV/cm and a total treatment time of 144 μs. For PEF inactivation data, symbols designate data points and lines represent these data after fitting with the second-order polynomial model. Symbols: +, total count during growth (data points for Scott A and OSY-8578 overlapped; therefore, both strains were represented by the same symbol); ○, PEF inactivation of Scott A; and ♦, PEF inactivation of OSY-8578.

FIG. 4.

Banding profiles of L. monocytogenes strains when genomic DNA was amplified by AP-PCR, with the primers PJ108 (A) and PJ118 (B). The PCR products were separated on agarose gel by electrophoresis. Tracks and the corresponding isolates are as follows: 1, 1-kb DNA ladder; 2, Scott A; 3, California; 4, V7; 5, OSY-8517; 6, OSY-8576; 7, OSY-8578; 8, OSY-8579; 9, OSY-8580; 10, OSY-8732; and 11, negative control.

FIG. 5.

Dendrogram demonstrating the genetic relationship of the nine L. monocytogenes strains, based on DNA macrorestriction with ApaI (A) and SmaI (B).