Optimizing Detection of Heat-Injured Listeria monocytogenes in Pasteurized Milk

Abstract

Optimal conditions for the detection of heat-injured cells of Listeria monocytogenes in modified Pennsylvania State University (mPSU) broth were determined using a response surface design generated by a computer program, EChip. Different combinations of incubation temperatures and lithium, magnesium, and D-serine concentrations were evaluated to determine the optimum conditions for the detection of heat-injured L. monocytogenes in filter-sterilized whole milk inoculated with selected problematic background microflora. A concentration of 212 mM lithium chloride completely inhibited the growth of Enterococcus faecium while permitting recovery and detection of L. monocytogenes. A concentration of 15.8 mM MgSO4 was found to be optimum for the recovery and detection of L. monocytogenes. A concentration of 140.2 mM D-serine was found to completely inhibit the germination of Bacillus subtilis var. globii spores but not recovery and detection of L. monocytogenes. Under optimum concentrations of LiCl, MgSO4, and D-serine and in the absence of background microflora, the effect of incubation temperature on percentage detection was described by a second-order polynomial model, and 28 degrees C was determined to be optimal. In the presence of background microflora, the effect of incubation temperature on percentage detection of heat-injured cells was described by a third-order polynomial model, and 30 degrees C was found to be optimal. Optimizing the levels of highly specific and selective agents, nutrients, and incubation temperature in one recovery enrichment system dramatically increased the Listeria/background microflora ratio. This resulting medium, optimized PSU (oPSU) broth, greatly improved the detection of heat-injured and nonheat-injured L. monocytogenes by both conventional and molecular methods (Oxoid's Listeria Rapid Test, Gen-Probe's Accuprobe Listeria monocytogenes Culture Identification Test, and Qualicon's BAX for screening Listeria monocytogenes).

Characterization and resuscitation of ‘non-culturable’ cells of Legionella pneumophila

“…There have been numerous reports that apparently dead cells (injured cells) could be reactivated by inclusion of ROS scavengers in agar plates [26-35]. We therefore added various concentrations of compounds that degrade or block the formation of ROS to standard medium (BCYE) (Table 1).…”

“…Keto acids prevent the toxic effects of light by inhibiting superoxide production and inhibit the rate of cysteine oxidation, an amino acid present in excess in the medium because of the cysteine auxotrophy of L. pneumophila species [46]. The presence of glutamate as well as pyruvate may lead to the formation of antioxidant compounds that directly or indirectly help a subpopulation of injured cells to recover during the plating procedure [26-35]. However, when other antioxidant compounds, including ascorbic acid, propyl gallate or α-ketoglutarate, were added to the standard medium, they failed to significantly restore the culturability of non-culturable L. pneumophila cells (Table 1).…”

“…It has been reported that apparently dead cells could be restored to viability on agar plates supplemented with compounds that degrade or block the formation of reactive oxygen species (ROS) [26-35]. Various stresses, including starvation, hypochlorous acid (HOCl) and heat shock, may leave cells in a vulnerable physiological state (injured state) in which atmospheric oxygen, during the plating procedure, may amplify cellular damage leading to an artifactual loss of culturability [26-35].…”

“…Various stresses, including starvation, hypochlorous acid (HOCl) and heat shock, may leave cells in a vulnerable physiological state (injured state) in which atmospheric oxygen, during the plating procedure, may amplify cellular damage leading to an artifactual loss of culturability [26-35]. In other words, cells detected as VBNC may be A-VBNC cells or D-VBNC cells or cells in an injured state.…”

Cross-protective effects of temperature, pH, and osmotic and starvation stresses in Escherichia coli O157:H7 subjected to pulsed electric fields in milk

“…Although similar studies were carried out with other processing technologies (Hayman, Anantheswaran, & Knabel, 2007;Teo, Ziegler, & Knabel, 2001), few studies have examined the exposure of microorganisms to sub-optimal growth conditions and their subsequent resistance to PEF inactivation.…”