Influence of Stress on Single-Cell Lag Time and Growth Probability for Listeria monocytogenes in Half Fraser Broth

The impacts of 12 common food industry stresses on the single-cell growth probability and single-cell lag time distribution of Listeria monocytogenes were determined in half Fraser broth, the primary enrichment broth of the International Organization for Standardization detection method. First, it was determined that the ability of a cell to multiply in half Fraser broth is conditioned by its history (the probability for a cell to multiply can be decreased to 0.05), meaning that, depending on the stress in question, the risk of false-negative samples can be very high. Second, it was established that when cells are injured, the single-cell lag times increase in mean and in variability and that this increase represents a true risk of not reaching the detection threshold of the method in the enrichment broth. No relationship was observed between the impact on single-cell lag times and that on growth probabilities. These results emphasize the importance of taking into account the physiological state of the cells when evaluating the performance of methods to detect pathogens in food.Listeria monocytogenes has been involved in severe food-borne outbreaks with high mortality rates. This pathogen is widespread in many environments (16) and can be isolated from a large variety of foods which are the major routes of infection in humans. Ready-to-eat foods that can support the growth of L. monocytogenes may pose a major risk for public health, and the European Union legislation generally requires absence in 25 g at the production stage as a food safety criterion for this type of food (4).In food, L. monocytogenes is often affected by one or more stresses caused by a variety of processing treatments, including heating, freezing, and exposure to acids and to high osmotic pressures (15, 25, 29, 39). Recovering stressed L. monocytogenes from food is of great importance in food safety since sublethally injured bacteria may repair themselves under suitable conditions and regain or even increase their pathogenicity (19, 30).The injury of microbial cells has two major consequences for pathogen behavior in enrichment broths. First, injured cells become sensitive to selective components present in enrichment broths to which they normally show resistance (9, 10, 11, 42). Therefore, some cells of the stressed bacterial population do not initiate growth in enrichment broth, eventually resulting in an inefficient detection of pathogenic bacteria in food samples (50). This phenomenon can explain results obtained in several studies showing the effect of inoculum size on the growth limits of bacterial populations (26, 27, 37). Second, due to repair time, stressed cells show a longer lag phase than do healthy cells (5, 7, 37). This situation results in a true risk of not reaching the bacterial concentration necessary for the detection of the pathogen (in the range of 10² to 10⁴ CFU ml⁻¹) within the enrichment duration.The recent development of gene-based or immunologically based procedures, such as PCR, gene probes, and enzyme-linked immunosorbent assay, has facilitated the development of more-rapid methods which can identify positive samples in considerably shorter time periods. Nevertheless, these relatively rapid tests also require efficient enrichment steps to increase target organism numbers to detectable levels.At the moment of pathogen detection, low numbers of sublethally injured cells, as often encountered in naturally contaminated foods, show a wide distribution of lag-phase durations (45) and may not be able to multiply in broth containing selective components (11, 42). The challenge of the enrichment stage is to obtain appropriate enrichment conditions (2) which will favor pathogen resuscitation and limit the food microflora growth.In our study, we have focused on the primary enrichment phase of the International Organization for Standardization 11290-1 L. monocytogenes detection method (3), i.e., the half Fraser broth (1/2FB). The objectives were to investigate the impact of 12 different stresses on the single-cell growth probability and single-cell lag time of L. monocytogenes in 1/2FB. The intraspecific variability and the impact of food components and background microflora on single-cell growth probability were also studied.