Isolation and quantification of highly acid resistant variants of Listeria monocytogenes

Heterogeneity in stress response of bacteria is one of the biggest challenges posed by minimal processing, which aims at finding the balance between microbiologically stable foods while maintaining the characteristics of fresh products. In this study, exposure of Listeria monocytogenes LO28 to acid stress, which can be encountered in the food processing environment as well as in the human body upon ingestion, led to inactivation kinetics showing considerable tailing, which was described by a biphasic inactivation model. Stable acid resistant variants of L. monocytogenes LO28 were isolated after exposure of late-exponential phase cells to pH 3.5 for 90 min. The resulting 23 stable resistant isolates could be divided in three groups: (a) highly increased acid resistance (<1 log₁₀ reduction, n=16), (b) slightly increased acid resistance (1-3 log₁₀ reduction, n=6), and (c) one isolate showing a variable acid stress response. The highly acid resistant group showed increased resistance to the tested pH range of 2.5 to 3.5 in both late-exponential and stationary phase. Increased acid resistance showed to be significantly correlated to reduced growth rate. The Weibull model was reparameterized, resulting in improved parameter estimation, and was used to estimate the inactivation kinetics at mild pH. Studying the growth boundaries of the wild type and a representative set of variants indicated that the increased resistance of the variants was only related to survival of severe pH stress but did not allow for better growth or survival at mild pH stress. This study shows that acid exposure of late-exponential phase cells reveals the presence of acid resistant subpopulations and that there is a phenotypic diversity amongst them. The occurrence of heterogeneity and stress resistant subpopulations may lead to a higher number of surviving microorganisms than expected. Also, stress resistant subpopulations can become part of the domestic flora in a food production line. The currently isolated acid resistant variants are a new group of stress resistant variants and underline the importance of gaining more insight in the mechanisms underlying this heterogeneity and increased resistance.