Study of dormancy states in Listeria monocytogenes

Listeria monocytogenes is a ubiquitous, Gram-positive foodborne pathogen capable of causing severe, potentially fatal invasive listeriosis in vulnerable populations (young, elderly, pregnant, and immunocompromised), with a case-fatality rate of 20-30%. The bacterium is highly resilient to food preservation stresses, displaying a psychrotrophic nature, high salinity tolerance, and the ability to form persistent biofilms on food contact surfaces. To survive prolonged adverse conditions, L. monocytogenes can undergo physiological reprogramming to enter phenotypic dormancy states, such as the viable but non-culturable (VBNC) state or transiently antibiotic tolerant persister cell state. These dormant populations pose severe food safety risks because they escape detection by conventional culturability-based methods, leading to false-negative assessments. This study aimed to characterise the dormancy states (VBNC and persister cells) and biofilm formation in L. monocytogenes strain 113 under environmental stress. The strain (a persistent lineage II strain) was subjected to prolonged nutrient starvation in Minimal Broth Medium (MBM) at 30°C over a 60-day period. Triplicate sampling was performed at days T0, T7, T14, T28, and T60 to examine the relationship between biofilm formation, biomass dynamics and dormancy persistence. Culturability was assessed via standard plate counting on ALOA and PCA agar. Total viable cells were quantified using the photoreactive dye PMAxx combined with real time qPCR, from which VBNC cell counts were estimated. Persister cells were enumerated by treating samples with gentamicin to eliminate metabolically active cells, followed by removal of the stress and plating to recover surviving cells. Biofilm biomass was quantified using crystal violet staining. Finally, resuscitation of VBNC cells was attempted using Listeria Repair Broth (LRB), nutrient-rich broth, and Tryptic Soy Broth supplemented with 0.6% Yeast Extract (TSB+YE). Plate counts revealed a progressive decline in culturability in both fractions. The supernatant fraction decreased from 5.04 Log₁₀ CFU/mL at T0 to undetectable levels by T28, while the biofilm fraction declined from 2.84 Log₁₀ CFU/mL at T7 to undetectable by T60. Conversely, PMAxx qPCR demonstrated a strong inverse correlation. In the supernatant fraction, VBNC populations increased from 0.10 Log₁₀ cells/mL at T0 to 4.62 Log₁₀ cells/mL at T60. In the biofilm fraction, VBNC populations increased from undetectable at T0 to 3.93 Log₁₀ cells/mL at T60, confirming a widespread transition into dormancy rather than cell death in both fractions. No persister cells were detected following gentamicin challenge at any time point, suggesting that entry into the VBNC state is the primary evolutionary survival response to nutrient starvation for this strain. All recovery attempts across multiple specialized formulations failed to resuscitate the VBNC cells at T28 and T60, highlighting a deep, potentially irreversible state of dormancy under the tested criteria. This study demonstrates that conventional culture-based testing significantly underestimates viable L. monocytogenes populations because they can transition to a VBNC state during starvation. While PMAxx-qPCR serves as a robust alternative tool to capture this hidden viability, the induced VBNC state represents a profound physiological shift that resists conventional resuscitation triggers. These findings underscore the need to incorporate viability-based diagnostic tools into food industry surveillance protocols to prevent persistent reservoirs of non-culturable yet physically intact pathogenic populations.