Resuscitation and growth kinetics of sub-lethally injured Listeria monocytogenes strains following fluorescence activated cell sorting (FACS)

Abstract

This study investigated the effect of acid (pH4.2), osmotic (10% NaCl) and heat (55°C for 30min) stress induced injury on Listeria monocytogenes strains ATCC19115, 69, 159/10 and 243 using differential plating and flow cytometry coupled with membrane integrity indicators, thiazole orange (TO) and propidium iodide (PI) staining. Growth kinetics of injured cells sorted by fluorescence activated cell sorting (FACS) were studied at 4, 25 and 37°C. The percentage of cell injury detectable by both flow cytometry and differential plating varied significantly among strains and stress treatments (p<0.0001). Based on flow cytometry and TO/PI staining, acid stress caused the highest level of injury followed by heat and osmotic stress. Following cell sorting, acid and osmotic stress injured cells were capable of resuscitation and re-growth while heat injured cells (except for strain 69) were incapable of re-growth despite having a high level of membrane intact cells. The lag phase duration (λ) of sorted stress injured cells resuscitated in brain heart infusion (BHI) broth was significantly influenced by strain variations (p<0.0001), stress treatments (p=0.007) and temperature of resuscitation (p≤0.001). Following repair, the maximum specific growth rate (μ_max) of resuscitated cells was not different from untreated control cells regardless of strain differences and stress treatments. Only temperature had a significant effect (p<0.0001) on growth rate. Sorted cells were also capable of growth at 4°C, with the time to detectable growth (≥1.40Log₁₀CFUml^-1) ranging from 3 to 15days. Overall, re-growth potential of sorted cells showed that while membrane integrity was a good indicator of cell injury and viability loss for acid and osmotic stress, it was not a sufficient indicator of heat stress injury. Once injured cells repair the cellular damage, their growth rate is not different from non-injured cells regardless of form of stress and strain differences. Thus highlighting the potential food safety risks of stress injured L. monocytogenes cells.

Keywords: Cell sorting; Flow cytometry; Injury; Listeria monocytogenes; Resuscitation; Stress.