Long-term-survival phase Salmonella enterica: Life cycle comparison with a high persister mutant, and tolerance to atmospheric cold plasma

Abstract

Persister cells and long-term survival (LTS) cells are epigenetic variants that demonstrate a tolerance towards stressful environmental conditions. Persisters can survive exposure to antibiotics while LTS cells can tolerate adverse conditions of the natural environment for extended periods. Bacteria in the LTS phase typically exhibit a decreased vulnerability to physical or chemical antimicrobial treatments compared to exponential- and stationary phase cells. To determine if these two phenomena have a common genetic basis, we measured the viability of a high persister mutant of Salmonella Typhimurium (AS13) throughout four phases of its life cycle under the premise that its high persistence would cause it to enter LTS phase at a higher concentration than the isogenic wild-type strain LT2 or Salmonella Typhimurium ATCC 14028 (Sal 3). All strains were grown in Lysogeny Broth (LB) at 35oC and were initially subjected to a persister cell assay. Over the course of 168 days, a standard plate count was performed at various times to obtain viable cell concentrations of the pathogen at exponential, stationary, death and LTS phases of the life cycle. Counts at 14 days and 140 days represented viable cell concentrations of the S. Typhimurium upon entry in the LTS phase and within the LTS phase, respectively. Stationary phase cells of AS13 had a significantly higher (p<0.05) concentration of persister cells compared to LT2 and Sal3. However, viable counts (log CFU/mL) of AS13, LT2 and Sal 3 were 6.79, 6.94 and 6.11 (day 14) and 4.74, 4.64 and 5.02 (day 140), respectively, indicating that AS13 did not enter into the LTS phase or survive in the LTS phase at a significantly higher concentration (p>0.05) compared to LT2 or Sal3. Based on this result we conclude that the genetic change responsible for the high persister phenotype of AS13 does not increase the viable cell concentration in the LTS phase.

We further evaluated the role of naturally occurring persister cells in the STAT and LTS phase cells of Salmonella Enteritidis ATCC 13076 and evaluated the antimicrobial efficacy of high voltage atmospheric cold plasma (HVACP) against LTS phase and stationary phase (STAT) cells of the pathogen in a model system (PBS, pH 7.0 45kV for 1-4 min) and on shell eggs (60kV for 1-5 min). The effect of HVACP treatments on selected quality characteristics of shell eggs was also evaluated. The S. Enteritidis was grown in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) at 35oC for 20 h (STAT) and 21days (LTS). A significantly higher persister cell concentration (p<0.05) was found in the LTS phase (4.22log CFU/mL) compared to STAT (1.72 log CFU/mL). A significantly lower (p<0.05) inactivation of LTS phase cells of S. Enteritidis occurred in both the PBS and shell eggs compared to STAT cells. In PBS, microbial reductions (log CFU/mL) of STAT cells were 1.0, 0.95, 1.45, and 1.44 after HVACP treatment for 1.0, 2.0, 3.0, and 4.0 min, respectively. In contrast, reductions (log CFU/mL) of LTS cells were significantly lower (p<0.05) at 0.04 (1 min), 0.06 (2 min) 0.01 (3 min), and 0.11 (4 min). A similar pattern was also observed for shell eggs whereby LTS cells exhibited substantially higher tolerance to HVACP than STAT cells (p < 0.05). No differences among eggshell strength and yolk color between HVACP-treated and untreated eggs were observed. Based on these results, we conclude that LTS cells of S. Enteritidis are more tolerant to HVACP than STAT cells and should be considered in the development of Salmonella inactivation protocols involving HVACP treatment of shell eggs. In conclusion, while high persistence does not improve LTS, the state of persistence appears to contribute to the ability of the pathogen survive HVACP treatment. These results suggest that the phenomenon of persistence should be considered in greater depth when considering treatments to improve food safety.