Two Subpopulations of Listeria monocytogenes Occur at Subinhibitory Concentrations of Leucocin 4010 and Nisin

151

111

We investigated the degree of physiological damage to bacterial cells caused by optical trapping using a 1,064-nm laser. The physiological condition of the cells was determined by their ability to maintain a pH gradient across the cell wall; healthy cells are able to maintain a pH gradient over the cell wall, whereas compromised cells are less efficient, thus giving rise to a diminished pH gradient. The pH gradient was measured by fluorescence ratio imaging microscopy by incorporating a pH-sensitive fluorescent probe, green fluorescent protein or 5(6)-carboxyfluorescein diacetate succinimidyl ester, inside the bacterial cells. We used the gram-negative species Escherichia coli and three gram-positive species, Listeria monocytogenes, Listeria innocua, and Bacillus subtilis. All cells exhibited some degree of physiological damage, but optically trapped E. coli and L. innocua cells and a subpopulation of L. monocytogenes cells, all grown with shaking, showed only a small decrease in pH gradient across the cell wall when trapped by 6 mW of laser power for 60 min. However, another subpopulation of Listeria monocytogenes cells exhibited signs of physiological damage even while trapped at 6 mW, as did B. subtilis cells. Increasing the laser power to 18 mW caused the pH gradient of both Listeria and E. coli cells to decrease within minutes. Moreover, both species of Listeria exhibited morepronounced physiological damage when grown without shaking than was seen in cells grown with shaking, and the degree of damage is therefore also dependent on the growth conditions.

Section: Discussionmentioning

confidence: 99%

Optical Tweezers Cause Physiological Damage to Escherichia coli and Listeria Bacteria

Rasmussen

Oddershede

Siegumfeldt

2008

151

111

“…In the present study, we did not detect any more tolerant subpopulation, neither in the planktonic nor in the attached population, since the decrease in pH i was very homogenous for all cells over time. Earlier, the FRIM method had successfully been used to detect subpopulations in L. monocytogenes cultures exposed to bacteriocins (12). Similarly, L. monocytogenes cells previously grown on agar plates were heterogeneous with respect to sensitivity to nisin when measuring pH i , and some of the cells appeared to be more tolerant than others (4).…”

Section: Discussionmentioning

confidence: 99%

“…Also, the dissipation of the pH gradient in L. monocytogenes after exposure to different bacteriocins has been determined with FRIM (4, 12). Hornbaek et al (12) found that treatment with subinhibitory concentrations of leucocin and nisin gave rise to two subpopulations: one consisting of cells with a dissipated pH gradient (⌬pH) and the other consisting of cells that maintained ⌬pH, which could indicate phenotypic heterogeneity.…”

mentioning

confidence: 99%

Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

Kastbjerg

Nielsen

Arneborg

et al. 2009

Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pH i ) over time by fluorescence ratio imaging microscopy. pH i values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pH i was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pH i 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.

“…1). Furthermore, the high frequency of revertance to a wild-type phenotype of some of the isolated resistant variants might explain the difficulty of isolating resistant L. monocytogenes variants, as reported before by various research groups (5,16).…”

Section: Discussionmentioning

confidence: 97%

“…The occurrence of stress-resistant variants in monoclonal populations of bacteria remains an important subject of debate, as some research groups have reported the isolation of such variants while others failed to demonstrate their presence and questioned their existence (1,3,5,16,17,19,24,29,31). In this study, we used a new strategy involving a kinetic modeling-based sampling scheme and an optimized stress challenge cycle method for the isolation of stress-resistant variants of L. monocytogenes.…”

Section: Discussionmentioning

confidence: 99%

Isolation of Highly Heat-Resistant Listeria monocytogenes Variants by Use of a Kinetic Modeling-Based Sampling Scheme

Boeijen

Francke

Moezelaar

et al. 2011

Stable high-hydrostatic-pressure (HHP)-resistant Listeria monocytogenes LO28 variants were previously isolated and characterized. These HHP variants were also more resistant to heat. In addition, nonlinear heat inactivation kinetics pointed toward the existence of heat-resistant variants, although these could not be isolated so far. In this study, we used kinetic modeling of inactivation curves of two isolated HHP variants and their wild type, and this revealed that the probability of finding resistant variants should depend on the nature of the inactivation treatment and the time of exposure. At specific heat and HHP conditions, resistant LO28 and EGDe variants were indeed isolated. Resistant LO28 variants were even isolated after a heat inactivation at 72°C in milk, and these variants showed high resistance to standard pasteurization conditions. The increased resistance of part of the isolated LO28 and EGDe variants was due to mutations in their ctsR genes. For the variants whose ctsR genes and upstream regions were not altered, the mechanisms leading to increased resistance remain to be elucidated. This research showed the strength of kinetic modeling in unraveling the causes of nonlinear inactivation and facilitating the isolation of heat-resistant L. monocytogenes variants.Listeria monocytogenes can cause listeriosis in animal and human populations. Human listeriosis is a rare but severe disease and is one of the leading causes of death among patients with food-borne diseases in the United States. The estimated annual rate of invasive listeriosis in the United States is 3 cases and for countries within the European Union is 2 to 10 cases per million people per year (33). Recently, several European countries experienced an apparent increase in the incidence of listeriosis (13).A specific characteristic of L. monocytogenes that appears to be critical to its ability to cause human food-borne illness is its capacity to survive under harsh conditions. The occurrence of variants and the generation of population heterogeneity are factors that may contribute to the survival capacity of L. monocytogenes. Previous research showed that heterogeneity in L. monocytogenes populations (strains EGDe, LO28, and Scott A) affects resistance to high hydrostatic pressure (HHP). Inactivation of such heterogeneous populations resulted in survival curves with significant tailing, indicating the presence of an HHP-sensitive and an HHP-resistant fraction (32). Analysis of the cells that survived such HHP treatments revealed that the higher resistance of LO28 (32) and Scott A (17, 18) was a stable feature for part of the resistant fraction. Contrary to these results, no stable HHP-resistant isolates were obtained for EGDe (32). A significant fraction of the stably resistant variants of both Scott A and LO28 had an altered ctsR gene. This gene encodes CtsR, a DNA binding protein that regulates class III heat shock genes (7). The observed alterations in ctsR resulted not only in increased resistance to high pressure but also in increas...